Biology subjects

Pohl, M.

Publications and source records attributed to Pohl, M..

7 recordsLinked to original sources

bioRxiv2025

Influence of ionic liquids on enzymatic asymmetric carboligations

The asymmetric mixed carboligation of aldehydes catalyzed by thiamine diphosphate (ThDP)-dependent enzymes provides a sensitive system for monitoring changes in activity, chemo-, and enantioselectivity. While previous studies have shown that organic cosolvents influence these parameters, we now demonstrate that similar effects occur upon addition of water-miscible ionic liquids (ILs). In this study, six ThDP-dependent enzymes were analyzed in the presence of 14 ILs under comparable conditions to assess their influence on enzymatic carboligation reactions yielding 2-hydroxy ketones. ILs exerted a moderate to strong influence on activity and, more notably, altered enantioselectivity. (R)-selective reactions were generally stable upon IL addition, while (S)-selective reactions frequently showed reduced selectivity or even inversion to the (R)-enantiomer. The most significant change was observed for the ApPDC_E469G variant of pyruvate decarboxylase from Acetobacter pasteurianus, where the enantiomeric excess shifted from 86% (S) to 60% (R) in the presence of 9% (w/v) Ammoeng 102. Control experiments indicated that this shift was primarily due to the Ammoeng cation rather than the anion. To explore the molecular basis of this phenomenon, all-atom molecular dynamics (MD) simulations were performed on wild-type ApPDC and the E469G variant in Ammoeng 101 and Ammoeng 102. The simulations revealed that hydrophobic and hydrophilic regions of the Ammoeng cations interact with the (S)-selective binding pocket, thereby favoring formation of the (R)-product. These results highlight the potential of solvent engineering for modulating enzyme selectivity and demonstrate that MD simulations can capture functionally relevant enzyme-solvent interactions at the atomic level.

bioengineering↗

bioRxiv2025

A staged approach using ants as test organism in ecotoxicology

Although ants (Hymenoptera: Formicidae) are ecological key components, they are currently not included in ecotoxicity testing. Based on their social organization, ecology and exposure routes to pesticides, we propose a multilevel testing scheme for ants starting from isolated workers (level-1), workers and brood (level-2) and founding queens or entire colonies (level-3). To evaluate feasibility, we tested three ant species (Camponotus maculatus, Crematogaster sp., Lasius niger) using the neonicotinoid imidacloprid as a test substance. Ants were orally exposed through liquid food. At level-1, worker survival showed a clear concentration response and allowed to estimate LC50 with sufficiently narrow confidence intervals. In level-2, worker mortality and sublethal effects negatively affected larval survival and development, e.g. occurrence of naked pupae, with a lower NOEC for larvae (<0.05 mg/L feeding solution) than workers (1.7 mg/L). At level-3, the test substance significantly reduced the reproductive output of newly mated L. niger queens over 21 days after a single exposure to 0.5 mg/L. Our results confirm that ants can be easily handled and tested in the laboratory, making them a valuable extension for ecotoxicity testing. Given their ecological relevance, easy culture in laboratory settings and promising first results, we recommend further research using our new testing scheme, e.g. testing other stressors, species and endpoints. Establishing a robust test protocol for ants would provide coverage of a new and ecologically important group of non-target insects, which contributes to a broader protection of biodiversity and ecosystems. HighlightsO_LIAnts play key roles in many ecosystems but are not covered in ecotoxicity testing. C_LIO_LIWe propose a testing scheme for ants with 3 different levels of complexity. C_LIO_LIAnts can be cultured and tested cost- and space-efficient in the laboratory. C_LIO_LIThree ant species were tested using imidacloprid in parts of our testing scheme. C_LIO_LILethal and sublethal endpoints can be linked to individual and colony fitness. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/641392v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@11387feorg.highwire.dtl.DTLVardef@1456368org.highwire.dtl.DTLVardef@8171b5org.highwire.dtl.DTLVardef@504536_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2024

Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet -granules and released from activated endothelial cells, interacts with factor H (FH) and von Willebrand factor (vWF). However, direct regulatory interaction with the complement cascade has not yet been described. We could show that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins, inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. Complement-regulatory function is validated in blood samples from patients with primary complement defects. Physiological relevance of TSP-1 is demonstrated in ANCA-vasculitis patients by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis following TSP-1 deficiency in an ANCA-vasculitis model. The newly described complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia, complement and platelets. In particular, the interplay between released TSP-1, vWF and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

molecular biology↗

bioRxiv2024

Dependence of aerosol-borne influenza A virus infectivity on relative humidity and aerosol composition

