One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species

Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct T_m (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255bp, Sudan 211bp, Reston 192bp, Taï Forest 166bp, Bundibugyo 146bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.     

Oligodeoxynucleotides containing conformationally constrained abasic sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure

The synthesis and incorporation into oligodeoxy­nucleotides of two novel, conformationally restricted abasic (AB) site analogs are described. The stability of oligonucleotide 18mer duplexes containing one such AB site opposite any of the four natural DNA bases was investigated by UV melting curve analysis and compared to that of duplexes containing a conformationally flexible propanediol unit 1 or a tetrahydrofuran unit 2 as an AB site analog. No major differences in the melting temperatures (ΔT_m 0–3°C) between the different abasic duplexes were observed. All AB duplexes were found to have T_ms that were lower by 9–15°C relative to a fully matched 18mer control duplex, and by 4–10°C relative to the corresponding 19mer duplexes in which the AB site is replaced by a mismatched nucleobase. Thus we conclude that the loss of stability of a duplex that is encountered by removal of a nucleobase from the stack cannot be compensated with conformational restriction of the AB site. From the van’t Hoff transition enthalpies obtained from the melting curves, it appears that melting cooperativity is higher for the duplexes containing the conformationally rigid AB sites. Fluorescence quenching experiments with duplexes containing the fluorescent base 2-amino­purine (2AP) opposite the AB sites showed a weak tendency towards more efficient stacking of this base in duplexes containing the conformationally constrained AB sites. Thus, such AB sites may structurally stabilize the cavity formed by the removal of a base. Potential applications emerging from the properties of such conformationally constrained AB sites in DNA diagnostics are discussed.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Effect of insulin-induced hypoglycemia on the serum concentrations of thyroxine,triiodothyronine and reverse triiodothyronine

The effect of insulin-induced hypoglycemia on serum thyroid hormone concentrations was studied in nine healthy individuals. Before, during and after the hypoglycemia blood samples were taken for measurement of the concentrations of glucose, thyroxine (T₄), triiodothyronine (T₃), reverse triiodothyronine (rT₃), catecholamines and pituitary hormones.There was no change in the mean serum T₄ level (± the standard error of the mean) of 67 ± 2 μg/l. However, the T₃ concentrations rose from a mean basal level of 1.86 ± 0.06 μg/l to a mean peak of 2.51 ± 0.21 μg/l (P < 0.01) at 45 minutes after the insulin injection, and the rT₃ concentrations fell from a mean basal level of 0.184 ± 0.008 μg/l to a mean nadir of 0.171 ± 0.022 μg/l (not a significant change). The mean peak epinephrine level was 545 ± 103 ng/l and it occurred between 30 and 45 minutes after the insulin injection; the mean peak norepinephrine level was 584 ± 114 ng/l and it occurred between 30 and 90 minutes after the injection. The growth hormone levels reached a mean peak of 26.1 ± 4.8 μg/l and the plasma cortisol levels rose to 215 ± 9 μg/l. The mean basal prolactin level was 8.5 ± 0.9 μg/l; in five subjects there was a rise to a mean peak of 50.6 ± 14.6 μg/l, whereas in the remaining four no significant increase occurred. No correlation was found between the changes in the serum T₃ concentration and any of the other factors studied.It was concluded that acute hypoglycemia is associated with a rapid increase in the serum T₃ concentration.     

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Kv7 K⁺-channel subunits differ in their apparent affinity for PIP₂ and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP₂ affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M₁ receptors by oxo-M leads to PIP₂ depletion through G_q and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M₁ receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC₅₀ for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhib_max) was 74 ± 3% (n = 5–7). When tonic PIP₂ abundance was increased by overexpression of PIP 5-kinase, the EC₅₀ was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhib_max (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3^T) that increases whole-cell currents by 30-fold without any change in apparent PIP₂ affinity. Kv7.3^T currents had a slightly right-shifted EC₅₀ as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhib_max (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhib_max was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP₂ by coexpressing them with Kv7.3^T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3^T) or higher affinity (Kv7.2 (R463E)/Kv7.3^T) channels, the EC₅₀ and Inhib_max were similar to Kv7.4 or Kv7.3^T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3^T. The resulting heteromer displayed a very low EC₅₀ for inhibition (32 ± 8 nM) and a slightly increased Inhib_max (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP₂ hydrolysis, PIP₂ binding to Kv7-channel subunits, and K⁺ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP₂ affinities.     

