bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of ATP in the regulation of intracellular Ca2+ release in single fibres of mouse skeletal muscle.
Authors: Allen D G , Lännergren J , Westerblad H
Published: December 2014

1. Single fibres were dissected from mouse flexor brevis muscle and injected with indo‐1 and the P3‐1 (2‐nitrophenyl)ethyl ester of ATP (caged ATP). Myoplasmic calcium concentration ([Ca2+]i) and force were monitored during single tetani or tetani repeated until force was reduced to about 30% of control values. In vitro experiments showed that an intense, brief ultraviolet illumination (a flash) photolysed 12% of the caged ATP to ATP. 2. Fibres that had been injected with caged ATP showed concentration‐dependent changes. High concentrations of caged ATP caused a reduction in [Ca2+]i during tetani (tetanic [Ca2+]i), a reduction in force in unfatigued tetani and the fibres fatigued more rapidly when stimulated repeatedly. 3. Photolytic release of ATP in unfatigued fibres caused a concentration‐dependent increase in tetanic [Ca2+]i and in force. 4. When ATP was released by photolysis in a fibre fatigued by repeated tetani, it produced a concentration‐dependent increase in tetanic [Ca2+]i and force. The increase in tetanic [Ca2+]i was small (63 nM per 100 microM increase in ATP) and could explain some, but not all, the increase in force. However, taking into account the fact that control flashes in the absence of caged ATP caused a small decrease in tetanic [Ca2+]i, we believe that the increase in force may be explained by the increase in tetanic [Ca2+]i. There was no evidence of changes in the sarcoplasmic reticulum Ca2+ pump rate after photolysis of caged ATP. 5. Caged ATP affects some site(s) involved in excitation‐contraction coupling and the consequences are similar to muscle fatigue. When a small fraction of this caged ATP is photolysed to ATP, the consequences of fatigue are partially reversed. These observations suggest that site(s) which either bind ATP or depend on ATP hydrolysis have a key role in excitation‐contraction coupling and in muscle fatigue.
G‐protein modulation of N‐type calcium channel gating current in human embryonic kidney cells (HEK 293).
Authors: Jones L P , Patil P G , Snutch T P , Yue D T
Published: December 2014

1. Voltage‐dependent inhibition of N‐type calcium currents by G‐proteins contributes importantly to presynaptic inhibition. To examine the effect of G‐proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from recombinant N‐type channels (alpha 1B, beta 1b and alpha 2) expressed in transiently transfected human embryonic kidney cells (HEK 293). Recombinant expression of a homogeneous population of channels provided a favourable system for rigorous examination of the mechanisms underlying G‐protein modulation. 2. During intracellular dialysis with GTP gamma S to activate G‐proteins, ionic currents demonstrated classic features of voltage‐dependent inhibition, i.e. strong depolarizing prepulses increased ionic currents and produced hyperpolarizing shifts in the voltage‐dependent activation of ionic current. No such effects were observed with GDP beta S present to minimize G‐protein activity. 3. Gating currents were clearly resolved after ionic current blockade with 0.1 mM free La3+, enabling this first report of gating charge translocation arising exclusively from N‐type channels. G‐proteins decreased the amplitude of gating currents and produced depolarizing shifts in the voltage‐dependent activation of gating charge movement. However, the greatest effect was to induce a approximately 20 mV separation between the voltage‐dependent activation of gating charge movement and ionic current. Strong depolarizing prepulses largely reversed these effects. These modulatory features provide telling clues about the kinetic steps affected by G‐proteins because gating currents arise from the movement of voltage sensors that trigger channel activation. 4. The mechanistic implications of concomitant G‐protein‐mediated changes in gating and ionic currents are discussed. We argue that G‐proteins act to inhibit both voltage‐sensor movement and the transduction of voltage‐sensor activation into channel opening.
Exotoxin‐insensitive G proteins mediate synaptically evoked muscarinic sodium current in rabbit sympathetic neurones.
Authors: Delmas P , Gola M
Published: December 2014

1. The involvement of G proteins in the transduction pathway that links muscarinic receptors to the low‐threshold voltage‐dependent sodium current (INa,M) was studied in neurones from intact sympathetic prevertebral ganglia using the whole‐cell configuration of the patch‐clamp technique. Experiments were performed in the presence of the nicotinic receptor antagonists hexamethonium (50 microM) and d‐tubocurarine (50 microM). 2. INa,M was activated by either bath‐applying muscarinic agonists or stimulating the preganglionic splanchnic nerves. Synaptically and agonist‐mediated INa,M did not display significant run‐down or changes in their properties in cells tested, irrespective of whether the pipette solutions contained GTP. 3. Dialysis of sympathetic neurones with GDP beta S (500‐750 microM) decreased the amplitude of INa,M by approximately 65% compared with control neurones within 30 min. 4. In the absence of muscarinic receptor stimulation, intracellular dialysis with GTP gamma S (500 microM) for 10 min slowly and slightly (20‐25%) activated INa,M. GTP gamma S dialysis markedly slowed down the decay of INa,M after its transient activation with carbachol pulses (10‐20 s) or nerve stimulation (3‐5 s). The INa,M activation became fully irreversible 2.9 min after the start of GTP gamma S dialysis. Dialysing cells with the G protein activator AIF4‐led to a rapid but transient activation of INa,M. 5. Synaptically and agonist‐evoked INa,M were not affected in ganglia treated with 0.5‐1 microgram ml‐1 pertussis toxin (PTX) for 7‐24 h at 37 degrees C. Control experiments showed that this treatment severely reduced the PTX‐sensitive inhibition of N‐type calcium currents induced by carbachol (CCh) and noradrenaline. Application of NEM (N‐ethylmaleimide) for 2 min depressed the INa,M evoked in response to bath‐applied CCh by only 27%. 6. Incubating ganglia with 5‐10 micrograms ml‐1 of cholera toxin for 7 h had no effect on the carbachol‐induced INa,M but greatly potentiated (approximately 250%) the synaptically evoked INa,M, presumably via a presynaptic mechanism. 7. These results show that the coupling between muscarinic receptors and NaM channels is mediated by pertussis toxin‐ and cholera toxin‐insensitive G proteins, possibly of the Gq/11 or G12 class.
Osmolarity modulates K+ channel function on rat hippocampal interneurons but not CA1 pyramidal neurons.
Authors: Baraban S C , Bellingham M C , Berger A J , Schwartzkroin P A
Published: December 2014

1. Whole‐cell and single‐channel recording methods were used in conjunction with infrared video microscopy techniques to examine the properties of voltage‐activated potassium channels in hippocampal neurons during the application of hyposmolar solutions to hippocampal slices from rats. 2. Hyposmolar external solutions (osmolarity reduced by 10% to 267 mosmol l‐1) produced a significant potentiation of voltage‐activated K+ current on lacunosum/moleculare (L/M) hippocampal interneurons, but not on CA1 and subiculum pyramidal neurons. Hyperpolarization‐activated (IH) and leak currents were not altered during the application of hyposmolar solutions in all cell types. 3. Mean channel open time and the probability of channel opening were dramatically increased under hyposmolar recording conditions for outside‐out patches from L/M interneurons; no changes were observed for patches from CA1 pyramidal neurons. Mean current amplitude and the threshold for channel activation were not affected by hyposmotic challenge. 4. Hyposmolar external solutions produced a significant reduction in the firing frequency of L/M interneurons recorded in current‐clamp mode. Hyposmolar solutions had no effect on resting membrane potential, action potential amplitude or duration, and spike after‐hyperpolarization amplitude. 5. These results indicate that selective modulation of interneuron ion channel activity may be a critical mechanism by which osmolarity can regulate excitability in the central nervous system.
A comparative study of cardiovascular, endocrine and behavioural effects of betamethasone and dexamethasone administration to fetal sheep.
Authors: Derks J B , Giussani D A , Jenkins S L , Wentworth R A , Visser G H , Padbury J F , Nathanielsz P W
Published: December 2014

1. Chronically instrumented, late‐gestation fetal sheep were prepared to: (1) characterize cardiovascular, endocrine and behavioural effects of fetal treatment with clinical doses of betamethasone and dexamethasone; (2) define specific differences, if any, in the actions of betamethasone and dexamethasone of measured fetal responses; and (3) assess the contribution of changes in peripheral vascular resistance to the glucocorticoid‐induced hypertension. 2. Following baseline, either saline (n = 9), betamethasone (n = 9), or dexamethasone (n = 6) was infused for 48 h in fetal sheep commencing at 125 days of gestation. A pronounced increase in fetal blood pressure occurred following both betamethasone and dexamethasone treatment. The nature and magnitude of this increase was similar following treatment with either glucocorticoid. 3. To address possible mechanisms contributing to the glucocorticoid‐induced fetal hypertension, fetal plasma catecholamine levels and changes in fetal femoral haemodynamics were assessed following fetal glucocorticoid treatment. A fall in fetal plasma noradrenaline and adrenaline concentrations occurred during betamethasone and dexamethasone treatment. In contrast, a progressive femoral vasoconstriction occurred during betamethasone treatment. 4. A modest fall in the incidence of fetal breathing movements occurred during fetal treatment with either betamethasone or dexamethasone. The magnitude of this reduction was similar with treatment of either glucocorticoid. The fall in fetal breathing during betamethasone and dexamethasone treatment was not associated with a fall in the incidence of fetal low voltage electrocortical activity. 5. Our results indicate that prenatal betamethasone and dexamethasone treatment of late‐gestation fetal sheep, in doses similar to those employed clinically, is associated with fetal cardiovascular, endocrine and behavioural effects. Both betamethasone and dexamethasone induce similar increases in fetal blood pressure and similar falls in the incidence of fetal breathing movements. The pronounced betamethasone‐induced fetal hypertension is associated with an increase in fetal femoral vascular resistance.
Ca(2+)‐induced Ca2+ release mediates Ca2+ transients evoked by single action potentials in rabbit vagal afferent neurones.
Authors: Cohen A S , Moore K A , Bangalore R , Jafri M S , Weinreich D , Kao J P
Published: December 2014

