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Different forms of life are known to express RNA ligases that catalyse the condensation of a 3’-hydroxy group and a 5’-terminal phosphate of RNA. No such RNA ligases have yet been identified in vertebrates. Here, we report that the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29), which we identified by a chemical proteomics approach, is a 5’-3’ RNA ligase. C12orf29 catalyses RNA ligation via auto-AMPylation of a critical lysine residue by using ATP as a cosubstrate and subsequent AMP transfer to the 5’-phosphate of an RNA substrate followed by phosphodiester bond formation. Studies at the cellular level reveal the involvement of C12orf29 in maintaining RNA integrity upon cellular stress induced by reactive oxygen species. These findings highlight the importance of RNA ligation for cellular fitness.

Cosmic-ray neutron albedo sensing (CRNS) is a modern technology that can be used to continuously measure the average water content in the environment (i.e., in soil, snow, or vegetation). The sensor footprint encompasses an area of 10-15 hectares and extends to 20-50 decimeters deep into the soil. This method might be an alternative to conventional in-situ sensors or to expensive sampling of soil or snow. It also has the potential to bridge the scale gap between point-scale measurements and remote-sensing data in both, the horizontal and the vertical domain.Currently, more than 200 sensors are operated in the growing networks of national and continental observatories. CRNS stations are continuously monitoring the local water dynamics at various field sites worldwide. They require almost no maintenance over the years due to a solar module, battery and telemetry. Since the method works non-invasively, the soil is left undisturbed. The passive sensing technique measures natural cosmogenic background radiation which interacts with hydrogen in the ground independent of temperature, frost, or wind effects. CRNS can also be used on mobile platforms for on-demand soil moisture mapping at the field- or regional scale. The sensors are rapidly operational on any ground- or airborne vehicle.In this presentation we will show various examples of stationary CRNS and their performance compared to traditional sensors at various sites in Germany, Europe, and beyond. We will discuss applications for hydrological modeling and new spatial mapping approaches. The data is particularly useful to study hydrological extreme events, droughts, heatwaves, floods, snow melt/accumulation, and it can be applied as a lower boundary condition in atmospheric models, in hydrological models, or agricultural irrigation management.

Introduction: Mucin 5AC (MUC5AC) belongs to the family of secreted gel-forming mucins. It is physiologically expressed in some normal mucin producing epithelial cells but also in pancreatic, ovarian, and colon cancer cells. The role of MUC5AC expression in cancer is not fully understood. This study was designed to explore the role of MUC5AC for pancreatic cancer progression, its association to microsatellite instability, and its diagnostic utility. Methods: Mucin 5AC expression was studied immunohistochemically in a tissue microarray (TMA) from 532 pancreatic cancers, 61 cancers of the ampulla Vateri, six acinar cell carcinomas and 12 large sections of pancreatitis. Results: Mucin 5AC staining was interpretable in 476 of 599 (79%) arrayed cancers. Staining was completely absent in normal pancreas and pancreatitis, but frequent in pancreatic cancer. Membranous and cytoplasmic MUC5AC expression was most common in pancreatic adenocarcinomas (71% of 423), followed by carcinomas of the ampulla Vateri (43% of 47), and absent in six acinar cell carcinomas. Mucin 5AC expression was unrelated to tumor phenotype (tumor stage, tumor grade, lymph node, and distant metastasis), and microsatellite instability in ductal adenocarcinomas and carcinomas of the ampulla Vateri. Conclusion: Our study indicates that MUC5AC is an excellent biomarker for pancreatic cancer diagnosis, especially to support the sometimes-difficult diagnosis on small biopsies. Mucin 5AC expression is unrelated to pancreatic cancer aggressiveness.

DNA polymerases can process a wide variety of structurally diverse nucleotide substrates, but the molecular basis by which the analogs are processed is not completely understood. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of DNA polymerases in real time and at the atomic level. The nucleotide analogs containing a selenophene, benzofuran, or benzothiophene moiety at the C5 position of 2′-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. KlenTaq (A family DNA polymerase) is sensitive to the size of the modification as it incorporates only one heterobicycle-modified nucleotide into the growing polymer, whereas it efficiently incorporates the selenophene-modified nucleotide analog at multiple positions. Notably, in the single nucleotide incorporation assay, irrespective of the heterocycle size, it exclusively adds a single nucleotide at the 3′-end of a primer, which enabled devising a simple two-step site-specific ON labeling technique. KOD and Vent(exo-) DNA polymerases, belonging to the B family, tolerate all the three modified nucleotides and produce ONs with multiple labels. Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real-time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides. published

