Tine Nielsen

Direct binding between BubR1 and B56–PP2A phosphatase complexes regulate mitotic progression

Summary BubR1 is a central component of the spindle assembly checkpoint that inhibits progression into anaphase in response to improper kinetochore–microtubule interactions. In addition, BubR1 also helps stabilize kinetochore–microtubule interactions by counteracting the Aurora B kinase but the mechanism behind this is not clear. Here we show that BubR1 directly binds to the B56 family of protein phosphatase 2A (PP2A) regulatory subunits through a conserved motif that is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and polo-like kinase 1 (Plk1). Two highly conserved hydrophobic residues surrounding the serine 670 Cdk1 phosphorylation site are required for B56 binding. Mutation of these residues prevents the establishment of a proper metaphase plate and delays cells in mitosis. Furthermore, we show that phosphorylation of serines 670 and 676 stimulates the binding of B56 to BubR1 and that BubR1 targets a pool of B56 to kinetochores. Our data suggest that BubR1 counteracts Aurora B kinase activity at improperly attached kinetochores by recruiting B56–PP2A phosphatase complexes.

Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

Background: The biological function of the ASB family of ubiquitin ligases is poorly characterized. Results: A family-wide analysis of ASB-associated proteins by mass spectrometry identified novel interactors. Conclusion: ASB proteins can associate in multimeric complexes. Asb11 ubiquitinates Ribophorin 1 and regulates its turnover. Significance: This data resource will facilitate the characterization of the biological function of ASB proteins. The ankyrin and SOCS (suppressor of cytokine signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting 18 members in humans, the identity of the physiological targets of the Asb proteins remains largely unexplored. To increase our understanding of the function of ASB proteins, we conducted a family-wide SILAC (stable isotope labeling by amino acids in cell culture)-based protein/protein interaction analysis. This investigation led to the identification of novel as well as known ASB-associated proteins like Cullin 5 and Elongins B/C. We observed that several proteins can be bound by more than one Asb protein. The additional exploration of this phenomenon demonstrated that ASB-Cullin 5 complexes can oligomerize and provides evidence that Cullin 5 forms heterodimeric complexes with the Cullin 4a-DDB1 complex. We also demonstrated that ASB11 is a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex. Moreover, expression of ASB11 can increase Ribophorin 1 protein turnover in vivo. In summary, we provide a comprehensive protein/protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases.

Outer membrane vesicles reflect environmental cues in Gallibacterium anatis

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg-production worldwide. Increased knowledge of the pathogenesis and virulence factors is important to better understand and prevent the negative effects of G. anatis. To this end outer membrane vesicles (OMVs) are natural secretion products of Gram-negative bacteria, displaying an enormous functional diversity and promising results as vaccine candidates. This is the first study to report that G. anatis secretes OMVs during in vitro growth. By use of transmission electron microscopy (TEM) and SDS-PAGE, we showed that changes in in vitro growth conditions, including incubation time, media composition and temperature, affected the OMV production and protein composition. A large protein band was increased in its concentration after prolonged growth. Analysis by LC-MS/MS indicated that the band contained two proteins; the 320.1 kDa FHA precursor, FhaB, and a 407.8 kDa protein containing a von Willebrand factor type A (vWA) domain. Additional two major outer-membrane (OM) proteins could be identified in all samples; the OmpH-homolog, OmpC, and OmpA. To understand the OMV formation better, a tolR deletion mutation (ΔtolR) was generated in G. anatis. This resulted in a constantly high and growth-phase independent production of OMVs, suggesting that depletion of peptidoglycan linkages plays a role in the OMV formation in G. anatis. In conclusion, our results show that G. anatis produce OMVs in vitro and the OMV protein profile suggests that the production is an important and well-regulated ability employed by the bacteria, which may be used for vaccine production purposes.

SUBSTANCE USE DURING PREGNANCY AND POSTNATAL OUTCOMES

Substance exposure in utero has been associated with physical birth defects and increased risk of regulatory and neuropsychological difficulties. The aims of this study were to describe women who use substances and are in treatment with respect to the type and number of substances used during pregnancy, as well as their background, and to examine the effect substance use has on gestational age, birth weight, and the development of neonatal abstinence syndrome at birth. A sample of 161 pregnant women and their 163 newborn children were included. The results indicate that the children whose mothers continued to use substances throughout their pregnancies were born at a lower gestational age (Chi-Square = 15.1(2), P < .01); children exposed to poly-substances in utero were more affected than those exposed to only alcohol and those with no substance exposure. The same children were more vulnerable to the development of neonatal abstinence syndrome at birth (Chi-Square = 51.7(2), P < .001). Newborns who were exposed primarily to alcohol in utero were at a significant risk of being born with low birth weight (Chi-Square = 8.8(2), P < .05) compared with those exposed to other types of substances. More than 50% of the mothers ceased using any substances (with the exception of tobacco) by birth, indicating that the treatment program did have an interventional effect on the mothers. The mothers’ ability to either cease or decrease the use of substances during pregnancy appears to have direct positive effect on their newborns.

Biochemical characterization of human gluconokinase and the proposed metabolic impact of gluconic acid as determined by constraint based metabolic network analysis.

The metabolism of gluconate is well characterized in prokaryotes where it is known to be degraded following phosphorylation by gluconokinase. Less is known of gluconate metabolism in humans. Human gluconokinase activity was recently identified proposing questions about the metabolic role of gluconate in humans. Here we report the recombinant expression, purification and biochemical characterization of isoform I of human gluconokinase alongside substrate specificity and kinetic assays of the enzyme catalyzed reaction. The enzyme, shown to be a dimer, had ATP dependent phosphorylation activity and strict specificity towards gluconate out of 122 substrates tested. In order to evaluate the metabolic impact of gluconate in humans we modeled gluconate metabolism using steady state metabolic network analysis. The results indicate that significant metabolic flux changes in anabolic pathways linked to the hexose monophosphate shunt (HMS) are induced through a small increase in gluconate concentration. We argue that the enzyme takes part in a context specific carbon flux route into the HMS that, in humans, remains incompletely explored. Apart from the biochemical description of human gluconokinase, the results highlight that little is known of the mechanism of gluconate metabolism in humans despite its widespread use in medicine and consumer products.

In silico prediction of Gallibacterium anatis pan-immunogens

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

Cognitive, emotional and social development in adolescents born to substance using women

The aim of this article is to investigate the long-term developmental consequences of being born to a substance-using mother, focusing on cognitive functions, attention, emotional and social development. The longitudinal sample comprised 48 adolescents aged 12-16 at the time of follow-up assessments, which included the Wechsler Intelligence Scale for Children-III, the Test of Everyday Attention for Children, The Tower of London test and the Strengths and Difficulties Questionnaire. The adolescents scored significantly lower than the norms on Wechslers subtests and Full-Scale IQ, and on The Everyday Attention test. There were few differences on the Strengths and Difficulties Questionnaire. The girls reported significantly more hyperactivity than the British norms, and the teachers reported higher impact scores in boys, compared to the British norms. Thus, the results on cognitive consequences of maternal substance use appear to be very substantial while the emotional and social consequences do not. The results suggest serious negative effects of substance exposure in utero on attention and cognitive functioning in general.

The predictive validity of using admissions testing and multiple mini-interviews in undergraduate university admissions

ABSTRACT The aim of this study was to evaluate the predictive validity of a two-step admissions procedure that included a cognitive ability test followed by multiple mini-interviews (MMIs) used to assess non-cognitive skills, compared to grade-based admissions relative to subsequent drop-out rates and academic achievement after one and two years of study. The participants consisted of the entire population of 422 psychology students who were admitted to the University of Southern Denmark between 2010 and 2013. The results showed significantly lower drop-out rates after the first year of study, and non-significant lower drop-out rates after the second year of study for the admission procedure that included the assessment of non-cognitive skills though the MMI and the admissions test. Furthermore, this admission procedure resulted in a significant lower risk of failing the final exam after the first and second year of study, compared to the grade-based admissions procedure. Finally, students admitted through the MMI and the admissions test scored significantly higher on academic learning self-efficacy and critical thinking compared to students selected by grades. The implications for higher education admissions are discussed.

Structural Insights into the Recognition of Phosphopeptide by the FHA Domain of Kanadaptin

Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins and represents the first evidence that kanadaptin is likely involved in a phosphorylation-mediated signaling pathway. These results will be of use for designing experiments to further probe the function of kanadaptin.

Gender Fairness in Self-Efficacy? A Rasch-Based Validity Study of the General Academic Self-Efficacy Scale (GASE)

ABSTRACT Studies have reported gender differences in academic self-efficacy. However, how and if academic self-efficacy questionnaires are gender-biased has not been psychometrically investigated. The psychometric properties of a general version of The Physics Self-Efficacy Questionnaire – the General Academic Self-Efficacy Scale (GASE) – were analyzed using Rasch measurement models, with data from 1018 Danish university students (psychology and technical), focusing on gender invariance and the sufficiency of the score. The short 4-item GASE scale was found to be essentially objective and construct valid and satisfactorily reliable, though differential item functioning was found relative to gender and academic discipline, and can be used to assess students’ general academic self-efficacy. Research on gender and self-efficacy needs to take gender into account and equate scores appropriately for unbiased analysis within academic disciplines.