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Context. The initial particle growth in protoplanetary disks is limited by a bouncing barrier at submillimeter wavelengths. Bouncing leads to tribocharging and the electrostatic attraction of tribocharged aggregates may eventually draw them into large clusters. A charge- mediated growth phase allows for the formation of larger entities, namely, clusters of aggregates that are more prone to further particle concentrations, such as the streaming instability. Aims. We aim to quantify the strength of the electrostatic forces. Methods. In laboratory experiments, we used an acoustic trap to levitate small aggregates of tribocharged submm grains. These aggregates spin up within the trap until they lose grains. Thus, we used the centrifugal force as a measure of the local force. Results. Grains are regularly bound strongly to their neighbors. In comparison, the force at ejection can be stronger than the attractive scattering forces of the trap and can therefore be several orders of magnitude larger than expected. We note that these forces are long- ranging, compared to van der Waals forces. Thus, charged aggregates are much more stable than uncharged ones. Conclusions. Particle aggregates in disks might grow to centimeter clusters or larger as tribocharging increases the effective binding forces. This allows for hydrodynamic concentration and planetesimal formation to eventually take place throughout a wide part of the disk. [ABSTRACT FROM AUTHOR]

Chromatin configurations of cattle oocytes from different follicle sizes were classified in three chromatin categories. Early diplotenes, dictyate-like structures, more progressed diplotenes and early diakinesis and metaphases were found. In follicle group A (0.4-0.9 mm) immature diplotenes predominated (82.3%), and a rather low maturation competence was observed (12.1%). With increasing follicle size the maturation competence was improved to 47.8 % metaphases II in follicle group C (3-8 mm). Impressive CMA3 positive blocks were visualized especially in early diplotenes. Heterochromatic CMA3 positive clusters surrounded the nucleolus. Zusammenfassung Maturationskompetenz und Chromatinverhalten von teilweise und vollkommen entwickelten Rinderoozyten Die Rinderoozyten aus verschieden großen Follikeln wurden auf Grund ihrer Chromatincharakterisierung in 3 Gruppen eingeteilt. Diplotäne, Dictyotän-ähnliche Strukturen, weiter fortgeschrittene Diplotäne sowie frühe Diakinesen wurden gefunden. In der Follikelgruppe A (0,4-0,9 mm) dominierten unreife Diplotäne (82,3%) verbunden mit einer niederen Maturationskompetenz (12,1%). Mit ansteigender Follikelgröße erhöhte sich auch die Maturationskompetenz auf 47,8% Metaphasen II in Follikelgruppe C (3-8 mm). CMA3-positive Blockformationen konnten speziell in frühen Diplotänen sichtbar gemacht werden; heterochromatische CMA3-positive Areale wurden auch unmittelbar rund um den Nukleolus gefunden. [ABSTRACT FROM AUTHOR]

Maturation competence of pig oocytes possessing a well established cumulus was correlated with increasing follicle size. In follicle group A (0.4 mm - 0.9 mm) a very low maturation competence was observed (16.1 %). With increasing follicle size, the maturation competence was improved to 53.6 % metaphase II in follicle group C (2.0 mm - 8 mm). Distamicyn A3-DAPI positive blocks were observed in the nuclei of oocytes from small follicles. Zusammenfassung Maturationsfähigkeit und Chromatin-Konfiguration von Schweineoozyten: Die Maturationsfähigkeit von Schweineoozyten mit einem gut ausgebildeten Kumulus verbesserte sich mit steigender Follikelgröße. In Follikelgruppe A 0,4 mm - 0,9 mm wurde eine sehr niedrige Maturationskompetenz beobachtet (16,1 %). In Follikelgröße C 2,0 mm - 8,0 mm wurden 53,6 % Metaphase II erreicht. In den Zellkernen der Oozyten die aus kleinen Follikeln stammten, konnten Distamycin A3-DAPI positive Blöcke gefunden werden. [ABSTRACT FROM AUTHOR]

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An important aspect in the development of small molecules as drugs or agrochemicals is their systemic availability after intravenous and oral administration. The prediction of the systemic availability from the chemical structure of a potential candidate is highly desirable, as it allows to focus the drug or agrochemical development on compounds with a favorable kinetic profile. However, such predictions are challenging as the availability is the result of the complex interplay between molecular properties, biology and physiology and training data is rare. In this work we improve the hybrid model developed earlier (Schneckener in J Chem Inf Model 59:4893-4905, 2019). We reduce the median fold change error for the total oral exposure from 2.85 to 2.35 and for intravenous administration from 1.95 to 1.62. This is achieved by training on a larger data set, improving the neural network architecture as well as the parametrization of mechanistic model. Further, we extend our approach to predict additional endpoints and to handle different covariates, like sex and dosage form. In contrast to a pure machine learning model, our model is able to predict new end points on which it has not been trained. We demonstrate this feature by predicting the exposure over the first 24 h, while the model has only been trained on the total exposure., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Pharmacokinetics (PK) is the result of a complex interplay between compound properties and physiology, and a detailed characterization of a molecule's PK during preclinical research is key to understanding the relationship between applied dose, exposure, and pharmacological effect. Predictions of human PK based on the chemical structure of a compound are highly desirable to avoid advancing compounds with unfavorable properties early on and to reduce animal testing, but data to train such models are scarce. To address this problem, we combine well-established physiologically based pharmacokinetic models with Deep Learning models for molecular property prediction into a hybrid model to predict PK parameters for small molecules directly from chemical structure. Our model predicts exposure after oral and intravenous administration with fold change errors of 1.87 and 1.86, respectively, in healthy subjects and 2.32 and 2.23, respectively, in patients with various diseases. Unlike pure Deep Learning models, the hybrid model can predict endpoints on which it was not trained. We validate this extrapolation capability by predicting full concentration-time profiles for compounds with published PK data. Our model enables early selection and prioritization of the most promising drug candidates, which can lead to a reduction in animal testing during drug discovery and development., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: all authors are employees of Bayer AG., (Copyright © 2023. Published by Elsevier Inc.)

Background: Recently, bacterial components were shown to enhance immune responses by shifting immune cell metabolism towards glycolysis and lactic acid production, also known as the Warburg Effect. Currently, the effect of allergen products for immunotherapy (AIT) and commercial vaccines on immune cell metabolism is mostly unknown., Objective: To investigate the effect of AIT products (adjuvanted with either MPLA or Alum) on myeloid dendritic cell (mDC) metabolism and activation., Methods: Bone marrow-derived mDCs were stimulated with five allergoid-based AIT products (one adjuvanted with MPLA, four adjuvanted with Alum) and two MPLA-adjuvanted vaccines and analyzed for their metabolic activation, expression of cell surface markers, and cytokine secretion by ELISA. mDCs were pre-incubated with either immunological or metabolic inhibitors or cultured in glucose- or glutamine-free culture media and subsequently stimulated with the MPLA-containing AIT product (AIT product 1). mDCs were co-cultured with allergen-specific CD4+ T cells to investigate the contribution of metabolic pathways to the T cell priming capacity of mDCs stimulated with AIT product 1., Results: Both the MPLA-containing AIT product 1 and commercial vaccines, but not the Alum-adjuvanted AIT products, activated Warburg metabolism and TNF-α secretion in mDCs. Further experiments focused on AIT product 1. Metabolic analysis showed that AIT product 1 increased glycolytic activity while also inducing the secretion of IL-1β, IL-10, IL-12, and TNF-α. Both rapamycin (mTOR-inhibitor) and SP600125 (SAP/JNK MAPK-inhibitor) dose-dependently suppressed the AIT product 1-induced Warburg Effect, glucose consumption, IL-10-, and TNF-α secretion. Moreover, both glucose- and glutamine deficiency suppressed secretion of all investigated cytokines (IL-1β, IL-10, and TNF-α). Glucose metabolism in mDCs was also critical for the (Th1-biased) T cell priming capacity of AIT product 1-stimulated mDCs, as inhibition of mTOR signaling abrogated their ability to induce Th1-responses., Conclusion: The AIT product and commercial vaccines containing the adjuvant MPLA were shown to modulate the induction of immune responses by changing the metabolic state of mDCs. Better understanding the mechanisms underlying the interactions between cell metabolism and immune responses will allow us to further improve vaccine development and AIT., Competing Interests: The authors are employees of the German Federal Institute for Vaccines and Biomedicines. The Paul-Ehrlich-Institut (PEI) is an Agency of the German Federal Ministry of Health. In relation to the present publication, the authors consider themselves not having a conflict of interest. Opinions expressed in the paper are personal views of the authors, not necessarily reflecting an official opinion of the PEI or the German Federal Ministry of Health., (Copyright © 2022 Zimmermann, Goretzki, Meier, Wolfheimer, Lin, Rainer, Krause, Wedel, Spies, Führer, Vieths, Scheurer and Schülke.)