We describe a novel biosafety aerosol chamber equipped with state-of-the-art instrumentation for bubble-bursting aerosol generation, size distribution measurement, and condensation-growth collection to minimize sampling artifacts when measuring virus infectivity in aerosol particles. Using this facility, we investigated the effect of relative humidity (RH) in very clean air without trace gases (except [~]400 ppm CO2) on the preservation of influenza A virus (IAV) infectivity in saline aerosol particles. We characterized infectivity in terms of 99%-inactivation time, t99, a metric we consider most relevant to airborne virus transmission. The viruses remained infectious for a long time, namely t99 > 5 h, if RH < 30% and the particles effloresced. Under intermediate conditions of humidity (40% < RH < 70%), the loss of infectivity was the most rapid (t99 {approx} 15-20 min, and up to t99 {approx} 35 min at 95% RH). This is more than an order of magnitude faster than suggested by many previous studies of aerosol-borne IAV, possibly due to the use of matrices containing organic molecules, such as proteins, with protective effects for the virus. We tested this hypothesis by adding sucrose to our aerosolization medium and, indeed, observed protection of IAV at intermediate RH (55 %). Interestingly, the t99 of our measurements are also systematically lower than those in 1-L droplet measurements of organic-free saline solutions, which cannot be explained by particle size effects alone.

microbiology↗

bioRxiv2023

Salt-mediated inactivation of influenza A virus in 1-ul droplets exhibits exponential dependence on NaCl molality

Influenza A virus (IAV) spreads through exhaled aerosol particles and larger droplets. Estimating the stability of IAV is challenging and depends on factors such as the respiratory matrix and drying kinetics. Here, we combine kinetic experiments on millimeter-sized saline droplets with a biophysical aerosol model to quantify the impact of NaCl on IAV stability. We show that IAV inactivation is determined by NaCl concentration, which increases during water evaporation and then decreases again when efflorescence occurs. When drying in air with relative humidity RH = 30%, inactivation follows an inverted sigmoidal curve, with inactivation occurring most rapidly when the NaCl concentration exceeds 20 molal immediately prior to efflorescence. Efflorescence reduces the NaCl molality to saturated conditions, resulting in a significantly reduced inactivation rate. We demonstrate that the inactivation rate k depends exponentially on NaCl molality, and after the solution reaches equilibrium, the inactivation proceeds at a first-order rate. Introducing sucrose, an organic co-solute, attenuates IAV inactivation via two mechanisms, firstly by decreasing the NaCl molality during the drying phase, and secondly by a protective effect against the NaCl-induced inactivation. For both pure saline and sucrose-containing droplets, our biophysical model ResAM accurately simulates the inactivation when NaCl molality is used as the only inactivating factor. This study highlights the role of NaCl molality in IAV inactivation and provides a mechanistic basis for the observed inactivation rates. SYNOPSIS: This work quantifies the dependence of influenza A virus stability on salt molality in drying droplets and furthers the understanding of airborne virus transmission.SYNOPSIS: This work quantifies the dependence of influenza A virus stability on salt molality in drying droplets and furthers the understanding of airborne virus transmission.

microbiology↗

bioRxiv2023

Co-inhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription

Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic strategies results in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC impacts initiation, development, and survival of this tumor type. Using patient-derived xenografts of pancreatic carcinoma driven by KRAS and MYC oncogenic transcription, we show that co-inhibition of Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) synergistically induce tumor regression through targeting promoter pause-release, a rate-limiting step in transcription elongation. By comparing the nascent transcriptome with the recruitment of elongation and termination factors along genes, we found that co-inhibition of TOP1 and BRD4, while globally impairing RNA production, disturbs recruitment of proteins involved in termination. Thus, RNA polymerases continue transcribing downstream of genes for hundreds of kilobases leading to readthrough transcription. This pervasive transcription also occurs during replication, perturbing replisome progression and leading to DNA damage. The synergistic effect of TOP1 and BRD4 inhibition is specific for cancer cells leaving normal cells unharmed, highlighting the sensitivity of the tumor to these transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of high transcription and replication. One Sentence SummaryTOP1 and BRD4 inhibitors synergize to selectively kill pancreatic cancer in vivo via readthrough transcription without emergence of drug resistance

cancer biology↗

bioRxiv2022

Inactivation of Influenza A virus by pH conditions encountered in expiratory aerosol particles results from localized conformational changes within Haemagglutinin and Matrix 1 proteins.

Multiple respiratory viruses including Influenza A virus (IAV) can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. For indoor air, small exhaled aerosols undergo rapid acidification to pH [~]4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes, however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers that was mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Changes to HA were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 seconds post-exposure to acidic conditions. In addition, virion integrity was partially but irreversibly affected by acidic conditions. This was attributed to a progressive unfolding of the internal matrix protein 1 (M1), and aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques such as X-ray crystallography, highlighting the usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future.

microbiology↗