Time kill-assays of antibiotic combinations for multidrug resistant clinical isolates of OXA-48 carbapenemase producing Klebsiella pneumoniae.

Treatment of infections caused by OXA-48 carbapenemase producing multidrug-resistant isolates often necessitates combination therapy. In vitro effect of different antibiotic combinations against multidrug-resistant (MDR) Klebsiella pneumoniae isolates were evaluated in this study.Meropenem-tobramycin (MER+TOB), meropenem-ciprofloxacin (MER+CIP), colistin-meropenem (COL+MER), colistin-ciprofloxacin (COL+CIP) and colistin-tobramycin (COL+TOB) combinations were tested by time kill-assays. Each antibiotic alone and in combination at their Cmax values were tested against 4 clinical K. pneumoniae isolates at 1, 2, 4, 6, 8, 12 and 24 h. Effect of colistin and its associations were also assessed at 30 min. Bactericidal activity was defined as ≥3log10 CFU mL-1 decrease compared with initial inoculum. Synergy was defined as ≥2log10CFU mL-1 decrease by the combination compared with the most active single agent. Presence of blaOXA-48, blaNDM, blaVIM, blaIMP, blaKPC and blaCTX-M-1 genes was screened by PCR using specific primers.The blaOXA-48 gene was identified together with blaCTXM-1 group gene in all isolates. COL+MER demonstrated to be synergistic and bactericidal. MER+TOB showed synergistic and bactericidal effect on two strains although, regrowth was seen on other two strains at 24 h. MER+CIP exhibited indifferent effect on the strains.Combination therapy could be a potential alternative to treat MDR K. pneumoniae infections. This combination might prevent resistance development and secondary effects of colistin monotherapy. MER+TOB and MER+CIP might have an isolate-dependent effect, that may not always result in synergism.

Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1.

Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determined the kinetics of CRT-1 in real time by relying on electrophysiological recordings of transport-associated currents. Our analysis revealed that CRT-1 harvested the concentration gradient of NaCl and the membrane potential but not the potassium gradient to achieve a very high concentrative power. We investigated the mechanistic basis for the ability of CRT-1 to maintain the forward cycling mode in spite of high intracellular concentrations of creatine: this is achieved by cooperative binding of substrate and co-substrate ions, which, under physiological ion conditions, results in a very pronounced (i.e. about 500-fold) drop in the affinity of creatine to the inward-facing state of CRT-1. Kinetic estimates were integrated into a mathematical model of the transport cycle of CRT-1, which faithfully reproduced all experimental data. We interrogated the kinetic model to examine the most plausible mechanistic basis of cooperativity: based on this systematic exploration, we conclude that destabilization of binary rather than ternary complexes is necessary for CRT-1 to maintain the observed cytosolic creatine concentrations. Our model also provides a plausible explanation why neurons, heart and skeletal muscle cells must express a creatine releasing transporter to achieve rapid equilibration of the intracellular creatine pool.

Comparison of Four Phenotypic Assays and Check-Direct CPE for Detection of Carbapenemases-Producing Enterobacterales.

