Structure of HI-6•Sarin-Acetylcholinesterase Determined by X-Ray Crystallography and Molecular Dynamics Simulation: Reactivator Mechanism and Design

Organophosphonates such as isopropyl metylphosphonofluoridate (sarin) are extremely toxic as they phosphonylate the catalytic serine residue of acetylcholinesterase (AChE), an enzyme essential to humans and other species. Design of effective AChE reactivators as antidotes to various organophosphonates requires information on how the reactivators interact with the phosphonylated AChEs. However, such information has not been available hitherto because of three main challenges. First, reactivators are generally flexible in order to change from the ground state to the transition state for reactivation; this flexibility discourages determination of crystal structures of AChE in complex with effective reactivators that are intrinsically disordered. Second, reactivation occurs upon binding of a reactivator to the phosphonylated AChE. Third, the phosphorous conjugate can develop resistance to reactivation. We have identified crystallographic conditions that led to the determination of a crystal structure of the sarinnonaged-conjugated mouse AChE in complex with [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride (HI-6) at a resolution of 2.2 Å. In this structure, the carboxyamino-pyridinium ring of HI-6 is sandwiched by Tyr124 and Trp286, however, the oxime-pyridinium ring is disordered. By combining crystallography with microsecond molecular dynamics simulation, we determined the oxime-pyridinium ring structure, which shows that the oxime group of HI-6 can form a hydrogen-bond network to the sarin isopropyl ether oxygen, and a water molecule is able to form a hydrogen bond to the catalytic histidine residue and subsequently deprotonates the oxime for reactivation. These results offer insights into the reactivation mechanism of HI-6 and design of better reactivators

Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer's disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators

Transthyretin ur ett strukturellt perspektiv

Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation. Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation. We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils. The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases

På Kungsbäck blir alla Gävlebor : - En fallstudie av ledarskap och kulturkrockar

Syfte: Syftet med denna uppsats är att studera och analysera lärare och studenter med olika kulturella bakgrunder på Högskolan i Gävle för att undersöka om kulturkrockar uppstår studenter emellan samt mellan studenter och lärare. En ytterligare aspekt var även att utröna lärarnas ledarstilar. Metod: Vi har valt att genomföra en kvalitativ fallstudie, där det empiriska materialet baseras på nio intervjuer med lärare och studenter på Högskolan i Gävle, samt två egna observationer. Sekundärdata har insamlats genom relevant litteratur och artiklar inom ämnesområdet som ligger till grund för den teoretiska referensramen. Resultat och slutsats: Vi har kommit fram till att högskolans lärare är medvetna om att kulturella skillnader råder, men att de trots detta inte gör något för att lösa eventuella problem som uppstår. Då det råder en saknad av mer auktoritär ledarstil ges vissa studenter möjlighet att utnyttja högskolans system. Vidare har vi kunnat dra den slutsats att färre studenter i framtiden kan komma att välja Högskolan i Gävle som sitt lärosäte om högskoleledningen inte åtgärdar de problem som finns. Förslag till fortsatt forskning: Vi tycker att det vore intressant om denna studie utfördes på en annan högskola i Sverige för att se vilket resultat som skulle utrönas där. Vidare anser vi att fördjupning inom ett av de problemområden som vi kommit fram till i denna studie, skulle vara intressant att djupare utforska. Uppsatsens bidrag: Med denna uppsats vill vi framhäva vilka kulturella skillnader som kan finnas på en mångkulturell högskola och hur eventuella problem tas om hand. Studien riktar sig till högskolestudenter, samt högskolelärare som möter olika kulturer dagligen. Nyckelord: Kultur, kulturella skillnader, kulturkrockar, ledarskap, ledarstilar, Högskolan i Gävle.Aim: The purpose of this thesis is to observe and analyze students and teachers with different cultural backgrounds at the University of Gävle, to determine if there are cultural clashes between students, as well as students and teachers. A further aspect was to explore the teachers leadership styles. Method: We have chosen to perform a qualitative case study, where the empirical material is based on nine interviews with teachers and students at the University of Gävle, and two observations. Secondary data, such as literature and articles have been collected and reviewed within the subject areas in order to gather the facts needed for the theoretical references. Result and conclusion: We have come to the conclusion that the university teachers are aware of the cultural differences, but despite the fact they don’t actually aim to solve the problems that arise. Due to the lack of a more authoritative leadership, the students are given the opportunity of taking advantage of the university system. We have further concluded that future students might not choose University of Gävle as their core of education if the board of education does not address the existing issues. Suggestion for future research: We think that it would be interesting if this study was concluded at a different university in Sweden in order to see what the results would be there. Furthermore, we believe that analyzing one of the problem areas in more depth would be a more efficient and interesting approach. Contribution of the thesis: Our goal with this study has been to highlight the cultural differences at a multicultural university and how the arising problems are solved. Our main target are the university students as well as the teachers that come across cultural differences on a daily bases. Keywords: Culture, cultural differences, cultural clashes, leadership, leadership styles, University of Gävle