Synthesis of 2, 3-dinor-PGFα metabolites

Thir report described the preparation of various 2,3-dinor-PGFα prostaglandins. Of particular importance is the synthesis of 2,3-dinor-15(S)-15-methyl PGF₂α, the primary metabolite in the enzymatic degradation of 15(S)-15-methyl-PGF₂α (1). Introduction of the three carbon β,γ-unsaturated carboxyl side chain was achieved in a one-step Wittig reaction. The 2,3-dinor structural assignments were established by carbon magnetic resonance (crm) spectroscopy.     

Limited heat tolerance in an Arctic passerine: Thermoregulatory implications for cold‐specialized birds in a rapidly warming world

1. Arctic animals inhabit some of the coldest environments on the planet and have evolved physiological mechanisms for minimizing heat loss under extreme cold. However, the Arctic is warming faster than the global average and how well Arctic animals tolerate even moderately high air temperatures (T _a) is unknown.
2. Using flow‐through respirometry, we investigated the heat tolerance and evaporative cooling capacity of snow buntings (Plectrophenax nivalis; ≈31 g, N = 42), a cold specialist, Arctic songbird. We exposed buntings to increasing T _a and measured body temperature (T _b), resting metabolic rate (RMR), rates of evaporative water loss (EWL), and evaporative cooling efficiency (the ratio of evaporative heat loss to metabolic heat production).
3. Buntings had an average (±SD) T _b of 41.3 ± 0.2°C at thermoneutral T _a and increased T _b to a maximum of 43.5 ± 0.3°C. Buntings started panting at T _a of 33.2 ± 1.7°C, with rapid increases in EWL starting at T _a = 34.6°C, meaning they experienced heat stress when air temperatures were well below their body temperature. Maximum rates of EWL were only 2.9× baseline rates at thermoneutral T _a, a markedly lower increase than seen in more heat‐tolerant arid‐zone species (e.g., ≥4.7× baseline rates). Heat‐stressed buntings also had low evaporative cooling efficiencies, with 95% of individuals unable to evaporatively dissipate an amount of heat equivalent to their own metabolic heat production.
4. Our results suggest that buntings’ well‐developed cold tolerance may come at the cost of reduced heat tolerance. As the Arctic warms, and this and other species experience increased periods of heat stress, a limited capacity for evaporative cooling may force birds to increasingly rely on behavioral thermoregulation, such as minimizing activity, at the expense of diminished performance or reproductive investment.

     

Pharmacokinetics and Bioequivalence of Two Formulations of Febuxostat 40-Mg and 80-Mg Tablets: A Randomized,Open-Label, 4-Way Crossover Study in Healthy Chinese Male Volunteers

The present study aimed to investigate the pharmacokinetic properties of febuxostat in healthy Chinese male volunteers and evaluate whether the two formulations of febuxostat 40-mg and 80-mg tablets are bioequivalent. A randomized, open-label, 4-way crossover study was conducted in healthy Chinese male volunteers under fasting conditions. 24 eligible subjects were randomized in a 1:1:1:1 ratio to receive a single dose of test or reference formulation of febuxostat 40-mg or 80-mg tablet. The washout period between each administration was 1 week. Plasma febuxostat was quantified by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Tolerability was evaluated by monitoring adverse events, physical examinations, 12-lead ECG and laboratory tests. After single-dosing of 1 tablet of 40-mg febuxostat, the pharmacokinetic parameters of test and reference formulations were: T_max 1.22±0.87 and 1.85±1.03 h, C_max 1689.16±461.31 and 1613.80±608.43 ng·mL^-1, AUC_0-t 5139.87±1349.28 and 5517.91±2024.26 ng·mL^-1·h, AUC_0−∞ 5263.06±1339.16 and 5640.48±2040.22 ng·mL^-1·h, t_1/2 4.82±2.61 and 4.85±1.78 h, respectively. After single-dosing of 1 tablet of 80-mg febuxostat, the pharmacokinetic parameters of test and reference formulations were: T_max 1.71±1.21 and 2.23±1.55 h, C_max 2744.47±1157.44 and 2998.17±1200.13 ng·mL^-1, AUC_0-t 9634.03±2768.25 and 10467.95±3501.65 ng·mL^-1·h, AUC_0−∞ 9834.32±2730.51 and 10626.63±3504.08 ng·mL^-1·h, t_1/2 6.25±2.44 and 5.46±1.65 h, respectively. For single-dosing of 1 tablet of 40-mg febuxostat, 90% CIs for the test/reference ratio of AUC_0-t, AUC_0−∞ and C_max were 89.79 to 102.55, 90.14 to 102.56 and 93.99 to 129.63, respectively. For single-dosing of 1 tablet of 80-mg febuxostat, 90% CIs for the test/reference ratio of AUC_0-t, AUC_0−∞ and C_max were 86.67 to 100.00, 87.50 to 100.51 and 79.48 to 105.99, respectively. This single dose study revealed similar pharmacokinetic properties in healthy Chinese male volunteers as those found in Caucasic population. The test and reference febuxostat tablets formulations met the regulatory criteria for bioequivalence at 40-mg and 80-mg strengths in fasting healthy Chinese male volunteers.Trial Registration: Chictr.org ChiCTR-TTRCC-14004288     

