Re-emergence of dengue, chikungunya, and Zika viruses in 2021 after a 10-year gap in Gabon.

Mosquito-borne viral infections are a major concern in endemic areas, such as Africa. Although outbreaks have been reported throughout Africa, only a few surveillance studies have been conducted in Gabon since the outbreaks of dengue virus (DENV) and chikungunya virus (CHIKV) in 2010. Therefore, the current situation is unknown. This study aimed to investigate the presence of arboviruses, especially DENV (serotypes 1-4), CHIKV, and Zika virus (ZIKV), in Gabon, Central Africa. Between 2020 and 2021, we collected 1060 serum samples from febrile patients and screened them against viruses using reverse transcription-quantitative PCR. We detected two DENV serotypes 1 (DENV-1), one CHIKV, and one ZIKV, and subsequently analyzed the genome sequences. To determine the genetic diversity and transmission route of the viruses, phylogenetic analysis was performed using complete or partial genome sequences. The DENV-1 and CHIKV strains detected in this study were closely related to the previous Gabonese strains, whereas the recent ZIKV strain was genetically different from a strain detected in 2007 in Gabon. This study provides new genomic information on DENV-1, CHIKV, and ZIKV that were detected in Gabon and insight into the circulation of the viruses in the country and their introduction from neighboring African countries.

Identification of novel orthonairoviruses from rodents and shrews in Gabon, Central Africa.

In Africa, several emerging zoonotic viruses have been transmitted from small mammals such as rodents and shrews to humans. Although no clinical cases of small mammal-borne viral diseases have been reported in Central Africa, potential zoonotic viruses have been identified in rodents in the region. Therefore, we hypothesized that there may be unrecognized zoonotic viruses circulating in small mammals in Central Africa. Here, we investigated viruses that have been maintained among wild small mammals in Gabon to understand their potential risks to humans. We identified novel orthonairoviruses in 24.6 % of captured rodents and shrews from their kidney total RNA samples. Phylogenetic analysis revealed that the novel viruses, Lamusara virus (LMSV) and Lamgora virus, were closely related to Erve virus, which was previously identified in shrews of the genus Crocidura and has been suspected to cause neuropathogenic diseases in humans. Moreover, we show that the LMSV ovarian tumour domain protease, one of the virulence determination factors of orthonairoviruses, suppressed interferon signalling in human cells, suggesting the possible human pathogenicity of this virus. Taken together, our study demonstrates the presence of novel orthonairoviruses that may pose unrecognized risks of viral disease transmission in Gabon.

Creatine kinase-(MB) and hepcidin as candidate biomarkers for early diagnosis of pulmonary tuberculosis: a proof-of-concept study in Lambaréné, Gabon.

BACKGROUND: The present study aimed to evaluate the diagnostic utility of creatine kinase-MB (CK-MB), hepcidin (HEPC), phospholipase A2 group IIA (PLa2G2A), and myosin-binding protein C (MYBPC1) for tuberculosis (TB). These four biomarkers are differentially regulated between quiescent Mycobacterium tuberculosis (Mtb) infected individuals (non-progressors to TB disease) and Mtb-infected TB disease progressors 6 months before the onset of symptoms. METHODS: We enrolled samples from patients experiencing moderate-to-severe pulmonary infections diseases including 23 TB cases confirmed by smear microscopy and culture, and 34 TB-negative cases. For each participant, the serum levels of the four biomarkers were measured using ELISA. RESULTS: The levels of CK-MB and HEPC were significantly reduced in patients with active TB disease. CK-MB median level was 2045 pg/ml (1455-4000 pg/ml) in active TB cases and 3245 pg/ml (1645-4000 pg/ml) in non-TB pulmonary diseases. Using the receiver operating characteristic curve (ROC) analysis, HEPC and CK-MB had the Area Under the Curve (AUC) of 79% (95% CI 67-91%) and 81% (95% CI 69-93%), respectively. Both markers correlated with TB diagnosis as a single marker. PLa2G2A and MYBPC1 with AUCs of 48% (95% CI 36-65%) and 62% (95% CI 48-76%) did not performed well as single biomarkers. The three markers'model (CK-MB-HEPC-PLa2G2A) had the highest diagnostic accuracy at 82% (95% CI 56-82%) after cross-validation. CONCLUSION: CK-MB and HEPC levels were statistically different between confirmed TB cases and non-TB cases. This study yields promising results for the rapid diagnosis of TB disease using a single marker or three biomarkers model.

Identification of potential novel hosts and the risk of infection with lymphocytic choriomeningitis virus in humans in Gabon, Central Africa.