The transcriptional coactivator YAP1 controls the balance between cell proliferation and apoptosis. YAP1 overexpression is linked to poor prognosis in many cancer types, yet its role in prostate cancer is unknown. Here, we applied YAP1 immunohistochemistry to a tissue microarray containing 17,747 clinical prostate cancer specimens. Cytoplasmic and nuclear YAP1 staining was seen in 81% and 63% of tumours. For both cytoplasmic and nuclear YAP1 staining, high levels were associated with advanced tumour stage, classical and quantitative Gleason grade, positive nodal stage, positive surgical margin, high KI67 labelling index, and early biochemical recurrence (p TMPRSS2:ERG fusion (p PTEN and 8p deletions (p

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a ubiquitous RNA splicing factor that is overexpressed and prognostically relevant in various human cancer types. To study the impact of hnRNPA1 expression in prostate cancer, we analyzed a tissue microarray containing 17,747 clinical prostate cancer specimens by immunohistochemistry. hnRNPA1 was expressed in normal prostate glandular cells but often overexpressed in cancer cells. hnRNPA1 immunostaining was interpretable in 14,258 cancers and considered strong in 33.4%, moderate in 45.9%, weak in 15.3%, and negative in 5.4%. Moderate to strong hnRNPA1 immunostaining was strongly linked to adverse tumor features including high classical and quantitative Gleason score, lymph node metastasis, advanced tumor stage, positive surgical margin, and early biochemical recurrence (p

Protein phosphatase 1 nuclear-targeting subunit (PNUTS) is ubiquitously expressed and associates with PTEN and protein phosphatase 1 (PP1) to control its activity. The role of PNUTS overexpression has hardly been studied in cancer. In this study, we used immunohistochemistry to quantitate PNUTS expression on a tissue microarray containing 17,747 clinical prostate cancer specimens. As compared to normal prostate epithelium, PNUTS expression was often higher in cancer. Among 12,235 interpretable tumors, PNUTS staining was negative in 21%, weak in 34%, moderate in 35%, and strong in 10% of cases. High PNUTS expression was associated with higher tumor stage, classical and quantitative Gleason grade, nodal stage, surgical margin, Ki67 labeling index, and early biochemical recurrence (p<0.0001 each). PNUTS expression proved to be a moderate prognostic parameter with a maximal univariable Cox proportional hazard for PSA recurrence-free survival of 2.21 compared with 5.91 for Gleason grading. It was independent from established prognostic parameters in multivariable analysis. Comparison with molecular data available from earlier studies using the same TMA identified associations between high PNUTS expression and elevated androgen receptor expression (p<0.0001), presence of TMPRSS2:ERG fusion (p<0.0001), and 8 of 11 chromosomal deletions (3p13, 5q21, 8p21, 10q23, 12p13, 13q14, 16q24, and 17p13; p<0.05 each). Particularly strong associations with PTEN and 12p13 deletions (p<0.0001 each) may indicate a functional relationship, which has already been established for PNUTS and PTEN. PNUTS had no additional role on outcome in PTEN-deleted cancers. In conclusion, the results of our study identify high PNUTS protein levels as a predictor of poor prognosis possibly linked to increased levels of genomic instability. PNUTS measurement, either alone or in combination, might be of clinical utility in prostate cancers.

The active enantiomer R-Praziquantel (PZQ) shows a clinically lower relative exposure when administered enantiomerically pure compared with a racemic form. We investigated the hypothesis that enantiomer-enantiomer interactions on cytochrome P450 (P450) enzymes could explain this observation and aimed to further deepen the understanding of PZQ metabolism. First, in an in vitro metabolite profiling study, the formation of multiple metabolites per P450, together with an observed interconversion of cis-4'-OH-PZQ to trans-4'-OH-PZQ in human hepatocytes, pointed out the inadequacy of measuring metabolite formation in kinetic studies. Thus, a substrate depletion approach to study PZQ enantiomeric interactions was applied. Second, an abundant CYP3A4 metabolite found in previous studies was structurally characterized. Third, substrate depletion methodologies were applied to determine P450 enzyme kinetics of PZQ and to further estimate enantiomer-enantiomer inhibitory parameters. A competitive inhibition between PZQ enantiomers for CYP2C9, 2C19, 3A4, and 3A5 was revealed. Analyses considering the clearance of only one or both enantiomers provided comparable enantiomer-enantiomer inhibition estimates. To conclude, this paper provides new insights into PZQ metabolic profile to enable a better understanding of enantioselective pharmacokinetics using substrate depletion-based methods. SIGNIFICANCE STATEMENT: In this study, enantiomer-enantiomer interactions of praziquantel on cytochrome P450 metabolizing enzymes are investigated via substrate depletion measurement using modeling methods. Together with new insights into the praziquantel metabolism, this work provides a novel data set to understand its pharmacokinetics.

