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Purpose: In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake.Experimental Design: Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines.Results: Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations.Conclusions: These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance. Clin Cancer Res; 20(4); 985–94. ©2013 AACR.

PDF file 64K, Supplementary Table S1. Overview of conflicting reports regarding the importance of OCT1 genetics, OCT1 expression, or cellular imatinib uptake for imatinib pharmacokinetics and response Supplementary Table S2. Multiple reaction monitoring transitions and MS parameters for determination of imatinib, N-desmethyl imatinib and their internal standards with LC-MS-MS Supplementary Table S3. Characteristics of SLC family members whose mRNA expression has been quantified by real-time PCR in the present study

PDF file 79K, Supplementary Figure S1: Uptake of imatinib and OCT1 probe substrate TEA into OCT1-expressing cells and vector-transfected control cells Supplementary Figure S2: Cellular uptake of the OCT1 probe substrate TEA and imatinib into mOct1- or mOct2-expressing HEK293 cells Supplementary Figure S3: Cellular uptake of imatinib into the CML cell lines Meg-01, LAMA-84 and K562 as well as into OCT1-expressing HEK cells and vector-transfected control cells measured after an incubation period of 120 minutes Supplementary Figure S4: Cellular uptake of imatinib into HEK cells expressing variant V408 in comparison to HEK cells expressing reference sequence M408 and vector-transfected control cells measured after an incubation period of 10 minutes Supplementary Figure S5: Knockdown of OCT1 protein expression by the used shRNA

PDF file 65K, Supplementary methods including chemicals, generation of a cell line expressing OCT1 variant p.V408, transport studies using oocytes and cell lines, LC-MS-MS analysis of imatinib, RNA isolation and quantification, shRNA knockdown, flow cytometry and confocal laser scanning microscopy

Background Slick, a sodium-activated potassium channel, has been recently identified in somatosensory pathways, but its functional role is poorly understood. The authors of this study hypothesized that Slick is involved in processing sensations of pain and itch. Methods Immunostaining, in situ hybridization, Western blot, and real-time quantitative reverse transcription polymerase chain reaction were used to investigate the expression of Slick in dorsal root ganglia and the spinal cord. Mice lacking Slick globally (Slick–/–) or conditionally in neurons of the spinal dorsal horn (Lbx1-Slick–/–) were assessed in behavioral models. Results The authors found Slick to be enriched in nociceptive Aδ-fibers and in populations of interneurons in the spinal dorsal horn. Slick–/– mice, but not Lbx1-Slick–/– mice, showed enhanced responses to noxious heat in the hot plate and tail-immersion tests. Both Slick–/– and Lbx1-Slick–/– mice demonstrated prolonged paw licking after capsaicin injection (mean ± SD, 45.6 ± 30.1 s [95% CI, 19.8 to 71.4]; and 13.1 ± 16.1 s [95% CI, 1.8 to 28.0]; P = 0.006 [Slick–/– {n = 8} and wild-type {n = 7}, respectively]), which was paralleled by increased phosphorylation of the neuronal activity marker extracellular signal–regulated kinase in the spinal cord. In the spinal dorsal horn, Slick is colocalized with somatostatin receptor 2 (SSTR2), and intrathecal preadministration of the SSTR2 antagonist CYN-154806 prevented increased capsaicin-induced licking in Slick–/– and Lbx1-Slick–/– mice. Moreover, scratching after intrathecal delivery of the somatostatin analog octreotide was considerably reduced in Slick–/– and Lbx1-Slick–/– mice (Slick–/– [n = 8]: 6.1 ± 6.7 bouts [95% CI, 0.6 to 11.7]; wild-type [n =8]: 47.4 ± 51.1 bouts [95% CI, 4.8 to 90.2]; P = 0.039). Conclusions Slick expressed in a subset of sensory neurons modulates heat-induced pain, while Slick expressed in spinal cord interneurons inhibits capsaicin-induced pain but facilitates somatostatin-induced itch. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack−/−) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6–P14) Slack−/− lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack−/−, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack−/−neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack−/−. We additionally detect a lack of mGluR-induced LTD in infant Slack−/−, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack−/−. This together with normalized expression levels of GluN2B and Rab4 hints to developmental “restoration” of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities.

The 3',5'-cGMP pathway triggers cytoprotective responses and improves cardiomyocyte survival during myocardial ischaemia and reperfusion (I/R) injury. These beneficial effects were attributed to NO-sensitive GC induced cGMP production leading to activation of cGMP-dependent protein kinase I (cGKI). cGKI in turn phosphorylates many substrates, which eventually facilitate opening of mitochondrial ATP-sensitive potassium channels (mitoKATP ) and Ca2+ -activated potassium channels of the BK type (mitoBK). Accordingly, agents activating mitoKATP or mitoBK provide protection against I/R-induced damages. Here, we provide an up-to-date summary of the infarct-limiting actions exhibited by the GC/cGMP axis and discuss how mitoKATP and mitoBK, which are present at the inner mitochondrial membrane, confer mito- and cytoprotective effects on cardiomyocytes exposed to I/R injury. In view of this, we believe that the functional connection between the cGMP cascade and mitoK+ channels should be exploited further as adjunct to reperfusion therapy in myocardial infarction.

Reportedly, the intermediate-conductance Ca

Ca

The transient receptor potential (TRP) ankyrin type 1 (TRPA1) channel is highly expressed in a subset of sensory neurons where it acts as an essential detector of painful stimuli. However, the mechanisms that control the activity of sensory neurons upon TRPA1 activation remain poorly understood. Here, using in situ hybridization and immunostaining, we found TRPA1 to be extensively co-localized with the potassium channel Slack (K

In light of the increasing evidence supporting a link between hearing loss and dementia, it is critical to gain a better understanding of the nature of this relationship. We have previously observed that following cochlear synaptopathy, the temporal auditory processing (e.g., auditory steady state responses, ASSRs), is sustained when reduced auditory input is centrally compensated. This central compensation process was linked to elevated hippocampal long-term potentiation (LTP). We further observed that, independently of age, central responsiveness to cochlear synaptopathy can differ, resulting in either a low or high capacity to compensate for the reduced auditory input. Lower central compensation resulted in poorer temporal auditory processing, reduced hippocampal LTP, and decreased recruitment of activity-dependent brain-derived neurotrophic factor (BDNF) expression in hippocampal regions (

Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

Neutrophils are the most numerous cells in the leukocyte population and essential for innate immunity. To limit their effector functions, neutrophils are able to modulate glycolysis and other cellular metabolic pathways. These metabolic pathways are essential not only for energy usage, but also for specialized effector actions, such as the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). It has been demonstrated that activated viable neutrophils can produce NETs, which consists of a DNA scaffold able to bind granule proteins and microorganisms. The formation of NETs requires the availability of increased amounts of adenosine triphosphate (ATP) as it is an active cellular and therefore energy-dependent process. In this article, we discuss the glycolytic and other metabolic routes in association with neutrophil functions focusing on their role for building up NETs in the extracellular space. A better understanding of the requirements of metabolic pathways for neutrophil functions may lead to the discovery of molecular targets suitable to develop novel anti-infectious and/or anti-inflammatory drugs.

Background and purpose Pore-forming α subunits of the voltage- and Ca2+ -activated K+ channel with large conductance (BKα) promote malignant phenotypes of breast tumour cells. Auxiliary subunits such as the leucine-rich repeat containing 26 (LRRC26) protein, also termed BKγ1, may be required to permit activation of BK currents at a depolarized resting membrane potential that frequently occur in non-excitable tumour cells. Experimental approach Anti-tumour effects of BKα loss were investigated in breast tumour-bearing MMTV-PyMT transgenic BKα knockout (KO) mice, primary MMTV-PyMT cell cultures, and in a syngeneic transplantation model of breast cancer derived from these cells. The therapeutic relevance of BK channels in the context of endocrine treatment was assessed in human breast cancer cell lines expressing either low (MCF-7) or high (MDA-MB-453) levels of BKα and BKγ1, as well as in BKα-negative MDA-MB-157. Key results BKα promoted breast cancer onset and overall survival in preclinical models. Conversely, lack of BKα and/or knockdown of BKγ1 attenuated proliferation of murine and human breast cancer cells in vitro. At low concentrations, tamoxifen and its major active metabolites stimulated proliferation of BKα/γ1-positive breast cancer cells, independent of the genomic signalling controlled by the oestrogen receptor. Finally, tamoxifen increased the relative survival time of BKα KO but not of wild-type tumour cell recipient mice. Conclusion and implications Breast cancer initiation, progression, and tamoxifen sensitivity depend on functional BK channels thereby providing a rationale for the future exploration of the oncogenic actions of BK channels in clinical outcomes with anti-oestrogen therapy.

Identification of prognostic or predictive molecular markers in glioblastoma resection specimens may lead to strategies for therapy stratification and personalized treatment planning. Here, we analyzed in primary glioblastoma stem cell (pGSC) cultures the mRNA abundances of seven stem cell (MSI1, Notch1, nestin, Sox2, Oct4, FABP7 and ALDH1A3), and three radioresistance or invasion markers (CXCR4, IK

The key auditory signature that may associate peripheral hearing with central auditory cognitive defects remains elusive. Suggesting the involvement of stress receptors, we here deleted the mineralocorticoid and glucocorticoid receptors (

The 3',5'-cGMP pathway triggers cytoprotective responses and improves cardiomyocyte survival during myocardial ischaemia and reperfusion (I/R) injury. These beneficial effects were attributed to NO-sensitive GC induced cGMP production leading to activation of cGMP-dependent protein kinase I (cGKI). cGKI in turn phosphorylates many substrates, which eventually facilitate opening of mitochondrial ATP-sensitive potassium channels (mitoK

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

Mammalian inner hair cells (IHCs) transduce sound into depolarization and transmitter release. Big conductance and voltage- and Ca2+-activated K+ (BK) channels are responsible for fast membrane rep...

International audience; Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide–gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3–/–) mice to obtain triallelic Cnga3+/– Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.

The neuronal Na

Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack−/−) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6–P14) Slack−/− lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack−/−, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack−/−neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack−/−. We additionally detect a lack of mGluR-induced LTD in infant Slack−/−, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack−/−. This together with normalized expression levels of GluN2B and Rab4 hints to developmental “restoration” of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03953-0.

Additional file 3. Comparison of the justification categories between 2016 and 2018 in calves with diarrhea. Absolute numbers used to calculate proportions and 95% confidence intervals.

Background Sodium-activated potassium channels (KNa) Slack (sequence like a calcium-activated potassium channel, aka Slo 2.2, KNa1.1 or KCNT1) are widely expressed throughout neuronal tissue, whereas their presence and function in the cardiovascular system is not well understood. Due to high intracellular sodium concentrations ([Na+]i) necessary to induce half-maximal channel activation, we hypothesized that Slack function is attributed to pathophysiological conditions such as myocardial ischemia. Purpose To elucidate the putative functions of Slack in the murine heart and in cardiomyocytes (CMs) and to explore whether the ischemia and reperfusion (I/R)-induced cardiac damage is affected by endogenous Slack channel activity. Methods I/R injury was evaluated in global and CM-specific Slack knockout mice (Slack gKO, CM Slack KO) and compared to litter-matched controls (Slack gWT, CM Slack CTR) by applying an in vivo model of acute myocardial infarction (MI). Infarct size (IS) was assessed at baseline, after ischemic pre- (iPre) and postconditioning (iPost) and in response to cinaciguat (CIN), a cGMP-elevating agent. Moreover, Slack expression and function in CMs was studied by biochemical and electrophysiological means and by utilizing the newly developed FRET-based K+ probe GEPII 1.0. Results IS in Slack gKO mice was increased in comparison to gWT littermates. In addition, the cardioprotection afforded by iPost was attenuated in the absence of Slack. To test if the increased vulnerability to I/R injury of the Slack gKO mouse model was originating from Slack activity in CMs, we subjected CM-specific Slack CTR and KO mutants to an identical MI procedure. IS measurements confirmed increased cardiac damage at baseline and reduced cardioprotective effects afforded by iPre and iPost in CM Slack KO mice. Interestingly, CIN (i.p., 30 min prior to I/R) reduced IS to a similar extent in both genotypes, suggesting that Slack functions in a cGMP-independent manner. Whole-cell patch clamp experiments on CMs demonstrated a reduction of the KNa-inhibitors clofilium- and chinidine-sensitive K+ outward currents in Slack gKO CM. Extracellular potassium ([K+]ex) accumulation measured with GEPII 1.0 was lower in Slack gKO versus gWT CM pools exposed to membrane permeabilizing agent digitonin. Accordingly, [K+]ex evoked by the Slack activators niclosamide and bithionol was lower in the absence of functional Slack in CMs. Conclusion The presented findings establish an important role of Slack channels for cardioprotective signalling mechanisms during I/R in vivo and for mediating beneficial effects of mechanical conditioning on IS. Corroborating in vitro studies on adult CMs exhibit an impaired [K+]ex dynamic in response to genetic or pharmacological modulation of Slack activity. Thus, we conclude that Slack-dependent K+ signalling pathways in CMs may represent a promising drug target that renders the heart muscle less vulnerable to the I/R-induced damage. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Work in the authors' laboratories is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (to R.L.) and the DFG Research Unit 2060, “cGMP Signaling in Cell Growth and Survival” (to R.L. and P.R.).

Background: The nitric oxide–sensitive guanylyl cyclase/cGMP-dependent protein kinase type I signaling pathway can afford protection against the ischemia/reperfusion injury that occurs during myocardial infarction. Reportedly, voltage and Ca 2+ -activated K + channels of the BK type are stimulated by cGMP/cGMP-dependent protein kinase type I, and recent ex vivo studies implicated that increased BK activity favors the survival of the myocardium at ischemia/reperfusion. It remains unclear, however, whether the molecular events downstream of cGMP involve BK channels present in cardiomyocytes or in other cardiac cell types. Methods: Gene-targeted mice with a cardiomyocyte- or smooth muscle cell–specific deletion of the BK (CMBK or SMBK knockouts) were subjected to the open-chest model of myocardial infarction. Infarct sizes of the conditional mutants were compared with litter-matched controls, global BK knockout, and wild-type mice. Cardiac damage was assessed after mechanical conditioning or pharmacological stimulation of the cGMP pathway and by using direct modulators of BK. Long-term outcome was studied with respect to heart functions and cardiac fibrosis in a chronic myocardial infarction model. Results: Global BK knockouts and CMBK knockouts, in contrast with SMBK knockouts, exhibited significantly larger infarct sizes compared with their respective controls. Ablation of CMBK resulted in higher serum levels of cardiac troponin I and elevated amounts of reactive oxygen species, lower phosphorylated extracellular receptor kinase and phosphorylated AKT levels and an increase in myocardial apoptosis. Moreover, CMBK was required to allow beneficial effects of both nitric oxide–sensitive guanylyl cyclase activation and inhibition of the cGMP-degrading phosphodiesterase-5, ischemic preconditioning, and postconditioning regimens. To this end, after 4 weeks of reperfusion, fibrotic tissue increased and myocardial strain echocardiography was significantly compromised in CMBK-deficient mice. Conclusions: Lack of CMBK channels renders the heart more susceptible to ischemia/reperfusion injury, whereas the pathological events elicited by ischemia/reperfusion do not involve BK in vascular smooth muscle cells. BK seems to permit the protective effects triggered by cinaciguat, riociguat, and different phosphodiesterase-5 inhibitors and beneficial actions of ischemic preconditioning and ischemic postconditioning by a mechanism stemming primarily from cardiomyocytes. This study establishes mitochondrial CMBK channels as a promising target for limiting acute cardiac damage and adverse long-term events that occur after myocardial infarction.

Intermediate conductance calcium-activated potassium channels (KCa3.1) have been recently implicated in pain processing. However, the functional role and localization of KCa3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking KCa3.1 (KCa3.1-/-) in various pain models and analyzed the expression pattern of KCa3.1 in dorsal root ganglia (DRG) and the spinal cord. KCa3.1-/- mice demonstrated normal behavioral responses in models of acute nociceptive, persistent inflammatory, and persistent neuropathic pain. However, their behavioral responses to noxious chemical stimuli such as formalin and capsaicin were increased. Accordingly, formalin-induced nociceptive behavior was increased in wild-type mice after administration of the KCa3.1 inhibitor TRAM-34. In situ hybridization experiments detected KCa3.1 in most DRG satellite glial cells, in a minority of DRG neurons, and in ependymal cells lining the central canal of the spinal cord. Together, our data point to a specific inhibitory role of KCa3.1 for the processing of noxious chemical stimuli.

Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst-evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC β1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer-based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.

Gain-of-function mutations in KCNT1, the gene encoding Slack (KNa1.1) channels, result in epilepsy of infancy with migrating focal seizures (EIMFS) and several other forms of epilepsy associated with severe intellectual disability. We have generated a mouse model of this condition by replacing the wild type gene with one encoding Kcnt1R455H, a cytoplasmic C-terminal mutation homologous to a human R474H variant that results in EIMFS. We compared behavior patterns and seizure activity in these mice with those of wild type mice and Kcnt1−/− mice. Complete loss of Kcnt1 produced deficits in open field behavior and motor skill learning. Although their thresholds for electrically and chemically induced seizures were similar to those of wild type animals, Kcnt1−/− mice were significantly protected from death after maximum electroshock-induced seizures. In contrast, homozygous Kcnt1R455H/R455H mice were embryonic lethal. Video-EEG monitoring of heterozygous Kcnt1+/R455H animals revealed persistent interictal spikes, spontaneous seizures and a substantially decreased threshold for pentylenetetrazole-induced seizures. Surprisingly, Kcnt1+/R455H mice were not impaired in tasks of exploratory behavior or procedural motor learning. These findings provide an animal model for EIMFS and suggest that Slack channels are required for the development of procedural learning and of pathways that link cortical seizures to other regions required for animal survival.

Mammalian inner hair cells (IHCs) transduce sound into depolarization and transmitter release. Big conductance and voltage- and Ca

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface‐bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal‐pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface‐bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT‐29. We found that TAs and DOP are agonists of the α2‐adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F‐actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F‐actin formation without decreasing the cytoplasmic cAMP level. The neurochemical‐induced internalisation in host cells is independent of the fibronectin‐binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

KCa3.1 K+ channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of KCa3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed KCa3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological KCa3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional KCa3.1 for therapy response. The KCa3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. KCa3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca2+ signals and DNA repair pathways. Consistently, KCa3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that KCa3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since KCa3.1 is druggable, KCa3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.

Summary Primary electroencephalographic (EEG) features of sleep arise in part from thalamocortical neural assemblies, and cortical potassium channels have long been thought to play a critical role. We have exploited the regionally dynamic nature of sleep EEG to develop a novel screening strategy and used it to conduct an adeno-associated virus (AAV)-mediated RNAi screen for cellular roles of 31 different voltage-gated potassium channels in modulating cortical EEG features across the circadian sleep-wake cycle. Surprisingly, a majority of channels modified only electroencephalographic frequency bands characteristic of sleep, sometimes diurnally or even in specific vigilance states. Confirming our screen for one channel, we show that depletion of the KCa1.1 (or “BK”) channel reduces EEG power in slow-wave sleep by slowing neuronal repolarization. Strikingly, this reduction completely abolishes transcriptomic changes between sleep and wake. Thus, our data establish an unexpected connection between transcription and EEG power controlled by specific potassium channels. We postulate that additive dynamic roles of individual potassium channels could integrate different influences upon sleep and wake within single neurons.

K+ channels crosstalk with biochemical signaling cascades and regulate virtually all cellular processes by adjusting the intracellular K+ concentration, generating the membrane potential, mediating cell volume changes, contributing to Ca2+ signaling, and directly interacting within molecular complexes with membrane receptors and downstream effectors. Tumor cells exhibit aberrant expression and activity patterns of K+ channels. The upregulation of highly "oncogenic" K+ channels such as the Ca2+-activated IK channel may drive the neoplastic transformation, malignant progression, metastasis, or therapy resistance of tumor cells. In particular, ionizing radiation in doses used for fractionated radiotherapy in the clinic has been shown to activate K+ channels. Radiogenic K+ channel activity, in turn, contributes to the DNA damage response and promotes survival of the irradiated tumor cells. Tumor-specific overexpression of certain K+ channel types together with the fact that pharmacological K+ channel modulators are already in clinical use or well tolerated in clinical trials suggests that K+ channel targeting alone or in combination with radiotherapy might become a promising new strategy of anti-cancer therapy. The present article aims to review our current knowledge on K+ channel signaling in irradiated tumor cells. Moreover, it provides new data on molecular mechanisms of radiogenic K+ channel activation and downstream signaling events.

