Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [H-3]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (-14 +/- 1 mV, n = 12, mean +/- S.E., substituting choline for Na+) or hyperpolarization (-46 +/- 3 mV, n = 12, mean +/- S.E., substituting nitrate for Cl-). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate-albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [H-3]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [H-3]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl- ion. This work contrasts with that for the extraction of [H-3]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [H-3]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [H-3]-palmitate extraction fraction in the IPL.

Tailoring surface properties to build colloidal diagnostic devices: Controlling interparticle associations

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (VIES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.

Concreteness effects in different tasks: implications for models of short-term memory

This study investigates concreteness effects in tasks requiring short-term retention. Concreteness effects were assessed in serial recall, matching span, order reconstruction, and free recall. Each task was carried out both in a control condition and under articulatory suppression. Our results show no dissociation between tasks that do and do not require spoken output. This argues against the redintegration hypothesis according to which lexical-semantic effects in short-term memory arise only at the point of production. In contrast, concreteness effects were modulated by task demands that stressed retention of item versus order information. Concreteness effects were stronger in free recall than in serial recall. Suppression, which weakens phonological representations, enhanced the concreteness effect with item scoring. In a matching task, positive effects of concreteness occurred with open sets but not with closed sets of words. Finally, concreteness effects reversed when the task asked only for recall of word positions (as in the matching task), when phonological representations were weak (because of suppression), and when lexical semantic representations overactivated (because of closed sets). We interpret these results as consistent with a model where phonological representations are crucial for the retention of order, while lexical-semantic representations support maintenance of item identity in both input and output buffers.

Product variety and just-in-time conflict and challenge

This paper addresses the question of how just-in-time can be implemented within high variety manufacture. To illustrate some of the principles in relation to the high variety-low volume situation the case of a computer manufacturer is considered in detail. For contrast the paper also considers the case of the manufacture of highly-configured four wheel drive vehicles where both variety and volumes are high. The most important issue in high variety/low volume production is that JIT operation should be seen in terms of the tactical holding of inventory in upstream buffers within the supply chain so that value is not added to work in progress prematurely. Tactical buffers ensure that service levels are maintained and the risk of stock-outs is minimized. In high variety/high volume production schedule integrity is the key factor, unreliable schedules being a major inhibitor to the introduction of JIT.

Distributed control of computer communication systems

Flow control in Computer Communication systems is generally a multi-layered structure, consisting of several mechanisms operating independently at different levels. Evaluation of the performance of networks in which different flow control mechanisms act simultaneously is an important area of research, and is examined in depth in this thesis. This thesis presents the modelling of a finite resource computer communication network equipped with three levels of flow control, based on closed queueing network theory. The flow control mechanisms considered are: end-to-end control of virtual circuits, network access control of external messages at the entry nodes and the hop level control between nodes. The model is solved by a heuristic technique, based on an equivalent reduced network and the heuristic extensions to the mean value analysis algorithm. The method has significant computational advantages, and overcomes the limitations of the exact methods. It can be used to solve large network models with finite buffers and many virtual circuits. The model and its heuristic solution are validated by simulation. The interaction between the three levels of flow control are investigated. A queueing model is developed for the admission delay on virtual circuits with end-to-end control, in which messages arrive from independent Poisson sources. The selection of optimum window limit is considered. Several advanced network access schemes are postulated to improve the network performance as well as that of selected traffic streams, and numerical results are presented. A model for the dynamic control of input traffic is developed. Based on Markov decision theory, an optimal control policy is formulated. Numerical results are given and throughput-delay performance is shown to be better with dynamic control than with static control.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

All-optical regenerative memory using a single device

In recent years the optical domain has been traditionally reserved for node-to-node transmission with the processing and switching achieved entirely in the electrical domain. However, with the constantly increasing demand for bandwidth and the resultant increase in transmission speeds, there is a very real fear that current electronic technology as used for processing will not be able to cope with future demands. Fuelled by this requirement for faster processing speeds, considerable research is currently being carried out into the potential of All-optical processing. One of the fundamental obstacles in realising All-optical processing is the requirement for All-optical buffering. Without all-optical buffers it is extremely difficult to resolve situations such as contention and congestion. Many devices have been proposed to solve this problem however none of them provide the perfect solution. The subject of this research is to experimentally demonstrate a novel all-optical memory device. Unlike many previously demonstrated optical storage devices the device under consideration utilises only a single loop mirror and a single SOA as its switch, whilst providing full regenerative capabilities required for long-term storage. I will explain some of the principles and characteristics of the device, which will then be experimentally demonstrated. The device configuration will then be studied and investigated as to its suitability for Hybrid Integrated Technology.

