A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection

PMCID: PMC3393739This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

A Tale of Switched Functions: From Cyclooxygenase Inhibition to M-Channel Modulation in New Diphenylamine Derivatives

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K+ channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K+ channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel

The trend of susceptibilities to amphotericin B and fluconazole of Candida species from 1999 to 2002 in Taiwan

BACKGROUND: Candida species have various degrees of susceptibility to common antifungal drugs. The extent of resistance to amphotericin B and fluconazole of Candida glabrata isolates causing candidemia has been reported. Active surveillance may help us to monitor the trend of susceptibility to antifungal drugs and to determine if there is an emerging co-resistance to both drugs of Candida species, specifically, of C. glabrata in Taiwan. METHODS: The susceptibilities to amphotericin B and fluconazole of Candida species collected in 1999 and 2002 of the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) were determined by the microdilution method. RESULTS: The antifungal susceptibilities of 342 and 456 isolates collected from 11 hospitals participating in both TSARY 1999 and TSARY 2002, respectively, have been determined. The resistance rate to amphotericin B has increased from 0.3% in the TSARY1999 to 2.2% in the TSARY 2002. In contrast, the resistance rate to fluconazole has decreased from 8.8% to 2.2%. Nevertheless, significantly more C. glabrata isolates were not susceptible to fluconazole in the TSARY 2002 (47.4%) than that in the TSARY 1999 (20.8%). There were 9.8% and 11% of C. glabrata isolates having susceptible-dose dependent and resistant phenotype to fluconazole in the TSARY 1999, verse 45.3% and 2.1% in the TSARY 2002. CONCLUSION: There was an increase of resistance rate to amphotericin B in C. glabrata. On the other hand, although the resistance rate to fluconazole has decreased, almost half of C. glabrata isolates were not susceptible to this drug. Hence, continuous monitoring the emerging of co-resistance to both amphotericin B and fluconazole of Candida species, specifically, of C. glabrata, will be an important early-warning system

Arrhythmia Caused by a Drosophila Tropomyosin Mutation Is Revealed Using a Novel Optical Coherence Tomography Instrument

Background: Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows in vivo measurement of cardiac phenotypes and a detecting instrument capable of recording multiple phenotype parameters. Methodology and Principal Findings: With a simple heart, a transparent body surface at larval stages and available genetic tools we chose Drosophila melanogaster as our model organism and developed for it a dual en-face/Doppler optical coherence tomography (OCT) instrument capable of recording multiple aspects of heart activity, including heart contraction cycle dynamics, ostia dynamics, heartbeat rate and rhythm, speed of heart wall movement and light reflectivity of cardiomyocytes in situ. We applied this OCT instrument to a model of Tropomyosin-associated DCM established in adult Drosophila. We show that DCM pre-exists in the larval stage and is accompanied by an arrhythmia previously unidentified in this model. We also detect reduced mobility and light reflectivity of cardiomyocytes in mutants. Conclusion: These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity i

Differential Changes in QTc Duration during In-Hospital Haloperidol Use

Aims: To evaluate changes in QT duration during low-dose haloperidol use, and determine associations between clinical variables and potentially dangerous QT prolongation. Methods: In a retrospective cohort study in a tertiary university teaching hospital in The Netherlands, all 1788 patients receiving haloperidol between 2005 and 2007 were studied; ninety-seven were suitable for final analysis. Rate-corrected QT duration (QTc) was measured before, during and after haloperidol use. Clinical variables before haloperidol use and at the time of each ECG recording were retrieved from hospital charts. Mixed model analysis was used to estimate changes in QT duration. Risk factors for potentially dangerous QT prolongation were estimated by logistic regression analysis. Results: Patients with normal before-haloperidol QTc duration (male <= 430 ms, female <= 450 ms) had a significant increase in QTc duration of 23 ms during haloperidol use; twenty-three percent of patients rose to abnormal levels (male >= 450 ms, female >= 470 ms). In contrast, a significant decrease occurred in patients with borderline (male 430-450 ms, female 450-470 ms) or abnormal before-haloperidol QTc duration (15 ms and 46 ms, respectively); twenty-three percent of patients in the borderline group, and only 9% of patients in the abnormal group obtained abnormal levels. Potentially dangerous QTc prolongation was independently associated with surgery before haloperidol use (OR(adj) 34.9, p = 0.009) and before-haloperidol QTc duration (OR(adj) 0.94, p = 0.004). Conclusion: QTc duration during haloperidol use changes differentially, increasing in patients with normal before-haloperidol QTc duration, but decreasing in patients with prolonged before-haloperidol QTc duration. Shorter before-haloperidol QTc duration and surgery before haloperidol use predict potentially dangerous QTc prolongatio

Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay

Mutations in the potassium channel subunit KCNE1 are associated with early-onset familial atrial fibrillation

<p>Abstract</p> <p>Background</p> <p>Atrial fibrillation (AF) is the most common arrhythmia. The potassium current I_Ksis essential for cardiac repolarization. Gain-of-function mutations in K_V7.1, the pore-forming α-subunit of the I_Kschannel, have been associated with AF. We hypothesized that early-onset lone AF is associated with mutations in the I_Kschannel regulatory subunit KCNE1.</p> <p>Methods</p> <p>In 209 unrelated early-onset lone AF patients (< 40 years) the entire coding sequence of <it>KCNE1 </it>was bidirectionally sequenced. We analyzed the identified KCNE1 mutants electrophysiologically in heterologous expression systems.</p> <p>Results</p> <p>Two non-synonymous mutations G25V and G60D were found in <it>KCNE1 </it>that were not present in the control group (n = 432 alleles) and that have not previously been reported in any publicly available databases or in the exom variant server holding exom data from more than 10.000 alleles. Proband 1 (female, age 45, G25V) had onset of paroxysmal AF at the age of 39 years. Proband 2 (G60D) was diagnosed with lone AF at the age of 33 years. The patient has inherited the mutation from his mother, who also has AF. Both probands had no mutations in genes previously associated with AF. In heterologous expression systems, both mutants showed significant gain-of-function for I_Ksboth with respect to steady-state current levels, kinetic parameters, and heart rate-dependent modulation.</p> <p>Conclusions</p> <p>Mutations in K_V7.1 leading to gain-of-function of I_Kscurrent have previously been described in lone AF, yet this is the first time a mutation in the beta-subunit <it>KCNE1 </it>is associated with the disease. This finding further supports the hypothesis that increased potassium current enhances AF susceptibility.</p

Targeted Deletion of Kcne2 Causes Gastritis Cystica Profunda and Gastric Neoplasia

Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 α subunit and the KCNE2 β subunit provide a K+ efflux current to facilitate gastric acid secretion by the apical H+K+ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2−/− mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2−/−mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia