SVD-clustering, a general image-analyzing method explained and demonstrated on model and Raman micro-spectroscopic maps

An image analyzing method (SVD-clustering) is presented. Amplitude vectors of SVD factorization (V1…Vi) were introduced into the imaging of the distribution of the corresponding Ui basis-spectra. Since each Vi vector contains each point of the map, plotting them along the X, Y, Z dimensions of the map reconstructs the spatial distribution of the corresponding Ui basis-spectrum. This gives valuable information about the first, second, etc. higher-order deviations present in the map. We extended SVD with a clustering method, using the significant Vi vectors from the VT matrix as coordinates of image points in a ne-dimensional space (ne is the effective rank of the data matrix). This way every image point had a corresponding coordinate in the ne-dimensional space and formed a point set. Clustering was applied to this point set. SVD-clustering is universal; it is applicable to any measurement where data are recorded as a function of an external parameter (time, space, temperature, concentration, species, etc.). Consequently, our method is not restricted to spectral imaging, it can find application in many different 2D and 3D image analyses. Using SVD-clustering, we have shown on models the theoretical possibilities and limitations of the method, especially in the context of creating, meaning/interpreting of cluster spectra. Then for real-world samples, two examples are presented, where we were able to reveal minute alterations in the samples (changing cation ratios in minerals, differently structured cellulose domains in plant root) with spatial resolution. © 2020, The Author(s)

Membrane dynamics as seen by Fourier transform infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803 The effects of lipid unsaturation and the protein-to-lipid ratio

AbstractThe roles of lipid unsaturation and lipid-protein interactions in maintaining the physiologically required membrane dynamics were investigated in a cyanobacterium strain, Synechocystis PCC 6803. The specific effects of lipid unsaturation on the membrane structure were addressed by the use of desaturase-deficient (desA−/desD−) mutant cells (which contain only oleic acid as unsaturated fatty acid species) of Synechocystis PCC 6803. The dynamic properties of the membranes were determined from the temperature dependence of the symmetric CH2 stretching vibration frequency, which is indicative of the lipid fatty acyl chain disorder. It was found that a similar membrane dynamics is maintained at any growth temperature, in both the wild-type and the mutant cell membranes, with the exception of mutant cells grown at the lower physiological temperature limit. It seems that in the physiological temperature range the desaturase system of the cells can modulate the level of lipid desaturation sufficiently to maintain similar membrane dynamics. Below the range of normal growth temperatures, however, the extent of lipid disorder was always higher in the thylakoid than in the cytoplasmic membranes prepared from the same cells. This difference was attributed to the considerable difference in protein-to-lipid ratio in the two kinds of membranes, as determined from the ratio of the intensities of the protein amide I band and the lipid ester CO vibration. The contributions to the membrane dynamics of an ab ovo present ‘structural’ lipid disorder due to the protein–lipid interactions and of a thermally induced ‘dynamic’ lipid disorder could be distinguished

Fehérjék membránba ágyazódásának, szerveződésének és lipidekkel való kölcsönhatásának biofizikája = Biophysics of protein insertion and folding in membranes and their interaction with lipids

1. A vakuólum ATPáz (V-ATPáz). Ez a membránkötött molekuláris motor az ATP hidrolízisből nyert kémiai energia révén protonokat pumpál a membránon keresztül, ami miatt a csontritkulás potenciális terápiájában ez az egyik kulcs target enzim. Meghatároztuk szintetikus V-ATPáz gátlóanyagok membránbeli lokalizációját, ami segíti az enzimen található kötőhelyeik azonosítását. Kimutattuk, hogy a V-ATPáz c alegységét funkcionálisan helyettesíteni képes langusztából izolált membránfehérje kétértékű kation kötőhelyet tartalmaz, aminek valószínűleg szerepe van a V-ATPáz c alegységének hatos gyűrűbe való rendeződésében. 2. Fehérjék és polipeptidek membránlipidekkel való kölcsönhatása. Foszfolipid membránok összetételének és fizikai állapotának a hidrofób gramicidin A és vízoldékony lizozim antibiotikumok membrán-kötődésére, -orientációjára és termikus kitekeredésére gyakorolt hatását tanulmányoztuk. A kapott eredmények új adatokat szolgáltatnak a nem kovalens lipid-fehérje kölcsönhatások szerepére az antibiotikus folyamatokban. A membránfehérjékkel kölcsönható határfelületi és kofaktor lipidek konformációját és a kölcsönhatás sztöchiometriáját tanulmányoztuk atomi felbontásban, publikált kristályszerkezetek felhasználásával. Az eredmények fontosak a lipid-fehérje kölcsönhatás natív biomembránokban tapasztalható funkcionális jelentőségének megértése szempontjából. A munkaterv némileg módosult a szerződött pályázatból való utólagos elvonások miatt. | 1. The vacuolar proton-ATPase (V-ATPase). This membranous molecular motor uses energy from ATP hydrolysis to drive proton transfer across membranes, hence it is a key potential target enzyme in osteoporosis therapy. Synthetic V-ATPase inhibitors were located in model membranes aiding the identification of their binding sites on the enzyme. A divalent cation binding site has been identified on a membrane protein isolated from lobster that is able to functionally substitute the V-ATPase subunit c. This binding site is likely to be related to the hexameric assembly of the subunit c ring of V-ATPase. 2. Protein- and polypeptide-membrane lipid interactions. The effect of the composition and the physical state of the phospholipid bilayer on the membrane-binding, -orientation and thermal unfolding of the hydrophobic gramicidin A and the water-soluble lysozyme hydrolase antibiotics were studied. The results provide new data on the significance of non-covalent lipid-protein interactions in anti-microbial processes. The conformation and stoichiometry of both annular and co-factor lipids interacting with membrane proteins were studied at atomic resolution using published crystal structures. The results are essential for the understanding the functional significance of lipid-protein interactions in native biomembranes. The grant suffered from some post-contract fund with-drawals