BACKGROUND: Early and accurate detection of carbapenemase-producing Enterobacterales (CPE) is fundamental to prevent their spread in hospital environment. Our objective was to compare between four commonly used phenotypic assays and Check-Direct CPE (CDCPE) multiplex PCR in CPE detection. We examined stool samples or rectal swabs for CPE, samples collected from 23 Jan 2017 to 23 Jul 2017 from patients in intensive-care units (ICUs) of our hospital. METHODS: A panel of 98 non-repetitive Enterobacterales isolates with reduced susceptibility to carbapenems were analyzed by means of (i) Modified Hodge Test (MHT), (ii) Blue Carba test (BCT), (iii) Combined Disc Test (CDT), and (iv) The Carbapenem Inactivation Method (CIM). All these phenotypic tests compared with CDCPE. Confirmation and validation of results was achieved by classical PCR and sequencing. RESULTS: Of the 98 non-repetitive Enterobacterales isolates, ninety-one were K. pneumoniae (93%), three K. oxytoca (3%), three E. cloacae (3%) and one E. coli (1%). By classic PCR the carbapenem resistance genes in K. pneumoniae isolates distributed as the followings; 49 blaOXA-48, 34 both blaOXA-48 and blaNDM-1, seven blaNDM-1 and one blaKPC. K. oxytoca; two blaOXA-48, one blaNDM-1. E. cloacae; two blaOXA-48, one blaNDM-1. E. coli; one isolate with both blaOXA-48 and blaNDM-1. The most common carbapenemase gene detected was blaOXA-48 rate of 54% (n = 53) followed by a combination of both blaOXA-48 and blaNDM-1 with rate of 36% (n = 35), only blaNDM-1 9% (n = 9) and blaKPC 1% (n = 1). Among phenotypic tests, we found CIM, MHT, and BCT correctly identified carbapenemase producers with sensitivity of 100%, 98%, and 90.8%, respectively. CONCLUSIONS: Rapid and accurate detection of CRE can be achieved by combination of both phenotypic and molecular tests. Surveillance studies are important both in terms of epidemiology and regulation of the treatment of patients.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Clonal distribution of vancomycin-resistant Enterococcus faecium in Turkey and the new singleton ST733.

BACKGROUND: The aim of this study was to provide information about the spread and characteristics of the vancomycin-resistant Enterococcus faecium isolates (VREfm) in Turkey. METHODS: Seventy-one nonduplicate consecutive isolates of VREfm were obtained from various clinical specimens of inpatients treated at university or training hospitals in seven regions of Turkey. Further characteristics included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, and multilocus sequence typing (MLST) of selected isolates. The presence of vancomycin resistance and virulence genes (esp and hyl) was investigated by polymerase chain reaction (PCR). RESULTS: All VREfm isolates had MICs to vancomycin of ≥32 mg/L and contained the vanA gene. The presence of esp gene was identified in 64 and hyl in eight VREfm isolates. All VREfm showed the multiresistance phenotype, including ampicillin (99%), penicillin (99%), imipenem (99%), ciprofloxacin (87%), moxifloxacin (87%), erythromycin (97%), streptomycin (86%), gentamicin (82%), tetracycline (70%), and teicoplanin (99%). All were susceptible to tigecycline while quinupristin-dalfopristin (97%) and linezolid (93%) were the most active other agents. Analysis of the PFGE profiles showed that 53 (74.6%) VREfm isolates shared a similar electrophoretic profile, designed as type 1, and were closely related (>85%). The sequence type was identified by MLST in 44 VRE isolates with unrelated or closely related PFGE patterns. MLST revealed that nosocomial spread of VREfm resulted from dissemination of lineage C1 E faecium clones. Sequence types ST78, ST203, and ST117 were the most frequently isolated. This is the first report of ST733 around the world. CONCLUSIONS: Lineage C1 clones are disseminated among clinical VREfm isolates in seven different regions in Turkey. Regarding VREfm isolates, the worldwide epidemic strains are in circulation in Turkey.

In vitro evaluation of double carbapenem and colistin combinations against OXA-48, NDM carbapenemase-producing colistin-resistant Klebsiella pneumoniae strains.

BACKGROUND: Treatment of pandrug-resistant isolates often necessitates combination therapy. Checkerboard synergy and time-killing assay tests were performed to evaluate the benefits of a triple combination with meropenem, ertapenem, and colistin against 10 colistin-resistant K. pneumoniae clinical isolates harboring different ß-lactamases. (blaOXA-48, blaNDM). MATERIALS AND METHODS: In this study, ertapenem and meropenem (ERT/MEM), meropenem and colistin (MEM/COL), ertapenem, meropenem and colistin (ERT/MEM/COL) combinations were tested using checkerboard techniques and time-kill assays of each antibiotic alone and in combination against 10 colistin-resistant clinical K. pneumoniae isolates. An analysis of K. pneumoniae isolate B6 using a scanning electron microscope revealed morphologic changes in the cell surface after treatment with each antibiotic both alone and in combination. The whole genome of K. pneumoniae KPNB1 was sequenced using an Ion Torrent PGM sequencer. RESULTS: According to the checkboard results, synergistic combinations were observed with ertapenem/meropenem (5/10 isolates), meropenem/colistin (7/10) and ertapenem/meropenem/colistin (9/10); no antagonism was observed for all combinations. For the time-kill assay results; synergism and bactericidal effects were observed with meropenem/colistin (10/10) and with ertapenem/meropenem/colistin (10/10) combinations, and an indifference effect was observed with the ertapenem and meropenem (10/10) combination. Strain number 1 was found 100% identical to Klebsiella pneumoniae subsp. pneumoniae HS11286 according to the outcomes of complete genome sequence analysis, and the strain carried the genes blaOXA-181, blaCTXM-15, blaNDM, arr-3, aac (6')-Ib-cr, rmtF, and catB1. CONCLUSION: Using double carbapenem antibiotics with colistin could be a potential alternative to treat colistin and carbapenem-resistant K. pneumoniae. The present study is the first Turkish report of OXA-181-type carbapenemase causing colistin resistance.