Background: Manifestation of respiratory allergy to American cockroach ( Periplaneta americana ) is prominent in the subtropical and tropical areas. However, co-existing perennial indoor inhalant allergies frequently compromise clinical diagnosis of cockroach allergy, and the analysis of sensitization pattern is limited by the lack of Periplaneta allergens widely available for component-resolved diagnostics (CRD). Objective: To evaluate a collection of previously described recombinant Periplaneta allergens for CRD in cockroach allergy. Methods: A panel of nine recombinant Periplanet a allergens (Per a 1-5, 7-10) was generated, purified, and subjected to physicochemical characterization by applying circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), amino acid (AA) analysis, and mass spectrometry (MS). Patients ( n = 117) from India, Korea, Venezuela, and Iran, reporting perennial respiratory indoor allergies with IgE sensitization to cockroach ( P. americana and/or Blattella germanica ), were included. The sensitization profile was monitored by the experimental ImmunoCAP testing. Results: ImmunoCAP testing confirmed IgE sensitization to Periplaneta and/or Blattella extract in 98 of 117 patients ( r = 0.95). Five out of 117 patients were sensitized to only one of the two cockroach species. Within the whole study group, the prevalence of sensitization to individual allergens varied from 4% (Per a 2) to 50% (Per a 9), with the highest IgE values to Per a 9. Patients from four countries displayed different sensitization profiles at which Per a 3 and Per a 9 were identified as major allergens in India and Korea. Periplaneta -derived lipocalin and myosin light chain were characterized as new minor allergens, designated as Per a 4 and Per a 8. Periplaneta extract showed higher diagnostic sensitivity than all individual components combined, suggesting the existence of allergens yet to be discovered. Conclusion: Utilization of a panel of purified Periplaneta allergens revealed highly heterogeneous sensitization patterns and allowed the classification of lipocalin and myosin light chain from Periplaneta as new minor allergens., Competing Interests: JL was employed by company Thermo Fisher Scientific. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wangorsch, Jamin, Eichhorn, Pablos, Sharma, Schweidler, Kastner, Wildner, Saloga, Führer, Reyna Orozco, Sherkat, Sadeghi, Teifoori, Park, Briza, Vieths, Ferreira, Arora, Lidholm, Gadermaier and Scheurer.)

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Activating chemical bonds through external triggers and understanding the underlying mechanism are at the heart of developing molecules with catalytic and switchable functions. Thermal, photochemical, and electrochemical bond activation pathways are useful for many chemical reactions. In this Article, a series of Ru(II) complexes containing a bidentate and a tripodal ligand were synthesized. Starting from all-pyridine complex 1(2+), the pyridines were stepwise substituted with "click" triazoles (2(2+)-7(2+)). Whereas the thermo- and photoreactivity of 1(2+) are due to steric repulsion within the equatorial plane of the complex, 3(2+)-6(2+) are reactive because of triazoles in axial positions, and 4(2+) shows unprecedented photoreactivity. Complexes that feature neither steric interactions nor axial triazoles (2(2+) and 7(2+)) do not show any reactivity. Furthermore, a redox-triggered conversion mechanism was discovered in 1(2+), 3(2+), and 4(2+). We show here ligand design principles required to convert a completely inert molecule to a reactive one and vice versa, and provide mechanistic insights into their functioning. The results presented here will likely have consequences for developing a future generation of catalysts, sensors, and molecular switches.

Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 μg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.