1. Standard intracellular recording techniques with ‘sharp’ micropipettes were used to evoke action potentials (APs) in acutely dissociated adult nodose neurones. 2. APs induced a transient increase in [Ca2+]i (a calcium transient), recorded with fura‐2, that was dependent upon [Ca2+]o and the number of APs. Over the range of one to sixty‐five APs, the relation between the amplitude of the calcium transient and the number of APs was well fitted by a rectangular hyperbola (chi 2 = 3.53, r = 0.968). From one to four APs, the calcium transient‐AP relation can be described by a line with a slope of 9.6 nM AP‐1 (r = 0.999). 3. Charge movement corresponding to Ca2+ influx evoked by a single AP was 39 +/‐ 2.8 pC (mean +/‐ S.E.M.) and did not change significantly during trains of one to thirty‐one APs (P < 0.05). 4. Caffeine (10 mM), a known agonist of the ryanodine receptor, produced an increase in [Ca2+]i. The caffeine‐induced rise in [Ca2+]i was attenuated (by > 90%) by lowering [Ca2+]o, and by ryanodine (10 microM), 2,5‐di(t‐butyl)hydroquinone (DBHQ, 10 microM), or thapsigargin (100 nM). 5. Neurones incubated with ryanodine, DBHQ or thapsigargin required at least eight APs to evoke a detectable calcium transient. These reagents did not significantly affect Ca2+ influx (P < 0.05). In the presence of these inhibitors, the calcium transient‐AP relation exhibited slopes of 1.2, 1.1 and 1.9 nM AP‐1 for ryanodine, DBHQ and thapsigargin, respectively. When compared with the slope of 9.6 nM AP‐1 in non‐treated neurones, it appears that Ca2+ influx produced by a single AP is amplified by ca 5‐ to 10‐fold.
Alpha 1‐adrenoceptor activation of a non‐selective cation current in rabbit portal vein by 1,2‐diacyl‐sn‐glycerol.
Authors: Helliwell R M , Large W A
Published: December 2014

1. The transduction mechanisms involved in the activation and modulation of the noradrenaline‐activated cation current (Icat) were investigated with whole‐cell patch clamp techniques in rabbit portal vein smooth muscle cells. 2. Intracellular application of guanosine 5‐O‐(3‐thiotriphosphate) (GTP gamma S, 500 microM) evoked a ‘noisy’ inward current at ‐50 mV with a similar current‐voltage relationship and reversal potential to the current evoked by bath application of noradrenaline (100 microM). Guanosine 5‐O‐(2‐thiodiphosphate) (GDP beta S, 1 mM) markedly inhibited noradrenaline‐activated Icat. 3. The phospholipase C (PLC) inhibitor U73122 inhibited the amplitude of the noradrenaline‐activated Icat in a concentration‐ and time‐dependent manner and the IC50 was about 180 nM. U73122 had similar effects on the cation current evoked by GTP gamma S. 4. Intracellular application of myo‐inositol 1,4,5‐trisphosphate (IP3, 100 microM) from the patch pipette did not activate any membrane current in cells where intracellular calcium concentration ([Ca2+]i) was buffered to 14 nM, but subsequent addition of noradrenaline evoked Icat. 5. Bath application of the 1,2‐diacyl‐sn‐glycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 10 microM) activated Icat, whereas the phorbol ester phorbol 12,13‐dibutyrate (PDBu, 0.1‐5 microM) failed to activate Icat, in every cell examined. Icat activated by OAG after bath application of PDBu was not significantly different from OAG‐activated Icat in the absence of PDBu. The DAG lipase inhibitor RHC80267 (10 microM) activated Icat in some cells, whereas the DAG kinase inhibitor R59949 (10 microM) never activated Icat. 6. Bath application of the protein kinase C inhibitor chelerythrine (1‐10 microM) had no effect on either OAG‐or noradrenaline‐activated Icat. 7. It is concluded that noradrenaline activates Icat via a G‐protein coupled to PLC and that the resulting DAG product plays a central role in the activation of cation channels via a protein kinase C‐independent mechanism.
Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10‐CFTR2.
Authors: MacVinish L J , Gill D R , Hyde S C , Mofford K A , Evans M J , Higgins C F , Colledge W H , Huang L , Sorgi F , Ratcliff R , Cuthbert A W
Published: December 2014

1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10‐CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10‐CFTR2, but not pTrial10, demonstrated a cAMP‐dependent anion conductance, measured by fluorescence microscopy using a halide‐sensitive probe, SPQ. 4. The CFTR‐dependent, cAMP‐sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium‐dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)‐ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild‐type and CF‐null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild‐type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR‐dependent current of 11.2 microA cm‐2. 5. Transfection of the airways with pTrial10‐CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR‐dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra‐tracheal installation rather than nasal insufflation was used to deliver the plasmids.
Antagonists of the cGMP‐gated conductance of vertebrate rods block the photocurrent in scallop ciliary photoreceptors.
Authors: Gomez M P , Nasi E
Published: December 2014