Oral batyl alcohol supplementation rescues decreased cardiac conduction in ether phospholipid-deficient mice.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2.

Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl- Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na+, and Cl- in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl- on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstrate ions and substrate such that their forward transport mode was maintained even under conditions of elevated intracellular Na+ or Cl- The most important differences in the transport cycle of GlyT1 and GlyT2 arose from the kinetics of charge movement and the resulting voltage-dependent rate-limiting reactions: the kinetics of GlyT1 were governed by transition of the substrate-bound transporter from outward- to inward-facing conformations, whereas the kinetics of GlyT2 were governed by Na+ binding (or a related conformational change). Kinetic modeling showed that the kinetics of GlyT1 are ideally suited for supplying the extracellular glycine levels required for NMDA receptor activation.

Characterization of Resistance Genes and Polymerase Chain Reaction-Based Replicon Typing in Carbapenem-Resistant Klebsiella pneumoniae.

Background: Fifty isolates of Klebsiella pneumoniae isolated from clinical samples between 2012 and 2016 that were found to be resistant to carbapenems were included in this study. Materials and Methods: Resistance genes were investigated by performing PCR. Plasmid typing was performed using PCR-based replicon typing. The clonal relationships between the strains were investigated using pulsed-field gel electrophoresis (PFGE). Results: OXA-48-type carbapenemase genes were detected in 86% (n = 43/50) of K. pneumoniae isolates, whereas NDM-type carbapenemase genes were detected in 14% (n = 7/50) of the isolates. blaTEM was detected 60% (n = 30) of the strains, blaSHV in 78% (n = 39), blaCTX-M-1 in 48% (n = 24), and blaCTX-M-2-type ß-lactamase in 10% (n = 5). blaCTX-M-1 and blaSHV were concomitantly distributed in 40% (n = 20) of the strains, blaTEM and blaSHV in 54% (n = 27), blaTEM, blaSHV, and blaCTX-M-1 in 32% (n = 16) and blaCTX-M-1 and blaCTX-M-2 in 10% (n = 5). Strain numbers 66, 69, 76, 77, and 78 coproduced carbapenemases, blaCTX-M-1 and blaCTX-M-2 in addition to blaOXA-48 or blaNDM-1 that were described as hybrid strains. IncR-type replicon was found in 50% (n = 25) of 50 isolates with plasmid typing, whereas IncA/C-type replicon was detected in 40% (n = 20) and IncFIIK-type replicon in 18% (n = 9) of the isolates. Outcomes of the transformation experiments showed that the OXA-48 gene was carried to the receiver cell on FII plasmids. No dominant epidemic clone was detected through PFGE. Conclusion: OXA-48 carbapenemase was found to be the most prevalent type of enzyme in our hospital, and the presence of NDM-1-type carbapenemase-carrying strain and an increase in their rate were detected.

Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity.

Several protein kinases, including protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.

The First Enterobacter cloacae Co-Producing NDM and OXA-48 Carbapenemases and Interhospital Spread of OXA-48 and NDM-Producing Klebsiella pneumoniae in Turkey.