“When preparing for crises, it is instructive to recall that Noah started building the ark before it began to rain” - Norman Augustine : En kvalitativ studie om hur svenska herrelitfotbollsföreningar hanterar kriser

Inledning: Under år 2020 har samtliga svenska herrelitfotbollsföreningar stött på en rad olika utmaningar på grund av Covid-19. Det är känt att fotbollsföreningar dagligen kan stöta på olika problem. Är föreningen inte förberedd på att hantera utmanande situationer kan dessa förvärras och i värsta fall utvecklas till en kris. För att en fotbollsförening ska kunna överleva och göra bra ifrån sig är bra sportsliga och ekonomiska resultat av allra största vikt. Förutsättningarna kan dock förändras när en kris uppstår, speciellt eftersom kriser kan se väldigt olika ut. Syfte: Syftet med studien är att undersöka svenska herrelitfotbollsföreningars hantering av Covid-19 under 2020. Metod: En kvalitativ metod med semistrukturerade intervjuer har genomförts med tre olika herrelitfotbollsföreningar. En komparativ disposition har använts för att undersöka och jämföra fenomenen i sin naturliga kontext. Urvalet är tre manliga sportchefer som är och har varit verksamma i Allsvenskan och Superettan. Analysen av resultatet gjordes med hjälp av axial kodning för att identifiera mönster och teman. Resultat: Resultatet visar att herrelitfotbollsföreningarna vill vara proaktiva i sin krishantering, men till följd av Covid-19s oberäkneliga karaktär har föreningarna varit tvungna att vara reaktiva i krishanteringen. Föreningarna har anpassat olika delar av verksamheten på grund av pandemins konsekvenser. En av föreningarna lyckades till viss del arbeta proaktivt. Covid-19 har haft och fortsätter att ha negativ påverkan på föreningarna. Främst ekonomiskt på grund av bortfall i diverse intäkter i form av publik och sponsoravtal men även sportsligt som exempelvis att spelare och personal insjuknat i Covid-19. Föreningarna har upplevt händelser som påverkat och hotat elitfotbollföreningarnas fortsatta verksamhet. Samtliga föreningar har permitterat och sagt upp personal för att minska på kostnader. Detta eftersom Covid-19 är komplext och är en ny kris som samtliga föreningar inte bemött tidigare. Slutsats: Svenska herrelitfotbollsföreningar bör ha en framförhållning i att systematiskt utveckla åtgärder som de kan vända sig till när kriser väl uppstår. Alla tre herrelitfotbollsföreningar har implementerat ett reaktivt tillvägagångssätt under hanteringen av Covid-19. Den proaktiva krishanteringen har sina begränsningar i förhållande till krisers komplexitet och kunde i förhållande till Covid-19 endast minimera och stävja dess påverkan till en viss nivå

Field testing of transgenic aspen from large greenhouse screening identifies unexpected winners

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy