Browsing by Subject "CHANNEL"

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  • Gal-Or, Eran; Gershoni, Yaniv; Scotti, Gianmario; Nilsson, Sofia Märta Elisabeth; Saarinen, Jukka Kalle Samuel; Jokinen, Ville Petteri; Strachan, Clare Joanna; Boije af Gennäs, Per Gustav; Yli-Kauhaluoma, Jari Tapani; Kotiaho, Ahti Antti Tapio (2019)
    Additive manufacturing (3D printing) is a disruptive technology that is changing production systems globally. In addition, microfluidic devices are increasingly being used for chemical analysis and continuous production of chemicals. Printing of materials such as polymers and metals is already a reality, but additive manufacturing of glass for microfluidic systems has received minor attention. We characterize microfluidic devices (channel cross-section dimensions down to a scale of 100 mm) that have been produced by additive manufacturing of molten soda-lime glass in tens of minutes and report their mass spectrometric and Raman spectroscopic analysis examples. The functionality of a microfluidic glass microreactor is shown with online mass spectrometric analysis of linezolid synthesis. Additionally, the performance of a direct infusion device is demonstrated by mass spectrometric analysis of drugs. Finally, the excellent optical quality of the glass structures is demonstrated with in-line Raman spectroscopic measurements. Our results promise a bright future for additively manufactured glass microdevices in diverse fields of science.
  • Mirzalieva, Oygul; Jeon, Shinhye; Damri, Kevin; Hartke, Ruth; Drwesh, Layla; Demishtein-Zohary, Keren; Azem, Abdussalam; Dunn, Cory D.; Peixoto, Pablo M. (2019)
    The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology to investigate the role of Mgr2p on the channel activity of TIM23. Deletion of Mgr2p decreased normal channel frequency and increased occurrence of a residual TIM23 activity. The residual channel lacked gating transitions but remained sensitive to synthetic import signal peptides. Similarly, a G145L mutation in Tim23p displaced Mgr2p from the import complex leading to gating impairment. These results suggest that Mgr2p regulates the gating behavior of the TIM23 channel.
  • Björk, Susann; Ojala, Elina A.; Nordstrom, Tommy; Ahola, Antti; Liljestrom, Mikko; Hyttinen, Jari; Kankuri, Esko; Mervaala, Eero (2017)
    Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the I-Ks potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings.
  • Ali-Sisto, Toni; Tolmunen, Tommi; Viinamäki, Heimo; Mäntyselkä, Pekka; Valkonen-Korhonen, Minna; Koivumaa-Honkanen, Heli; Honkalampi, Kirsi; Ruusunen, Anu; Nandania, Jatin; Velagapudi, Vidya; Lehto, Soili M. (2018)
    Background: Major depressive disorder (MDD) is characterized by increased oxidative and nitrosative stress. We compared nitric oxide metabolism, i.e., the global arginine bioavailability ratio (GABR) and related serum amino acids, between MDD patients and non-depressed controls, and between remitted and non-remitted MDD patients. Methods: Ninety-nine MDD patients and 253 non-depressed controls, aged 20-71 years, provided background data via questionnaires. Fasting serum samples were analyzed using ultra-performance liquid chromatography coupled to mass spectrometry to determine the serum levels of ornithine, arginine, citrulline, and symmetric and asymmetric dimethylarginine. GABR was calculated as arginine divided by the sum of ornithine plus citrulline. We compared the above measures between: 1) MDD patients and controls, 2) remitted (n= 33) and non-remitted (n = 45) MDD patients, and 3) baseline and follow-up within the remitted and non-remitted groups. Results: Lower arginine levels (OR 0.98, 95% CI 0.97-0.99) and lower GABR (OR 0.13, 95% CI 0.03-0.50) were associated with the MDD vs. the non-depressed group after adjustments for potential confounders. The remitted group showed a decrease in GABR, arginine, and symmetric dimethylarginine, and an increase in ornithine after the follow-up compared with within-group baseline values. The non-remitted group displayed an increase in arginine and ornithine levels and a decrease in GABR. No significant differences were recorded between the remitted and non-remitted groups. Limitations: The MDD group was not medication-free. Conclusions: Arginine bioavailability may be decreased in MDD. This could impair the production of nitric oxide, and thus add to oxidative stress in the central nervous system.
  • Lahtinen, Annukka M.; Marjamaa, Annukka; Swan, Heikki; Kontula, Kimmo (2011)
  • Eskelin, Katri; Oksanen, Hanna M; Poranen, Minna (2021)
    Asymmetrical flow field-flow (AF4) fractionation aims in separation of sample components to yield elution of homogenous fractions identified as well-defined peaks in the chromatograms. Separation that occurs in matrixfree open channel potentiates high recovery that can be close to 100%. However, sample properties and separation conditions may induce carryover of sample components during AF4 analysis and in sample sequences. This compromises the quality of the data collected from the online detectors and the downstream offline analytics of the collected fractions. In this study, we followed sample carryover in AF4 using model viruses and analyzed various cleaning solutions and rinse methods to reduce carryover. We introduce an SDS-NaOH -based rinsing and decontamination protocol for the AF4 instrument enabling high-quality data collection.
  • The CMS collaboration; Sirunyan, A. M.; Eerola, P.; Kirschenmann, H.; Pekkanen, J.; Voutilainen, M.; Havukainen, J.; Heikkilä, J. K.; Järvinen, T.; Karimäki, V.; Kinnunen, R.; Lampén, T.; Lassila-Perini, K.; Laurila, S.; Lehti, S.; Lindén, T.; Luukka, P.; Mäenpää, T.; Siikonen, H.; Tuominen, E.; Tuominiemi, J.; Tuuva, T. (2018)
    A search is presented for new high-mass resonances decaying into electron or muon pairs. The search uses proton-proton collision data at a centre-of-mass energy of 13TeV collected by the CMS experiment at the LHC in 2016, corresponding to an integrated luminosity of 36 fb(-1). Observations are in agreement with standard model expectations. Upper limits on the product of a new resonance production cross section and branching fraction to dileptons are calculated in a model-independent manner. This permits the interpretation of the limits in models predicting a narrow dielectron or dimuon resonance. A scan of different intrinsic width hypotheses is performed. Limits are set on the masses of various hypothetical particles. For the Z(SSM)' (Z(psi)') particle, which arises in the sequential standard model (superstring-inspired model), a lower mass limit of 4.50 (3.90) TeV is set at 95% confidence level. The lightest Kaluza-Klein graviton arising in the Randall-Sundrum model of extra dimensions, with coupling parameters k/(M) over bar (Pl) of 0.01, 0.05, and 0.10, is excluded at 95% con fi dence level below 2.10, 3.65, and 4.25TeV, respectively. In a simpli fi ed model of dark matter production via a vector or axial vector mediator, limits at 95% con fi dence level are obtained on the masses of the dark matter particle and its mediator.
  • Hiekkala, Marjo Eveliina; Vuola, Pietari; Artto, Ville; Häppölä, Paavo; Häppölä, Elisa; Vepsäläinen, Salli; Cuenca-Leon, Ester; Lal, Dennis; Gormley, Padhraig; Hämäläinen, Eija; Ilmavirta, Matti; Nissilä, Markku; Säkö, Erkki; Sumelahti, Marja-Liisa; Harno, Hanna; Havanka, Hannele; Keski-Säntti, Petra; Färkkilä, Markus; Palotie, Aarno; Wessman, Maija; Kaunisto, Mari Anneli; Kallela, Mikko (2018)
    Objective To study the position of hemiplegic migraine in the clinical spectrum of migraine with aura and to reveal the importance of CACNA1A, ATP1A2 and SCN1A in the development of hemiplegic migraine in Finnish migraine families. Methods The International Classification of Headache Disorders 3rd edition criteria were used to determine clinical characteristics and occurrence of hemiplegic migraine, based on detailed questionnaires, in a Finnish migraine family collection consisting of 9087 subjects. Involvement of CACNA1A, ATP1A2 and SCN1A was studied using whole exome sequencing data from 293 patients with hemiplegic migraine. Results Overall, hemiplegic migraine patients reported clinically more severe headache and aura episodes than non-hemiplegic migraine with aura patients. We identified two mutations, c.1816G>A (p.Ala606Thr) and c.1148G>A (p.Arg383His), in ATP1A2 and one mutation, c.1994C>T (p.Thr665Met) in CACNA1A. Conclusions The results highlight hemiplegic migraine as a clinically and genetically heterogeneous disease. Hemiplegic migraine patients do not form a clearly separate group with distinct symptoms, but rather have an extreme phenotype in the migraine with aura continuum. We have shown that mutations in CACNA1A, ATP1A2 and SCN1A are not the major cause of the disease in Finnish hemiplegic migraine patients, suggesting that there are additional genetic factors contributing to the phenotype.