BACKGROUND: The aim of this study was to investigate the occurrence of carbapenemase-producing Enterobacteriaceae. METHODS: A total of 54 carbapenem nonsusceptible Enterobacteriaceae (CRE) isolates were recovered from clinical samples sent to the Dr. Lutfi Kirdar Kartal Training and Research Hospital from the period 2011 through 2014. Forty-four isolates were Klebsiella pneumoniae (CRKP) and the other 10 were Enterobacter cloacae (CREC).The isolate identifications and antibiotic sensitivity tests were performed using a Vitek2 automatic system. The clonality of isolates was determined using rep-PCR Diversilab. Presence of blaOXA-48, blaNDM, blaVIM, blaIMP, and blaKPC genes were screened using polymerase chain reaction (PCR) with specific primers. RESULTS: CRKP were isolated from blood, urine, wounds, catheter tips, and tracheal aspirate samples; a total 44 isolates were evaluated. All isolates were nonsusceptible to ertapenem/imipenem or meropenem. Eighteen percent of the isolates were resistant to colistin. CREC were isolated from blood, urine, cerebrospinal fluid and sputum; a total of 10 isolates were evaluated. They were resistant to all carbapenems and 90% were resistant to cefoperazone/sulbactam and trimethoprim/sulfamethoxazole, and 50 - 70% isolates were resistant to gentamicin, amikacin, and ciprofloxacin. Thirty-three (75%) OXA-48 producing CRKP were identified. Thirteen (29.5%) were positive and two (4.5%) NDM-producing K. pneumoniae were co-producing OXA-48. Of the ten CREC strains tested, eight were positive for blaNDM, one isolate was positive for blaVIM and another for blaIMP genes. rep-PCR typing revealed the presence of a clonal dissemination in CRKP and CREC in the hospital. CONCLUSIONS: To our knowledge, this is the first identification of blaNDM in E. cloacae isolates in Turkey. These findings describe an interhospital spread of CRKP-producing OXA-48 and NDM carbapenemases that started in 2011. Continuous monitoring is necessary to better understand their dissemination in the hospital, which probably occurred as a result of transmission from an environmental reservoir. These findings emphasize the need for intensive surveillance and precautions.

Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage-Gated Kv7.2 Potassium Channels.

Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signaling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP2 ), Ca2+ /calmodulin, and phosphorylation. Recent studies found that the PIP2 sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP2 -binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST-fusion proteins exposed to recombinant protein kinases by using LC-MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3ß could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein-protein and protein-lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567.

Evaluation of Diastolic Function in Patients with Normal Perfusion and Type 2 Diabetes Mellitus with Gated Single-photon Emission Computed Tomography.

Early identification of diastolic dysfunction of patients with diabetes is important in preventing cardiac events. In this study, we aimed to show that both myocardial perfusion and diastolic function parameters can be evaluated in diabetic patients with possible silent cardiac symptoms using gated single-photon emission computed tomography (G-SPECT). We examined eighty patients: Forty with and forty without diabetes. The patients were compared in terms of systolic and diastolic parameters obtained using G-SPECT. 99mTc-sestamibi was used to obtain 8-frame images in each cardiac cycle, with calculation of the left ventricular ejection fraction (LVEF), peak filling rate (PFR), mean filling rate during the first third of diastolic time (MFR/3), and time to peak filling (TTPF) using the QGS software. G-SPECT results were compared in forty diabetic and forty nondiabetic patients of similar age and sex. Of the diastolic function parameters, PFR was found to be lower in patients with than without diabetes (2.31 ± 0.68 vs. 2.76 ± 0.68, respectively; P = 0.004). The TTPF and MFR/3 in both groups were similar. PFR was negatively correlated with end-diastolic volume and end-systolic volume (ESV) and positively correlated with LVEF. This correlation was stronger in patients with diabetes. The diastolic parameter PFR, obtained using G-SPECT, was significantly lower in patients with than without diabetes. We believe that these parameters should be noted for the early diagnosis or prevention of heart disease in patients with a risk of diastolic dysfunction.

Comparison of the Novel Oxa-48 and Kpc K-SeT Assay, and Blue-Carba Test for the Detection of Carbapenemase-Producing Enterobacteriaceae Using PCR as a Reference Method.