A Passive Heat Maintenance Strategy Implemented during a Simulated Half-Time Improves Lower Body Power Output and Repeated Sprint Ability in Professional Rugby Union Players

Reduced physical performance has been observed following the half-time period in team sports players, likely due to a decrease in muscle temperature during this period. We examined the effects of a passive heat maintenance strategy employed between successive exercise bouts on core temperature (T_core) and subsequent exercise performance. Eighteen professional Rugby Union players completed this randomised and counter-balanced study. After a standardised warm-up (WU) and 15 min of rest, players completed a repeated sprint test (RSSA 1) and countermovement jumps (CMJ). Thereafter, in normal training attire (Control) or a survival jacket (Passive), players rested for a further 15 min (simulating a typical half-time) before performing a second RSSA (RSSA 2) and CMJ’s. Measurements of T_core were taken at baseline, post-WU, pre-RSSA 1, post-RSSA 1 and pre-RSSA 2. Peak power output (PPO) and repeated sprint ability was assessed before and after the simulated half-time. Similar T_core responses were observed between conditions at baseline (Control: 37.06±0.05°C; Passive: 37.03±0.05°C) and for all other T_core measurements taken before half-time. After the simulated half-time, the decline in T_core was lower (-0.74±0.08% vs. -1.54±0.06%, p<0.001) and PPO was higher (5610±105 W vs. 5440±105 W, p<0.001) in the Passive versus Control condition. The decline in PPO over half-time was related to the decline in T_core (r = 0.632, p = 0.005). In RSSA 2, best, mean and total sprint times were 1.39±0.17% (p<0.001), 0.55±0.06% (p<0.001) and 0.55±0.06% (p<0.001) faster for Passive versus Control. Passive heat maintenance reduced declines in T_core that were observed during a simulated half-time period and improved subsequent PPO and repeated sprint ability in professional Rugby Union players.     

P relaxation responses associated with n(2)/o(2) diffusion in soybean nodule cortical cells and excised cortical tissue

N₂-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O₂ followed by N₂ or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (δ 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O₂ perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T₁), and spin-spin relaxation time (T₂), under perfusion with N₂ or Ar as opposed to O₂, indicated that loss of signal was due to T₁ saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T₁, values derived from biexponential relaxation processes under perfusing O₂ were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N₂ these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T₁ relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T₂ values for cortical tissue were also biexponential but were essentially the same under O₂ (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N₂ (39% 0.07 ± 0.01 s and 61% 0.37 ± 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T₁ decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T₁ experiments demonstrate that O₂ does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O₂ perfusion) is responsible for the observed relaxation changes.     

Highly Sensitive Detection of Melamine Using a One-Step Sample Treatment Combined with a Portable Ag Nanostructure Array SERS Sensor

There is an urgent need for rapid and reliable methods able to detect melamine in animal feed. In this study, a quick, simple, and sensitive method for the determination of melamine content in animal feed was developed using surface-enhanced Raman spectroscopy on fabricated Ag nanorod (AgNR) array substrates with a one-step sample extraction procedure. The AgNR array substrates washed by HNO₃ solvent (10⁻⁷ M) and methanol and showed the good stability within 6 months. The Raman shift at △ν = 682 cm⁻¹ was used as the characteristic melamine peak in the calculations. Sufficient linearity was obtained in the 2–200 μg·g⁻¹ range (R² = 0.926). The limits of detection and quantification were 0.9 and 2 μg·g⁻¹, respectively. The recovery rates were 89.7–93.3%, with coefficients of variation below 2.02%. The method showed good accuracy compared with the tradition GC-MS analysis. This new protocol only need 2 min to fininsh the detection which could be developed for rapid onsite screening of melamine contamination in quality control and market surveillance applications.     

