Search

Summary. The research deals with the investigation of one alternative of carbon-dioxide utilization – underground storage – from a petroleum geoscience point of view. The basic assumptions and the results of the laboratory studies to be carried out later are based on a specific hydrocarbon field in Hungary. The previously measured and studied geological and petrophysical parameters of the reservoir (porosity, permeability, saturation, capacity, etc.) will be restudied and specified, based on the results of the new concept of laboratory experiments. By defining these parameters, a 3D geological model, a “Dynamic model” will be created to understand the effect of carbon-dioxide injection on the dynamic behavior of a moderate-tight sandstone reservoir. Based on the results of the dynamic model, the storage capacity will be defined. The carbon-dioxide injection laboratory experiments will contribute to understanding these underground geo-chemical reactions (e.g. carbonation ability, compositional variation) and flow characteristics. Összefoglalás. Korunk egyik legmeghatározóbb problémája az üvegházhatású gázok, különösen a szén-dioxid kibocsátásának csökkentése. Alapvetően két fő ágra oszlanak ezen törekvések: egyrészt a kibocsátásért felelős technológiák optimalizálására/„zöldítésére”, másrészt a már kibocsátott szén-dioxid hasznosítására (CCU), illetve befogására és letárolására (CCS). A kutatás a szén-dioxid hasznosítás egyik alternatívájával, a föld alatti letárolás lehetőségének vizsgálatával foglalkozik földtudományi aspektusból. A kézirat alap felvetései és a későbbiekben magmintákon elvégzendő laborkísérletek eredményei egy konkrét, Magyarországon található szénhidrogén mezőre vonatkoznak. Emiatt más szén-dioxid tárolására alkalmas földtani képződmény (sókaverna, széntelep, illetve sósvizes aquifer) vizsgálatára a tanulmány nem tér ki. A laborkísérletek eredményeinek segítségével az előzetesen a mérnöki gyakorlatban használt és ismert tárolói paraméterek (porozitás, permeabilitás, kapacitás, telítettség stb.) kerülnek pontosításra. Az elvégzendő labormérések: higany besajtolásos porozitás vizsgálat, centrifugális kapilláris nyomásgörbe és relatív permeabilitási görbék meghatározása, röntgendiffrakciós anyagvizsgálat. Ezen paraméterek ismeretében egy pontosított földtani modell kerül megalkotásra. A kutatás első fázisában a már meglévő, ipari gyakorlatban alkalmazott kőzetvizsgálati módszerek kerültek felülvizsgálatra, és egy új szemléletű, az eddigi módszereket pontosító eljárás került kidolgozásra a hazai geológiai formációkra vonatkoztatva. A későbbiekben a kőzetmintákon végzett tárolói paramétereket szimuláló szén-dioxid besajtolási kísérletek a föld alatti reakciók (pl. karbonátosodási képesség) és az áramlástani sajátosságok megértéséhez és modellezéséhez is hozzájárulhatnak. Feltételezhetően a föld alatti reakcióknak köszönhetően egy, a korábbi becslésektől eltérő tárolótérfogatot lehet meghatározni. Megállapításra került, hogy a szén-dioxid szuperkritikus állapotban besajtolva, rétegvízzel rendelkező zárt rétegekben egybefüggő „csóvaként” vándorol a porózus kőzeteken keresztül, amely a gravitációs szegregáció következtében vertikálisan kitágul a fedőkőzet alatt. Az oldalirányú szén-dioxid expanzió folyamatát a folyadékok csapdázódása korlátozza. Ezért a többfázisú áramlás és csapdázódás alapos vizsgálata elengedhetetlen a tárolókapacitás pontos meghatározásának érdekében. A szén-dioxid besajtolhatóságát és a tárolási kapacitást nagymértékben befolyásolja a szén-dioxiddal telített sóoldat relatív permeabilitása, amely erősen függ a kőzet heterogenitásától. Miljkovic (2006) sóoldattal telített homokkő minták szimulációs méréseit hasonlította össze, amelyek csak a heterogenitás tekintetében tértek el egymástól. Megmutatta, hogy a kis strukturálatlan heterogenitás, úgy tűnik, nem befolyásolja jelentősen a CO2 telítettségét és ennek következtében a tárolási kapacitást. Ezzel ellentétben Kuo és társai (2011) kimutatták, hogy a telítési profilt erősen befolyásolja a mag heterogenitása, és nagy injektálási sebességre van szükség ahhoz, hogy a heterogén közeg relatív állandó telítettségét elérje a homogénhez képest. Hozzájuk hasonlóan Shi és társai (2009) heterogén homokkő magokon szimulálták a szén-dioxiddal telített sóoldat elvezetését és beszívódását. Kimutatták, hogy a porozitás változása szignifikáns hatással volt a CO2 migrációs mintázatára alacsony kiszorítási sebesség mellett és ez fokozatosan eltűnik az injektálási sebesség növelésével. Fontos célkitűzésként jelenik meg a kutatásokban ezen tézisek vizsgálata, valamint a többfázisú áramlási kísérletek elvégzése a magyarországi CCS potenciális jelöltjeként számon tartott tároló magmintáin. A víz-gáz elvezetési relatív permeabilitási vizsgálatok szimulált tározókörülmények között szintén fontos új információkat fognak szolgáltatni, melyek lehetőséget teremtenek a CO2 front végső eloszlásának meghatározására, valamint javaslattételre a tárolási kapacitás pontosítására és a geokémiai változásokra az adott tárolórétegekre jellemző heterogenitás függvényében.

Elméleti háttér: Az olyan válsághelyzetekben, mint a COVID-19, kiemelt hangsúlyt kell kapjon a segítő szakmákban dolgozó szakemberek pszichés támogatása a szolgáltatások fenntarthatósága és a kiégés megelőzése szempontjából. Különösen fontos a kiégés hátterében felismerhető vitális kimerültség mérése. Cél: Keresztmetszeti vizsgálatunk célja a segítő területen dolgozók vitális kimerültségének és ennek különböző háttérváltozókkal való összefüggéseinek vizsgálata a COVID-19 első hullámában. Módszerek: Online kérdőívünket az egészségügy, a szociális ellátás, az oktatás és a hitélet területén dolgozó szakemberek (n = 931) töltötték ki. A kérdőív a vitális kimerültség felmérése mellett kitért a szociodemográfiai, fizikai (egészség-magatartással kapcsolatos), munkahelyi és családi jellemzők feltérképezésére. Eredmények: Megállapítottuk, hogy az egészségügyi és szociális dolgozók kimerültebbek, mint mások (F(1, 929) = 16,801; p < 0,001). A vitális kimerültség alakulásában a következő változóknak tulajdoníthattunk nagyobb prediktív hatást: az alvás minőségének változása ( β = 0,292; p < 0,01), a család és munka egyensúlyának változása ( β = –0,238; p < 0,01) és a szubjektív egészségi állapot ( β = –0,201; p < 0,01). Közepes erősségű összefüggést láttunk az étkezések gyakoriságának változása és az alvásminőség változása között (χ2(4, n = 931) = 144,8; p < 0,001), valamint a család-munka egyensúly változása és az alvás minőségének változása között (χ2(12, n = 788) = 171,4; p < 0,001). Következtetések: Az alvás megfelelő minőségének és mennyiségének, a rendszeres étkezésnek és a fizikai aktivitásnak nemcsak megelőző szerepe, hanem stabilizáló ereje van akut krízisekben is. Az egyéni tényezőkön túl a munkáltatóknak is fontos szerepük van az alkalmazottak jóllétének megőrzésében, hiszen a munkahelyi kommunikáció és a munkakörülmények is jelentős mértékben befolyásolják azt.Theoretical background: In a critical situations such as COVID-19, priority should be given to the psychological support of helping professionals regarding the sustainability of services and prevention of burnout. It’s exceptionally important to measure the vital exhaustion that might be a reason of burnout. Aim: The purpose of this study was to investigate helping professionals’ vital exhaustion in relation to different groups of background variables during the first period of the COVID-19 pandemic. Methods: Helping professionals from health and social care, education, and the field of religion (n = 931) were contacted with an online questionnaire in Hungary in the spring of 2020. After calculating descriptive statistics of vital exhauson, we examined the sociodemographic, physical (related to health behavior), professional and personal background variables. Results: Health and social care workers were more exhausted than members of other helping professions (F(1, 929) = 16.801, p < 0.001). In the development of vital exhaustion, we could attribute a greater predictive effect to the following variables: change in the quality of sleep ( β = 0.292, p < 0.01), change in family-work balance ( β = –0.238, p < 0.01), and subjective health ( β = –0.201, p < 0.01). We could attribute a moderate correlation between changes in the frequency of meals and changes in sleep quality (χ2(4, n = 931) = 144.8, p < 0.001), and changes in family-work balance and changes in sleep quality (χ2(12, n = 788) = 171.4, p < 0.001). Conclusions: Proper quality and quantity of sleep, regular meals, and physical activity not only have preventive significance but they also have a stabilizing effect in acute crises. Work conditions and communication at work can have a major influence on wellbeing. Therefore beyond the individual factors employers also play an important role to maintain their employees’ wellbeing.

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

Boda Zsolt: Ki dönt? Kormányzási stílusok és közpolitikai változás Magyarországon 2002–2014. Budapest,Gondolat Kiadó–TK, 2020

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

Perfluorosulfonic acid (PFSA) polymers such as Nafion are the most frequently used Proton Exchange Membrane (PEM) in PEM fuel cells. Nafion XL is one of the most recently developed membranes designed to enhance performance by employing a mechanically reinforced layer in the architecture and a chemical stabilizer. The influence of the water and acid pre-treatment process on the physicochemical properties of Nafion XL membrane and Membrane Electrode Assembly (MEA) was investigated. The obtained results indicate that the pre-treated membranes have higher water uptake and dimensional swelling ratios, i.e., higher hydrophilicity, while the untreated membrane demonstrated a higher ionic exchange capacity. Furthermore, the conductivity of the acid pre-treated Nafion XL membrane was ~ 9.7% higher compared to the untreated membrane. Additionally, the maximum power densities obtained at 80 °C using acid pre-treatment were ~ 0.8 and 0.93 W/cm2 for re-cast Nafion and Nafion XL, respectively. However, the maximum generated powers for untreated membranes at the same condition were 0.36 and 0.66 W/cm2 for re-cast Nafion and Nafion XL, respectively. The overall results indicated that the PEM’s pre-treatment process is essential to enhance performance.

Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis.

Az Innovációs és Technológiai Minisztérium (ITM) 2020. év elején közzétette az elfogadott energia- és klímapolitikai stratégiai dokumentumokat, amelyek meghatározzák Magyarország rövid- és hosszútávú energiastratégiáját. A stratégia alapvető célkitűzései megfelelnek az Európai Tanács 2019. december 12-i következtetéseiben foglaltakkal, miszerint fokozni kell a globális éghajlat-politikai fellépést. Magyarország Kormánya a megfogalmazott akciótervekkel igyekszik megvalósítani azt a célkitűzést, hogy az Európai Unió és a világ többi vezető gazdasági hatalma, a Párizsi Megállapodásban kitűzött célokkal összhangban, legkésőbb 2050-re klímasemlegessé váljon. Ez a vállalás fogja meghatározni a magyarországi energiaszektor, és a legnagyobb energiaszükségletű iparágak elkövetkező évtizedeit. Cikkemben bemutatom az eddigi energetikai trendek és mutatók alakulását, hogy mélyebb betekintést nyerjen az olvasó a szintén elemezni kívánt hat, úgynevezett „zászlóshajó-projektek”-be, amelyek az ITM által beazonosításra kerültek és a legfontosabb beavatkozási területeket fedik le.

The proton exchange membrane (PEM) represents a pivotal material and a key challenge in developing fuel cell science and hydrogen technology. Nafion is the most promising polymer which will lead to its commercialisation. Hybrid membranes of nanosized tungsten oxide (WO

The design and construction process of the unique cycling bridges at Lake Tisza are presented in this article. The 4 new bridges are parts of the closing segment of the cycle route around the artificial reservoir, which is a popular tourist destination in Hungary, and part of the UNESCO World Heritage. The proximity of the natural environment motivated the use of organic, flowing shapes.The unique Eger- and Szomorka bridges are independent continuous half-through arch bridges, 8 spans with a total length of 308.46m, and 3 spans with a total length of 86.30 m, respectively. The bridge over River Tisza is a 5 span bridge with a total length of 279.47 m, which is placed on the extended piers of the existing roadway bridge. It consists of 2 deck truss bridges on the side-spans and 3 network arch bridges in the mid- spans. A 5.70 m span bascule bridge over one of the draining canals of the lake was also accomplished as part of the project.The Eger and Szomorka bridges are internationally unique due to the fact that the Designers have dreamed a continuous sinusoid wave on the supports; which, by twirling under and above the deck, results in a continuous structure. The successful construction of the Tisza River Bridge also required some special and unprecedented construction methods.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