1. Hyperpolarizing scallop photoreceptors, like vertebrate rods, use cGMP as an internal messenger and their light‐sensing structure is also of ciliary origin. To ascertain possible functional similarities between the light‐sensitive conductances in the two classes of visual cells, we examined in scallop photoreceptors the effects of several antagonists of the photocurrent of rods. 2. Extracellular application of L‐cis‐diltiazem rapidly and reversibly suppressed the photocurrent. The effect was stereospecific and dose dependent, with a K1/2 of approximately 400 microM. Intracellular dialysis at lower doses (100‐200 microM) also induced a substantial inhibition. 3. L‐cis‐Diltiazem reduced the light‐activated conductance without shifting the intensity‐response curve. Furthermore, the drug also blocked the current directly evoked by application of cGMP. These observations indicate that the inhibitory effects result from blockage of the conductance, rather than from impairment of the activating cascade. 4. The fractional blockage increased e‐fold per approximately 55 mV depolarization, regardless of the side of drug application, as if the charged form of L‐cis‐diltiazem can only access the blocking site from the intracellular compartment. 5. The amiloride derivative 3',4'‐dichlorobenzamil potently suppressed the photocurrent (K1/2 approximately 5 microM), without affecting its kinetics or operating range. Amiloride itself was also effective at higher concentrations. 6. The pharmacological resemblance of these light‐dependent channels to those of rods and cones indicates that significant aspects of the transduction cascade are conserved across disparate sensory cells of ciliary origin.
pH gradient effects on chloride transport across basolateral membrane vesicles from guinea‐pig jejunum.
Authors: Touzani K , Alvarado F , Vasseur M
Published: December 2014

1. The effects of alkaline‐inside pH gradients on 36Cl‐ uptake were quantified by using brush‐border membrane (BBM) and basolateral membrane (BLM) vesicles from guinea‐pig jejunum. 2. With BBM vesicles, a pHo/pHi gradient of 5.0/7.5 yielded fast overshoots involving a random, non‐obligatory Cl(‐)‐H+ symport, strongly inhibited by CCCP. In contrast, BLM vesicles responded to similar pH gradients with much smaller, delayed overshoots, unaffected by CCCP. 3. The initial Cl‐ entry rates into BLM vesicles were a function of each pHo, pHi and delta pH value. They were stimulated by valinomycin in the presence of inward‐directed K+ gradients. Short‐circuiting the membrane potential with equilibrated K+ and valinomycin inhibited pH gradient‐dependent Cl‐ uptake, but only partially. 4. Taken together, these results indicate that guinea‐pig jejunal BLM vesicles possess both Cl‐ conductance and Cl(‐)‐H+ symport activities. 5. Even when different, the BBM and the BLM symporters are mechanistically similar. Neither of them involves a Cl(‐)‐OH‐ antiport, nor a simultaneous Cl(‐)‐anion exchange mechanism. Rather, for each membrane, all of these activities (symport, anion exchange) can be explained in terms of a single mobile carrier acting as a random, non‐obligatory Cl(‐)‐H+ symporter where exchange occurs simply by counterflow. Net Cl‐ translocation via either the ternary (Cl(‐)‐C‐H+) or the binary (Cl(‐)‐C) complexes accounts, respectively, for the existence of two, operationally distinct, electroneutral and rheogenic components. 6. The BBM symporter appears to involve an AE2 protein, but the molecular identity of the BLM one remains to be established.
Cushing's syndrome: from physiological principles to diagnosis and clinical care
Authors: Raff Hershel , Carroll Ty
Published: December 2014

The physiological control of cortisol synthesis in the adrenal cortex involves stimulation of adrenocorticotrophic hormone (ACTH) by hypothalamic corticotrophin‐releasing hormone (CRH) and then stimulation of the adrenal by ACTH. The control loop of the hypothalamic–pituitary–adrenal (HPA) axis is closed by negative feedback of cortisol on the hypothalamus and pituitary. Understanding this system is required to master the diagnosis, differential diagnosis and treatment of endogenous hypercortisolism – Cushing's syndrome. Endogenous Cushing's syndrome is caused either by excess ACTH secretion or by autonomous cortisol release from the adrenal cortex. Diagnosis of cortisol excess exploits three physiological principles: failure to achieve the normal nadir in the cortisol diurnal rhythm, loss of sensitivity of ACTH‐secreting tumours to cortisol negative feedback, and increased excretion of free cortisol in the urine. Differentiating a pituitary source of excess ACTH (Cushing's disease) from an ectopic source is accomplished by imaging the pituitary and sampling for ACTH in the venous drainage of the pituitary. With surgical removal of ACTH or cortisol‐secreting tumours, secondary adrenal insufficiency ensues because of the prior suppression of the HPA axis by glucocorticoid negative feedback. Medical therapy is targeted to the anatomical location of the dysregulated component of the HPA axis. Future research will focus on new diagnostics and treatments of Cushing's syndrome. These are elegant examples of translational research: understanding basic physiology informs the development of new approaches to diagnosis and treatment. Appreciating pathophysiology generates new areas for inquiry of basic physiological and biochemical mechanisms.
Synthesis and Analysis of Soft Parallel Robots Comprised of Active Constraints
Authors: Hopkins Jonathan B. , Rivera Jordan , Kim Charles , Krishnan Girish
Published: December 2014