OBJECTIVES: Lymphocytic choriomeningitis virus (LCMV), a human pathogenic arenavirus, is distributed worldwide. However, no human cases have been reported in Africa. This study aimed to investigate the current situation and potential risks of LCMV infection in Gabon, Central Africa. METHODS: A total of 492 human samples were screened to detect LCMV genome RNA and anti-LCMV IgG antibodies using reverse transcription-quantitative PCR and enzyme-linked immunosorbent assay (ELISA), respectively. ELISA-positive samples were further examined using a neutralization assay. Viral RNAs and antibodies were also analyzed in 326 animal samples, including rodents, shrews, and bushmeat. RESULTS: While no LCMV RNA was detected in human samples, the overall seroprevalence was 21.5% and was significantly higher in male and adult populations. The neutralization assay identified seven samples with neutralizing activity. LCMV RNA was detected in one species of rodent (Lophuromys sikapusi) and a porcupine, and anti-LCMV IgG antibodies were detected in four rodents and three shrews. CONCLUSIONS: This study determined for the first time the seroprevalence of LCMV in Gabon, and revealed that local rodents, shrews, and porcupines in areas surrounding semi-urban cities posed an infection risk. Hence, LCMV infection should be considered a significant public health concern in Africa.

Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome.

Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships. Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27-37 µM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 µM) and Q52R (IC50 55 µM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2-3 µM), while Q52R inhibition was 15-20% at 10 µM. Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity.

LUF7244 plus Dofetilide Rescues Aberrant Kv11.1 Trafficking and Produces Functional IKv11.1.

Voltage-gated potassium 11.1 (Kv11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-Kv11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 µM dofetilide or 10 µM dofetilide + 5 µM LUF7244. In trafficking defective G601S-Kv11.1 cells, dofetilide (10 µM) or dofetilide + LUF7244 (10 + 5 µM) also restored Kv11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 µM) increased IKv 11.1 despite the presence of dofetilide (1 µM) in WT Kv11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 µM) and dofetilide (1 µM) increased IKv11.1 compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1 Thus, combined administration of LUF7244 and an IKv11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects. SIGNIFICANCE STATEMENT: Decreased levels of functional Kv11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of Kv11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired Kv11.1 trafficking defects, resulting in functional Kv11.1 current.

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification.

KIR2.1 potassium channels, producing inward rectifier potassium current (I K1 ), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the "epestfind" webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1 , however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels.

LUF7244, an allosteric modulator/activator of Kv 11.1 channels, counteracts dofetilide-induced torsades de pointes arrhythmia in the chronic atrioventricular block dog model.

BACKGROUND AND PURPOSE: Kv 11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv 11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model. EXPERIMENTAL APPROACH: LUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide-induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block. KEY RESULTS: LUF7244 (0.5-10 µM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 µM) had no effects on IKIR2.1 , INav1.5 , ICa-L , and IKs , doubled IKr , shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide-induced early afterdepolarizations. LUF7244 (2.5 mg·kg-1 ·15 min-1 ) in dogs with sinus rhythm was not proarrhythmic and shortened, non-significantly, repolarization parameters (QTc: -6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide-induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 µM after chronic atrioventricular block. CONCLUSIONS AND IMPLICATIONS: LUF7244 counteracted dofetilide-induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv 11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

Computational Identification of Novel Kir6 Channel Inhibitors.

KATP channels consist of four Kir6.x pore-forming subunits and four regulatory sulfonylurea receptor (SUR) subunits. These channels couple the metabolic state of the cell to membrane excitability and play a key role in physiological processes such as insulin secretion in the pancreas, protection of cardiac muscle during ischemia and hypoxic vasodilation of arterial smooth muscle cells. Abnormal channel function resulting from inherited gain or loss-of-function mutations in either the Kir6.x and/or SUR subunits are associated with severe diseases such as neonatal diabetes, congenital hyperinsulinism, or Cantú syndrome (CS). CS is an ultra-rare genetic autosomal dominant disorder, caused by dominant gain-of-function mutations in SUR2A or Kir6.1 subunits. No specific pharmacotherapeutic treatment options are currently available for CS. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for CS, particular for mutations, which lack high affinity for sulfonylurea inhibitor glibenclamide. By applying a combination of computational methods including atomistic MD simulations, free energy calculations and pharmacophore modeling, we identified several novel Kir6.1 inhibitors, which might be possible candidates for drug repurposing. The in silico predictions were confirmed using inside/out patch-clamp analysis. Importantly, Cantú mutation C166S in Kir6.2 (equivalent to C176S in Kir6.1) and S1020P in SUR2A, retained high affinity toward the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point toward developing novel therapies for Cantú disease.