Background The lectin pathway serine protease mannan-binding lectin-associated serine protease 1 (MASP-1) has been demonstrated to be a major link between complement and coagulation, yet little is known about its interactions with the fibrinolytic system. The aim of this work was to assess the effects of MASP-1 on fibrin clot lysis in different experimental settings. Methods Rotational thrombelastometry was used to evaluate the effect of MASP-1 on the lysis of clots formed in whole blood under static conditions. Whole blood clots were also formed in the presence and absence of MASP-1 under flow conditions in the Chandler loop and their lysis was analysed separately by fluorescence release of incorporated labelled fibrin. Real-time observation by laser scanning confocal microscopy was used to investigate the lysis of plasma clots where MASP-1 was present either during clot formation or lysis. Cleavage of tPA or plasminogen by MASP-1 was analysed by gel electrophoresis. We performed a turbidimetric clot lysis assay in the presence and absence of the MASP-1 inhibitor SGMI-1 (Schistocerca gregaria protease inhibitor (SGPI)-based MASP inhibitor-1) to evaluate the effect of endogenous MASP-1 in normal plasma and plasma samples from sepsis patients. Results In the thrombelastometric experiments, where MASP-1 was present during the entire clotting and lysis process, MASP-1 had a significant profibrinolytic effect and accelerated clot lysis. When clots were formed in the presence of MASP-1 under flow in the Chandler loop, the effects on fibrinolysis were heterogenous with impaired fibrinolysis in some individuals (n = 5) and no (n = 3) or even the opposite effect (n = 2) in others. In plasma clot lysis observed by confocal microscopy, lysis was prolonged when MASP-1 was added to the lysis solution, yet there was no difference in lysis time when MASP-1 was present during clot formation. When MASP-1 was incubated with tPA or plasminogen, respectively, cleavage of single-chain tPA into two-chain tPA and a slight reduction of plasminogen were observed. SGMI-1 significantly prolonged clot lysis in the turbidimetric clot lysis assay suggesting that MASP-1 accelerated lysis in plasma samples. Conclusion MASP-1 is able to alter the susceptibility of blood clots to the fibrinolytic system. MASP-1 has complex, mostly promoting effects on fibrinolysis with high inter-individual variation. Interactions of MASP-1 with the fibrinolytic system may be relevant in the development and therapy of cardiovascular and thrombotic diseases.

The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin–associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Reversible serine proteinase inhibitors comprise 18 unrelated families. Each family has a distinct representative structure but contains a surface loop that adopts the same, canonical conformation in the enzyme–inhibitor complex. The Laskowski mechanism universally applies for the action of all canonical inhibitors independent of their scaffold, but it has two nontrivial extrapolations. Intrascaffolding additivity states that all enzyme-contacting loop residues act independently of each other, while interscaffolding additivity claims that these residues act independently of the scaffold. These theories have great importance for engineering proteinase inhibitors but have not been comprehensively challenged. Therefore, we tested the interscaffolding additivity theory by hard-randomizing all enzyme-contacting canonical loop positions of a Kazal- and a Pacifastin-scaffold inhibitor, displaying the variants on M13 phage, and selecting the libraries on trypsin and chymotrypsin. Directed evolution delivered different patterns on both scaffolds against both enzymes, which contradicts interscaffolding additivity. To quantitatively assess the extent of non-additivity, we measured the affinities of the optimal binding loop variants and their binding loop-swapped versions. While optimal variants have picomolar affinities, swapping the evolved loops results in up to 200,000-fold affinity loss. To decipher the underlying causes, we characterized the stability, overall structure and dynamics of the inhibitors with differential scanning calorimetry, circular dichroism and NMR spectroscopy and molecular dynamic simulations. These studies revealed that the foreign loop destabilizes the lower-stability Pacifastin scaffold, while the higher-stability Kazal scaffold distorts the foreign loop. Our findings disprove interscaffolding additivity and show that loop and scaffold form one integrated unit that needs to be coevolved to provide high-affinity inhibition.

