Developing Small-Molecule Inhibitors of HECT-Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities

The ubiquitin system regulates countless physiological and disease-associated processes and has emerged as an attractive entryway for therapeutic efforts. With over 600 members in the human proteome, ubiquitin ligases are the most diverse class of ubiquitylation enzymes and pivotal in encoding specificity in ubiquitin signaling. Although considerable progress has been made in the identification of small molecules targeting RING ligases, relatively little is known about the “druggability” of HECT (homologous to E6AP C terminus) ligases, many of which are critically implicated in human pathologies. A major obstacle to optimizing the few available ligands is our incomplete understanding of their inhibitory mechanisms and the structural basis of catalysis in HECT ligases. Here, we survey recent approaches to manipulate the activities of HECT ligases with small molecules to showcase the particular challenges and opportunities these enzymes hold as therapeutic targets

Atmospheric CO2 availability induces varying responses in net photosynthesis, toxin production and N2 fixation rates in heterocystous filamentous Cyanobacteria (Nostoc and Nodularia)

Heterocystous Cyanobacteria of the genus Nodularia form major blooms in brackish waters, while terrestrial Nostoc species occur worldwide, often associated in biological soil crusts. Both genera, by virtue of their ability to fix N2 and conduct oxygenic photosynthesis, contribute significantly to global primary productivity. Select Nostoc and Nodularia species produce the hepatotoxin nodularin and whether its production will change under climate change conditions needs to be assessed. In light of this, the effects of elevated atmospheric CO2 availability on growth, carbon and N2 fixation as well as nodularin production were investigated in toxin and non-toxin producing species of both genera. Results highlighted the following:Biomass and volume specific biological nitrogen fixation (BNF) rates were respectively almost six and 17 fold higher in the aquatic Nodularia species compared to the terrestrial Nostoc species tested, under elevated CO2 conditions.There was a direct correlation between elevated CO2 and decreased dry weight specific cellular nodularin content in a diazotrophically grown terrestrial Nostoc species, and the aquatic Nodularia species, regardless of nitrogen availability.Elevated atmospheric CO2 levels were correlated to a reduction in biomass specific BNF rates in non-toxic Nodularia species.Nodularin producers exhibited stronger stimulation of net photosynthesis rates (NP) and growth (more positive Cohen’s d) and less stimulation of dark respiration and BNF per volume compared to non-nodularin producers under elevated CO2 levels. This study is the first to provide information on NP and nodularin production under elevated atmospheric CO2 levels for Nodularia and Nostoc species under nitrogen replete and diazotrophic conditions

Exploring the influence of atmospheric CO2 and O2 levels on the utility of nitrogen isotopes as proxy for biological N2 fixation

Funding: M.M.G. was funded by the DFG: SPP1833 grants GE2558/3-1 and GE2558/4-1. E.E.S. acknowledges funding from an NERC Frontiers grant (NE/V010824/1). Funding for open access publication was awarded by the Federal Government, the state of Mecklenburg-Vorpommen (FKZ VIII-0639-INP00-2023/004-002), and the Leipzig Institute for PlasmaScience and Technology to N.W.Biological N2 fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δ15N) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δ15N has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric pO2 was lower and pCO2 was higher. Here, we explore δ15N signatures under three atmospheres with (i) elevated CO2 and no O2 (Archean), (ii) present-day CO2, and O2 and (iii) future elevated CO2, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δ15N and the associated fractionation factor epsilon (ε = δ15Nbiomass – δ15NN2) during BNF in Archaea and Bacteria, including cyanobacteria, and habitats. The ε ranges between 3.70‰ and −4.96‰ with a mean ε value of −1.38 ± 0.95‰, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set revealed correlations of isotopic fractionation of BNF with CO2 concentrations, toxin production, and light, although within 1‰. Moreover, correlation showed significant dependency of ε to species type, C/N ratios and toxin production in cyanobacteria, albeit it within a small range (−1.44 ± 0.89‰). We therefore conclude that δ15N is likely robust when applied to the pre-Cambrian-like atmosphere, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower ε) observed in the toxin-producing Nodularia and Nostoc spp. suggests a heretofore unknown role of toxins in modulating nitrogen isotopic signals that warrants further investigation.Peer reviewe

How are oxygen budgets influenced by dissolved iron and growth of oxygenic phototrophs in an iron-rich spring system? Initial results from the Espan Spring in Fürth, Germany