The application of surfactants in liquid protein formulation is a common practice to protect proteins from liquid-air interface-induced protein aggregation. Typically, Polysorbate 20 or 80 are used, but degradation of these surfactants can result in particle formation and/or protein degradation. The purpose of the current study was to directly compare three alternative protein stabilizing molecules - Poloxamer 188, hydroxypropyl-cyclodextrin and a trehalose-based surfactant - to Polysorbate 80 for their capacities to reduce agitation-induced protein aggregation and particle formation; and furthermore, investigate their underlying protein stabilizing mechanisms. To this end, a small-volume, rapid agitation stress approach was used to quantify the molecules' abilities to stabilize two model proteins. This assay was presented to be a powerful tool to screen the protein stabilizing capability of surfactants using minimum of material and time. SEC, turbidity measurements and particle analysis showed an efficient protein stabilization of all tested surfactants as well as cyclodextrin. STD-NMR and dynamic surface tension measurements indicated the competitive surface adsorption to be the main protein stabilizing mechanism of the three surfactants tested. It might also play a role to some extent in the protein stabilization by HPβCD. However, additional mechanisms might also contribute to protein stabilization leaving room for further investigations.

The 2018-2020 consecutive drought events in Germany resulted in impacts related with several sectors such as agriculture, forestry, water management, industry, energy production and transport. The key to increase preparedness for extreme drought events are high-resolution information systems. A major national operational drought information system is the German Drought Monitor (GDM), launched in 2014 [1]. It provides daily soil moisture (SM) simulated with the mesoscale hydrological model (mHM) and its related soil moisture index [2] at a spatial resolution of 4×4 km². The release of the new soil map BUEK200 allowed us to increase its model resolution to ≈1.2×1.2 km², which is used now for the second version of the GDM [3]. To explore the ability of the GMD-v2 to provide drought information at one-kilometer scale, we evaluated mHM soil moisture simulations against an unprecedented large sample of soil moisture observations from 40 locations across Germany. These SM observations are obtained from single profile measurements, spatially distributed sensor networks, cosmic-ray neutron stations, and lysimeters over a wide range of climatic conditions, vegetation types and soil depths. Specifically, the study aimed at answering two research questions: 1) how well do high-resolution German-wide soil moisture simulations capture the dynamics in observed soil moisture that constitute the basis for the near real-time soil moisture drought monitoring system? 2) Does the mHM simulations obtained with the high spatial resolution data set provide soil moisture estimates with greater model efficiency than those obtained in the coarser resolution? The results showed that the agreement of simulated and observed SM dynamics is especially high during the vegetation period (0.84 median Spearman correlation(r)) and lower in winter (0.59 median r). Moderate but significant improvements between the low- and high-resolution GDM versions to observed SM were found in correlations for autumn (+0.07 median r) and winter (+0.12 median r). The spatially distributed sensor networks outperformed single profile measurements with higher than average correlation values especially for the 25–60 cm depth, which supports the closer scale match of spatially distributed measurements to the simulations. The results indicate areas for potential improvement and shows limitations from both: model parameterization (e.g., improvement of local scale hydrological processes) and observations methodology (e.g., reduction of measurement errors). Finally, the results of this study underline the fact that nationwide drought information systems depend both on appropriate simulations of the water cycle and a broad, high-quality observational soil moisture database.References:[1] Zink, M. et al. doi: 10.1088/1748-9326/11/7/074002 , 2016[2] Samaniego et al. doi: 10.1175/jhm-d-12-075.1 2013[3] Boeing, et al. doi: 10.5194/hess-2021-402 2021 (in revision)

Operational hydrological modelling and forecasts are based on complex simulation workflows that include, a.o. input data acquisition, pre-processing, hydrologic simulations, post-processing, publication and dissemination of the results. Stakeholders expect regular updates of the information at specified times and in high quality. Therefore, it must be ensured that in the event of an interruption in the workflow, the error can be quickly identified and rectified. Simultaneously, practitioners have high expectations of the model results, that should profit from continuous development of the hydrologic model and other components.The open-source mesoscale Hydrologic Model mHM (mhm-ufz.org) is a spatially distributed hydrologic model that conceptualizes dominant hydrological processes on the land surface. The unique feature of mHM is the Multiscale Parameter Regionalization (MPR) [1] that relates geophysical properties of the land (e.g., soil and land cover properties) to model parameters via transfer functions at a high spatial resolution (typically less than 250 m cell size). Subsequently, model parameters are aggregated to the spatial resolution at which the model runs are conducted (over 1 km). MPR allows seamless model application at different spatial resolutions and model parameters to be transferred in space [2]. mHM has been applied at different scales ranging from catchments to continents ([3], [4], [5]). mHM is written in Fortran programming language and is available under the GNU Lesser General Public License v3.mHM has been in continuous development for more than a decade now. In the past year, the following technical and methodological features have been added to the model:Installation via conda: mHM installation can be cumbersome because a Fortran compiler and netCDF4 library is required. We have now created a conda package (ananconda.org) for mHM that allows installing release versions of mHM. Reading of hourly meteorological input files: Traditionally, mHM was designed to read daily meteorological files. However, its internal time step is hourly. As higher resolved observational datasets become available, mHM can now read hourly data. This feature is critical for flood forecasting. Recently, mHM was applied globally at 0.1 deg grid resolution within the EU Copernicus-funded ULYSSES project. It took 36 hours to simulate 1.4 million grid cells for 30 years of daily values at 18 compute cores (using OpenMP parallelization). Although the run time provides an acceptable CO2 footprint of the simulations, it was challenging to organize a 51 member global hydrological forecast ensemble of six terrestrial environmental variables (Q, ET, SM, SWE, PET, GWR). We used the ecFlow workflow manager (https://confluence.ecmwf.int/display/ECFLOW) to submit the simulations to an HPC cluster. ecFlow allows to monitor the status of jobs and build complex workflows that include various tasks. Using a workflow manager like ecFlow allows creating reproducible simulation results more easily. We developed a general-purpose python package (ecPy) to interact with ecFlow functionalities for a wide range of software applications. We will present these new features and design of ecPy in this presentation.References:[1] https://doi.org/10.1029/2008WR007327[2] https://doi.org/10.5194/gmd-2021-103[3] https://doi.org/10.1002/wrcr.20431[4] https://doi.org/10.1175/bams-d-17-0274.1[5] https://doi.org/10.1061/(asce)he.1943-5584.0002097