// Lena Edalat 1, 2, * , Benjamin Stegen 2, * , Lukas Klumpp 2, 5 , Erik Haehl 2 , Karin Schilbach 3 , Robert Lukowski 1 , Matthias Kuhnle 4 , Gunther Bernhardt 4 , Armin Buschauer 4 , Daniel Zips 2 , Peter Ruth 1 , Stephan M. Huber 2 1 Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tubingen, Tubingen, Germany 2 Department of Radiation Oncology, University of Tubingen, Tubingen, Germany 3 Department of General Pediatrics, Oncology/Hematology, University of Tubingen, Tubingen, Germany 4 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, Regensburg, Germany 5 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tubingen, Tubingen, Germany * These authors contributed equally to this work Correspondence to: Peter Ruth, e-mail: peter.ruth@uni-tuebingen.de Stephan M. Huber, e-mail: stephan.huber@uni-tuebingen.de Keywords: glioma, radiation therapy, patch-clamp recording, fura-2 Ca2 + imaging, transfilter migration Received: October 20, 2015 Accepted: January 29, 2016 Published: February 16, 2016 ABSTRACT Infiltration of the brain by glioblastoma cells reportedly requires Ca 2+ signals and BK K + channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca 2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca 2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo . Importantly, paxilline blocked IR-induced migration in vivo . Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K + channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo . This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.

Background: Removal of nonvital tissue is an accepted method to eradicate biofilms and to stimulate wound healing. Debridement using a monofilament polyester fiber pad has clinically been shown to be effective as well as pain and trauma free. Methods: For in vitro determination of the cleansing capacity of this product compared to gauze swabs, a wound debridement model was used with glass plates coated with a bovine serum albumin solution, stained with hematoxylin. Both products were moistened and fixed to a weight connected to a regulated motor and were then pulled over the holding device with the coated glass plate under standardized conditions (power = 0.067 N/cm2, velocity = 1.6 cm/s). Results: At a low coating concentration (0.5%) both products were equally effective, but at a high concentration (1.5%) cleansing did not occur after 5 wipes. When wiping the plates 15 times, the debridement pad cleansed significantly (p < 0.001) better than gauze. When consecutively wiping 4 coated plates with a single debridement pad or swab, the pad exhibited and maintained a significantly higher cleansing capacity while gauze quickly lost its effect. Conclusion: Our in vitro test results indicated a higher cleansing capacity of the debridement pad compared to gauze swabs.

Objective— Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca 2+ -activated K + (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (I SK4 ) in proliferating VSMC and neointima formation. Approach and Results— Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. I SK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased I SK4 , whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in I SK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that I SK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced I SK4 by 50% suggesting a constitutive activation of I SK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. Conclusions— NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

Kcnt1 encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels.