In this paper, we introduce a new type of spatial parallel robot that is comprised of soft inflatable constraints called trichamber actuators (TCAs). We extend the principles of the freedom and constraint topologies (FACT) synthesis approach to enable the synthesis and analysis of this new type of soft robot. The concepts of passive and active freedom spaces are introduced and applied to the design of general parallel systems that consist of active constraints (i.e., constraint that can be actuated to impart various loads onto the system's stage) that both drive desired motions and guide the system's desired degrees of freedom (DOFs). We provide the fabrication details of the TCA constraints introduced in this paper and experimentally determine their appropriate FACT-based constraint model. We fabricate a soft parallel robot that consists of three TCA constraints and verify and validate its FACT-predicted performance using finite element analysis (FEA) and experimental data. Other such soft robots are synthesized using FACT as case studies.
Rapid Computation of Statistically Stable Particle/Feature Ratios for Consistent Substrate Stresses in Printed Flexible Electronics
Authors: Zohdi T. I.
Published: December 2014

This paper develops a statistically based computational method to rapidly determine stresses in flexible substrates during particle printing processes. Specifically, substrate stresses due to multiple surface particle contact sites are statistically computed by superposing point load solutions for different random particle realizations (sets of random loading sites) within a fixed feature boundary. The approach allows an analyst to rapidly determine the number of particles in a surface feature needed to produce repeatable substrate stresses, thus minimizing the deviation from feature to feature and ensuring consistent production. Three-dimensional examples are provided to illustrate the technique. The utility of the approach is that an analyst can efficiently ascertain the number of particles needed within a feature, without resorting to computationally intensive numerical procedures, such as the finite element method.
Experimental Investigation of Degree of Premixing on Isothermal Air-Water Two-Phase Flow Through Minichannel
Authors: Mehta Hemant B. , Banerjee Jyotirmay
Published: December 2014

The study on the effect of degree of premixing on isothermal gas-liquid two-phase flow pattern and flow pattern maps in microscale channels is inadequate. The present paper discusses the experimental investigation on air-water two-phase flow through 2.15 mm horizontal circular minichannel. Two type of inlet channel geometry, T-junction (TJ) and Y-junction (YJ), are considered. Six different flow patterns are observed and flow pattern maps are developed. The developed flow pattern maps for TJ and YJ channels are compared with the maps available in the literature. The surface tension dominated region is found to have major effect of inlet channel geometry. The results obtained for minichannel can be guidelines to analyze and design diverse two-phase microfluidic applications.
Flow Boiling Heat Transfer Enhancement in Subcooled and Saturated Refrigerants in Minichannel Heat Sinks
Authors: Yakhshi-Tafti Ehsan , Pearlman Howard , You Seung M.
Published: December 2014

Forced two-phase cooling is investigated for handling high power electronics and laser applications having high heat flux and isothermality requirements. Experimental results are reported for minichannel heat sinks with and without enhanced boiling coatings showing increased heat transfer coefficients and higher critical heat flux for coated versus uncoated surfaces.
Analysis of Conjugate Heat Transfer in Microchannels: Solution via Hybrid Analytical-Numerical Method
Authors: Braga N. R. , Sphaier L. A.
Published: December 2014

This paper considers the conjugate problem of convective heat transfer in microchannel coupled to the conduction problem within the channel wall thickness. A Graetz-type simplification is assumed, for which the flow is dynamically developed, while being thermally developing. The boundary conditions for the problem are applied within the outer thickness of the channel wall, and both a constant heat flux condition and a constant are analyzed. The solution procedure is based on the Generalized Integral Transform Technique, by means of which, the temperature field within the fluid and solid wall are expressed in terms of orthogonal eigenseries expansions. The solution is implemented within a computational framework and verified against previously published results and analyses of simple limiting cases. After verification, the effects of the velocity profile and wall thickness on the Nusslet numb are investigated.
Two-Phase Pressure Drop Characteristics During Low Temperature Transients in PEMFCs
Authors: Banerjee Rupak , Kandlikar Satish G.
Published: December 2014

Proton Exchange Membrane fuel cells are being considered as the powertrain of choice for automotive applications. Automotive fuel cells experience transients during start-up, shut-down and changing load conditions, which constitute a significant part of the drive cycle. Transient behavior of PEMFCs can be classified into three categories: electrochemical, thermal and two-phase flow. Two-phase transients require a longer time to return to steady state than the electrochemical transient (which typically requires less than 1 second). Experiments have shown two-phase transients to be more prominent at the lower temperatures due to the increased presence of liquid water.