At present most knowledge on the impact of iron on 18O / 16O ratios (i.e. δ18O) of dissolved oxygen (DO) under circum-neutral conditions stems from experiments carried out under controlled laboratory conditions. These showed that iron oxidation leads to an increase in δ18ODO values. Here we present the first study on effects of elevated Fe(II) concentrations on the δ18ODO in a natural, iron-rich, circum-neutral watercourse. Our results show that iron oxidation was the major factor for rising dissolved oxygen isotope compositions in the first 85 m of the system in the cold season (February) and for the first 15 m during the warm season (May). Further along the course of the stream, the δ18ODO decreased towards values known for atmospheric equilibration around +24.6 ‰ during both seasons. Possible drivers for these changes may be reduced iron oxidation, increased atmospheric exchange and DO production by oxygenic phototrophic algae mats. In the cold season, the δ18ODO values stabilized around atmospheric equilibrium, whereas in the warm season stronger influences by oxygenic photosynthesis caused values down to +21.8 ‰. In the warm season from 145 m downstream of the spring, the δ18ODO increased again until it reached atmospheric equilibrium. This trend can be explained by respiratory consumption of DO combined with a relative decrease in photosynthetic activity and increasing atmospheric influences. Our study shows that dissolved Fe(II) can exert strong effects on the δ18ODO of a natural circum-neutral spring system even under constant supply of atmospheric O2. However, in the presence of active photosynthesis, with supply of O2 to the system, direct effects of Fe oxidation on the δ18ODO value become masked. Nonetheless, critical Fe(II) concentrations may indirectly control DO budgets by enhancing photosynthesis, particularly if cyanobacteria are involved.</p

How to develop, externally validate, and update multinomial prediction models

Multinomial prediction models (MPMs) have a range of potential applications across healthcare where the primary outcome of interest has multiple nominal or ordinal categories. However, the application of MPMs is scarce, which may be due to the added methodological complexities that they bring. This article provides a guide of how to develop, externally validate, and update MPMs. Using a previously developed and validated MPM for treatment outcomes in rheumatoid arthritis as an example, we outline guidance and recommendations for producing a clinical prediction model using multinomial logistic regression. This article is intended to supplement existing general guidance on prediction model research. This guide is split into three parts: 1) Outcome definition and variable selection, 2) Model development, and 3) Model evaluation (including performance assessment, internal and external validation, and model recalibration). We outline how to evaluate and interpret the predictive performance of MPMs. R code is provided. We recommend the application of MPMs in clinical settings where the prediction of a nominal polytomous outcome is of interest. Future methodological research could focus on MPM-specific considerations for variable selection and sample size criteria for external validation

Prognostic factors associated with failure of total elbow arthroplasty

AimsThe aims of this study were to identify and evaluate the current literature examining the prognostic factors which are associated with failure of total elbow arthroplasty (TEA).MethodsElectronic literature searches were conducted using MEDLINE, Embase, PubMed, and Cochrane. All studies reporting prognostic estimates for factors associated with the revision of a primary TEA were included. The risk of bias was assessed using the Quality In Prognosis Studies (QUIPS) tool, and the quality of evidence was assessed using the modified Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) framework. Due to low quality of theevidence and the heterogeneous nature of the studies, a narrative synthesis was used.ResultsA total of 19 studies met the inclusion criteria, investigating 28 possible prognostic factors. Most QUIPS domains (84%) were rated as moderate to high risk of bias. The quality of the evidence was low or very low for all prognostic factors. In low-quality evidence, prognostic factors with consistent associations with failure of TEA in more than one study were: the sequelae of trauma leading to TEA, either independently or combined with acute trauma, and male sex. Severalother studies investigating sex reported no association. The evidence for other factors was of very low quality and mostly involved exploratory studies.ConclusionThe current evidence investigating the prognostic factors associated with failure of TEA is of low or very low quality, and studies generally have a moderate to high risk of bias. Prognostic factors are subject to uncertainty, should be interpreted with caution, and are of little clinical value. Higher-quality evidence is required to determine robust prognostic factors for failure of TEA

The Role of Millimeter-Waves in the Distance Measurement Accuracy of an FMCW Radar Sensor