Vascular tone can be regulated via nucleotides like ATP and UTP that are derived from erythrocytes or platelets or released from the endothelium (Ralevic & Burnstock 2003). It is known that ATP is an agonist of P2Y2 receptors and UTP is an agonist of P2Y2/4 receptors (Abbracchio et al. 2006). A decrease in blood pressure most likely results from ATP inducing endothelium-dependent relaxation (von Kugelgen et al. 1987, von Kugelgen & Starke 1990, Ralevic & Burnstock 1991a, 2003). Evidence suggests that this vasodilatory response can be mediated by nitric oxide (NO; Ralevic & Burnstock 1991a), endothelial-derived hyperpolarization (EDH; Malmsjo et al. 1999, 2002, Wihlborg et al. 2003) and/or prostacyclin (Hammer et al. 2003, Wihlborg et al. 2003). P2Y2 receptors are found on the endothelium and it was proposed that their activation stimulates the synthesis and release of NO (Ralevic & Burnstock 1991b, Buvinic et al. 2002, Burnstock 2009). Moreover, aortic rings from P2Y2 receptor knockout mice (P2Y2−/−) exhibit impaired vasorelaxation in response to ATP, which suggests that NO release is subsequent to P2Y2 receptor activation (Guns et al. 2005, 2006). Of note, mechanical destruction of the endothelium abolishes the ATP induced vasodilatory effect and produces a direct vasoconstrictory response on vascular smooth muscle cells (Kennedy & Burnstock 1985, Kennedy et al. 1985, Ralevic & Burnstock 1996b). Activation of P2Y4 receptors is associated with vasoconstriction (Dietrich et al. 1996, McMillan et al. 1999, Rubino et al. 1999). In contrast to ATP causing vasodilation, UTP was found to vasoconstrict mouse aortic rings (Boarder & Hourani 1998, Kauffenstein et al. 2010) and rabbit inner ear arteries (von Kugelgen et al. 1987). However, depending on route of administration, species, localization within the vascular tree or vessel type, UTP can cause vasoconstriction, vasodilation or both (Ralevic & Burnstock 1996a,b, Janigro et al. 1997, Horiuchi et al. 2001, Guns et al. 2005, Rayment et al. 2007, Inscho 2009). In recent studies, we demonstrated that P2Y2 receptors play a physiological role in blood pressure regulation and, as a consequence, P2Y2−/− mice were found to have salt-resistant hypertension (Rieg et al. 2007a, Pochynyuk et al. 2010). Direct intra-arterial blood pressure measurements indicated that the blood pressure responses to a P2Y2/4 agonist result in vasodilation via a NO-independent mechanism that possibly involves EDH release subsequently to P2Y2 receptor activation. This was concluded because endothelial NO synthase knockout mice (eNOS−/−) showed an identical blood pressure effect in response to P2Y2/4 receptor activation compared to WT mice. In contrast to WT mice, P2Y2−/− mice responded to P2Y2/4 receptor activation with an increase in blood pressure, possibly a direct effect of P2Y4 receptor activation on vascular smooth muscle cells, which is independent of P2Y2 receptors (Rieg et al. 2011). The vasodilation mediated by EDH requires activation of calcium-activated potassium channels, including KCa2.3 (small-conductance) and KCa3.1 (intermediate-conductance), which are expressed in most endothelial cells (Kohler & Ruth 2010). In contrast, KCa1.1 (big-conductance) is expressed in vascular smooth muscle cells (Feletou 2009, Kohler & Ruth 2010). The activation of calcium-activated potassium channels is speculated to produce hyperpolarization of the endothelium, which is then transmitted (possibly via connexins, see below) to underlying vascular smooth muscle cells causing vasodilation via EDH. Functional studies employing blockers of calcium-activated potassium channels have demonstrated the role of these channels in EDH and subsequent vascular smooth muscle relaxation (Adeagbo & Triggle 1993, Holzmann et al. 1994, Waldron & Garland 1994, Zygmunt & Hogestatt 1996, Eichler et al. 2003). Vascular smooth muscle cells and endothelial cells are functionally linked, and the point of contact between the two cells, the myoendothelial gap junction (MEGJ), plays a key role in the regulation of vascular function (Figueroa & Duling 2009). Initially, it was assumed that a diffusible endothelial factor was the mechanism resulting in hyperpolarization; however, this view was later questioned by experiments demonstrating the involvement of the MEGJ (Griffith et al. 2002, Dora et al. 2003, Chaytor et al. 2005, Mather et al. 2005, Sokoya et al. 2006). It was concluded from these studies that EDH is transferred from the endothelium to the smooth muscle by direct charge transfer through the MEGJ (de Wit & Wolfle 2007, Feletou & Vanhoutte 2009, Grgic et al. 2009, Edwards et al. 2010, de Wit & Griffith 2010, Garland et al. 2011). Gap junction proteins found in the vasculature include as follows: Cx37, Cx40, Cx43 and Cx45 (Figueroa et al. 2004, Lohman et al. 2012); however, the MEGJ is specifically comprised of Cx37 and Cx40 which are speculated to conduct the EDH response as an electrical signal between endothelial and vascular smooth muscle cells (Chaytor et al. 2005, Isakson & Duling 2005, Haddock et al. 2006). To further define the underlying mechanism(s) involved in the P2Y2 receptor-initiated blood pressure responses, possibly via EDH, we studied blood pressure and heart rate in WT, Cx37, Cx40, KCa1.1 and KCa3.1 knockout (−/−) mice in response to systemic application of a P2Y2/4 receptor agonist. We report that P2Y2/4 receptor activation generates a similar biphasic blood pressure response in all mice except for KCa3.1−/− and Cx37−/− mice, which show impaired vascular reactivity. This implies that both KCa3.1 and Cx37 are required for full vascular reactivity in response to P2Y2/4 receptor activation.

