Az ischaemiás agykárosodásban szerepet játszó TrpM2 kationcsatorna szerkezet-funkció vizsgálata = Structure-function studies of the TrpM2 cation channel involved in ischaemic brain damage.

Megállapítottuk, hogy a csatornát 4 Ca2+ ion kötődése aktiválja. Az aktiváció a Monod-Wymann-Changeux mechanizmust követi, 1 Ca2+ kötődése ~33-szorosra, a 4 ion összesen ~10^6-szorosra, növeli a nyitott-csukott egyensúlyi állandót. A Ca2+ kötőhelyei a kaputól intracelluláris irányban találhatók egy védett üregben, közel a pórus nyílásához. A nyitott póruson beáramló Ca2+ telítésben tartja az aktiváló helyeket, ezért intakt sejtekben, ADPR jelenlétében, egy rövid Ca2+ szignál is elnyújtott TRPM2 aktivitást válthat ki. Megállapítottuk, hogy az ADPR nagy affinitással (K1/2=1uM) aktivál, de az ADPR-hidrolízisnek a csatorna csukódásában játszott szerepét nem tudtuk tisztázni. Az ADPRázok konzervált ""Nudix-box"" motívuma (REFXEE) a TRPM2 NUDT9-H doménjében atípusos (RILRQE). A NUDT9 enzimben az EF->IL mutáció 1%-ára csökkenti, az EE->KK mutáció felfüggeszti az ADPRáz aktivitást. Létrehoztunk egy ""inaktív"" QE->KK és egy ""hiperaktív"" IL->EF TRPM2 mutánst, de e 4 egyedi és 2 dupla mutáció egyike sem befolyásolta az ADPR iránti affinitást illetve a csukódási sebességet. Intakt sejtekben az AMP gátolta, míg a H2O2, a ciklikus ADPR (cADPR), és a nikotinsav-adenin-dinukleotid-foszfát (NAADP) aktiválták a TRPM2-t, és fokozták ADPR iránti érzékenységét. Izolált membrán patch-ben megállapítottuk, hogy a H2O2, az AMP, és a cADPR közvetlenül nem hatnak a TRPM2-re, míg az NAADP és az NAAD kis affinitású parciális agonisták. Tehát intakt sejtekben e modulátorok hatásai közvetettek. | We have revealed that the channel is activated by binding of 4 Ca2+ ions, following the Monod-Wymann-Changeux mechanism. Binding of 1 Ca2+ increases the closed-open equilibrium constant by ~33-fold, the 4 ions altogether by ~10^6-fold. The Ca2+ binding sites are found intracellularly of the gate, in a protected crevice, near the pore entrance, and are kept saturated by Ca2+ flowing through the open pore. Thus, in intact cells, in the presence of ADPR, a single brief Ca2+ spark can elicit prolonged TRPM2 channel activity. We have shown that ADPR activates the channel with high affinity (K1/2=1 uM), but could not clarify the role of ADPR hydrolysis in channel closure. The conserved ADPRase ""Nudix-box"" motif (REFXEE) is atypical in the NUDT9-H domain of TRPM2 (RILRQE). The EF->IL mutation decreases ADPRase activity of the NUDT9 enzyme to ~1%, while the EE->KK mutation completely abolishes it. We constructed an ""inactive"" QE?KK and a ""hiperactive"" IL->EF TRPM2 mutant, but neither of the 4 single and 2 double mutants affected ADPR affinity or channel closing rate. In intact cells AMP inhibits, while H2O2, cyclic ADPR (cADPR), and nicotinic acid-adenin-dinucleotide-phosphate (NAADP) activate TRPM2, and enhance its sensitivity to ADPR. We have found that direct application of H2O2, AMP, and cADPR in isolated patches does not affect the channels, while NAADP and NAAD are low-affinity partial agonists. Thus, in intact cells the effects of these modulators are indirect

Insula Insolita – Szigetköz és Bős-Nagymaros párhuzamos története

A közlemény célja a szigetközi ártér és a Bős-Nagymaros Vízlépcsőrendszer (BNV) párhuzamos történetének bemutatása és a történet tanulságainak levonása. A BNV-t megelőző idők árvízvédelmi és folyószabályozási munkáinak mellékhatásaként, továbbá a Duna fokozott szennyezőanyag terhelése miatt, súlyos degradációs folyamatok indultak be a Szigetközben, melyek megállítása és visszafordítása a BNV egyik fontos célkitűzése volt. A múlt század nyolcvanas éveiben kialakult mozgalmak a környezetvédelem jelszavát hirdetve, ám hamis érvekre hivatkozva a BNV megvalósításának megakadályozását tűzték ki célul. Ezek a mozgalmak, köszönhetően a rendszerváltás idejére kiépített politikai befolyásuknak, végül elérték a céljukat: a BNV beruházását magyar részről egyoldalúan felfüggesztették, majd végleg leállították. Szlovákia azonban, mint a nemzetközi beruházás másik résztvevője, nem adta fel a projektet és a magyar fél kiszállásából adódó kényszerhelyzethez alkalmazkodva, egyoldalúan kivitelezte azt. Ennek következménye lett a szigetközi Duna szakasz 30 évvel ezelőtti elterelése. A BNV magyar részről történő leállítása súlyos károkat okozott Magyarországnak: az anyagi, szellemi és erkölcsi károkozáson túl, az ország de facto elvesztette a beruházás tárgyában Szlovákia ellen indított nemzetközi pert és még az elkészült mű által megtermelt megújuló energiából sem részesedik. Az elmúlt évtizedek során végzett monitoring tevékenység egyértelműen cáfolta a vízlépcsőellenes mozgalom ökológiai katasztrófát vizionáló álláspontját. A szigetközi hullámtér talaj- és felszíni vizeinek minőségi és mennyiségi viszonyaiban degradáció helyett javulás történt ‒ még a BNV előtti időkhöz képest is. Ez a javulás a Duna vízminőségében beállt pozitív változásoknak, valamint a Szigetközben kiépült vízpótló-rendszernek köszönhető. A vízpótló-rendszer kiépítésével a magyar vízügyi szolgálat újfent bebizonyította, hogy képes nagyszerű teljesítmények elérésére

Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the ~3 A° resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post- filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2

The relevance of neck linker docking in the motility of kinesin

Conventional kinesin is a motor protein, which is able to walk along a microtubule processively. The exact mechanism of the stepping motion and force generation of kinesin is still far from clear. In this paper we argue that neck linker docking is a crucial element of this mechanism, without which the experimentally observed dwell times of the steps could not be explained under a wide range of loading forces. We also show that the experimental data impose very strict constraints on the lengths of both the neck linker and its docking section, which are compatible with the known structure of kinesin.Comment: Accepted for publication in BioSystems as part of the proceedings of BIOCOMP 200

Neck linker docking coordinates the kinetics of kinesin's heads

Conventional kinesin is a two-headed homodimeric motor protein, which is able to walk along microtubules processively by hydrolyzing ATP. Its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. Although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. Considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, here we develop the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads (including their gating mechanisms) during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). Besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion.Comment: to appear in the Biophysical Journa

Application of Electronic Tongue to Soya Drink Discrimination

The objective of the research was to compare the taste attributes of different commercial soya drinks. Furthermore, the task was to determine the effect of different ingredients and processing technologies on the taste attributes of the product. Based on the results of electronic tongue measurements the instrument is able to determine the effect of the applied technology and to distinguish soya juice samples according to sensory preferences. Canonical discriminant analysis showed that the groups of two measurements of the same products were overlapping. Therefore, the electronic tongue measurements are supposed to be of acceptable repeatability. The canonical discriminant analysis showed that the taste attributes of soya juice made of hulled soybeans was beneficial for the taste attributes relative to that of the juice made of not-hulled soybeans. Three main groups could be observed from the analyzed six commercial soya drink samples based on canonical discriminant analysis. There is a group of top market brands having definite taste improver additives and another one containing three products having low amount of additives. However, the group of samples made of soybean and rice is located between the above-mentioned two groups in the discriminant score plot

A Novel Immune-Related Gene Prognostic Index (IRGPI) in Pancreatic Adenocarcinoma (PAAD) and Its Implications in the Tumor Microenvironment

Purpose: Pancreatic adenocarcinoma (PAAD) is one of the most lethal malignancies, with less than 10% of patients surviving more than 5 years. Existing biomarkers for reliable survival rate prediction need to be enhanced. As a result, the objective of this study was to create a novel immune-related gene prognostic index (IRGPI) for estimating overall survival (OS) and to analyze the molecular subtypes based on this index. Materials and procedures: RNA sequencing and clinical data were retrieved from publicly available sources and analyzed using several R software packages. A unique IRGPI and optimum risk model were developed using a machine learning algorithm. The prediction capability of our model was then compared to that of previously proposed models. A correlation study was also conducted between the immunological tumor microenvironment, risk groups, and IRGPI genes. Furthermore, we classified PAAD into different molecular subtypes based on the expression of IRGPI genes and investigated their features in tumor immunology using the K-means clustering technique. Results: A 12-gene IRGPI (FYN, MET, LRSAM1, PSPN, ERAP2, S100A1, IL20RB, MAP3K14, SEMA6C, PRKCG, CXCL11, and GH1) was established, and verified along with a risk model. OS prediction by our model outperformed previous gene signatures. According to the findings of our correlation studies, different risk groups and IRGPI genes were found to be tightly related to tumor microenvironments, and PAAD could be further subdivided into immunologically distinct molecular subtypes based on the expression of IRGPI genes. Conclusion: The current study constructed and verified a unique IRGPI. Furthermore, our findings revealed a connection between the IRGPI and the immunological microenvironment of tumors. PAAD was differentiated into several molecular subtypes that might react differently to immunotherapy. These findings could provide new insights for precision and translational medicine for more innovative immunotherapy strategies

Application of Electronic Tongue for Distinguishing Coffee Samples and Predicting Sensory Attributes

Efforts have been made to predict the sensory profile of coffee samples by instrumental measurement results. The objective of the work was to evaluate the most important sensory attributes of coffee samples prepared from ground roasted coffee by electronic tongue and by sensory panel. Further aim was to predict the Arabica concentration and the main sensory attributes of the different coffee blends by electronic tongue and to analyze the sensitivity of the electronic tongue to the detection of poor quality coffee samples. Five coffee blends with known Arabica and Robusta concentration ratio, five commercially available coffee blends and a poor quality coffee were analyzed. The electronic tongue distinguished the coffee samples according to the Arabica and Robusta content. The sensory panel was able to discriminate the samples based on global aroma, bitterness and coffee aroma intensity (p < 0.01). The Arabica concentration was predicted from the electronic tongue results by PLS with close correlation and low prediction error. Models were developed to predict sensory attributes of the tested coffee samples from the results obtained by the electronic instrument