Biology subjects

Spiegel, H.

Publications and source records attributed to Spiegel, H..

5 recordsLinked to original sources

bioRxiv2026

Antibody-mediated inhibition of ADAMTS-7 reduces experimental atherosclerosis

Objective: One of the strongest genetic associations with coronary artery disease (CAD) risk maps to the metalloproteinase 'a disintegrin and metalloproteinase with thrombospondin motifs 7' (ADAMTS-7) locus. The protein was shown to promote plaque formation and instability. We aimed to generate and evaluate an antibody-based strategy targeting ADAMTS-7 therapeutically to reduce atherosclerotic plaque formation. Approach and Results: A truncated form of human ADAMTS-7 was produced in Nicotiana benthamiana and used as antigen for antibody generation by hybridoma technology. Eight monoclonal antibodies (mAbs) were screened, among which ADAMTS-7-mAb32 (mAb32) demonstrated the highest affinity, as confirmed by surface plasmon resonance analyses and immunoblotting against full-length ADAMTS-7. In vitro, mAb32 inhibited interactions of ADAMTS-7 with its substrates TIMP-1 and SVEP1 in a dose- and time-dependent manner, as assessed by time-resolved Forster resonance energy transfer assays. To assess therapeutic efficacy in vivo, Apoe-/- mice were fed a Western diet for ten weeks and treated with weekly injections of mAb32 or control IgG over the last six weeks. En face aortic Oil Red O staining revealed significantly reduced plaque area in the treatment group, without changes in plasma cholesterol levels or body weight. No evidence of liver or kidney toxicity was observed. Conclusion: Monoclonal antibody-based inhibition of ADAMTS-7 reduced atherosclerotic burden in vivo without affecting lipid metabolism, supporting ADAMTS-7 as a viable therapeutic target in CAD. Further development of mAb32 may provide a cholesterol-independent treatment strategy for atherosclerosis.

physiology↗

bioRxiv2026

Beyond efficacy: Persistence and off-target effects of three biological nitrification inhibitors in two contrasting agricultural soils

The use of nitrogen (N) fertilizers to meet global food demands is expected to continue rising. However, up to 70% of N applied to agricultural soils is lost through microbially mediated processes such as nitrification. Inhibiting nitrification is thus a key strategy to reduce N losses and improve fertilizer N use efficiency. Various plant-derived compounds, termed biological nitrification inhibitors (BNIs), have been shown to reduce accumulation of nitrification products, intermediates, and byproducts (nitrite, nitrate, nitric and nitrous oxides). However, the mechanisms by which BNIs affect nitrifiers, along with their specificity and persistence in soil are not well understood. Here, we evaluated the effects of three BNIs: methyl 3-(4-hydroxyphenyl) acrylate (MHPA), 6-methoxy-2(3H)-benzoxazolone (MBOA), and limonene, on ammonia-oxidizing, total microbial, and fungal communities in two soils with contrasting pH. Their persistence in each soil was also evaluated. Although ammonia-oxidizing archaea initially dominated nitrifier communities in both soils, their bacterial counterparts significantly increased after mineral N addition but also were more sensitive to BNI application. Limonene and the synthetic inhibitor DMPP stimulated ammonium immobilization, as total soil mineral N was significantly reduced. Limonene and MHPA had the strongest off-target effects, increasing the relative abundance of hydrocarbon-degrading bacteria and potential fungal pathogens, respectively. In contrast, MBOA inhibited nitrification with minimal off-target effects. Among the tested BNIs, MBOA was also the most persistent in the high-pH, high-nitrification-rate soil. Our results show that MBOA is a promising biological inhibitor and highlight the importance of understanding BNIs ecological effects to develop targeted and sustainable N management strategies.

microbiology↗

bioRxiv2026

Potential of exogenous biological nitrification inhibitor addition to improve soil nitrogen availability for crop growth.

Modern agriculture is characterized by substantial fertilizer nitrogen (N) losses from soils, resulting in low crop N-use efficiency. Biological nitrification inhibitors (BNIs) are studied as a strategy to improve N retention in soils by suppressing nitrification. However, the impacts of applying exogenous BNIs to crops with unknown intrinsic BNI capacity remain poorly understood. In this study, we evaluated the impacts of adding three BNIs (methyl 3-(4-hydroxyphenyl) acrylate [MHPA], 6-methoxy-2(3H)-benzoxazolone [MBOA], and limonene), their mixture, and the synthetic nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on barley (Hordeum vulgare L.) growth, plant and soil N dynamics, and soil microbial communities. Using a rhizobox system with planted and bare-soil compartments, combined with 15N isotope tracing and molecular microbial community analyses, we assessed the spatio-temporal dynamics of N transformations, losses, plant N uptake, and microbial community responses in an alkaline agricultural soil. Independent of inhibitor application, the applied fertilizer N was lost primarily through NO- leaching (3-9% of the applied N). In contrast, N2O emissions represented only 0.001-0.028% of the applied N and varied with inhibitor type. MHPA increased dissolved inorganic N soil pools without affecting plant biomass or 15N uptake or strongly shifting microbial community composition. MBOA reduced NO3- concentrations in soil pore water without influencing plant growth or N uptake but shifted soil microbial community composition. In contrast, limonene reduced plant growth and 15N uptake and most significantly altered microbial community composition, without significantly changing N availability. Applying a BNI mixture, as well as limonene alone, was detrimental to plant growth and 15N uptake. DMPP showed only minor effects on N pools, plant growth, plant N uptake and microbial community composition. Overall, our results reveal both the potential and limitations of exogenous BNI application for improving N retention in crop systems.

microbiology↗

bioRxiv2022

ALiCE: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as ALiCE(R), engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 L microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer(R) CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

biochemistry↗

bioRxiv2020

Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies

SARS-CoV-2 causes ongoing infections worldwide, and identifying people with immunity is becoming increasingly important. Available point-of-care diagnostic systems as lateral flow assays have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity or possibility for quantitative measurements. Here, a new point-of-care approach for SARS-CoV-2 specific antibody detection in human serum based on magnetic immuno-detection is described and compared to standard ELISA. For magnetic immuno-detection, immunofiltration columns were coated with a SARS-CoV-2 spike protein peptide. SARS-CoV-2 peptide reactive antibodies, spiked at different concentrations into PBS and human serum, were rinsed through immunofiltration columns. Specific antibodies were retained within the IFC and labelled with an isotype specific biotinylated antibody. Streptavidin-functionalized magnetic nanoparticles were applied to label the secondary antibodies. Enriched magnetic nanoparticles were then detected by means of frequency magnetic mixing detection technology, using a portable magnetic read-out device. Measuring signals corresponded to the amount of SARS-CoV-2 specific antibodies in the sample. Our preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader detection range and was four times faster than ELISA. Further optimizations could reduce assay times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-of-care measurements without expensive lab equipment.

molecular biology↗