Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition

Studier av metoder som kan reducera utsläpp av vattenburet fosfor från Korsnäsverken

Föreliggande examensarbete på 20 högskolepoäng har utförts i samarbete mellan Korsnäs ABoch Kungliga tekniska högskolan. Arbetets syfte var att:Utifrån kunskaper om fosforströmmarna, i Korsnäsverkets externa vattenreningssystem,föreslå lämpliga vägar att reducera utsläppen av vattenburet fosfor till recipienten. Pappersbruket Korsnäsverken är lokaliserat strax utanför Gävle och ägs av Korsnäs AB somär en del av Kinnevik AB. Detta pappersbruk har Östersjön som recipient där fosfor är det begränsade tillväxtämnet. Då Korsnäsverken släpper ut fosfor bidrar de därmed till Östersjönspågående eutrofieringen. För att klara miljödomstolens satta utredningsvillkor, angående fosforreducering i Östersjön, måste Korsnäsverken halvera sina fosforutsläpp.Under detta arbete inriktades de utförda laborationerna mot kemisk fällning, som reduceringsmetod. De övriga beskrivna reduceringsmetoderna har undersökts genom litteraturstudier. Genom tidigare arbeten har de vattenburna fosforutsläppen lokaliserats tillvissa avloppsvattenströmmar inom Korsnäsverkens externa rening. De strömmar med mestfosfor härstammar från blekeriet, renseriet och fiberförande linjen i tillverkningsprocessen.Detta examensarbete koncentrerades på de två punkter, med högst koncentration av fosfor,som möjliga fällningspunkter. Dessa var Blekeribassängen (AVB) ochSlutsedimenteringsbassängen (Sed.zon). Arbetsmomenten under arbetet delades upp i sjudelar som byggde på respektive tidigare moments resultat: 1. Verifierande analyser 2. Metoduppläggningsförsök 3. Systemscreeningsförsök 4. Optimeringsförsök 5. Sammanfattning av resultat 6. Kostnads- och konsekvensanalys 7. Jämförelse av resultat med dagens utsläpp, BAT och krav De tre första momenten var tillsammans förberedande för det fjärde där laborationsförsök gjordes med de fällningskemikalier som gett högst reducering under tidigare försök. Underdet fjärde momentet testades dessutom sedimenteringstidens påverkan vid användning avendast polymer i Sed.zon. Laborationsmomentens resultat kunde sedan användas för att utföramoment 6. De beräknade kostnaderna och konsekvenserna sammanställdes i tabell 1.This Master’s thesis has been written in cooperation with the paper mill Korsnäsverken,owned by Korsnäs AB, which is located in Gävle, Sweden. The aim of the project: On the basis of earlier received knowledge, from the currents in the external wastewatertreatment system at Korsnäsverken, propose suitable ways of reducing the emissions ofdissolved phosphorus to the recipient. Since emissions of the growth-regulating nutrient phosphorus from Korsnäsverken may cause eutrophication, in their recipient the Baltic Sea, the regional environmental court has decided that the paper mill must reduce their phosphorous emissions by half. The experiments performed during this thesis were focused on chemical reduction. The other reducing methods described have been examined by literature studies. The water dissolved phosphorous has, in an earlier Master’s thesis, been located to certain currents in the external wastewater treatment system of Korsnäsverken and was further examined in the experimentsof this thesis. The used water in the experiments was taken from the bleach pond (AVB) andthe end sedimentation pond (Sed.zon). The results, from chemical reducing experiments in Sed.zon, showed that Korsnäsverkencould probably meet the condition set by the regional environmental court and reduce the emissions of phosphorus with 50 %. (Red bars in figure 1.) It furthermore showed that chemical reduction with polymer alone in Sed.zon could probably also reduce phosphorus by50 %. In AVB the result of chemical reduction was positive, but not as efficient as that of Sed.zon.www.ima.kth.s

"Jag bara kan!" Ett undersökande arbete om hur barn upplever sitt eget lärande och pedagogers syn på barns lärande. "I just know!" A study of how children experience their own learning and teachers view of children´s learning

Abstract Holmgren, Maria & Åstrand, Ingvor (2008). ”Jag bara kan!” Ett undersökande arbete om hur barn upplever sitt eget lärande och pedagogers syn på barns lärande. Malmö: Lärarutbildningen: Malmö Högskola Examensarbetet handlar om hur sex barn upplever sitt eget lärande. Arbetet handlar även om hur fyra pedagoger ser på barns lärande. Samtliga förskolor som informanterna befinner sig på ligger centralt i en storstad. Syftet med examensarbetet är att synliggöra barnens beskrivningar av hur de upplever sitt eget lärande. Syftet är även att synliggöra fyra pedagogers syn på barnens lärande, miljöns fysiska och sociala betydelse för lärandet och synen som de fyra pedagogerna har på det livslånga lärandet. De frågeställningar vi utgått ifrån är: Hur upplever barnen sitt eget lärande på förskolan? Hur ser pedagogerna på barns lärande? De metoder som använts för att besvara frågeställningarna har varit kvalitativa intervjuer, intervjuer i form av samtal. Teoretiska utgångspunkter i examensarbetet har varit Jean Piagets konstruktivistiska synsätt, Lev Vygotskijs sociokulturellt -historiska perspektiv och Ingrid Pramling Samuelssons teorier och forskning kring barns lärande. Resultatet pekar på att barnen har ett väldigt stort självförtroende i sitt lärande och över vad de kan. Barnen beskriver de situationer som de lär sig i och med vem de lär sig. Pedagogerna har insikter om att barnen lär hela tiden men att barnen inte förstår eller ser sin egen läroprocess. Miljön och personerna runt omkring barnen har stor betydelse för deras lärande. Nyckelord: Barns lärande, förskola, samspel, det livslånga lärandet, läromiljön, pedagogers barnsy