Factor D (FD) is an essential element of the alternative pathway of the complement system, and it circulates predominantly in cleaved, activated form in the blood. In resting blood, mannose-binding lectin–associated serine protease 3 (MASP-3) is the exclusive activator of pro-FD. Similarly to FD, MASP-3 also circulates mainly in the active form. It was not clear, however, how zymogen MASP-3 is activated. To decipher its activation mechanism, we followed the cleavage of MASP-3 in human hirudin plasma. Our data suggest that neither lectin pathway proteases nor any protease controlled by C1-inhibitor are required for MASP-3 activation. However, EDTA and the general proprotein convertase inhibitor decanoyl-RVKR-chloromethylketone completely prevented activation of exogenous MASP-3 added to blood samples. In this study, we show that proprotein convertase subtilisin/kexin (PCSK) 5 and PCSK6 are able to activate MASP-3 in vitro. Unlike PCSK5, PCSK6 was detected in human serum and plasma, and previously PCSK6 had also been shown to activate corin in the circulation. In all, PCSK6 emerges as the MASP-3 activator in human blood. These findings clarify the very first step of the activation of the alternative pathway and also connect the complement and the proprotein convertase systems in the blood.

The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding–associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface.

The aim of this study was to investigate the prevalence and pattern of psychoactive substances among suspected DUID (Driving Under the Influence of Drugs) drivers in Hungary in 2014 and 2015. Blood and/or urine samples of 1252 suspected drivers (600 in 2014 and 652 in 2015) were analyzed for classical illicit and licit drugs, stimulant designer drugs (SDDs), and for synthetic cannabinoids, with 78.3% and 79.6% positive cases for at least one substance in 2014, and 2015, respectively. Impairment was proven in 39.2% (2014) and 35.7% (2015) of all drivers tested, based on the legal criteria of Hungary. Classical illicit drugs were found to be present in blood or urine of 89-61%, drivers tested. Drivers also tested positive for legal medications in 20-22%, SDDs in 21-28%, and synthetic cannabinoids in 15-19% of all cases. This indicates a drop in prevalence for classical illicit drugs and a slight but statistically non-significant increase for the other three substance groups. The distribution of drug types in each category were: [1] classical illicit drugs: cannabis (432), amphetamine (321), and cocaine (79); [2] medicines: alprazolam (94) and clonazepam (36); [3] SDDs: pentedrone (137) and α-PVP (33); [4] synthetic cannabinoids: AB-CHMINACA (46) and MDMB-CHMICA (30). The average age of illicit drug and SDD users was 30 years, while legal medications users were 36 years old on average, and the mean age of synthetic cannabinoid users was 26.5 years. The presence of both alcohol and at least one drug in samples was found in about 10% of the cases, both years. The ratio of multi-drug use was 33.0% in 2014 and 41.3% in 2015. Compared to former years the number of drivers who tested positive for drugs doubled in Hungary, but it is still low compared to alcohol positive cases. The relatively low detected rate of DUID can be explained by (1) combined alcohol consumption masking drug symptoms, (2) the absence of road-side tests for illicit and designer drugs and, (3) police officers not adequately trained to recognize milder symptoms of impairment. Targeted education of police officers, prompt medical examination and the use of a symptom-focused on-site survey, could improve the efficacy of DUID investigations. Our findings are not comparable with drug consumption habits of the general driving population. The last roadside survey (DRUID EU-6 Project) was performed in Hungary in 2008-2009, prior to the mass spreading of designer drugs. As their appearance has drastically changed the pattern of drug consumption of the population, a new roadside survey, targeting general drivers, would be necessary.

Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

The Ca(2+) -binding protein S100A4 interacts with the C terminus of nonmuscle myosin IIA (NMIIA) causing filament disassembly, which is correlated with an increased metastatic potential of tumor cells. Despite high sequence similarity of the three NMII isoforms, S100A4 discriminates against binding to NMIIB. We searched for structural determinants of this selectivity. Based on paralog scanning using phage display, we identified a single position as major determinant of isoform selectivity. Reciprocal single amino acid replacements showed that at position 1907 (NMIIA numbering), the NMIIA/NMIIC-specific alanine provides about 60-fold higher affinity than the NMIIB-specific asparagine. The structural background of this can be explained in part by a communication between the two consecutive α-helical binding segments. This communication is completely abolished by the Ala-to-Asn substitution. Mutual swapping of the disordered tailpieces only slightly affects the affinity of the NMII chimeras. Interestingly, we found that the tailpiece and position 1907 act in a nonadditive fashion. Finally, we also found that the higher stability of the C-terminal coiled-coil region of NMIIB also discriminates against interaction with S100A4. Our results clearly show that the isoform-selective binding of S100A4 is determined at multiple levels in the structure of the three NMII isoforms and the corresponding functional elements of NMII act synergistically with one another resulting in a complex interaction network. The experimental and in silico results suggest two divergent evolutionary pathways: NMIIA and NMIIB evolved to possess S100A4-dependent and -independent regulations, respectively.

Pathologically increased vascular permeability is an important dysfunction in the pathomechanism of life-threatening conditions, such as sepsis, ischemia/reperfusion, or hereditary angioedema (HAE), diseases accompanied by uncontrolled activation of the complement system. HAE for example is caused by the deficiency of C1-inhibitor (the main regulator of early complement activation), which leads to edematous attacks threatening with circulatory collapse. We have previously reported that endothelial cells become activated during HAE attacks. A natural target of C1-inhibitor is mannan-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, which plays a key role in the activation of complement lectin pathway. We have previously shown that MASP-1 induces the pro-inflammatory activation of endothelial cells and in this study we investigated whether MASP-1 can directly affect endothelial permeability. All experiments were performed on human umbilical vein endothelial cells (HUVECs). Real-time micro electric sensing revealed that MASP-1 decreases the impedance of HUVEC monolayers and in a recently developed permeability test (XperT), MASP-1 dose-dependently increased endothelial paracellular transport. We show that protease activated receptor-1 mediated intracellular Ca2+-mobilization, Rho-kinase activation dependent myosin light chain (MLC) phosphorylation, cytoskeletal actin rearrangement, and disruption of interendothelial junctions are underlying this phenomenon. Furthermore, in a whole-transcriptome microarray analysis MASP-1 significantly changed the expression of 25 permeability-related genes in HUVECs—for example it up-regulated bradykinin B2 receptor expression. According to our results, MASP-1 has potent permeability increasing effects. During infections or injuries MASP-1 may help eliminate the microbes and/or tissue debris by enhancing the extravasation of soluble and cellular components of the immune system, however, it may also play a role in the pathomechanism of diseases, where edema formation and complement lectin pathway activation are simultaneously present. Our findings also raise the possibility that MASP-1 may be a promising target of anti-edema drug development.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin–associated serine protease (MASP)-1/3(−/−) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.

As a part of the new Danube bridge project in the Baja – Kalocsa region the Government of Hungary ordered a feasibility study and location plan to prepare the new bridge investment. Primary goal of the feasibility study was to explore all requirements of the development and subsequent operation of the Paks City Nuclear Powerplant, and according to these suggestions - analyze the technical, environmental, social and economic aspects - to give proposals for the new place and structural system of the new bridge, and proposals for the indispensable implementation of the transport network elements necessarily needed. The key task of the feasibility study was to investigate, and compare the possible structural systems at the chosen location, and to define the optimal variant – for further developments until the level of permition plan. The optional structural system is highly influenced by the possible spans, which is primarily specified by water management and shipping management criteria, so these conditions were basic parameters at each variants. The following structural system variants were elaborated and investigated in the study plans: girder bridge, cable-stayed bridge, truss bridge, extradosed bridge and arch bridge. The building system, possible special building technology needs, estimated building time, estimated costs of maintenance and operation, and the aesthetics of the designed bridge were taken into consideration at the comparison of the structural systems. Because of the simplicity of construction method, advanced technology and aesthetic appearance, the extradosed bridge were preferred with 200+220+200 m inner spans for implementation. When making the feasibility study, one of the Client's requirements was to invest the economical railway connection of the nearby power plant through the bridge, that's why economical alternatives were presented for possible railway line realizations on the structure. The paper mentions the design parameters, the advantages and disadvantages of the presented concepts in the feasibility study. It presents the difficulties and solutions during design and construction phase.

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin.MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is approximately 3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 A. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin.MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.