Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa

Human leukocyte receptor IIIa (hFcγRIIIa) plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. In previous studies, a major impact of the presence of carbohydrates at Asn-162 on the binding between the receptor and the Fc part of wild type fucosylated or glycoengineered nonfucosylated antibodies has been shown. In this study, we performed a site directed carbohydrate analysis at hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, respectively. Using mass spectrometry (MS) and a multienzyme protein digest, we analyzed the proteolysis-generated glycopeptides in detail. We could show that hFcγRIIIa expressed by HEK cells was mostly bearing multifucosylated biantennary Asn162-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could demonstrate that the glycan antennae with terminal GalNAc could be sialylated as indicated by a novel reporter ion HexNAcHexNAcNeuAc(+) (m/z 698.28) using a source induced dissociation (SID) scan in the MS cycle. In contrast to the hFcγRIIIa Asn-162 glycosylation pattern from HEK cells, the CHO cells derived receptor contains bi- and triantennary galactosylated and highly sialylated carbohydrates. Our data suggest that the type of expression host system was a dominating factor for formation of distinct glycopatterns of hFcγRIIIa, while the protein sequence and the site of glycosylation remained unchanged for both types of cells. Using surface plasmon resonance (SPR) interaction analysis, we show that the cell type and site specific glycosylation pattern of hFcγRIIIa influences its binding behavior to immunoglobulin molecules.     

Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens.     

Efficacy of a New Educational Tool to Improve Handrubbing Technique amongst Healthcare Workers: A Controlled,Before-After Study

Introduction

Hand hygiene is a key component of infection control in healthcare. WHO recommends that healthcare workers perform six specific poses during each hand hygiene action. SureWash (Glanta Ltd, Dublin, Ireland) is a novel device that uses video-measurement technology and immediate feedback to teach this technique. We assessed the impact of self-directed SureWash use on healthcare worker hand hygiene technique and evaluated the device''s diagnostic capacity.

Methods

A controlled before-after study: subjects in Group A were exposed to the SureWash for four weeks followed by Group B for 12 weeks. Each subject''s hand hygiene technique was assessed by blinded observers at baseline (T₀) and following intervention periods (T₁ and T₂). Primary outcome was performance of a complete hand hygiene action, requiring all six poses during an action lasting ≥20 seconds. The number of poses per hand hygiene action (maximum 6) was assessed in a post-hoc analysis. SureWash''s diagnostic capacity compared to human observers was assessed using ROC curve analysis.

Results

Thirty-four and 29 healthcare workers were recruited to groups A and B, respectively. No participants performed a complete action at baseline. At T₁, one Group A participant and no Group B participants performed a complete action. At baseline, the median number of poses performed per action was 2.0 and 1.0 in Groups A and B, respectively (p = 0.12). At T₁, the number of poses per action was greater in Group A (post-intervention) than Group B (control): median 3.8 and 2.0, respectively (p<0.001). In Group A, the number of poses performed twelve weeks post-intervention (median 3.0) remained higher than baseline (p<0.001). The area under the ROC curves for the 6 poses ranged from 0.59 to 0.88.

Discussion

While no impact on complete actions was demonstrated, SureWash significantly increased the number of poses per hand hygiene action and demonstrated good diagnostic capacity.     

ASIC2 Subunits Facilitate Expression at the Cell Surface and Confer Regulation by PSD-95

Acid-sensing ion channels (ASICs) are Na⁺ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2−/− neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.     

A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8⁺ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.KEY WORDS: EBV-associated cancers, Cell-based drug screening, EBNA1 GAr domain, Yeast-based models, Immune evasion, Doxorubicin, Daunorubicin, 5-fluorouracil     

Site-specific chlorophyll reference conditions for lakes in Northern and Western Europe

The Water Framework Directive (WFD) requires EU Member States to assess the “ecological status” of surface waters. As a component of ecological status, many European countries are developing a classification scheme for chlorophyll concentrations as a measure of phytoplankton biomass. The chlorophyll classification must be based on the degree of divergence of a water body from an appropriate baseline or ‘reference condition’. This article describes the development of a series of regression models for predicting reference chlorophyll concentrations on a site-specific basis. For model development, a large dataset of European lakes considered to be in reference condition, 466 lakes in total, was assembled. Data were included from 12 European countries, but lakes from Northern and Western Europe dominated and made up 92% of all reference lakes. Data have been collated on chlorophyll concentration, altitude, mean depth, alkalinity, humic type, surface area and geographical region. Regression models were developed for estimating site-specific reference chlorophyll concentrations from significant predictor ‘typology’ variables. Reference chlorophyll concentrations were found to vary along a number of environmental gradients. Concentrations increased with colour and alkalinity and decreased with lake depth and altitude. Forward selection was used to identify independent explanatory variables in regression models for predicting site-specific reference chlorophyll concentrations. Depth was selected as an explanatory variable in all models. Alkalinity was included in models for low colour and humic lakes and altitude was included in models for low colour and very humic lakes. Uncertainty in the models was quite high and arises from errors in the data used to develop the models (including natural temporal and spatial variability in data) and also from additional explanatory variables not considered in the models, particularly nutrient concentrations, retention time and grazing. Despite these uncertainties, site-specific reference conditions are still recommended in preference to type-specific reference conditions, as they use the individual characteristics of a site known to influence phytoplankton biomass, rather than adopt standards set to generally represent a large population of lakes of a particular type. For this reason, site-specific reference conditions should result in reduced error in ecological status classifications, particularly for lakes close to typology boundaries.     

Interaction ofH-2 and nonH-2 linked genes in the regulation of antibody response to a threshold dose of sheep erythrocytes

The antibody response to a threshold dose (¹⁰) of SE was studied in the High responder line (H) and the Low responder line (L) of mice obtained by bidirectional selective breeding for the character quantitative agglutinin response to an optimal dose of SE, and in interline hybrids: F₁, F₁ and both backcrosses. Whereas the interline difference in agglutinin responses at the optimal dose is due to the additive effect of about ten independently segregating loci, one of which isH-2 linked, the responsiveness to the threshold dose is determined by the effect of two loci. The direction of the dominance effect also varies with the antigen dose: high responsiveness is partially dominant at the optimal dose while at the threshold dose nonresponder character is partially dominant. The role of theH-2 linked locus was investigated. It has been demonstrated that on an identical background (equivalent to that of F₁ hybrids) this locus is responsible for 12% of the interline difference at the optimal antigen dose, and for 61% at the threshold antigen dose. For the two antigen doses, the quantitative effect of theH-2 locus is in agreement with the estimate of the number of loci obtained by variance analysis. The intervention of a second gene, non-H-2 linked, in the regulation of responsiveness to 10⁶ SE is demonstrated by appropriate assortative matings. The interaction between the two genes is discussed.