Will you tolerate this? The impact of affective commitment on complaint intention and postrecovery behavior

Successful complaint management primarily depends on customers' willingness to voice their complaints and on companies' ability to adequately deal with these complaints. This article investigates the impact of one relationship characteristic in the complaint management process: affective commitment. Based on two studies, the authors investigate whether affective commitment moderates the impact of complaint barriers on complaint intention (a) and whether it moderates the link between complaint satisfaction and purchase behavior after the complaint (b). Results show that affectively committed customers exhibit higher complaint intention irrespective of the level of complaint barriers. Furthermore, affectively committed customers display little change in their postrecovery behavior, even after a service failure followed by an unsatisfactory recovery attempt. It seems that these customers are tolerant and want to help the provider improve their business. Affective commitment seems to amplify willingness to help the company by means of voicing dissatisfaction despite considerable efforts in doing so. Moreover, affective commitment buffers the negative effects of service failures on postrecovery behavior. Findings have important implications for managers. They highlight the necessity to measure customers' affective commitment. Based on that, tailored complaint systems can be designed, which help in achieving a more effective allocation of resources for customer recovery.

Characterization of microvesicles released from human red blood cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

Efficient asynchronous scheduling algorithms for cost-effective buffered crossbar switches

Buffered crossbar switches have recently attracted considerable attention as the next generation of high speed interconnects. They are a special type of crossbar switches with an exclusive buffer at each crosspoint of the crossbar. They demonstrate unique advantages over traditional unbuffered crossbar switches, such as high throughput, low latency, and asynchronous packet scheduling. However, since crosspoint buffers are expensive on-chip memories, it is desired that each crosspoint has only a small buffer. This dissertation proposes a series of practical algorithms and techniques for efficient packet scheduling for buffered crossbar switches. To reduce the hardware cost of such switches and make them scalable, we considered partially buffered crossbars, whose crosspoint buffers can be of an arbitrarily small size. Firstly, we introduced a hybrid scheme called Packet-mode Asynchronous Scheduling Algorithm (PASA) to schedule best effort traffic. PASA combines the features of both distributed and centralized scheduling algorithms and can directly handle variable length packets without Segmentation And Reassembly (SAR). We showed by theoretical analysis that it achieves 100% throughput for any admissible traffic in a crossbar with a speedup of two. Moreover, outputs in PASA have a large probability to avoid the more time-consuming centralized scheduling process, and thus make fast scheduling decisions. Secondly, we proposed the Fair Asynchronous Segment Scheduling (FASS) algorithm to handle guaranteed performance traffic with explicit flow rates. FASS reduces the crosspoint buffer size by dividing packets into shorter segments before transmission. It also provides tight constant performance guarantees by emulating the ideal Generalized Processor Sharing (GPS) model. Furthermore, FASS requires no speedup for the crossbar, lowering the hardware cost and improving the switch capacity. Thirdly, we presented a bandwidth allocation scheme called Queue Length Proportional (QLP) to apply FASS to best effort traffic. QLP dynamically obtains a feasible bandwidth allocation matrix based on the queue length information, and thus assists the crossbar switch to be more work-conserving. The feasibility and stability of QLP were proved, no matter whether the traffic distribution is uniform or non-uniform. Hence, based on bandwidth allocation of QLP, FASS can also achieve 100% throughput for best effort traffic in a crossbar without speedup.

Synthesis of aromatic monothiols and aromatic dithiols to increase the folding rate and yield of disulfide containing proteins

Most pharmaceutically relevant proteins and many extracellular proteins contain disulfide bonds. Formation of the correct disulfide bonds is essential for stability in almost all cases. Disulfide containing proteins can be rapidly and inexpensively overexpressed in bacteria. However, the overexpressed proteins usually form aggregates inside the bacteria, called inclusion bodies, which contains inactive and non-native protein. To obtain native protein, inclusion bodies need to be isolated and resolubilized, and then the resulting protein refolded in vitro. In vitro protein folding is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. The most commonly used redox buffer contains reduced and oxidized glutathione. Recently, aliphatic dithiols and aromatic monothiols have been employed as redox buffers. Aliphatic dithiols improved the yield of native protein as compared to the aliphatic thiol, glutathione. Dithiols mimic the in vivo protein folding catalyst, protein disulfide isomerase, which has two thiols per active site. Furthermore, aromatic monothiols increased the folding rate and yield of lysozyme and RNase A relative to glutathione. By combining the beneficial properties of aliphatic dithiols and aromatic monothiols, aromatic dithiols were designed and were expected to increase in vitro protein folding rates and yields. Aromatic monothiols (1-4) and their corresponding disulfides (5-8), two series of ortho- and para-substituted ethylene glycol dithiols (9-15), and a series of aromatic quaternary ammonium salt dithiols (16-17) were synthesized on a multigram scale. Monothiols and disulfides (1-8) were utilized to fold lysozyme and bovine pancreatic trypsin inhibitor. Dithiols (11-17) were tested for their ability to fold lysozyme. At pH 7.0 and pH 8.0, and high protein concentration (1 mg/mL), aromatic dithiols (16, 17) and a monothiol (3) significantly enhanced the in vitro folding rate and yield of lysozyme relative to the aliphatic thiol, glutathione. Additionally, aromatic dithiols (16, 17) significantly enhance the folding yield as compared to the corresponding aromatic monothiol (3). Thus, the folding rate and yield enhancements achieved in in vitro protein folding at high protein concentration will decrease the volume of renaturation solution required for large scale processes and consequently reduce processing time and cost.

