A surface-focused biotinylation procedure identifies the Yersinia pestis catalase KatY as a membrane-associated but non-surface-located protein

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37 degrees C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10(9) cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

Phagocytosis and Respiratory Burst Activity in Lumpsucker (Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes.     

Arachidonic acid randomizes endothelial cell motion and regulates adhesion and migration

Cell adhesion and migration are essential for the evolution, organization, and repair of living organisms. An example of a combination of these processes is the formation of new blood vessels (angiogenesis), which is mediated by a directed migration and adhesion of endothelial cells (ECs). Angiogenesis is an essential part of wound healing and a prerequisite of cancerous tumor growth. We investigated the effect of the amphiphilic compound arachidonic acid (AA) on EC adhesion and migration by combining live cell imaging with biophysical analysis methods. AA significantly influenced both EC adhesion and migration, in either a stimulating or inhibiting fashion depending on AA concentration. The temporal evolution of cell adhesion area was well described by a two-phase model. In the first phase, the spreading dynamics were independent of AA concentration. In the latter phase, the spreading dynamics increased at low AA concentrations and decreased at high AA concentrations. AA also affected EC migration; though the instantaneous speed of individual cells remained independent of AA concentration, the individual cells lost their sense of direction upon addition of AA, thus giving rise to an overall decrease in the collective motion of a confluent EC monolayer into vacant space. Addition of AA also caused ECs to become more elongated, this possibly being related to incorporation of AA in the EC membrane thus mediating a change in the viscosity of the membrane. Hence, AA is a promising non-receptor specific regulator of wound healing and angiogenesis.     

Region-specific mechanical properties of the human patella tendon.

The present study investigated the mechanical properties of tendon fascicles from the anterior and posterior human patellar tendon. Collagen fascicles from the anterior and posterior human patellar tendon in healthy young men (mean +/- SD, 29.0 +/- 4.6 yr, n = 6) were tested in a mechanical rig. A stereoscopic microscope equipped with a digital camera recorded elongation. The fascicles were preconditioned five cycles before the failure test based on pilot data on rat tendon fascicle. Human fascicle length increased with repeated cycles (P < 0.05); cycle 5 differed from cycle 1 (P < 0.05), but not cycles 2-4. Peak stress and yield stress were greater for anterior (76.0 +/- 9.5 and 56.6 +/- 10.4 MPa, respectively) than posterior fascicles (38.5 +/- 3.9 and 31.6 +/- 2.9 MPa, respectively), P < 0.05, while yield strain was similar (anterior 6.8 +/- 1.0%, posterior 8.7 +/- 1.4%). Tangent modulus was greater for the anterior (1,231 +/- 188 MPa) than the posterior (583 +/- 122 MPa) fascicles, P < 0.05. In conclusion, tendon fascicles from the anterior portion of the human patellar tendon in young men displayed considerably greater peak and yield stress and tangent modulus compared with the posterior portion of the tendon, indicating region-specific material properties.     

Discovery and characterization of two new stem rust resistance genes in <Emphasis Type="Italic">Aegilops sharonensis</Emphasis>

Key message

We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen.

Abstract

Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F₆ recombinant inbred line and an F₂ population, two genes were identified that mapped to the short arm of chromosome 1S^sh, designated as Sr-1644-1Sh, and the long arm of chromosome 5S^sh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

     

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.     

Infections in temporal proximity to HPV vaccination and adverse effects following vaccination in Denmark: A nationwide register-based cohort study and case-crossover analysis

BackgroundPublic trust in the human papilloma virus (HPV) vaccination programme has been challenged by reports of potential severe adverse effects. The reported adverse symptoms were heterogeneous and overlapping with those characterised as chronic fatigue syndrome (CFS) and have been described as CFS-like symptoms. Evidence suggests that CFS is often precipitated by an infection. The aim of the study was to examine if an infection in temporal proximity to HPV vaccination is a risk factor for suspected adverse effects following HPV vaccination.Methods and findingsThe study was a nationwide register-based cohort study and case-crossover analysis. The study population consisted of all HPV vaccinated females living in Denmark, born between 1974 and 2006, and vaccinated between January 1, 2006 and December 31, 2017. The exposure was any infection in the period ± 1 month around time of first HPV vaccination and was defined as (1) hospital-treated infection; (2) redemption of anti-infective medication; or (3) having a rapid streptococcal test done at the general practitioner. The outcome was referral to a specialised hospital setting (5 national HPV centres opened June 1, 2015) due to suspected adverse effects following HPV vaccination. Multivariable logistic regression was used to estimate the association between infection and later HPV centre referral. The participants were 600,400 HPV-vaccinated females aged 11 to 44 years. Of these, 48,361 (9.7%) females had a hospital-treated infection, redeemed anti-infective medication, or had a rapid streptococcal test ± 1 month around time of first HPV vaccination. A total of 1,755 (0.3%) females were referred to an HPV centre. Having a hospital-treated infection in temporal proximity to vaccination was associated with significantly elevated risk of later referral to an HPV centre (odds ratio (OR) 2.75, 95% confidence interval (CI) 1.72 to 4.40; P < 0.001). Increased risk was also observed among females who redeemed anti-infective medication (OR 1.56, 95% CI 1.33 to 1.83; P < 0.001) or had a rapid streptococcal test (OR 1.45, 95% CI 1.10 to 1.93; P = 0.010). Results from a case-crossover analysis, which was performed to adjust for potential unmeasured confounding, supported the findings. A key limitation of the study is that the HPV centres did not open until June 1, 2015, which may have led to an underestimation of the risk of suspected adverse effects, but stratified analyses by year of vaccination yielded similar results.ConclusionsTreated infection in temporal proximity to HPV vaccination is associated with increased risk for later referral with suspected adverse vaccine effects. Thus, the infection could potentially be a trigger of the CFS-like symptoms in a subset of the referred females. To our knowledge, the study is the first to investigate the role of infection in the development of suspected adverse effects after HPV vaccination and replication of these findings are needed in other studies.

Lene Wulff Krogsgaard and co-workers study temporal associations between infections and HPV vaccination.