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents.

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM IKATP channel structures. Mg-ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 µmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 µmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.

Istaroxime, a positive inotropic agent devoid of proarrhythmic properties in sensitive chronic atrioventricular block dogs.

Current inotropic agents in heart failure therapy associate with low benefit and significant adverse effects, including ventricular arrhythmias. Istaroxime, a novel Na+/K+-transporting ATPase inhibitor, also stimulates SERCA2a activity, which would confer improved inotropic and lusitropic properties with less proarrhythmic effects. We investigated hemodynamic, electrophysiological and potential proarrhythmic and antiarrhythmic effects of istaroxime in control and chronic atrioventricular block (CAVB) dogs sensitive to drug-induced Torsades de Pointes arrhythmias (TdP). In isolated normal canine ventricular cardiomyocytes, istaroxime (0.3-10 µM) evoked no afterdepolarizations and significantly shortened action potential duration (APD) at 3 and 10 µM. Istaroxime at 3 µg/kg/min significantly increased left ventricular (LV) contractility (dP/dt+) and relaxation (dP/dt-) respectively by 81 and 94% in anesthetized control dogs (n = 6) and by 61 and 49% in anesthetized CAVB dogs (n = 7) sensitive to dofetilide-induced TdP. While istaroxime induced no ventricular arrhythmias in control conditions, only single ectopic beats occurred in 2/7 CAVB dogs, which were preceded by increase of short-term variability of repolarization (STV) and T wave alternans in LV unipolar electrograms. Istaroxime pre-treatment (3 µg/kg/min for 60 min) did not alleviate dofetilide-induced increase in repolarization and STV, and mildly reduced incidence of TdP from 6/6 to 4/6 CAVB dogs. In six CAVB dogs with dofetilide-induced TdP, administration of istaroxime (90 µg/kg/5 min) suppressed arrhythmic episodes in two animals. Taken together, inotropic and lusitropic properties of istaroxime in CAVB dogs were devoid of significant proarrhythmic effects in sensitive CAVB dogs, and istaroxime provides a moderate antiarrhythmic efficacy in prevention and suppression of dofetilide-induced TdP.

Selective late sodium current inhibitor GS-458967 suppresses Torsades de Pointes by mostly affecting perpetuation but not initiation of the arrhythmia.

BACKGROUND AND PURPOSE: Enhanced late sodium current (late INa ) in heart failure and long QT syndrome type 3 is proarrhythmic. This study investigated the antiarrhythmic effect and mode of action of the selective and potent late INa inhibitor GS-458967 (GS967) against Torsades de Pointes arrhythmias (TdP) in the chronic atrioventricular block (CAVB) dog. EXPERIMENTAL APPROACH: Electrophysiological and antiarrhythmic effects of GS967 were evaluated in isolated canine ventricular cardiomyocytes and CAVB dogs with dofetilide-induced early afterdepolarizations (EADs) and TdP, respectively. Mapping of intramural cardiac electrical activity in vivo was conducted to study effects of GS967 on spatial dispersion of repolarization. KEY RESULTS: GS967 (IC50 ~200nM) significantly shortened repolarization in canine ventricular cardiomyocytes and sinus rhythm (SR) dogs, in a concentration and dose-dependent manner. In vitro, despite addition of 1µM GS967, dofetilide-induced EADs remained present in 42% and 35% of cardiomyocytes from SR and CAVB dogs, respectively. Nonetheless, GS967 (787±265nM) completely abolished dofetilide-induced TdP in CAVB dogs (10/14 after dofetilide to 0/14 dogs after GS967), while single ectopic beats (sEB) persisted in 9 animals. In vivo mapping experiments showed that GS967 significantly reduced spatial dispersion of repolarization: cubic dispersion was significantly decreased from 237±54ms after dofetilide to 123±34ms after GS967. CONCLUSION AND IMPLICATIONS: GS967 terminated all dofetilide-induced TdP without completely suppressing EADs and sEB in vitro and in vivo, respectively. The antiarrhythmic mode of action of GS967, through the reduction of spatial dispersion of repolarization, seems to predominantly impede the perpetuation of arrhythmic events into TdP rather than their initiating trigger.

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression.

BACKGROUND: The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 µM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. METHODS: Molecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. RESULTS: PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 µM of PA-6 inhibited outward IK1 at -50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 µM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 µM). CONCLUSIONS: 1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Short-term Variability of Repolarization Is Superior to Other Repolarization Parameters in the Evaluation of Diverse Antiarrhythmic Interventions in the Chronic Atrioventricular Block Dog.

