Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis

Background: The thermostable beta-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e. g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-beta-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. Results: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on beta-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as T-m by differential scanning calorimetry (101.9 degrees C for wt), was kept in the mutated variants and significant decrease (Delta T of 5 -10 degrees C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in K-M and turnover. The K-M-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 x increase for pNPGlc, while the K-M decreased a corresponding extent for Q3. Turnover was only significantly changed using pNPGlc, and was decreased 2-3 x in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 x compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in K-M for this substrate. Conclusion: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both K-M and turnover. An affinity change, leading to a decreased K-M, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site

Risker med GMO i jordbruket

The world is growing more and more genetically modified (or engineered) crops (GM crops). In Argentina farmers grow almost only GM soy, and no conventional soy. It is a controversial subject, since genetic engineering has many advocates and critics. The positive sides of GM crops are that it can create higher yields and incomes for the farmers. Those are the main objects of growing GM crops. Critics, on the other hand, say that GM crops have bad effects on the environment, and the risks could be too high. For example GM crops can spread to neighboring farms, where organic crops are growing. Many scientists claim that nobody really have enough knowledge of the effects of growing GM crops, and therefore thorough risk assessments are needed to examine the effects and possible risks of growing GM crops before starting to grow them. Politicians, too often, make decisions on false scientific grounds, when they claim that genetic engineering is a safe and precise method. The large corporations developing GMOs are heavily influencing political decision making. Scientists have studied the genetic engineering process and the results show that the method is not precise. It is difficult to know exactly what gene is transferred, and where it ends up. It is also important to discuss the precautionary principle in the decision making process

Where have all the young men gone? Problematizing the small amount of adolescent men among the visitors to the youth health centres in Sweden

Syftet med studien var att problematisera det faktum att gruppen unga män utgör en minoritet av besökarna till Sveriges ungdomsmottagningar (UM). Problemområdet studerades ur epidemiologisk såväl som genusvetenskaplig synvinkel. Studien behandlade konsekvenser som unga mäns frånvaro på UM kan ha för klamydiaepidemin. Även snedfördelningen vad det gäller ansvarstagande för den sexuella hälsan män och kvinnor emellan problematiseras. Problemområdet belystes genom utvärdering av ett projekt som genomfördes av en enskild UM och som syftade till att få fler unga män att söka sig dit. Huvudsaklig metod var en enkätundersökning som kompletterades med ett antal intervjuer samt granskning av besökstatistik. Faktorer som hindrar respektive främjar unga män att besöka UM kartlades utifrån erhållna resultat och tidigare studier. Det rörde sig främst om frågor gällande tillgänglighet, personal, egna besökstider och kännedom om UM. De unga männen har lyst med sin frånvaro sedan första UM startade 1970. Trots att problemet lyfts gång på gång från flera instanser, saknas konkreta åtgärder för att påverka rådande situation. Vi tror att detta bottnar i brist på resurser såväl som i traditionella strukturer, oklar ansvarsfördelning och vaga målformuleringar hos Sveriges UM.Folkhälsovetenskapligt progra

Extra anpassningar i teori och praktik - En studie i hur begreppet extra anpassningar tolkas och tillämpas

Det framkommer i resultatet att extra anpassningar är ett implementerat begrepp i verksamheten. Informanterna i studien är av uppfattningen att det utförs inom ramen för den ordinarie undervisningen och i huvudsak av läraren själv. Sedan Skolverket 2014 utkom med sina allmänna råd om extra anpassningar har det blivit tydligare vad det är och vad som förväntas av läraren. På skolan finns numera en arbetsgång samt en gemensam checklista över extra anpassningar. Det framkommer att specialpedagogen har en viktig roll i arbetet genom att vägleda och stötta lärarna samt att följa upp arbetet så att det blir systematiskt

Identification of small molecules blocking the Pseudomonas aeruginosa type III secretion system protein PcrV

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that employs its type III secretion system (T3SS) during the acute phase of infection to translocate cytotoxins into the host cell cytoplasm to evade the immune system. The PcrV protein is located at the tip of the T3SS, facilitates the integration of pore-forming proteins into the eukaryotic cell membrane, and is required for translocation of cytotoxins into the host cell. In this study, we used surface plasmon resonance screening to identify small molecule binders of PcrV. A follow-up structure-activity relationship analysis resulted in PcrV binders that protect macrophages in a P. aeruginosa cell-based infection assay. Treatment of P. aeruginosa infections is challenging due to acquired, intrinsic, and adaptive resistance in addition to a broad arsenal of virulence systems such as the T3SS. Virulence blocking molecules targeting PcrV constitute valuable starting points for development of next generation antibacterials to treat infections caused by P. aeruginosa. Originally included in thesis in manuscript form.</p

Exploring resveratrol dimers as virulence blocking agent : Attenuation of type III secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa

Bacterial infections continue to threaten humankind and the rapid spread of antibiotic resistant bacteria is alarming. Current antibiotics target essential bacterial processes and thereby apply a strong selective pressure on pathogenic and non-pathogenic bacteria alike. One alternative strategy is to block bacterial virulence systems that are essential for the ability to cause disease but not for general bacterial viability. We have previously show that the plant natural product (-)-hopeaphenol blocks the type III secretion system (T3SS) in the Gram-negative pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa. (-)-Hopeaphenol is a resveratrol tetramer and in the present study we explore various resveratrol dimers, including partial structures of (-)-hopeaphenol, as T3SS inhibitors. To allow rapid and efficient assessment of T3SS inhibition in P. aeruginosa, we developed a new screening method by using a green fluorescent protein reporter under the control of the ExoS promoter. Using a panel of assays we showed that compounds with a benzofuran core structure i.e. viniferifuran, dehydroampelopsin B, anigopreissin A, dehydro-δ-viniferin and resveratrol-piceatannol hybrid displayed significant to moderate activities towards the T3SS in Y. pseudotuberculosis and P. aeruginosa

Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo.

Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1