SVD-clustering, a general image-analyzing method explained and demonstrated on model and Raman micro-spectroscopic maps

An image analyzing method (SVD-clustering) is presented. Amplitude vectors of SVD factorization (V1…Vi) were introduced into the imaging of the distribution of the corresponding Ui basis-spectra. Since each Vi vector contains each point of the map, plotting them along the X, Y, Z dimensions of the map reconstructs the spatial distribution of the corresponding Ui basis-spectrum. This gives valuable information about the first, second, etc. higher-order deviations present in the map. We extended SVD with a clustering method, using the significant Vi vectors from the VT matrix as coordinates of image points in a ne-dimensional space (ne is the effective rank of the data matrix). This way every image point had a corresponding coordinate in the ne-dimensional space and formed a point set. Clustering was applied to this point set. SVD-clustering is universal; it is applicable to any measurement where data are recorded as a function of an external parameter (time, space, temperature, concentration, species, etc.). Consequently, our method is not restricted to spectral imaging, it can find application in many different 2D and 3D image analyses. Using SVD-clustering, we have shown on models the theoretical possibilities and limitations of the method, especially in the context of creating, meaning/interpreting of cluster spectra. Then for real-world samples, two examples are presented, where we were able to reveal minute alterations in the samples (changing cation ratios in minerals, differently structured cellulose domains in plant root) with spatial resolution. © 2020, The Author(s)

Lipid-fehérje kölcsönhatás és dinamika vizsgálata modell és biológiai membránokban. Egy új, nanotechnológiás, membránfehérje vizsgáló rendszer kifejlesztése. = Lipid-protein interactions, and dynamics in model and biological membranes. The development of a new experimental nano-technologic device to study structure and functioning of membrane-proteins.

Összehasonlítva a biológiai membránokban a lipid-, illetve a fehérje-dinamika, illetve a fehérjék másodlagos szerkezetváltozásainak hőmérsékletfüggését, megállapítottuk, hogy alacsony hőmérsékleti stressz körülményei között elsősorban a lipidek, magas hőmérsékleti stressz esetén pedig a fehérjék dinamika/szerkezet változásai a meghatározóak a biológiai membrán viselkedésében. (PMC Biophysics (2009) 2:1, Biochemistry (2009) 48, 10120-10128) Polielektrolitokból készített, tetszőlegesen töltött felszíneken felépített egy-molekula vastagságú kazein rétegek adszorpciójának nyomon követésével meghatároztuk a Ca-foszfát szállításáért felelős kazein micellák kialakulásának mechanizmusát. Megmutattuk, hogy a kazein micellák belsejében levő ?-kazein molekulák foszfoszeril csoportjaihoz kapcsolódnak a Ca-foszfát nanoklaszterek, amikhez a további ?-kazeinek pozitív csoportjaikkal kötődnek. Az ?-kazeinek hidrofób csoportjainak kölcsönhatása révén, kis, kb. 20 nm-es aggregátumok alakulnak ki, amelyeket a Ca-foszfát nanoklaszterek kapcsolnak össze. A micellák felszínére ?-kazein molekulák tapadnak, amik “lágyabb“ szerkezetük révén befedik azokat a pontokat az ?-kazeinek felszínén, amik a további akár ?-kazein, akár Ca-foszfát kötődéshez kellenek, és így a micella képződése befejeződik, felszínét ?-kazein molekulák borítják, amik nem érzékenyek a Ca-ionokra, mint az ?-kazein, így a micellák nem csapódnak ki. ( JBC (2010) 285, 38811-38817, Eur Biophys J(2012) 41:959-968) | Using Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy, we have compared lipid and protein dynamics/structural changes as a function of the temperature in biological membranes. It has been shown that among low-temperature stress conditions the changes in the dynamics of the lipids, among high-temperature stress conditions the changes of the protein dynamics and secondary structure are dominant in the behaviour of the membranes. (PMC Biophysics (2009) 2:1, Biochemistry (2009) 48, 10120-10128) By preparing polyelectrolyte films having charged surfaces, and building onto them layer-by-layer aggregated casein-Ca-phosphate complexes, we determined the mechanism of the construction of the casein micelles, the essential components of Ca-phosphate transport in mammals. We have shown that the phosphoseryl residues on the ?-casein molecules are essential for binding the Ca-phosphate. The bound Ca-phosphate nanoclusters bind the next layer of the ?-caseins, as so on. Hydrophobic parts of the amphiphilic molecules organize ?-caseins into about 20 nm diameter aggregates, interconnected by Ca-phosphate nanoclusters. Termination of the micelle formation is achieved via the adsorption of ?-casein onto the micelle surface. Due to its “soft“ secondary structure, ?-casein can cover those parts of the ?-casein molecules, which are essential for Ca-phosphate binding and further aggregation of ?-caseins. (JBC (2010) 285, 38811-38817, Eur Biophys J(2012) 41:959-968

What have we learned from two-pore potassium channels? Their molecular configuration and function in the human heart

Two-pore domain potassium channels (K2P) control excitability, stabilize the resting membrane potential below firing threshold, and accelerate repolarisation in different cells. Until now, fifteen different genes for the six K2P channel subfamily were cloned. The pore-forming part is translated from two genes and they are built up from a dimer of two two-unit transmembrane domains functioning with a wide spectrum of physiological profiles. K2P ion channels were discovered in the last two decades and gave novel opportunity to recognize the complex molecular mechanism of the potassium ion flux, and may lead to the design of individual drug targeting in the future. In this review, we summarise the structure, function, channelopathies and pharmacological silhouette of the two-pore potassium channels in the human tissues. In addition, we present the computer model of the partially reconstructed wild type K2P1/TWIK1 lacking the intracellular C and N terminal loop

A foszfatidilglicerol szerepe fotoszintetikus membránok szerkezetének kialakításában = The essential role of phosphatidylglycerol in the formation of photosynthetic membrane structure

A negatív töltéssel rendelkező lipidek közül a foszfatidilglicerol (PG) jelentős szerepet tölt be. 1. Létrehoztuk egy FG szintézisben gátolt Synechocytis PCC6803 mutánst melyben megnéztük a CP43 kapcsolódását a kettes fotokémiai rendszer reakció centrumához. Ezt a mutánst egy, a fénybegyűjtő rendszer nélküli mutánssal hoztuk létre, a cdsA gén inaktiválásával. Megállapítottuk, hogy az FG kiürülés hatására a CP43 fehérje bekötődése gátolt. Megállapítottuk, hogy az FG kiürülés csökkenti a cianobaktérium sejtek oxigénfejlesztő képességét. 2. Megállapítottuk, hogy az FG kiürülés megváltoztatja a fotoszintetikus membrán felszíni töltését. 3. Az FG molekulák hatására bekövetkező fotoszintetikus aktivitást a reakció centrumok megváltozása okozza. A reakció centrumok fényérzékennyé vállnak. Ez ellen a sejtek megnövekedett karotin tartalommal védekeznek. A myxoxantin és az echinenon mennyisége nő meg. 4. A Syenechococcus PCC PCC7942 cdsA mutáns létrehozásával igazoltuk, hogy az FG molekulák szerepe a fotoszintetikus szervezetekben általánosítható. Megállapítottuk, hogy az FG kiürülés a különböző törzseken különböző mértékben hat. 5 Az FG molekulák kiürülésének hatására bekövetkező stressz elleni protekció a karotinoid molekulák megjelenése fotoszintetikus reakció centrumokbanmellett megfigyeltük azt is, hogy az FG molekulák zsírsav összetétele megváltozik. A molekulák átalakulását tömegspektometriásan igazoltuk. Az eredményeinket egy áttekintő cikkünkben foglaltuk össze. | Among the negatively charged lipids phosphatidylglycerol (PG) plays an important role. We used transformable cyanobacterial strains.1n We used a Phycobilisome-less mutant of Synechocytis PCC6803 was transformed with inactivated cdsA and the synthesis of PG was blocked. Binding of CP43 to the reaction center complex was investigated. The oxygen evolving activity of mutant was reduced. 2. The PG-depletion affected the surface charge of membranes. 3. PG-depletion induced sensitivity of cells which was compensated by elevated carotenoid content, mainly myxoxanthophyll and echinenone. 4. We generated Syenechococcus PCC PCC7942 cdsA mutant with which we could generalize the importance of PG in photosynthetic organizms. However, we could observe slight differencein different mutant. 5. Westudied PG remodeling by mas spectrometry