Overshoot / undershoot behavior of current and voltage has been observed during investigations of electrochemical transients. This study investigates similar overshoot / undershoot behavior in the two-phase pressure drop in the reactant channels. An increase in the current drawn from the PEMFC is accompanied by larger air flow rates and greater water generation. An in situ setup is utilized to measure the pressure drop in the reactant channels across the length of the channel, when the electrical load drawn from the PEMFC is changed. This pressure drop measurement along the length of the reactant channels is used to characterize the overshoot / undershoot behavior.

A parametric study is conducted to identify the factors which influence the overshoot / undershoot in two-phase flow pressure drop. The transient behavior is explored at the temperatures of 40, 60 and 80°C. Transient behavior is more pronounced at the lower temperature. Five different ramp rates have been used to show that faster ramp rates results in larger overshoot. The effect of magnitude of current change is investigated using four levels of load change. It was observed that increased magnitude of change results in increased overshoot behavior. However, no direct relationship has been observed between the magnitude of overshoot and the time required to return to steady state.
The Effect of Injection and Suction on the Interfacial Mass Transport Resistance in a Proton Exchange Membrane Fuel Cell Air Channel
Authors: Koz Mustafa , Kandlikar Satish G.
Published: December 2014

Proton exchange membrane fuel cells are efficient and environmentally friendly electrochemical engines. The present work focuses on air channels that bring the oxidant air into the cell. Characterization of the oxygen concentration drop from the channel to the gas diffusion layer (GDL)-channel interface is a need in the modeling community. This concentration drop is expressed with the non-dimensional Sherwood number (Sh). At the aforementioned interface, the air can have a non-zero velocity normal to the interface: injection of air to the channel and suction of air from the channel. A water droplet in the channel can constrict the channel cross section and lead to a flow through the GDL. In this numerical study, a rectangular air channel, GDL, and a stationary droplet on the GDL-channel interface are simulated to investigate the Sh under droplet induced injection/suction conditions. The simulations are conducted with a commercially available software package, COMSOL Multiphysics.
Optical emission spectroscopic studies and comparisons of CH3F/CO2 and CH3F/O2 inductively coupled plasmas
Authors: Lou Qiaowei , Kaler Sanbir , Donnelly Vincent M. , Economou Demetre J.
Published: December 2014

A CH3F/CO2 inductively coupled plasma (ICP), sustained in a compact plasma reactor, was investigated as a function of power (5–400 W) and feed gas composition, at a pressure of 10 mTorr, using optical emission spectroscopy and rare gas actinometry. Number densities of H, F, and O increased rapidly between 74% and 80% CO2, ascribed to the transition from polymer-covered to polymer-free reactor walls, similar to that found previously in CH3F/O2 ICPs at 48% O2. Below 40% O2 or CO2, relative emission intensity ratios were almost identical for most key species in CH3F/O2 and CH3F/CO2 ICPs except for higher OH/Xe (a qualitative measure of OH and H2O densities) over the full range of CH3F/O2 composition. The number density of H, F, and O increased with power in CH3F/CO2 (20%/80%) plasmas (polymer-free walls), reaching 4.0, 0.34, and 1.6 × 1013/cm3, respectively, at 300 W. The CO number density increased with power and was estimated, based on self-actinometry, to be 8.8 × 1013/cm3 at 300 W. The CO2 number density was independent of power below 40 W (where very little decomposition occurred), and then decreased rapidly with increasing power, reaching 2.8 × 1013/cm3 at 300 W, corresponding to 83% dissociation. Films deposited on p-Si, 10 cm from the open, downstream end of the plasma reactor, were analyzed by x-ray photoelectron spectroscopy. Between 10% and 40% CO2 or O2 addition to CH3F, film deposition rates fell and O content in the films increased. Faster deposition rates in CH3F/CO2 plasmas were ascribed mainly to a larger thermodynamic driving force to form solid carbon, compared with CH3F/O2 plasmas. Oxygen content in the films increased with increasing CO2 or O2 addition, but for the same deposition rate, no substantial differences were observed in the composition of the films.
Bacteriophage Resistance Mechanisms in the Fish Pathogen Flavobacterium psychrophilum: Linking Genomic Mutations to Changes in Bacterial Virulence Factors
Authors: Castillo Daniel , Christiansen Rói Hammershaimb , Dalsgaard Inger , Madsen Lone , Middelboe Mathias
Published: December 2014

