Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVκ3-huVH3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic application

Silicate condensation in Mira variables

Context. The formation of dust in winds of cool and highly evolved stars and the rate of injection of dust into the interstellar medium is not yet completely understood, despite the importance of the process for the evolution of stars and galaxies. This holds in particular for oxygen-rich stars, where it is still not known which process is responsible for the formation of the necessary seed particles of their silicate dust. Aims. We study whether the condensation of silicate dust in Mira envelopes could be caused by cluster formation by the abundant SiO molecules. Methods. We solve the dust nucleation and growth equations in the co-moving frame of a fixed mass element for a simplified model of the pulsational motions of matter in the outer layers of a Mira variable, which is guided by a numerical model for Mira pulsations. It is assumed that seed particles form through the clustering of SiO. The calculation of the nucleation rate is based on published experimental data. The quantity of dust formed is calculated via a moment method and the calculation of radiation pressure on dusty gas is based on a dirty silicate model. Results. Dust nucleation occurs in the model at the upper culmination of the trajectory of a gas parcel where it stays for a considerable time at low temperatures. Subsequent dust growth occurs during the descending part of the motion and continues after the next shock reversed motion. It is found that sufficient dust forms that radiation pressure exceeds the gravitational pull of the stars such that the mass element is finally driven out of the star. Conclusions. Nucleation of dust particles by clustering of the abundant SiO molecules could be the mechanism that triggers silicate dust formation in Miras

High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts

The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays

Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.

BackgroundIn May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests.MethodsWe analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level.Results141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each.ConclusionThe EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted

INSTAND Monkeypox EQA September 2022—all results.

This table contains the raw results of the EQA participants without any correction. Results that were excluded from evaluation are highlighted in orange. (XLSX)</p

Passing Bablok fit of laboratory-based Ct/Cq values (black dots) for all pairs of EQA samples, differing in concentration by a power of ten.

The Passing Bablok regression line (green) is shown with the lower and upper 95% confidence limits (red).</p

Fig 2 -

Analysis of Ct/Cq values for (A) monkeypox virus PCR results and (B) for orthopoxvirus PCR results for different test systems. The grey boxes display all results for the respective sample, and the distributions of specific manufacturer-based collectives are illustrated as smaller, colored box plots in overlay with the total results. For all boxes, the whiskers stretch from the 1st quartile—1.5*(interquartile range) to the 3rd quartile + 1.5*(interquartile range).</p

Fig 1 -

Distribution of the qualitative PCR results for the four samples of the monkeypox EQA survey for A) monkeypox virus (MPXV) and B) orthopoxvirus (OPXV). Numbers in the columns represent the actual number of results for the corresponding category.</p