Kinetics and Structure of Self-Assembled Flagellin Monolayers on Hydrophobic Surfaces in the Presence of Hofmeister Salts: Experimental Measurement of the Protein Interfacial Tension at the Nanometer Scale

In the present study, we monitor the adsorption–desorption kinetics and adsorbed layer structure of the bacterial protein flagellin in the presence of Hofmeister salts by a surface sensitive label-free optical biosensor (optical waveguide lightmode spectroscopy, OWLS). The recorded OWLS data were analyzed by a computer code using a set of coupled differential equations modeling the adsorption–desorption process. By supposing reversibly and irreversibly adsorbed protein states with different adsorption footprints, the kinetic data could be perfectly fitted. We revealed that the proteins adsorbing in the presence of kosmotropic salts had smaller footprints, leading to a more oriented and densely packed layer. Kosmotropic salts increased both the adsorption rate constant and the transition rate constants from the reversibly to the irreversibly adsorbed state. In contrast, chaotropic salts increased the desorption rate constant and led to decreased adsorbed mass and a more loosely packed film. Neither circular dichroism spectroscopy in bulk solutions or Fourier transform infrared spectroscopy of surface-adsorbed flagellins could reveal significant structural changes due to the presence of the Hofmeister salts, and supported our conclusions about the adsorption mechanism. On the basis of the measured kinetic and structural data (footprints of adsorbed proteins), we developed a model to calculate the protein–water-substrate interfacial tension in the presence of Hofmeister salts, and compared the experimentally obtained values with related literature data. The calculated values are consistent with previously published data of surface tension changes, andto the best of our knowledgerepresent the first experimental results for this quantity

Nanoparticles for drug delivery across the blood-brain barrier: a cell culture study

Background: Efficient drug delivery across the blood-brain barriers (BBB) is a central problem in pharmaceutical treatment of neurological diseases. Most pharmaceutical drug candidates including hydrophilic molecules, biopharmaceuticals, and efflux transporter ligands have a low permeability across barriers. To solve this unmet therapeutical need vesicular or solid nanoparticle drug delivery systems targeting physiological transporters of the BBB hold a great promise. Curcumin extracted from the plant turmeric possesses anti-oxidative, anti-inflammatory and neuroprotective properties and is a potential treatment for different cerebral diseases. However, the clinical application of this natural compound is hampered by its poor water solubility and absorption, rapid metabolism and systemic elimination resulting in low bioavailability. Nanosized, biocompatible and biodegradable vesicles containing Evans blue-albumin as a model molecule and a hydrophobic therapeutic biomolecule, curcumin were prepared and characterized. In addition, fluorescent solid nanoparticles were also examined. The aim of our study was to test the cellular toxicity and penetration of nanovesicles loaded with albumin or curcumin and fluorescent nanoparticles all containing ligands for transporters on culture models of the BBB. Methods: The nanovesicles and fluorescent solid nanoparticles were labelled with different ligands, biotin, a glucose analogue and glutathione. Primary rat and human hCMEC/D3 brain endothelial cells were used as in vitro model systems of the BBB. The cellular toxicity of the nanoparticles was measured by real-time cell microelectric sensing (RTCA-SP, ACEA Biosciences) and MTT assay. The permeability tests were performed on triple co-culture BBB model and hCMEC/D3 cells using Transwell inserts. Brain endothelial uptake of nanoparticles was quantified by fluorescent spectroscopy and visualized by confocal microscopy. Results: No toxicity for loaded or unloaded nanovesicles or solid nanoparticles was found by MTT assay and impedance measurements. The loading of curcumin into liposomes significantly decreased its toxicity for brain endothelial cells at high concentrations. The presence of a glucose analogue in nanovesicles increased the uptake of the model molecule to cultured brain endothelial cells. The brain endothelial uptake of both loaded nanovesicles and solid nanoparticles could be followed by confocal microscopy. Conclusion: Our data indicate that encapsulation of lipophilic or macromolecular drugs into nanovesicles may decrease cellular toxicity and increase uptake and transport at the BBB, however the type of the targeting ligand and its coupling to the nanoparticle may be crucial for efficacy