The tumor suppressor p53 is regulated by various posttranslational modifications including different types of ubiquitylation, which exert distinct effects on p53. While modification by ubiquitin chains targets p53 for degradation, attachment of single ubiquitin moieties (mono-ubiquitylation) affects the intracellular location of p53 and/or its interaction with chromatin. However, how this is achieved at the molecular level remains largely unknown. Similarly, since p53 can be ubiquitylated at different lysine residues, it remains unclear if the eventual effect depends on the position of the lysine modified. Here, we combined genetic code expansion with oxime ligation to generate p53 site-specifically mono-ubiquitylated at position 120. We found that mono-ubiquitylation at this position neither interferes with p53 ubiquitylation by the E3 ligases HDM2 and E6AP in complex with the viral E6 oncoprotein nor affects p53 binding to a cognate DNA sequence. Thus, ubiquitylation per se does not affect physiologically relevant properties of p53. published

Post-translational modification (PTM) with ADP-ribose and poly(ADP-ribose) using nicotinamide adenine dinucleotide (NAD+ ) as substrate is involved in the regulation of numerous cellular pathways in eukaryotes, notably the response to DNA damage caused by cellular stress. Nevertheless, due to intrinsic properties of NAD+ e.g., high polarity and associated poor cell passage, these PTMs are difficult to characterize in cells. Here, two new NAD+ derivatives are presented, which carry either a fluorophore or an affinity tag and, in combination with developed methods for mild cell delivery, allow studies in living human cells. We show that this approach allows not only the imaging of ADP-ribosylation in living cells but also the proteome-wide analysis of cellular adaptation by protein ADP-ribosylation as a consequence of environmental changes such as H2 O2 -induced oxidative stress or the effect of the approved anti-cancer drug olaparib. Our results therefore pave the way for further functional and clinical studies of the ADP-ribosylated proteome in living cells in health and disease. published

Background: Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein of widely unknown function that may play a role in tumor growth control. Because mesothelin is overexpressed in a variety of human cancer types including ovarian cancers, it represents an attractive target for novel therapies employing adaptive T-cell transfer in these tumors. However, tumor heterogeneity is a challenge for targeted therapies. Methods: To study the extent of heterogeneity of mesothelin overexpression in primary ovarian cancers and their peritoneal and lymph node metastases, a tissue microarray (TMA) was constructed from multiple sites of 220 ovarian cancers and analyzed by immunohistochemistry. One tissue core each was taken from up to 18 different tumor blocks per cancer, resulting in a total of 2,460 tissue spots from 423 tumor sites (188 primary cancers, 162 peritoneal carcinosis and 73 lymph node metastases). Results: Positive mesothelin expression was found in 2,041 of the 2,342 (87%) arrayed tissue spots and in 372 of the 392 (95%) tumor sites that were interpretable for mesothelin immunohistochemistry. Intratumoral heterogeneity was found in 23% of 168 primary cancer sites interpretable for mesothelin and decreased to 12% in 154 peritoneal carcinosis and to 6% in 71 lymph node metastases (p Conclusions: Our data demonstrate that mesothelin expression is frequent and highly homogeneous in ovarian cancer and prompt for future anti-mesothelin therapy studies in this tumor type. Citation Format: Sören Weidemann, Natalia Gorbokon, Maximilian Lennartz, Claudia Hube-Magg, Christoph Fraune, Christian Bernreuther, Till Clauditz, Frank Jacobsen, Kristina Jansen, Barbara Schmalfeldt, Linn Wölber, Peter Paluchowski, Enikö Berkes, Uwe Heilenkötter, Guido Sauter, Ria Uhlig, Waldemar Wilczak, Stefan Steurer, Ronald Simon, Till Krech, Andreas Marx, Eike Burandt. High homogeneity of mesothelin expressionin primary and metastatic ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3303.