  • Leinonen, Jaakko T.; Crotti, Lia; Djupsjobacka, Aurora; Castelletti, Silvia; Junna, Nella; Ghidoni, Alice; Tuiskula, Annukka M.; Spazzolini, Carla; Dagradi, Federica; Viitasalo, Matti; Kontula, Kimmo; Kotta, Maria-Christina; Widen, Elisabeth; Swan, Heikki; Schwartz, Peter J. (2018)
    Background: Ventricular fibrillation (VF) is a major cause of sudden cardiac death. In some cases clinical investigations fail to identify the underlying cause and the event is classified as idiopathic (IVF). Since mutations in arrhythmia-associated genes frequently determine arrhythmia susceptibility, screening for disease-predisposing variants could improve IVF diagnostics. Methods and results: The study included 76 Finnish and Italian patients with a mean age of 31.2 years at the time of the VF event, collected between the years 1996-2016 and diagnosed with idiopathic, out-of-hospital VF. Using whole-exome sequencing (WES) and next-generation sequencing (NGS) approaches, we aimed to identify genetic variants potentially contributing to the life-threatening arrhythmias of these patients. Combining the results from the two study populations, we identified pathogenic or likely pathogenic variants residing in the RYR2, CACNA1C and DSP genes in 7 patients (9%). Most of them(5, 71%) were found in the RYR2 gene, associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). These genetic findings prompted clinical investigations leading to disease reclassification. Additionally, in 9 patients (11.8%) we detected 10 novel or extremely rare (MAF <0.005%) variants that were classified as of unknown significance (VUS). Conclusion: The results of our study suggest that a subset of patients originally diagnosed with IVF may carry clinically-relevant variants in genes associated with cardiac channelopathies and cardiomyopathies. Although misclassification of other cardiac channelopathies as IVF appears rare, our findings indicate that the possibility of CPVT as the underlying disease entity should be carefully evaluated in IVF patients. (C) 2017 Elsevier B.V. All rights reserved.
  • d'Enterria, David; Krajczar, Krisztian; Paukkunen, Hannu (2015)
    Single and pair top-quark production in proton-lead (p-Pb) and lead-lead (Pb-Pb) collisions at the CERN Large Hadron Collider (LHC) and Future Circular Collider (FCC) energies, are studied with next-to-leading-order perturbative QCD calculations including nuclear parton distribution functions. At the LHC, the pair-production cross sections amount to sigma(t (t) over bar) = 3.4 mu b in Pb-Pb at root s(NN) = 5.5 TeV, and sigma(t (t) over bart) = 60 nb in p-Pb at root s(NN) = 8.8 TeV. At the FCC energies of root s(NN) = 39 and 63 TeV, the same cross sections are factors of 90 and 55 times larger respectively. In the leptonic final-state t (t) over bar --> W(+)b W-(b) over bar --> b (b) over bar ll nu nu with l = e(+/-), mu(+/-), after typical acceptance and efficiency cuts, one expects about 90 and 300 top-quarks per nominal LHC-year and 4.7 . 10(4) and 10(5) per FCC-year in Pb-Pb and p-Pb collisions respectively. The total t (t) over bar cross sections, dominated by gluon fusion processes, are enhanced by 3-8% in nuclear compared to p-p collisions due to an overall net gluon antishadowing, although different regions of their differential distributions are depleted due to shadowing or EMC-effect corrections. The rapidity distributions of the decay leptons in t (t) over bar processes can be used to reduce the uncertainty on the Pb gluon density at high virtualities by up to 30% at the LHC (full heavy-ion programme), and by 70% per FCC-year. The cross sections for single-top production in electroweak processes are also computed, yielding about a factor of 30 smaller number of measurable top-quarks after cuts, per system and per year. (C) 2015 The Authors. Published by Elsevier B.V.
  • Parmar, Mayuriben; Rawson, Shaun; Scarff, Charlotte A.; Goldman, Adrian; Dafforn, Timothy R.; Muench, Stephen P.; Postis, Vincent L.G. (2018)
    The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7 angstrom range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.