Flavobacterium psychrophilum
is an important fish pathogen in salmonid aquaculture worldwide. Due to increased antibiotic resistance, pathogen control using bacteriophages has been explored as a possible alternative treatment. However, the effective use of bacteriophages in pathogen control requires overcoming the selection for phage resistance in the bacterial populations. Here, we analyzed resistance mechanisms in
F. psychrophilum
after phage exposure using whole-genome sequencing of the ancestral phage-sensitive strain 950106-1/1 and six phage-resistant isolates. The phage-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage resistance and the genetic modifications were supported by direct measurements of bacteriophage adsorption rates, biofilm formation, and secretion of extracellular enzymes, which were all impaired in the resistant strains, probably due to superficial structural changes. The clustered regularly interspaced short palindromic repeat (CRISPR) region was unaffected in the resistant isolates and thus did not play a role as a resistance mechanism for
F. psychrophilum
under the current conditions. All together, the results suggest that resistance in
F. psychrophilum
was driven by spontaneous mutations, which were associated with a number of derived effects on the physiological properties of the pathogen, including reduced virulence under
in vitro
conditions. Consequently, phage-driven physiological changes associated with resistance may have implications for the impact of the pathogen in aquaculture, and these effects of phage resistance on host properties are therefore important for the ongoing exploration of phage-based control of
F. psychrophilum
.
Asexual Propagation of a Virulent Clone Complex in a Human and Feline Outbreak of Sporotrichosis
Authors: Teixeira Marcus de Melo , Rodrigues Anderson Messias , Tsui Clement K. M. , de Almeida Luiz Gonzaga Paulo , Van Diepeningen Anne D. , van den Ende Bert Gerrits , Fernandes Geisa Ferreira , Kano Rui , Hamelin Richard C. , Lopes-Bezerra Leila Maria , Vasconcelos Ana Tereza Ribeiro , de Hoog Sybren , de Camargo Zoilo Pires , Felipe Maria Sueli Soares
Published: December 2014

Sporotrichosis is one of the most frequent subcutaneous fungal infections in humans and animals caused by members of the plant-associated, dimorphic genus
Sporothrix
. Three of the four medically important
Sporothrix
species found in Brazil have been considered asexual as no sexual stage has ever been reported in
Sporothrix schenckii
,
Sporothrix brasiliensis
, or
Sporothrix globosa
. We have identified the mating type (
MAT
) loci in the
S. schenckii
(strain 1099-18/ATCC MYA-4821) and
S. brasiliensis
(strain 5110/ATCC MYA-4823) genomes by using comparative genomic approaches to determine the mating type ratio in these pathogen populations. Our analysis revealed the presence of a
MAT1-1
locus in
S. schenckii
while a
MAT1-2
locus was found in
S. brasiliensis
representing genomic synteny to other
Sordariomycetes
. Furthermore, the components of the mitogen-activated protein kinase (MAPK)-pheromone pathway, pheromone processing enzymes, and meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The ratio of
MAT1-1
to
MAT1-2
was not significantly different from 1:1 for all three
Sporothrix
species, but the population of
S. brasiliensis
in the outbreaks originated from a single mating type. We also explored the population genetic structure of these pathogens using sequence data of two loci to improve our knowledge of the pattern of geographic distribution, genetic variation, and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with a low level of haplotype diversity in
S. brasiliensis
isolates from different regions of sporotrichosis outbreaks in Brazil. In contrast,
S. schenckii
isolates demonstrated a high degree of genetic variability without significant geographic differentiation, indicating the presence of recombination. This study demonstrated that two species causing the same disease have contrasting reproductive strategies and genetic variability patterns.
An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei
Authors: McAdams Natalie M. , Ammerman Michelle L. , Nanduri Julee , Lott Kaylen , Fisk John C. , Read Laurie K.
Published: December 2014

In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing.
Trypanosoma brucei
RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an
in vitro
and
in vivo
characterization of TbRGG3 function in
T. brucei
mitochondria. We show that
in vitro
TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions.
In vivo
, inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage
T. brucei
. TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.
Dynamics of Lewis b Binding and Sequence Variation of the
babA
Adhesin Gene during Chronic Helicobacter pylori Infection in Humans
Authors: Nell Sandra , Kennemann Lynn , Schwarz Sandra , Josenhans Christine , Suerbaum Sebastian
Published: December 2014

Helicobacter pylori
undergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin of
H. pylori
is BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed paired
H. pylori
isolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons of
babA
identified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with a
babA
gene amplified from the binding strain,
H. pylori
strains with mosaic
babA
genes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane β-barrel domain, plays an essential role in the biogenesis of this protein.

IMPORTANCE
Helicobacter pylori
causes a chronic infection of the human stomach that can lead to ulcers and cancer. The bacterium can bind to gastric epithelial cells with specialized outer membrane proteins. The best-studied protein is the BabA adhesin which binds to the Lewis b blood group antigen. Since
H. pylori
is a bacterium with very high genetic variability, we asked whether
babA
evolves during chronic infection and how mutations or recombination in
babA
affect binding. We found that BabA-mediated adherence was stable in most individuals but observed a complete loss of binding or reduced binding in 22% of individuals. One strain pair in which binding was lost was used to generate
babA
sequences that were mosaics of a functional allele and a nonfunctional allele, and the mosaic sequences were used to identify amino acids critically involved in binding of BabA to Lewis b.
How equity is addressed in clinical practice guidelines: a content analysis
Authors: Shi Chunhu , Tian Jinhui , Wang Quan , Petkovic Jennifer , Ren Dan , Yang Kehu , Yang Yang
Published: December 2014

Objectives

Considering equity into guidelines presents methodological challenges. This study aims to qualitatively synthesise the methods for incorporating equity in clinical practice guidelines (CPGs).

Setting

Content analysis of methodological publications.

Eligibility criteria for selecting studies

Methodological publications were included if they provided checklists/frameworks on when, how and to what extent equity should be incorporated in CPGs.

Data sources

We electronically searched MEDLINE, retrieved references, and browsed guideline development organisation websites from inception to January 2013. After study selection by two authors, general characteristics and checklists items/framework components from included studies were extracted. Based on the questions or items from checklists/frameworks (unit of analysis), content analysis was conducted to identify themes and questions/items were grouped into these themes.

Primary outcomes

The primary outcomes were methodological themes and processes on how to address equity issues in guideline development.

Results

8 studies with 10 publications were included from 3405 citations. In total, a list of 87 questions/items was generated from 17 checklists/frameworks. After content analysis, questions were grouped into eight themes (‘scoping questions’, ‘searching relevant evidence’, ‘appraising evidence and recommendations’, ‘formulating recommendations’, ‘monitoring implementation’, ‘providing a flow chart to include equity in CPGs’, and ‘others: reporting of guidelines and comments from stakeholders’ for CPG developers and ‘assessing the quality of CPGs’ for CPG users). Four included studies covered more than five of these themes. We also summarised the process of guideline development based on the themes mentioned above.

Conclusions

For disadvantaged population-specific CPGs, eight important methodological issues identified in this review should be considered when including equity in CPGs under the guidance of a scientific guideline development manual.
Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control
Authors: Gray Allan , Wright Alex , Jackson Pete , Hale Mike , Treanor Darren
Published: December 2014

Aims

Histochemical staining of tissue is a fundamental technique in tissue diagnosis and research, but it suffers from significant variability. Efforts to address this include laboratory quality controls and quality assurance schemes, but these rely on subjective interpretation of stain quality, are laborious and have low reproducibility. We aimed (1) to develop a method for histochemical stain quantification using whole slide imaging and image analysis and (2) to demonstrate its usefulness in measuring staining variation.

Methods

A method to quantify the individual stain components of histochemical stains on virtual slides was developed. It was evaluated for repeatability and reproducibility, then applied to control sections of an appendix to quantify H&E staining (H/E intensities and H:E ratio) between automated staining machines and to measure differences between six regional diagnostic laboratories.

Results

The method was validated with <0.5% variation in H:E ratio measurement when using the same scanner for a batch of slides (ie, it was repeatable) but was not highly reproducible between scanners or over time, where variation of 7% was found. Application of the method showed H:E ratios between three staining machines varied from 0.69 to 0.93, H:E ratio variation over time was observed. Interlaboratory comparison demonstrated differences in H:E ratio between regional laboratories from 0.57 to 0.89.

Conclusions

A simple method using whole slide imaging can be used to quantify and compare histochemical staining. This method could be deployed in routine quality assurance and quality control. Work is needed on whole slide imaging devices to improve reproducibility.
Short term outcomes following clipping and coiling of ruptured intracranial aneurysms: does some of the benefit of coiling stem from less procedural impact on deranged physiology at presentation?
Authors: Mortimer Alex M , Bradford Celia , Steinfort Brendan , Faulder Ken , Assaad Nazih , Harrington Timothy
Published: December 2014

Background

Endovascular coiling (EVC) has been shown to yield superior clinical outcomes to surgical clipping (SC) in the treatment of ruptured cerebral aneurysms. The reasons for these differences remain obscure. We aimed to assess outcomes of EVC and SC relative to baseline physiological derangement.

Methods

This was an exploratory analysis of prospectively collected trial data. Physiological derangement was assessed using the Acute Physiology and Chronic Health Evaluation II (APACHE II) scoring system. Other contributory variables such as age, World Federation of Neurosurgical Societies (WFNS) grade, and development of complications, including hydrocephalus and vasospasm, were included in the analysis. Clinical outcome was independently assessed at 90 days using the modified Rankin Scale (mRS). Hospital stay, ventilated days, and total norepinephrine dose were also used as secondary outcomes. Multivariate analysis was performed using binary logistic regression.

Results

EVC was performed in 69 patients and SC in 66 patients. More profound physiological derangement (APACHE II score >15) was the strongest predictor of poor outcome in the overall cohort (OR 17.80, 95% CI 4.78 to 66.21, p<0.0001). For those with more deranged physiology (APACHE II score>15; 59 patients), WFNS grade ≥4 (OR 6.74, 1.43 to 31.75) and SC (OR 6.33, 1.27 to 31.38) were significant predictors of poor outcome (p<0.05). Favorable outcome (mRS 0–2) was seen in 11% of SC patients compared with 38% of EVC patients in this subgroup. SC patients had significantly increased total norepinephrine dose, ventilated days, and hospital stay (p<0.05).

Conclusions

More profound physiological derangement at baseline is a strong predictor of eventual poor outcome, and outcomes for patients with more profound baseline physiological derangement may be improved if undergoing a coiling procedure.