Background: Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine protease which is essential for the desquamation of corneocytes and thus plays a pivotal role in maintaining skin homeostasis. In cancer, KLK7 overexpression was suggested to represent a route for metastasis through cleavage of cell junction and extracellular matrix proteins of cancer cells. Methods: To comprehensively determine KLK7 protein expression in normal and neoplastic tissues, a tissue microarray containing 13,447 samples from 147 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Results: KLK7 positivity was found in 64 of 147 tumor categories, including 17 tumor categories with at least one strongly positive case. The highest rate of KLK7 positivity was found in squamous cell carcinomas from various sites of origin (positive in 18.1%-63.8%), ovarian and endometrium cancers (4.8%-56.2%), salivary gland tumors (4.8%-13.7%), bilio-pancreatic adenocarcinomas (20.0%-40.4%), and adenocarcinomas of the upper gastrointestinal tract (3.3%-12.5%). KLK7 positivity was linked to nodal metastasis (p=0.0005), blood vessel infiltration (p=0.0037), and lymph vessel infiltration (p Conclusions: These data provide a comprehensive overview on KLK7 expression in normal and neoplastic human tissues. The prognostic relevance of KLK7 expression and the possible role of KLK7 as a drug target need to be further investigated. Citation Format: Simon Kind, Carolina Castillo, Ria Uhlig, Natalia Gorbokon, Maximilian Lennartz, Sebastian Dwertmann Rico, Viktor Reiswich, Florian Viehweger, Martina Kluth, Claudia Hube-Magg, Christian Bernreuther, Franziska Büscheck, Till Clauditz, Christroph Fraune, Andrea Hinsch, Till Krech, Patrick Lebok, Stefan Steurer, Eike Burandt, Sarah Minner, Andreas Marx, Ronald Simon, Waldemar Wilczak, Guido Sauter, Anne Menz, Frank Jacobsen. KLK7 expression in human tumors: A tissue microarray study on 13,447 tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3302.

The 2018–2020 consecutive drought events in Germany resulted in impacts related with several sectors such as agriculture, forestry, water management, industry, energy production and transport. A major national operational drought information system is the German Drought Monitor (GDM), launched in 2014. It provides daily soil moisture (SM) simulated with the mesoscale hydrological model (mHM) and its related soil moisture index at a spatial resolution of 4 × 4 km2. Key to preparedness for extreme drought events are high-resolution information systems. The release of the new soil map BUEK200 allowed to increase the model resolution to ~1.2 × 1.2 km2, which is used in the second version of the GDM. In this paper, we explore the ability to provide drought information on the one-kilometer scale in Germany. Therefore, we compare simulated SM dynamics using homogenized and deseasonalized SM observations to evaluate the high-resolution drought simulations of the GDM. These SM observations are obtained from single profile measurements, spatially distributed sensor networks, cosmic-ray neutron stations and lysimeters at 40 sites in Germany. The results show that the agreement of simulated and observed SM dynamics is especially high in the vegetation period (0.84 median correlation R) and lower in winter (0.59 median R). Lower agreement in winter results from methodological uncertainties in simulations as well as in observations. Moderate but significant improvements between the first and second GDM version to observed SM were found in correlations for autumn (+0.07 median R) and winter (+0.12 median R). The annual drought intensity ranking and the spatial structure of drought events over the past 69 years is comparable for the two GDM versions. However, the higher resolution of the second GDM version allows a much more detailed representation of the spatial variability of SM, which is particularly beneficial for local risk assessments. Furthermore, the results underline that nationwide drought information systems depend both on appropriate simulations of the water cycle and a broad, high-quality observational soil moisture database.

Lipid based formulations (LBF) have shown to overcome food dependent bioavailability for some poorly water-soluble drugs. However, the utility of LBFs can be limited by low dose loading due to a low drug solubility in LBF vehicles. This study investigated the solubility and drug loading increases in LBFs using lipophilic counterions to form lipophilic salts of venetoclax. Venetoclax docusate was formed from venetoclax free base and verified by 1H NMR. Formation of stable venetoclax-fatty acid associations with either oleic acid or decanoic acid were attempted, however, the molecular associations were less consistent based on 1H NMR. Venetoclax docusate displayed a up to 6.2-fold higher solubility in self-emulsifying drug delivery systems (SEDDS) when compared to the venetoclax free base solubility resulting in a higher dose loading. A subsequent bioavailability study in landrace pigs demonstrated a 2.5-fold higher bioavailability for the lipophilic salt containing long chain SEDDS compared to the commercially available solid dispersion Venclyxto® in the fasted state. The bioavailability of all lipophilic salt SEDDS in the fasted state was similar to Venclyxto® in the fed state. This study confirmed that lipophilic drug salts increase the dose loading in LBFs and showed that lipophilic salt-SEDDS combinations may be able to overcome bioavailability limitations of drugs with low inherent dose loading in lipid vehicles. Furthermore, the present study demonstrated the utility of a LBF approach, in combination with lipophilic salts, to overcome food dependent variable oral bioavailability of drugs.

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B‐family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B‐family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’‐overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B‐family DNA polymerases., Due to their life in hot environments, archaea are increasingly affected by deamination of the bases cytosine and adenine. To prevent mutations, archaeal DNA polymerases seem to recognize these base changes by a so‐called “read‐ahead” mechanism and react accordingly. Here we show by means of crystal structures how Thermococcus kodakarensis (KOD) DNA polymerase binds uracil and hypoxanthine in a special binding pocket and thus can recognize these bases on the single stranded template overhang already six nucleotides before the active site.

Isothermal amplification reactions represent an important and exciting approach to achieve widespread, low cost, and easily implemented molecular diagnostics. This work presents a modified recombinase polymerase amplification (RPA) reaction, which can be directly coupled to a simple electrochemical measurement to ultimately allow development of a nucleic acid-based assay for antibiotic resistance genes. It is shown that use of reagents from a standard RPA reaction kit allows incorporation of horse radish peroxidase-labeled thymine nucleotides into amplified DNA strands, which can be detected via an amperometric signal readout for detection of important gene sequences. The assay is exemplified through detection of fragments of the oxacillin resistance gene in Escherichia coli cells bearing a drug resistance plasmid, achieving a potential limit of detection of 319 cfus/mL and an unoptimized time to result of 60 min. This work serves as a suitable demonstration of the potential for a system to deliver detection of key drug resistance genes at clinically relevant levels.

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques, such as nuclear magnetic resonance or liquid chromatography mass spectrometry were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (liquid chromatography mass spectrometry) or require large amount of sample material (NMR), which are not always available when

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies., Tumours that are deficient in mismatch-repair genes should, in theory, have higher evolvability. Here, the authors explore this theory in gastro-oesophageal tumours.

Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been developed whereby alternative hydrogen‐bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base‐pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications., Hydrogen bonding or rather more hydrophobic? Two different pairing concepts are the basis for the currently most promising unnatural base pair (UBP) candidates. This review mainly focusses on structural studies investigating UBP pairings in free duplex DNA or the active site of KlenTaq DNA polymerase, but also highlights the general mechanisms for the acceptance of these unnatural substrates by DNA polymerases.

Evaluation of tumor budding in colorectal cancer (CRC) may help to predict the tumors’ metastatic potential and patients with an aggressive tumor, although not yet metastasized at time of surgery might benefit from adjuvant therapy. The degree of intratumoral tumor budding (ITB) was classified as low, intermediate, and high grade according to the recommendations of the International Tumor Budding Consensus Conference (ITBCC) 2016 on H&E and pankeratin-stained TMA sections from 1262 CRC, no special type (NST), including 655 stage II CRC and was correlated to clinicopathological data and overall survival. Results show that higher ITB rates are significantly linked to higher tumor grade and stage, positive nodal status, lymphovascular invasion (P

Archaeal B-family DNA polymerases (DNA pols) are the driving force of cutting-edge biotechnological applications like next-generation sequencing. The acceptance of chemically modified nucleotides by DNA pols is key to these technologies. Until now, no structural data have been available for these DNA pols in complex with modified substrates, which could build the basis for understanding interactions between the enzyme and the chemically modified nucleotide and for the further development of next-generation nucleotides. For the first time, we crystallized an exonuclease-deficient variant of the wild-type B-family KOD DNA pol with a modified nucleotide in a closed, ternary complex. We also crystalized the A-family DNA pol KlenTaq with the same nucleotide. The reported structural data reveal how the protein and the DNA modulate two distinct conformations of the appended moiety in the A- and B-family DNA pols and how these influence the processing of the modified nucleotide. Overall, this study provides first insight into the interplay between B-family DNA pols and relevant modified substrates.

Archaeale B‐Familien‐DNA‐Polymerasen (DNA‐Pols) sind die treibende Kraft führender biotechnologischer Anwendungen wie moderne Sequenzierungsansätze. Die Akzeptanz chemisch modifizierter Nukleotide durch DNA‐Pols ist der Schlüssel zu diesen Technologien. Bis jetzt sind keine strukturellen Daten für diese DNA‐Pols in Komplexen mit modifizierten Substraten verfügbar, die zum Verständnis der Interaktionen zwischen Enzym und chemischer Modifikation sowie der Entwicklung von Nukleotiden der nächsten Generation beitragen könnten. Dafür haben wir eine Exonuklease‐defiziente Variante des Wildtyps der B‐Familien KOD‐DNA‐Pol mit einem modifizierten Nukleotid in einem geschlossenen, ternären Komplex kristallisiert. Zum Vergleich haben wir die A‐Familien‐DNA‐Pol KlenTaq mit demselben Nukleotid kristallisiert. Die beiden Kristallstrukturen erklären wie die Modifikation in den A‐ und B‐Familien‐DNA‐Pols durch das Protein und die DNA in zwei unterschiedliche Konformationen gelenkt wird. Außerdem erlauben die Strukturen Rückschlüsse darauf, wie die jeweilige Konformation die Prozessierung des modifizierten Nukleotids beeinflusst. Insgesamt bietet diese Studie erste Einblicke wie B‐Familien‐DNA‐Pols mit relevantem modifiziertem Substrat interagieren. published