Allergen products for specific immunotherapy of type I allergies were first authorized for the German market in the 1970s. In addition to finished products manufactured in advance and in batches, so-called named patient products have recently been defined as Medicinal Products by the German Medicinal Products Act ("Arzneimittelgesetz", AMG 14th Revision 2005). Some allergen products previously marketed as named patient products are now required to obtain marketing authorization according to the German ordinance for therapy allergens. Products have to be batch released by the competent German Federal Agency, the Paul-Ehrlich-Institut (PEI). Samples of product batches are delivered to the PEI in order to perform experimental quality controls. With regard to named patient products, PEI tests batch samples of the bulk extract preparations used for manufacturing of the respective, named patient products. The institute releases approximately 2,800 allergen product batches annually.

Control of cellular processes by regulated proteolysis is conserved among all organisms. FtsH, the only membrane-anchored AAA protease in bacteria, fulfills a variety of regulatory functions. This review focuses on soluble FtsH substrates in Escherichia coli and in other bacteria and outlines emerging substrate recognition principles.

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.

The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione-S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH.

Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane-bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co-purified with LpxC. However, degradation of LpxC was DnaK/J-independent in contrast to turnover of the heat shock sigma factor sigma32 (RpoH). The stability of LpxC in a bacterial one-hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C-terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half-life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C-terminus of LpxC contains a signal sequence necessary for FtsH-dependent degradation.

Rhodobacter capsulatus was shown to grow efficiently with taurine as sole source of sulfur. We identified a gene region exhibiting similarity to the Escherichia coli tauABC genes coding for a taurine-specific ABC transporter. The R. capsulatus tauABC genes were flanked by two putative operons (orf459-484-590 and cysE-srpI-nifS2) both reading in opposite direction relative to tauABC. Orf459 shows strong similarity to taurine:pyruvate aminotransferase (Tpa) from Bilophila wadsworthia catalyzing the initial transamination during anaerobic taurine degradation, and Orf590 exhibits clear similarity to sulfoacetaldehyde sulfo-lyase from Desulfonispora thiosulfatigenes probably catalyzing the step following the taurine:pyruvate aminotransferase (Tpa) reaction, whereas nifS2 might code for a putative cysteine desulfurase. Expression of R. capsulatus tauABC and nifS2 was inhibited by sulfate, suggesting that tauABC and nifS2 might belong to the same regulon. In contrast, transcription of orf459 was not inhibited by sulfate but was induced by taurine. A tauAB deletion mutant showed significantly reduced growth compared to the wild-type with taurine as sole sulfur source in the presence of serine as a nitrogen source, whereas normal growth was observed in the presence of taurine and ammonium. Deletion of orf459-484-590 completely abolished growth with taurine/serine. Single mutations in any of the three genes resulted in the same phenotype, indicating that all three genes of this putative operon are essential for taurine sulfur utilization in the presence of serine. A model for anaerobic taurine sulfur assimilation in R. capsulatus is discussed.

Seven microsatellite loci were evaluated for their suitability for parentage control. The polymerase chain reaction (PCR) was used to amplify the short tandem repeat (STR) loci in separate reactions. The microsatellite polymorphisms were visualized by radioisotopic autoradiographic detection. The microsatellite loci showed extensive polymorphism with allele numbers ranging from 4-23 and polymorphism information content (PIC) values in the range of 0.57-0.87. The analysis of these loci also revealed that they have a 99.9% combined probability of exclusion (PE) of erroneous parentage. The results of this study revealed that a very high probability of exclusion could be reached with only four microsatellite loci.

Chromatin configurations of cattle oocytes from different follicle sizes were classified in three chromatin categories. Early diplotenes, dictyate-like structures, more progressed diplotenes and early diakinesis and metaphases were found. In follicle group A (0.4-0.9 mm) immature diplotenes predominated (82.3%), and a rather low maturation competence was observed (12.1%). With increasing follicle size the maturation competence was improved to 47.8% metaphases II in follicle group C (3-8 mm). Impressive CMA3 positive blocks were visualized especially in early diplotenes. Heterochromatic CMA3 positive clusters surrounded the nucleolus.