BACKGROUND: Rapid, simple, and accurate laboratory detection of carbapenemases is very important for proper antibiotic therapy and infection control. METHODS: In this study, carbapenem-nonsusceptible Enterobacteriaceae (CRE) isolates were used to evaluate the performance of a new lateral flow immunochromatographic (IC) assay, the OXA-48 and KPC K-SeT assay, and modified Blue-Carba test (BCT) for the rapid detection of OXA-48 carbapenemase in comparison with polymerase chain reaction (PCR) amplification. These CREs of various enterobacterial species were isolated from various clinical samples including OXA-48 (47), NDM-1 (6), KPC-1 (1), IMP-1 (1), VIM-2,-4 (2), IMP-2 (1), OXA-51 (1), and OXA-23 (1) producers. RESULTS: The OXA-48 K-SeT test detected all OXA-48 carbapenemase producers with 100% sensitivity and specificity. The BCT detected carbapenemase producers with 93% sensitivity and 100% specificity. CONCLUSIONS: Both IC assays and BCT tests have good performance and are easy to perform, rapid, simple to interpret, and highly sensitive. We suggest that BCT can be used initially as an accurate, inexpensive, and rapid phenotypic confirmation test to identify Class A, B, and D carbapenemases in the routine diagnostic microbiology laboratory, thus allowing the detection of carbapenemase activity directly from bacterial cultures.

The N Terminus Specifies the Switch between Transport Modes of the Human Serotonin Transporter.

The serotonin transporter (SERT) and other monoamine transporters operate in either a forward transport mode where the transporter undergoes a full transport cycle or an exchange mode where the transporter seesaws through half-cycles. Amphetamines trigger the exchange mode, leading to substrate efflux. This efflux was proposed to rely on the N terminus, which was suggested to adopt different conformations in the inward facing, outward facing and amphetamine-bound states. This prediction was verified by tryptic digestion of SERT-expressing membranes: in the absence of Na+, the N terminus was rapidly digested. Amphetamine conferred protection against cleavage, suggesting a relay between the conformational states of the hydrophobic core and the N terminus. We searched for a candidate segment that supported the conformational switch by serial truncation removing 22 (ΔN22), 32 (ΔN32), or 42 (ΔN42) N-terminal residues. This did not affect surface expression, inhibitor binding, and substrate influx. However, amphetamine-induced efflux by SERT-ΔN32 or SERT-ΔN42 (but not by SERT-ΔN22) was markedly diminished. We examined the individual steps in the transport cycle by recording transporter-associated currents: the recovery rate of capacitive peak, but not of steady state, currents was significantly lower for SERT-ΔN32 than that of wild type SERT and SERT-ΔN22. Thus, the exchange mode of SERT-ΔN32 was selectively impaired. Our observations show that the N terminus affords the switch between transport modes. The findings are consistent with a model where the N terminus acts as a lever to support amphetamine-induced efflux by SERT.

The Effects of Ivabradine on Left Ventricular Synchronization and Tei Index in Patients with Systolic Heart Failure.

BACKGROUND: The aim of our study was to evaluate in stable outpatients with systolic heart failure (HF) the 3 months effect of ivabradine on LV synchronization and Tei index in stable outpatients with systolic HF. METHODS: We evaluated prospectively 40 (30 males, 10 females) patients with HF. All patients were evaluated before and after treatment by transthoracic M mode, two dimensional (2D), pulsed-wave (PW), continuous wave (CW), color flow and tissue Doppler imaging (TDI) and tissue synchronization imaging (TSI). Standard deviation of Ts of the 12 LV segments (Ts-SD-12) is the most widely used parameter of intra-LV asynchrony. RESULTS: Thirty men and 10 women with mean ± SD age of 64.7 ± 9.9 years were included in this study. Most of the patients benefitted from some degree of clinical improvement, 12/16 (75.0%) from NYHA III to II and 18/24 (75.0%) from II to I, respectively. Resting heart rate was significantly reduced after ivabradine treatment (84.3 ± 11.4 vs. 66.5 ± 11.5 bpm, p < 0.001). E/E' and Tei index were significantly changed after ivabradine treatment (17.3 ± 9.0 vs. 14.8 ± 7.1, p = 0.02 and 0.86 ± 0.74 vs. 0.81 ± 0.69, p = 0.02). Intra-LV synchrony parameters Ts-SD-12 and Ts-12 were significantly reduced after ivabradine (46.8 ± 13.6 vs. 42.7 ± 13.1, p = 0.01 and 142.5 ± 44.0 vs. 128.5 ± 45.2, p = 0.009). CONCLUSIONS: The present study demonstrated that adding ivabradine to the standard therapy reduced HR and significantly improved LV ventricular asynchrony and Tei index in systolic HF patients.

