Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction

Aerosol Formation During Processing of Potentially Infectious Samples on Roche Immunochemistry Analyzers (cobas e analyzers) and in an End-to-End Laboratory Workflow to Model SARS-CoV-2 Infection Risk for Laboratory Operators

AbstractBackgroundThis study assessed formation of potentially infectious aerosols during processing of infectious samples in a real-world laboratory setting, which could then be applied in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).MethodsThis two-part study assessed aerosol formation when using cobas e analyzers only and in an end-to-end laboratory workflow. To estimate aerosol formation, recombinant hepatitis B surface antigen (HBsAg) was used as a surrogate marker for infectious virus particles to evaluate the potential risk of SARS-CoV-2 infection to laboratory operators. Using the HBsAg model, air sampling was performed at different positions around the cobas e analyzers and in four scenarios reflecting critical handling and/or transport locations in an end-to-end laboratory workflow. Aerosol formation of HBsAg was quantified using the Elecsys® HBsAg II quant II assay. The model was then applied to a SARS-CoV-2 context using SARS-CoV-2 infection-specific parameters to calculate viral RNA copies.ResultsFollowing application to SARS-CoV-2, the mean HBsAg uptake per hour when recalculated into viral RNA copies was 1.9 viral RNA copies across the cobas e analyzers and 0.87 viral RNA copies across all tested scenarios in an end-to-end laboratory workflow. This corresponds to a maximum aspiration rate of &lt;16 viral RNA copies during an 8-hour shift when using cobas e analyzers and/or in an end-to-end laboratory workflow.ConclusionsThe low production of marker-containing aerosol when using cobas e analyzers and in an end-to-end laboratory workflow is consistent with a remote risk of laboratory-acquired SARS-CoV-2 infection for laboratory operators.SummaryThis study investigated the formation of potentially infectious aerosols during processing of infectious samples in a model using hepatitis B surface antigen (HBsAg) as a marker for infectious virus particles. The risk to laboratory operators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was then inferred. Air sampling was performed around cobas e analyzers and in an end-to-end laboratory workflow, after which HBsAg was quantified and applied to SARS-CoV-2 using SARS-CoV-2 infection-specific parameters. The maximum aspiration rate of &lt;16 viral RNA copies/8-hour shift, when applied to a SARS-CoV-2 context, poses a remote risk of SARS-CoV-2 infection to laboratory operators.</jats:sec

Phosphosite Mapping of HIP-55 Protein in Mammalian Cells

In the present study, hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa (HIP-55) protein was over-expressed in HEK293 cells, which was genetically attached with 6x His tag. The protein was purified by nickel-charged resin and was then subjected to tryptic digestion. The phosphorylated peptides within the HIP-55 protein were enriched by TiO(2) affinity chromatography, followed by mass spectrometry analysis. Fourteen phosphorylation sites along the primary structure of HIP-55 protein were identified, most of which had not been previously reported. Our results indicate that bio-mass spectrometry coupled with manual interpretation can be used to successfully identify the phosphorylation modification in HIP-55 protein in HEK293 cells

Data_Sheet_1_Aerosol formation during processing of potentially infectious samples on Roche immunochemistry analyzers (cobas e analyzers) and in an end-to-end laboratory workflow to model SARS-CoV-2 infection risk for laboratory operators.docx

ObjectivesTo assess aerosol formation during processing of model samples in a simulated real-world laboratory setting, then apply these findings to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to assess the risk of infection to laboratory operators.DesignThis study assessed aerosol formation when using cobas e analyzers only and in an end-to-end laboratory workflow. Recombinant hepatitis B surface antigen (HBsAg) was used as a surrogate marker for infectious SARS-CoV-2 viral particles. Using the HBsAg model, air sampling was performed at different positions around the cobas e analyzers and in four scenarios reflecting critical handling and/or transport locations in an end-to-end laboratory workflow. Aerosol formation of HBsAg was quantified using the Elecsys® HBsAg II quant II immunoassay. The model was then applied to SARS-CoV-2.ResultsFollowing application to SARS-CoV-2, mean HBsAg uptake/hour was 1.9 viral particles across the cobas e analyzers and 0.87 viral particles across all tested scenarios in an end-to-end laboratory workflow, corresponding to a maximum inhalation rate of ConclusionLow production of marker-containing aerosol when using cobas e analyzers and in an end-to-end laboratory workflow is consistent with a remote risk of laboratory-acquired SARS-CoV-2 infection for laboratory operators.</p