Short-term variability (STV), to quantify beat-to-beat variability of repolarization, is a surrogate parameter that reliably identifies proarrhythmic risk in preclinical models. Examples include not only the use in the chronic atrioventricular block (CAVB) dog model whereby it was developed but also in vulnerable patients with heart failure or drug-induced long QT syndrome. In the CAVB dog model, STV can specifically distinguish between safe and unsafe drugs in proarrhythmic screening. Conversely, this dog model also offers the possibility to evaluate antiarrhythmic strategies in a setting of Torsades de Pointes (TdP) induction with a standard IKr inhibitor. The different antiarrhythmic interventions studied in suppression and prevention of drug-induced TdP in vivo in the CAVB dog model and in vitro in canine ventricular cardiomyocytes are described in this overview. We provide evidence that STV predicts the magnitude of antiarrhythmic effect against TdP better than other repolarization parameters in both suppression and prevention conditions. Moreover, suppression and prevention experiments revealed the same level of antiarrhythmic efficacy, whereas cellular experiments seem more sensitive in comparison with drug testing in vivo. Together, these observations suggest that STV could be used as a consistent indicator to rank efficacy of antiarrhythmic interventions in a number of conditions.

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR 2.1 backward trafficking.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR 2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK1 ), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR 2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR 2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 µM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 µM, 24 hrs) and late-endosomal/lysosomal KIR 2.1 accumulation. Increased KIR 2.1 expression level was also observed in the presence of Nav 1.5 co-expression. Augmented KIR 2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv 11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at -120 mV, 5 µM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at -120 mV; 2 µM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR 2.1 degradation.

Calcium release near L-type calcium channels promotes beat-to-beat variability in ventricular myocytes from the chronic AV block dog.

Beat-to-beat variability of ventricular repolarization (BVR) has been proposed as a strong predictor of Torsades de Pointes (TdP). BVR is also observed at the myocyte level, and a number of studies have shown the importance of calcium handling in influencing this parameter. The chronic AV block (CAVB) dog is a model of TdP arrhythmia in cardiac hypertrophy, and myocytes from these animals show extensive remodeling, including of Ca(2+) handling. This remodeling process also leads to increased BVR. We aimed to determine the role that (local) Ca(2+) handling plays in BVR. In isolated LV myocytes an exponential relationship was observed between BVR magnitude and action potential duration (APD) at baseline. Inhibition of Ca(2+) release from sarcoplasmic reticulum (SR) with thapsigargin resulted in a reduction of [Ca(2+)]i, and of both BVR and APD. Increasing ICaL in the presence of thapsigargin restored APD but BVR remained low. In contrast, increasing ICaL with preserved Ca(2+) release increased both APD and BVR. Inhibition of Ca(2+) release with caffeine, as with thapsigargin, reduced BVR despite maintained APD. Simultaneous inhibition of Na(+)/Ca(2+) exchange and ICaL decreased APD and BVR to similar degrees, whilst increasing diastolic Ca(2+). Buffering of Ca(2+) transients with BAPTA reduced BVR for a given APD to a greater extent than buffering with EGTA, suggesting subsarcolemmal Ca(2+) transients modulated BVR to a larger extent than the cytosolic Ca(2+) transient. In conclusion, BVR in hypertrophied dog myocytes, at any APD, is strongly dependent on SR Ca(2+) release, which may act through modulation of the l-type Ca(2+) current in a subsarcolemmal microdomain.

III-V-on-silicon anti-colliding pulse-type mode-locked laser.

An anti-colliding pulse-type III-V-on-silicon passively mode-locked laser is presented for the first time based on a III-V-on-silicon distributed Bragg reflector as outcoupling mirror implemented partially underneath the III-V saturable absorber. Passive mode-locking at 4.83 GHz repetition rate generating 3 ps pulses is demonstrated. The generated fundamental RF tone shows a 1.7 kHz 3 dB linewidth. Over 9 mW waveguide coupled output power is demonstrated.

Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.

BACKGROUND: Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. METHOD: We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. RESULTS: Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). CONCLUSIONS: We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.

Narrow-linewidth short-pulse III-V-on-silicon mode-locked lasers based on a linear and ring cavity geometry.

Picosecond-pulse III-V-on-silicon mode-locked lasers based on linear and ring extended cavity geometries are presented. In passive mode-locked operation a 12 kHz -3dB linewidth of the fundamental RF tone at 4.7 GHz is obtained for the linear cavity geometry and 16 kHz for the ring cavity geometry. Stabilization of the repetition rate of these devices using hybrid mode-locking is also demonstrated.

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae).

Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6µM) inward rectifier currents.