High-accuracy, short-range distance measurement is required in a variety of industrial applications e.g., positioning of robots in a fully automated production process, level measurement of liquids in small containers. An FMCW radar sensor is suitable for this purpose, since many of these applications involve harsh environments. Due to the progress in the field of semiconductor technology, FMCW radar sensors operating in different millimeter-wave frequency bands are available today. An important question in this context, which has not been investigated so far is how does a millimeter-wave frequency band influence the sensor accuracy, when thousands of distance measurements are performed with a sensor. This topic has been dealt with for the first time in this paper. The method used for analyzing the FMCW radar signal combines a frequency- and phase-estimation algorithm. The frequency-estimation algorithm based on the fast Fourier transform and the chirp-z transform provides a coarse estimate of the target distance. Subsequently, the phase-estimation algorithm based on a cross-correlation function provides a fine estimate of the target distance. The novel aspects of this paper are as follows. First, the estimation theory concept of Cramér-Rao lower bound (CRLB) has been used to compare the accuracy of two millimeter-wave FMCW radars operating at 60 GHz and 122 GHz. In this comparison, the measurement parameters (e.g., bandwidth, signal-to-noise ratio) as well as the signal-processing algorithm used for both the radars are the same, thus ensuring an unbiased comparison of the FMCW radars, solely based on the choice of millimeter-wave frequency band. Second, the improvement in distance measurement accuracy obtained after each step of the combined frequency- and phase-estimation algorithm has been experimentally demonstrated for both the radars. A total of 5100 short-range distance measurements are made using the 60 GHz and 122 GHz FMCW radar. The measurement results are analyzed at various stages of the frequency- and phase-estimation algorithm and the measurement error is calculated using a nanometer-precision linear motor. At every stage, the mean error values measured with the 60 GHz and 122 GHz FMCW radars are compared. The final accuracy achieved using both radars is of the order of a few micrometers. The measured standard deviation values of the 60 GHz and 122 GHz FMCW radar have been compared against the CRLB. As predicted by the CRLB, this paper experimentally validates for the first time that the 122 GHz FMCW radar provides a higher repeatability of micrometer-accuracy distance measurements than the 60 GHz FMCW radar. View Full-Tex

Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study

Background: Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence. Methods: We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix- Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models. Results: Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), “K. quasivariicola” (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and “K. quasivariicola” (odds ratio = 0.17, p \u3c 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53]). Conclusions: Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options

Design Guidelines for interlocked stator cores made of CoFe sheets

CoFe lamination stacks used for high-performance electric motors can be manufactured economically in high volumes by interlocking. In order to ensure sufficient joint strength with minimized sheet thickness, a comprehensive knowledge of the influences of various process parameters, such as embossing depth, clearance and counter punch force, is essential. To analyze these parameters, which also influence the magnetic properties, experiments are carried out and resulting joint strengths are determined in top tensile tests. The negative influences of the cutting process on magnetic conductivity and thus hysteresis losses due to residual stresses and plastic deformation are well known. In the subsequent stacking step, an influence of embossing and pre-stresses on the material properties is expected. In addition, local electrical contacts between the sheets may occur due to the interlocking process, causing additional eddy currents. Loss measurements are conducted to investigate the effect of the joining process on the magnetic properties of the stack. In doing so, the influence of process parameters such as the embossing depth and clearance on eddy current power losses is analyzed

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53

Theresa Klett,1,&ast; Martin Schwer,1,&ast; Larissa N Ernst,1,&ast; Marc U Engelhardt,1 Simon J Jaag,2 Benedikt Masberg,2 Cornelius Knappe,2 Michael Lämmerhofer,2 Matthias Gehringer,3,4 Frank M Boeckler1,5 1Laboratory for Molecular Design & Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 2Pharmaceutical (Bio-) Analysis, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 3Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 4Medicinal Chemistry, Institute for Biomedical Engineering, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 5Interfaculty Institute for Biomedical Informatics (IBMI), Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany&ast;These authors contributed equally to this workCorrespondence: Frank M Boeckler, Laboratory for Molecular Design & Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8 (Haus B), Tübingen, D – 72076, Germany, Tel +49 7071 29 74567, Email [email protected]: Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN).Methods: After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5’-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS).Results: In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53.Conclusion: The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and − 9.1 °C. Keywords: covalent fragment-based drug discovery, differential scanning fluorimetry, 5,5’-dithiobis-(2-nitrobenzoic acid), intact protein mass spectrometry, glutathion