Warming Amplification of Minimum and Maximum Temperatures over High-Elevation Regions across the Globe

An analysis of the annual mean temperature (T_MEAN) (1961–2010) has revealed that warming amplification (altitudinal amplification and regional amplification) is a common feature of major high-elevation regions across the globe against the background of global warming since the mid-20th century. In this study, the authors further examine whether this holds for annual mean minimum temperature (T_MIN) and annual mean maximum temperature (T_MAX) (1961–2010) on a global scale. The extraction method of warming component of altitude, and the paired region comparison method were used in this study. Results show that a significant altitudinal amplification trend in T_MIN (T_MAX) is detected in all (four) of the six high-elevation regions tested, and the average magnitude of altitudinal amplification trend for T_MIN (T_MAX) [0.306±0.086 °C km^-1(0.154±0.213 °C km^-1)] is substantially larger (smaller) than T_MEAN (0.230±0.073 °C km^-1) during the period 1961–2010. For the five paired high- and low-elevation regions available, regional amplification is detected in the four high-elevation regions for T_MIN and T_MAX (respectively or as a whole). Qualitatively, highly (largely) consistent results are observed for T_MIN (T_MAX) compared with those for T_MEAN.     

Ditylenchus dipsaci Infestation of Trifolium repens. I. Temperature Effects,Seedling Invasion,and a Field Survey

Rates of development of stem nematode (Ditylenchus dipsaci) in white clover (Trifolium repens) seedlings were found to be linearly related to temperature. Basal developmental temperature (T_b) was 3 °C, and the thermal constant (S) for development of gravid adult females from freshly laid eggs was 270 accumulated day-degrees above the T_b. Only 12% at 20 °C and 4% at 4 °C of the gravid female nematodes inoculated into seedling axils successfully penetrated seedling epidermis. These nematodes slowly migrated within the seedling and after a lag of 5 days at 20 °C started to lay eggs. The maximal rate of egg production was temperature-dependent, being 0.8 and 3.1 eggs female⁻¹ day⁻¹ at 10 and 20 °C, respectively. Nematodes emigrated rapidly from infested stolons when they were immersed in water, with rates being highest at 25 °C and lowest at 4 °C. The sensitivity to temperature of many of the parameters that govern nematode population dynamics indicates that climatic changes will have a marked effect upon this host-parasite system. A study of infested stolons from the field indicated that nematode numbers increased up to 3,000 or more before tissue senesence, triggered by nematode damage, caused a mass emigration of nematodes from the stolon.     

Potential Impacts of Future Warming and Land Use Changes on Intra-Urban Heat Exposure in Houston,Texas

Extreme heat events in the United States are projected to become more frequent and intense as a result of climate change. We investigated the individual and combined effects of land use and warming on the spatial and temporal distribution of daily minimum temperature (T_min) and daily maximum heat index (HI_max) during summer in Houston, Texas. Present-day (2010) and near-future (2040) parcel-level land use scenarios were embedded within 1-km resolution land surface model (LSM) simulations. For each land use scenario, LSM simulations were conducted for climatic scenarios representative of both the present-day and near-future periods. LSM simulations assuming present-day climate but 2040 land use patterns led to spatially heterogeneous temperature changes characterized by warmer conditions over most areas, with summer average increases of up to 1.5°C (T_min) and 7.3°C (HI_max) in some newly developed suburban areas compared to simulations using 2010 land use patterns. LSM simulations assuming present-day land use but a 1°C temperature increase above the urban canopy (consistent with warming projections for 2040) yielded more spatially homogeneous metropolitan-wide average increases of about 1°C (T_min) and 2.5°C (HI_max), respectively. LSM simulations assuming both land use and warming for 2040 led to summer average increases of up to 2.5°C (T_min) and 8.3°C (HI_max), with the largest increases in areas projected to be converted to residential, industrial and mixed-use types. Our results suggest that urbanization and climate change may significantly increase the average number of summer days that exceed current threshold temperatures for initiating a heat advisory for metropolitan Houston, potentially increasing population exposure to extreme heat.     

Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (β_T) or γ (γ_T) subunits in Xenopus laevis oocytes. αβ and αβ_T channels (9.6 and 8.7 pS, respectively, with 150 mM Li⁺) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P _o) for αβ was 0.54 and for αβ_T was 0.50. In comparison, αγ and αγ_T channels exhibited different kinetics: αγ channels (6.7 pS in Li⁺) had either long open times with short closings, resulting in a high P _o (0.78), or short openings with long closed times, resulting in a low P _o (0.16). The mean P _o for all αγ channels was 0.48. αγ_T (6.6 pS in Li⁺) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P _o of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγ_T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβ_T (1 μM amiloride), and αγ_T (0.1 μM amiloride) channels. αβ and αβ_T had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγ_T was 10-fold slower than αβ_T. Our results indicate that subunit composition of ENaC is a main determinant of P _o. In addition, channel kinetics and P _o are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not P _o.     

Measurement of Pulmonary Flow Reserve and Pulmonary Index of Microcirculatory Resistance for Detection of Pulmonary Microvascular Obstruction

Background

The pulmonary microcirculation is the chief regulatory site for resistance in the pulmonary circuit. Despite pulmonary microvascular dysfunction being implicated in the pathogenesis of several pulmonary vascular conditions, there are currently no techniques for the specific assessment of pulmonary microvascular integrity in humans. Peak hyperemic flow assessment using thermodilution-derived mean transit-time (T_mn) facilitate accurate coronary microcirculatory evaluation, but remain unvalidated in the lung circulation. Using a high primate model, we aimed to explore the use of T_mn as a surrogate of pulmonary blood flow for the purpose of measuring the novel indices Pulmonary Flow Reserve [PFR = (maximum hyperemic)/(basal flow)] and Pulmonary Index of Microcirculatory Resistance [PIMR = (maximum hyperemic distal pulmonary artery pressure)×(maximum hyperemic T_mn)]. Ultimately, we aimed to investigate the effect of progressive pulmonary microvascular obstruction on PFR and PIMR.

Methods and Results

Temperature- and pressure-sensor guidewires (TPSG) were placed in segmental pulmonary arteries (SPA) of 13 baboons and intravascular temperature measured. T_mn and hemodynamics were recorded at rest and following intra-SPA administration of the vasodilator agents adenosine (10–400 µg/kg/min) and papaverine (3–24 mg). Temperature did not vary with intra-SPA sensor position (0.010±0.009 v 0.010±0.009°C; distal v proximal; p = 0.1), supporting T_mn use in lung for the purpose of hemodynamic indices derivation. Adenosine (to 200 µg/kg/min) & papaverine (to 24 mg) induced dose-dependent flow augmentations (40±7% & 35±13% T_mn reductions v baseline, respectively; p<0.0001). PFR and PIMR were then calculated before and after progressive administration of ceramic microspheres into the SPA. Cumulative microsphere doses progressively reduced PFR (1.41±0.06, 1.26±0.19, 1.17±0.07 & 1.01±0.03; for 0, 10⁴, 10⁵ & 10⁶ microspheres; p = 0.009) and increased PIMR (5.7±0.6, 6.3±1.0, 6.8±0.6 & 7.6±0.6 mmHg.sec; p = 0.0048).

Conclusions

Thermodilution-derived mean transit time can be accurately and reproducibly measured in the pulmonary circulation using TPSG. Mean transit time-derived PFR and PIMR can be assessed using a TPSG and adenosine or papaverine as hyperemic agents. These novel indices detect progressive pulmonary microvascular obstruction and thus have with a potential role for pulmonary microcirculatory assessment in humans.     

“First experience with JenaValve?: a single-centre cohort”

Aims

Since the introduction of transcatheter aortic valve implantation (TAVI), newer generation and novel devices such as the retrievable JenaValve™ have been developed. We evaluated the procedural and 6-month results of our first experience with implantation of the JenaValve™.