Background: Wound infection plays an important role in compromised wound healing. A high bioburden impairs healing and leads to formation of a chronic wound. Distinctly higher pH values were observed in chronic wounds compared to acute wounds. However, there is only limited knowledge of pH dependency on the antibacterial efficacy of common antimicrobial substances. Methods: This study investigated the pH influence on the antimicrobial efficacy of povidone (PVP)-iodine, silver nitrate, chlorhexidine, octenidine and polihexanide against Staphylococcus aureus and Pseudomonas aeruginosa using the agar diffusion test and microplate laser nephelometry. Results: The bactericidal activity of chlorhexidine and octenidine was mainly pH-independent in a pH range of 5.0-9.0. In contrast, polihexanide showed a significant efficacy increase at a higher pH. It was also found that the influence of the pH on antiseptics differs among species of bacteria. For instance, S. aureus exhibited an increasing sensitivity against silver nitrate with rising pH whereas the effect on P. aeruginosa was found to be distinctly decreased. The antimicrobial effect of PVP-iodine was strongly diminished with rising pH. Conclusions: The shift towards higher pH values in chronic wounds compared to acute wounds makes it imperative to know whether the antimicrobial efficacy of applied antimicrobial substances is altered by different pH levels. The results suggest that application of polihexanide might be advantageous for the management of wound infections, as both S. aureus and P. aeruginosa exhibited an increased susceptibility with rising pH.

AIMS: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo. METHODS AND RESULTS: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice. CONCLUSIONS: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.

Intermediate conductance calcium-activated potassium channels (K

Background Negative pressure wound therapy (NPWT) has advanced the management of the open abdomen and other wounds. However, using the correct wound filler is essential as the material needs to convey a heterogeneous pressure distribution and create the pressure gradients required to drain interstitial fluid and remove wound exudate. This study compared the differences in fluid distribution during NPWT using a large-pore PU-foam dressing (Suprasorb® CNP foam), drainage film (Suprasorb® CNP drainage film) and a specialized NPT dressing system (KCI ABThera™ NPT system) in vitro. Methods A tissue model was prepared from gelatine, powdered milk, and tissue marking dye. Each filler/dressing was placed on a tissue model which was connected to a fluid reservoir, linked to a TRAC™ Pad and sealed. The TRAC™ Pad was connected to a vacuum pump set to −120 mmHg for 8 h. An infra-red camera was used to record fluid transport in the dressings. Results All fillers/dressings exhibited a fast fluid distribution and transport during the first hour (gradients 0.97–1.31) and steady state was reached after approximately 160 min. The NPT dressing system demonstrated a distribution limited to 70%, while the PU-foam and the drainage film presented a uniform and complete dispersal of fluid. Conclusion The results demonstrate that using an appropriate filler/dressing can facilitate effective wound fluid transport and drainage of interstitial fluid by producing heterogeneous pressures at the wound bed.

The iberiotoxin-sensitive large conductance voltage- and Ca2+-activated potassium (BK) channels (maxi-K+-channels) hyperpolarize the cell membrane thus supporting Ca2+entry through Ca2+-release activated Ca2+channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca2+-insensitive BK channels (BKM513I+Δ899–903) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-functionV617FJAK2, or inactiveK882EJAK2. K+conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K+current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K+current in BKM513I+Δ899–903-expressing oocytes was significantly increased following coexpression of JAK2 orV617FJAK2 but notK882EJAK2. Coexpression of the BK channel withV617FJAK2 but notK882EJAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel andV617FJAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 μM) significantly decreased K+current. Inhibition of channel insertion by brefeldin A (5 μM) decreased the K+current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel andV617FJAK2. The iberiotoxin (50 nM)-sensitive K+current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 μM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. Significant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.

Negative-pressure wound therapy (NPWT) is an advantageous treatment option in wound management to promote healing and reduce the risk of complications. NPWT is mainly carried out using open-cell polyurethane (PU) foams that stimulate granulation tissue formation. However, growth of wound bed tissue into foam material, leading to disruption of newly formed tissue upon dressing removal, has been observed. Consequently, it would be of clinical interest to preserve the positive effects of open-cell PU foams while avoiding cellular ingrowth. The study presented analyzed effects of NPWT using large-pored PU foam, fine-pored PU foam, and the combination of large-pored foam with drainage film on human dermal fibroblasts grown in a collagen matrix. The results showed no difference between the dressings in stimulating cellular migration during NPWT. However, when NPWT was applied using a large-pored PU foam, the fibroblasts continued to migrate into the dressing. This led to significant breaches in the cell layers upon removal of the samples after vacuum treatment. In contrast, cell migration stopped at the collagen matrix edge when fine-pored PU foam was used, as well as with the combination of PU foam and drainage film. In conclusion, placing a drainage film between collagen matrix and the large-pored PU foam dressing reduced the ingrowth of cells into the foam significantly. Moreover, positive effects on cellular migration were not affected, and the effect of the foam on tissue surface roughness in vitro was also reduced.

K