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5'-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:16 ispartof: location:Switzerland status: published

Background The E-Cadherin gene (CDH1, Cadherin 1), located at 16q22.1 encodes for a calcium-dependent membranous glycoprotein with an important role in cellular adhesion and polarity maintenance. Methods To systematically determine E-Cadherin protein expression in normal and cancerous tissues, 14,637 tumor samples from 112 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Results E-Cadherin was strongly expressed in normal epithelial cells of most organs. From 77 tumor entities derived from cell types normally positive for E-Cadherin, 35 (45.5%) retained at least a weak E-Cadherin immunostaining in ≥99% of cases and 61 (79.2%) in ≥90% of cases. Tumors with the highest rates of E-Cadherin loss included Merkel cell carcinoma, anaplastic thyroid carcinoma, lobular carcinoma of the breast, and sarcomatoid and small cell neuroendocrine carcinomas of the urinary bladder. Reduced E-Cadherin expression was linked to higher grade (p = 0.0009), triple negative receptor status (p = 0.0336), and poor prognosis (p = 0.0466) in invasive breast carcinoma of no special type, triple negative receptor status in lobular carcinoma of the breast (p = 0.0454), advanced pT stage (p = 0.0047) and lymph node metastasis in colorectal cancer (p p p = 0.0104) and was associated with advanced tumor stage (p = 0.0276) and higher grade (p = 0.0035) in clear cell RCC, and linked to advanced tumor stage (p = 0.0424) and poor prognosis in papillary RCC (p ≤ 0.05). Conclusion E-Cadherin is consistently expressed in various epithelial cancers. Down-regulation or loss of E-Cadherin expression in cancers arising from E-Cadherin positive tissues as well as E-Cadherin neo-expression in cancers arising from E-Cadherin negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.

Purpose Expansion of CD8+ cytotoxic Tlymphocytes is a prerequisite for anti-cancer immune activity and has gained interest in the era of immune checkpoint therapy. Methods To understand the CD8+ T cell dynamics in the tumor microenvironment, we used multiplex fluorescence immunohistochemistry to quantitate CD8+ proliferation (Ki67 co-expression) in tissue microarrays from 1107 colorectal, 642 renal cell, 1066 breast, 375 ovarian, 451 pancreatic and 347 gastric cancer samples. Results The density and the percentage of proliferating (Ki67+) CD8+ T cells were both highly variable between tumor types as well as between patients with the same tumor type. Elevated density and percentage of proliferating CD8+ cytotoxic T cells were significantly associated with favorable tumor parameters such as low tumor stage, negative nodal stage (p ≤ 0.0041 each), prolonged overall survival (p ≤ 0.0028 each) and an inflamed immune phenotype (p = 0.0025) in colorectal cancer and, in contrast, linked to high tumor stage, advanced ISUP/Fuhrman/Thoenes grading (each p ≤ 0.003), shorter overall survival (p ≤ 0.0330 each) and an immune inflamed phenotype (p = 0.0094) in renal cell cancer. In breast, ovarian, pancreatic and gastric cancer the role of (Ki67+)CD8+ Tcells was not linked to clinicopathological data. Conclusion Our data demonstrate a tumor type dependent prognostic impact of proliferating (Ki67+)CD8+ Tcells and an inverse impact in colorectal and renal cell cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s13402-021-00601-4.

Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, d-fructose is more involved in glycoxidation than d-glucose favoring the formation of advanced glycation endproducts (AGEs). While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl),Nε-Boc]-OH (Fmoc-Lys(Glc,Boc)-OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions. Supplementary Information The online version contains supplementary material available at 10.1007/s00726-021-02989-7.

Agricultural production is highly dependent on the weather. The mechanisms of action are complex and interwoven, making it difficult to identify relevant management and adaptation options. The present study uses random forests to investigate such highly non-linear systems for predicting yield anomalies in winter wheat at district level in Germany. In order to take into account sub-seasonality, monthly features are used that explicitly take soil moisture into account in addition to extreme meteorological events. Clustering is used to show spatially different damage potentials, such as a higher susceptibility to drought damage from April to July in eastern Germany compared to the rest of the country. The variable that explains most differences is soil moisture in March, where higher soil moisture has a detrimental effect on crop yields. In general, soil moisture explains more yield variations than the meteorological variables, while the top 25 cm of soil moisture is a better yield predictor than the total soil column. The approach has proven to be suitable to explain historical extreme yield anomalies for years with exceptionally high losses (2003, 2018) and gains (2014) and the spatial distribution of these anomalies. The highest test R-square is about 0.70. Furthermore, the sensitivity of yield variations to soil moisture and extreme meteorological conditions, as shown by the visualisation of average marginal effects, contributes to the promotion of targeted decision support systems.