Phosphorylation regulates the sensitivity of voltage-gated Kv7.2 channels towards phosphatidylinositol-4,5-bisphosphate.

KEY POINTS: Phosphatidylinositol-4,5-bisphosphate (PIP2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2 . Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors. ABSTRACT: The function of numerous ion channels is tightly controlled by G protein-coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol-4,5-bisphosphate (PIP2 ). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography-coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP2 -mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage-sensitive phosphatase Dr-VSP than were wild-type channels. In vitro phosphorylation assays with the purified C-terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild-type but not mutant Kv7.2 channels towards PIP2 depletion via Dr-VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2 -binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR-mediated channel regulation.

Successful thrombolytic therapy in a patient with congenital corrected transposition of the great arteries.

OBJECTIVE: The aim of this report is to emphasize the importance of thrombolytic therapy in selected patients, such as those with congenital heart defects in whom a coronary artery anomaly can be observed. CASE REPORT: We present here a 63 year-old female patient who was admitted to our emergency department with ST segment elevation myocardial infarction and a history of a congenital heart defect. We treated the patient successfully with thrombolytic therapy instead of primary percutaneous intervention, because of the suspicion of a coronary artery anomaly. On the following day, we performed coronary angiography on the patient, which revealed the anomalous origin of the coronary arteries, with the left and right coronary arteries originating from the right sinus of Valsalva and the circumflex artery originating from the left sinus of Valsalva. This anomaly in this patient group is described for the first time. CONCLUSION: Coronary artery anomaly may be observed in patients with congenitally corrected transposition of the great arteries, and in the case of requiring emergency reperfusion, thrombolytic treatment can be an alternative strategy in this patient group.

A new diagnostic tool for masked hypertension: impaired sleep quality.

INTRODUCTION: The aim of this study was to evaluate the relationship between masked hypertension and impaired sleep quality. Additionally, we evaluated the diagnostic role and prevalence of poor sleep quality among patients with newly diagnosed masked hypertension. MATERIAL AND METHODS: A total of 112 individuals, 72 patients with newly diagnosed masked hypertension and 40 normotensive healthy volunteers, were included in this study. All patients underwent evaluation comprising 12-lead electrocardiography, transthoracic echocardiography, 24-hour Holter ECG, and basic laboratory tests. Additionally, all participants completed questionnaires, including the Pittsburgh Sleep Quality Index (PSQI). RESULTS: The total PSQI score was significantly higher in the masked hypertension group than in the normotensive healthy volunteers (4.13 ±2.43 vs. 2.33 ±1.67, p < 0.001). A PSQI score > 5 was found in 45.8% (n = 33) of patients in the masked hypertension group and 15% (n = 6) of patients in the normotensive group (p < 0.001). The non-dipper pattern was found in 17.5% of the healthy volunteer group and 59.94% (n = 41) of the masked hypertension group (p < 0.001). When we compared the dipping pattern of the masked hypertension groups, there was a significant difference in PSQI score between the dipper and non-dipper groups (4.87 ±3.21 vs. 3.58 ±2.33, p < 0.001). Multiple logistic regression analyses showed that masked hypertension, LV mass, and LV mass index score were independent predictors of poor PSQI. CONCLUSIONS: This study demonstrates impaired sleep quality in subjects with masked hypertension, particularly those with a non-dipper pattern. Additionally, this study indicates that impaired sleep quality may help diagnose masked hypertension, particularly in the non-dipper group.