Macroinvertebrate abundance in tropical Andean streams, Ecuador

Deforestation in the tropical Andes is affecting ecological conditions of streams, and determination of how much forest should be retained is a pressing task for conservation, restoration and management strategies. We calculated and analyzed eight benthic metrics (structural, compositional and water quality indices) and a physical-chemical composite index with gradients of vegetation cover to assess the effects of deforestation on macroinvertebrate communities and water quality of 23 streams in southern Ecuadorian Andes. Using a geographical information system (GIS), we quantified vegetation cover at three spatial scales: the entire catchment, the riparian buffer of 30 m width extending the entire stream length, and the local scale defined for a stream reach of 100 m in length and similar buffer width. Macroinvertebrate and water quality metrics had the strongest relationships with vegetation cover at catchment and riparian scales, while vegetation cover did not show any association with the macroinvertebrate metrics at local scale. At catchment scale, the water quality metrics indicate that ecological condition of Andean streams is good when vegetation cover is over 70%. Further, macroinvertebrate community assemblages were more diverse and related in catchments largely covered by native vegetation (>70%). Overall, our results suggest that retaining an important quantity of native vegetation cover within the catchments and a linkage between headwater and riparian forests help to maintain and improve stream biodiversity and water quality in Andean streams affected by deforestation. Also, this research proposes that a strong regulation focused to the management of riparian buffers can be successful when decision making is addressed to conservation/restoration of Andean catchments.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

Decolourisation of Acid orange 7 in a microbial fuel cell with a laccase-based biocathode: Influence of mitigating pH changes in the cathode chamber

Biocathodes may be a suitable replacement of platinum in microbial fuel cells (MFCs) if the cost of MFCs is to be reduced. However, the use of enzymes as bio-cathodes is fraught with loss of activity as time progresses. A possible cause of this loss in activity might be pH increase in the cathode as pH gradients in MFCs are well known. This pH increase is however, accompanied by simultaneous increase in salinity; therefore salinity may be a confounding variable. This study investigated various ways of mitigating pH changes in the cathode of MFCs and their effect on laccase activity and decolourisation of a model azo dye Acid orange 7 in the anode chamber. Experiments were run with catholyte pH automatically controlled via feedback control or by using acetate buffers (pH 4.5) of various strength (100 mM and 200 mM), with CMI7000 as the cation exchange membrane. A comparison was also made between use of CMI7000 and Nafion 117 as the transport properties of cations for both membranes (hence their potential effects on pH changes in the cathode) are different.

Mentale Veerkracht als Buffer tegen Docenten Stress-Reactiviteit: een ESM-studie

Om meer inzicht te krijgen in factoren die positief bijdragen aan de stress-reactiviteit van de docent wordt in deze studie onderzocht in hoeverre mentale veerkracht en docenten eigen effectiviteitsverwachting (DEE) een bufferende werking hebben in de samenhang tussen dagelijkse gebeurtenissen met stress en het positief (PA) en negatief affect (NA). Deze kennis kan gebruikt worden op zowel preventief (selectie) als curatief niveau. Het onderzoek is gedaan onder 41 docenten (leeftijd M=40, SD=9.2) uit verschillende typen onderwijs. Middels de experience sample methode werden gedurende een week activiteitsgerelateerde, sociale en gebeurtenisgerelateerde stress en de emotionele reactiviteit, geobjectiveerd in veranderingen in PA en NA gemeten. Vooraf de onderzoeksweek vulden zij twee vragenlijsten in betreffende mentale veerkracht en DEE. Het meten van positief en negatief affect (PA en NA), activiteitsgerelateerde, sociale en gebeurtenisgerelateerde stress gebeurde door middel van de Experience Sample Methode, zowel via papieren boekje als digitaal (smartphone, tablet, computer). Mentale veerkracht is gemeten met de RS-NL, DEE door middel van een vertaalde vragenlijst van de Teacher Self-Efficacy Scale. Door middel van een multi-level regressieanalysie werden tussen de drie stressmaten en de afhankelijke variabelen PA en NA zijn significante samenhangen gevonden. Volgens de verwachting speelt mentale veerkracht een modererende rol in de samenhang tussen de stressmaten en NA. DEE speelt een modererende rol in de samenhang tussen sociale stress en PA.