BackgroundCytokeratin 18 (CK18) is an intermediate filament protein of the cytokeratin acidic type I group and is primarily expressed in single-layered or “simple” epithelial tissues and carcinomas of different origin. MethodsTo systematically determine CK18 expression in normal and cancerous tissues, 11,952 tumor samples from 115 different tumor types and subtypes (including carcinomas, mesenchymal and biphasic tumors) as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. ResultsCK18 was expressed in normal epithelial cells of most organs but absent in normal squamous epithelium. At least an occasional weak CK18 positivity was seen in 90 of 115 (78.3%) tumor types. Wide-spread CK18 positivity was seen in 37 (31.9%) of tumor entities, including adenocarcinomas of the lung, prostate, colon and pancreas as well as ovarian cancer. Tumor categories with variable CK18 immunostaining included cancer types arising from CK18 positive precursor cells but show CK18 downregulation in a fraction of cases, tumor types arising from CK18 negative precursor cells occasionally exhibiting CK18 neo-expression, tumors derived from normal tissues with variable CK18 expression, and tumors with a mixed differentiation. CK18 downregulation was for example seen in renal cell cancers and breast cancers, whereas CK18 neo-expression was found in squamous cell carcinomas of various origins. Down-regulation of CK18 in invasive breast carcinomas of no special type and clear cell renal cell carcinomas (ccRCC) was related to adverse tumor features in both tumors (p≤0.001) and poor patient prognosis in ccRCC (p=0.0088). Up-regulation of CK18 in squamous cell carcinomas was linked to high grade and lymph node metastasis (p

Science advances 7(12), eabf1380 - (2021). doi:10.1126/sciadv.abf1380, For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution., Published by Assoc., Washington, DC [u.a.]

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins. Both ubiquitin and NEDD8 can be phosphorylated, but the biological role of NEDD8 phosphorylation remains unclear. Here, the authors identify similarities and differences of ubiquitin and NEDD8 phosphorylation, showing that phosphorylated NEDD8 has a distinct interactome and regulates HSP70 proteins.

The attachment of differently linked ubiquitin (Ub) chains of varying length to proteins is a prevalent posttranslational modification in eukaryotic cells. We established an affinity enrichment approach for the identification of linkage- and length-selective ubiquitin binding proteins and could show that in addition to the linkage, the length of a Ub chain is an important determinant for Ub chain recognition.

For the two proteins myoglobin (MB) and fluoroacetate dehalogenase (FAcD), we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of crystals, the same sample delivery device, as well as the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins reasonable data statistics can be obtained with approximately 5000 room temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that diffraction quality is rather linked to the properties of the crystals than to the radiation source. Time-resolved experiments can therefore be conducted at the source that best matches the desired time-resolution.

In this essay, I depict some personal thoughts and experience related to Chemical Biology. A particular emphasis is put on education and training of the new talents in the field and in consequence, I give the youngest generation the opportunity to express their opinion on Chemical Biology. published

The SARS-CoV-2 beta coronavirus is spreading globally with unprecedented consequences for modern societies. The early detection of infected individuals is a pre-requisite for all strategies aiming to contain the virus. Currently, purification of RNA from patient samples followed by RT-PCR is the gold standard to assess the presence of this single-strand RNA virus. However, these procedures are time consuming, require continuous supply of specialized reagents, and are prohibitively expensive in resource-poor settings. Here, we report an improved nucleic-acid-based approach to detect SARS-CoV-2, which alleviates the need to purify RNA, reduces handling steps, minimizes costs, and allows evaluation by non-specialized equipment. The use of unprocessed swap samples and the ability to detect as little as three viral genome equivalents is enabled by employing a heat-stable RNA- and DNA-dependent DNA polymerase, which performs the double task of stringent reverse transcription of RNA at elevated temperatures as well as PCR amplification of a SARS-CoV-2 specific target gene. As results are obtained within 2 hours and can be read-out by a hand-held LED-screen, this novel protocol will be of particular importance for large-scale virus surveillance in economically constrained settings.

LAMP is an approach for isothermal nucleic acids diagnostics with increasing importance but suffers from the need of tedious systems design and optimization for every new target. Here, we describe an approach for its simplification based on a single nucleoside-5&prime, O-triphosphate (dNTP) that is covalently modified with a DNA strand. We found that the DNA-modified dNTP is a substrate for DNA polymerases in versatile primer extension reactions despite its size and that the incorporated DNA indeed serves as a target for selective LAMP analysis.

Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment. published