Methods and results

From June 2012 to December 2013, 24 consecutive patients (mean age 80 ± 7 years, 42 % male) underwent an elective transapical TAVI with the JenaValve™. Device success was 88 %. The mortality rate was 4 % at 30 days and 31 % at 6 months. TAVI reduced the mean transvalvular gradient (44.2 ± 11.1 mmHg vs. 12.3 ± 4.3 mmHg, p < 0.001) and increased the mean aortic valve area (0.8 3 ± 0.23 to 1.70 ± 0.44 cm²). A mild paravalvular leakage (PVL) occurred in 4 patients (18 %) and a moderate PVL in 1 patient (4 %). Mean New York Heart Association Functional Class improved from 2.9 ± 0.5 to 2.0 ± 0.8 at 30 days.

Conclusion

TAVI using the JenaValve™ prosthesis seems adequate and safe in this first experience cohort.     

Evaluación de la efectividad de la prueba rápida OptiMAL-IT™ para el seguimiento de pacientes con diagnóstico de malaria en la Amazonía peruana

Introduction:

In Peru, optical microscopy with the thick smear test continues to be performed for the follow-up of malaria patients. This test is simple but it requires microscopic equipment and suitable staff to perform the reading of the samples. Studies suggest that the rapid OptiMAL-IT™ test is an option for follow-up.

Objective:

To evaluate the effectiveness of OptiMAL-IT™ as a follow-up test in malaria patients in endemic areas of Perú.

Materials and methods:

We conducted an observational, analytical cross-sectional study of diagnostic tests performed in patients with malaria. We selected all the patients attending different health facilities in the Peruvian departments of San Martín and Loreto who met the inclusion criteria. Optical microscopy with thick smear and OptiMAL-IT™ was used on days 2, 3, 7, and 14 for Plasmodium vivax and until day 21 of follow-up for Plasmodium falciparum. Percentages of correctly classified samples and predictive values were calculated, and the results were compared between the western jungle and the eastern jungle using Chi2 or Fisher''s exact tests.

Results:

We registered 262 patients from San Martín and 302 from Loreto. The percentage of correctly classified cases and the negative predictive value were higher than 92.0% and 93,0%, respectively, from the third day of follow-up; no statistical differences were found in the results obtained from the western jungle and those from the eastern jungle.

Conclusions:

The OptiMAL-IT™ test would be effective as a follow-up test in patients diagnosed with malaria in endemic areas of Perú.     

Flavobacterium plurextorum sp. nov. Isolated from Farmed Rainbow Trout (Oncorhynchus mykiss)

Five strains (1126-1H-08^T, 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08^T exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678^T and Flavobacterium pectinovorum DSM 6368^T (98.5% and 97.9% sequence similarity, respectively). DNA–DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08^T, selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678^T and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08^T was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C_15∶0 and C_15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08^T ( = CECT 7844^T = CCUG 60112^T).     

One-Step Transepithelial Topography-Guided Ablation in the Treatment of Myopic Astigmatism

Purpose

To evaluate one-step topography-guided transepithelial ablation in the treatment of low to moderate myopic astigmatism using a 1KHz excimer laser.

Methods

Retrospective study of 117 consecutive eyes available for evaluation 12 months after surgery. Pre- and post-operative visual and refractive data as well as post-operative pain and haze were analyzed. A novel technique integrating custom refractive- and epithelial- ablation in a single uninterrupted procedure was used.

Results

The mean pre-operative spherical equivalent (SE) and the mean cylinder were: –3.22 diopters (D) ±1.54 (SD) (range –0.63 to –7.25 D) and –0.77 D ±0.65 (range 0 to –4.50 D), respectively. At 12 months after surgery: no eyes lost ≥2 lines of corrected distant visual acuity (CDVA). Safety and efficacy indexes were 1.27 and 1.09, respectively. Uncorrected distant visual acuity (UDVA) was ≥20/20 in 96.6% of the eyes. Manifest refraction spherical equivalent was within ±0.5 D of the desired refraction in 93.2% of the eyes. Average root mean square (RMS) wavefront error measured at central 6 mm, increased from 0.38 pre-operatively to 0.47 µm post-operatively. Refractive stability was achieved and sustained 1 month after surgery. No visually significant haze was registered during the observation period. Post-operative pain was reported in 4.5% of patients.

Conclusions

One-step transepithelial topography-guided treatment for low to moderate myopia and astigmatism performed with a 1 KHz laser, provided safe, effective, predictable and stable results with low pain and no visually significant haze.