Impact of Biomechanical Forces on Antibiotics Release Kinetics from Hydroxyapatite Coated Surgical Fixation Pins

This work investigates the impact of biomechanical wear and abrasion on the antibiotic release profiles of hydroxyapa-tite (HA) coated fixation pins during their insertion into synthetic bone. Stainless steel fixation pins are coated with crystalline TiO2 by cathodic arc evaporation forming the bioactive layer for biomimetic deposition of Tobramycin con-taining HA. Tobramycin is either introduced by co-precipitation during HA formation or by adsorption-loading after HA deposition. The samples containing antibiotics are inserted into bone mimicking polyethylene foam after which the drug release is monitored using high performance liquid chromatography. This analysis shows that HA coating wear and delamination significantly decrease the amount of drug released during initial burst, but only marginally influence the sustained release period. Spalled coating fragments are found to remain within the synthetic bone material structure. The presence of HA within this structure supports the assumption that the local release of Tobramycin is not only ex-pected to eliminate bacteria growth directly at the pin interface but as well at some distance from the implant. Further-more, no negative effect of gamma sterilization could be observed on the drug release profile. Overall, the observed results demonstrate the feasibility of a multifunctional implant coating that is simultaneously able to locally deliver clinically relevant doses of antibiotics and an HA coating capable of promoting osteoconduction. This is a potentially promising step toward orthopaedic devices that combine good fixation with the ability to treat and prevent post-surgical infections

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

<div><p>The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.</p></div

Distal radius fractures and risk of incident neurocognitive disorders in older adults : a retrospective cohort study

Introduction: Distal radius fractures (DRF) are associated with increased risk of subsequent fractures and physical decline in older adults. This study aims to evaluate the risk cognitive decline following DRF and potential for timely screening and intervention. Methods: A cohort of 1046 individuals 50–75 years of age with DRF were identified between 1995 and 2015 (81.5% female; mean age 62.5 [± 7.1] years). A control group (N = 1044) without history of DRF was matched by age, sex, and fracture date (i.e., index). The incidence of neurocognitive disorders (NCD) in relation to DRF/index was determined. Group comparisons were adjusted by age and comorbidity measured by the Elixhauser index. Results: The DRF group had a greater incidence of NCD compared to the control group (11.3% vs. 8.2%) with a 56% greater relative risk (HR = 1.56, 95% Cl: 1.18, 2.07; p = 0.002) after adjusting for age and comorbidity. For every 10-year age increase, the DRF group was over three times more likely to develop a NCD (HR = 3.23, 95% Cl: 2.57, 4.04; p < 0.001). Conclusion: DRF in adults ages 50 to 75 are associated with increased risk of developing neurocognitive disorders. DRF may represent a sentinel opportunity for cognitive screening and early intervention. Summary: Distal radius fractures (DRF) have been associated with greater risk of future fractures and physical decline. This study reports that DRF are also associated with greater risk of developing neurocognitive disorders in older adults. Timely intervention may improve early recognition and long-term outcomes for older adults at risk of cognitive decline

Bacterial-display screening of variants identified after FACS.

<p>21 clones identified after rounds three and four of FACS sorting were screened for ERBB2-binding (<b>A</b>), HSA-binding (<b>B</b>) and ERBB2-binding in the presence of an excess of HSA (<b>C</b>). 5 nM ERBB2 was used in screen A and the binding signal divided by the surface expression level for each clone was normalized against the signal from ADAPT_ERBB2-1. 20 nM HSA was used in screen B and the signals were normalized to scaffold ABD. 5 nM ERBB2 together with 1 µM unlabeled HSA was used in screen C and ADAPT_ERBB2-1 was used for normalization. All measurements were done in duplicate on different days and the bars indicate the standard deviation.</p

Affinities of selected affinity matured ADAPTs after FACS.

<p>Kinetic parameters are presented as mean values with standard deviation. K_D was calculated from the rate constants (k_d/k_a) and the number of replicates for each variant is indicated within brackets. Ten variants were selected for kinetic analysis. Variants of the strongest binder, ADAPT_{ERBB2-FACS-12}, with different substitutions were also characterized. The binding kinetics and melting temperatures for some interactions were not determined (N.D.). No binding was observed to murine ERBB2.</p><p>*Determined by manual inspection of melting curve.</p