A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

Functional genomics of the yeast DNA-damage response

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Expression of human α1 antitrypsin in transgenic sheep

We have recently described the production of large amounts (< or = 65 grams per litre) of enzymatically active human alpha 1 antitrypsin in the milk of transgenic sheep (Wright et al., 1991). Here, we describe in more detail the expression of the human protein in the milk of these animals throughout the lactation period. Human alpha 1 antitrypsin is also found at much lower levels in the plasma of transgenic ewes before, during and after lactation. It is also detected in male plasma at very low levels. We have previously shown human alpha 1 antitrypsin purified from transgenic sheep milk to be indistinguishable from commercially available human plasma derived alpha 1 antitrypsin in terms of gross sugar content and in vitro activity. Here we extend this comparison to more detailed analyses of glycosylation state, amino-terminal sequence, pI value, and molecular weight determination by mass spectrometry.     

An organ culture system to model early degenerative changes of the intervertebral disc

Introduction  

Back pain, a significant source of morbidity in our society, is related to the degenerative changes of the intervertebral disc. At present, the treatment of disc disease consists of therapies that are aimed at symptomatic relief. This shortcoming stems in large part from our lack of understanding of the biochemical and molecular events that drive the disease process. The goal of this study is to develop a model of early disc degeneration using an organ culture. This approach is based on our previous studies that indicate that organ culture closely models molecular events that occur in vivo in an ex vivo setting.     

The settling of tsetse flies Glossina morsitans westwood (Diptera, Muscidae) on cloth screens

It has been found that tsetse flies prefer to settle on the darker of two screens but, contrary to expectation, they settle on the sunny side of a screen rather than on the shaded side. The attractiveness of a black and white striped screen diminished progressively as the width of the white stripes was increased but even narrow white stripes made a screen less attractive than an all-black control. This breaking up of the black screen was important since white adjacent to a black screen did not reduce its attractiveness.

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Zusammenfassung Die Wahl von verschieden hellen Stoffschirmen durch sich setzende Tsetsefliegen wurde in Freien beobachtet. Schwarze, dunkelgraue, hellgraue und weisse Schirme wurden verglichen; in allen Fällen wurde der dunklere Schirm vorgezogen. In ähnlicher Weise setzte sich eine grössere Anzahl von Fliegen auf einen schwarzen Schirm statt auf einen weissen desselben Formats, während ein schwarz-weiss gestreifter Schirm an Anziehungskraft um so mehr einbüsste, je breiter die weissen Streifen gemacht wurden. Fast alle Fliegen setzten sich auf die schwarzen Teile eines schwarz-weiss gestreiften Schirmes. Ein weisser Streifen neben einem schwarzen Schirm konnte jedoch die Anziehungskraft des letzteren nicht vermindern. Gegen alle Erwartungen zogen die sich setzenden Fliegen aber die sonnige Seite des Schirmes der schattigen vor.Es wird auf frühere Experimente hingewiesen, die Tsetsefliegen wie auch andere stechende Fliegen betreffen. Die Frage, warum Tsetsefliegen sich an Schirme setzen, wird erörtert und die Resultate der Arbeiten, über die hier berichtet wird, werden besprochen.