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Context.— Special AT-rich sequence–binding protein 2 (SATB2) induces local chromatin loops to facilitate transcription. SATB2 immunostaining is commonly used as a marker for colorectal adenocarcinoma and osteosarcoma. Objective.— To extend our knowledge on the diagnostic value of SATB2 analysis in a comprehensive set of human tumors. Design.— Tissue microarrays with 15 012 samples from 120 tumor types and 608 samples of 76 different normal tissues were analyzed. Results.— SATB2 positivity was found in 89 of 120 different tumor types (74%), including 59 of 120 (49%) with at least 1 moderately positive tumor and 38 of 120 tumor types (32%) with at least 1 strongly positive tumor. Expression was frequent in adenomas (44/42–47/44; 94%–96% positive), adenocarcinomas (1747 of 2023; 86%), and various subtypes of neuroendocrine neoplasms (3/7–12/12; 43%–100%) of the colorectum and appendix, Merkel cell carcinoma (25 of 34, 74%), osteosarcomas (15 of 25; 60%), and papillary renal cell carcinoma (RCC) (121 of 235; 52%). Associations to clinicopathologic tumor features were assessed in colorectal and kidney cancers. In colorectal cancer, weak SATB2 expression was linked to high pT (P < .001), nodal metastasis (P < .001), right-sided tumor location (P < .001), microsatellite instability (P < .001), and BRAF mutations (P = .02). In papillary RCC, low SATB2 expression was associated with high pT (P = .02), distant metastasis (P = .04), and reduced tumor-specific survival (P = .04). In clear cell RCC, low SATB2 expression was linked to high pT (P < .001), high Union for International Cancer Control stage (P < .001), high Thoenes grade (P = .02), and reduced recurrence-free survival (P = .02). Conclusions.— Strong SATB2 expression argues for a colorectal origin within adenocarcinomas and neuroendocrine neoplasms. Weak SATB2 expression reflects progression and poor prognosis in colorectal and kidney cancer.

The Sixth Assessment Report from the Intergovernmental Panel on Climate Change emphasized on the water cycle, and water-related disasters (i.e., water scarcity, droughts, floods) that impact all sectors and regions. Therefore, assessing future water availability is critical to develop mitigation strategies and formulate adaptation policies. While developing relevant information systems, it is critical to ensure the involvement of stakeholders in the field of policy development, communities impacted by future water availability (e.g., agriculture, fisheries, shipping industry), and private industries (e.g., paper and pulp, hydropower) to ensure that information presented can be useful to support decision making. Destination Earth (DestinE) aims to, among other products, to develop – on a global scale – a highly accurate digital model of the Earth to monitor and predict the interaction between natural phenomena and human activities. As part of the European Commission’s Green Deal and Digital Strategy, DestinE will contribute to achieving the objectives of the twin transition, green and digital. High resolution climate simulations (ICON and IFS climate models) are used as meteorological forcings for the mesoscale Hydrological Model (mHM) to produce high temporal (1 hour) and spatial resolution (5 km) streamflow estimates at a global scale. The impact model consists of the mHM model, which includes key hydrological processes e.g., run-off, soil moisture dynamics, fast and slow interflow processes to estimate river discharge. The application prototype will provide: 1) co-designing the indicators and indices as well as application functionalities together with relevant stakeholders. 2) downscaling of the essential climate variables 3) providing bias correction for the climate variables 4) running the mHM model under various climate scenarios. In addition, the application will receive data through direct streaming from the climate simulations thus ensuring interactivity of the application for the users. During the development phase of the DestinE digital twin, the climate simulations used in the current work are taken from the results of the NextGEMS project. They have been used to provide a proof of concept for the mHM model, and provide initial results for stakeholder engagement, and enable early involvement of stakeholders in the co-design of relevant applications.

Germany experienced exceptional multi-year water storage deficits starting in 2018. Recurring years below average precipitation and above average temperatures lead to water deficits that accumulated primarily in slow-response subsurface water storages such as (deep) soil moisture or groundwater levels. Total water storage (TWS) anomalies estimated by the GRACE satellite mission show a strong decline for Germany since measurements began in 2002, but these time series began with a relatively wet period, ended in an exceptional drought situation, and are still relatively short. Therefore the resulting trends are not representative for long-term TWS dynamics and should not be used to extrapolate the development into the future (Güntner et al, 2022). In addition, the GRACE signal does not allow partitioning of the water storage and flux components. Hydrological simulations provide a suitable tool to analyse the long-term dynamics of water storages. We analyse the long-term monthly TWS anomalies estimated with the hydrological model mHM (Samaniego et al. (2010), Kumar et al. (2013)) from a data reconstruction starting in 1766 (Rakovec et al, 2022). Comparison of the monthly total water storage estimates between the hydrological simulations and two GRACE solutions (JPL RL06M MSCNv02CRI, GFZ COST-G RL01) show good agreement for both anomalies (R²=0.78-0.84) and the residuals after removing the seasonal cycle (R²=0.69-0.72) on the scale of Germany (spatial mean). We specifically examine the periods of recovery from total water storage deficits from a water balance perspective. Besides precipitation (P) being the main driver of changes in the TWS, the progress of recovery from water storage deficits is controlled by the main water fluxes evapotranspiration (E) and runoff (Q) that are the loss terms in the water balance equation deltaTWS = P - E - Q. Decadal evaluations indicate that the water balance in Germany in recent decades has been increasingly driven by above-average E as a result of the temperature rise. While the cumulative precipitation deficits in the last decade 2011 - 2020 are less exceptional compared to other historical decades, cumulative E residuals account for a much larger part of the water deficits than in other historical decades. The results will contribute to an improved understanding how TWS deficit recovery are affected by long-term changes in the water balance. References: Güntner, A., Gerdener, H., Boergens, E., Kusche, J., Kollet, S., Dobslaw, H., Hartick, C., Sharifi, E., and Flechtner, F. (2022): Changes of water storage in Germany since 2002 observed with GRACE/GRACE-FO, GRACE/GRACE-FO Science Team Meeting 2022, Potsdam, Germany, 18–20 Oct 2022, GSTM2022-93, https://doi.org/10.5194/gstm2022-93. Rakovec, O., Samaniego, L., Hari, V., Markonis, Y., Moravec, V., Thober, S., Hanel, M., & Kumar, R. (2022): The 2018–2020 Multi‐Year Drought Sets a New Benchmark in Europe. In Earth’s Future (Vol. 10, Issue 3). American Geophysical Union (AGU). https://doi.org/10.1029/2021ef002394 Samaniego L., R. Kumar, S. Attinger (2010): Multiscale parameter regionalization of a grid-based hydrologic model at the mesoscale. Water Resour. Res., 46,W05523, doi:10.1029/2008WR007327 Kumar, R., L. Samaniego, and S. Attinger (2013): Implications of distributed hydrologic model parameterization on water fluxes at multiple scales and locations, Water Resour. Res., 49, doi:10.1029/2012WR012195

The tumor suppressor protein fragile histidine triad (Fhit) is known to be associated with genomic instability and apoptosis. The tumor-suppressive function of Fhit depends on the interaction with the alarmone diadenosine triphosphate (Ap3A), a noncanonical nucleotide whose concentration increases upon cellular stress. How the Fhit–Ap3A complex exerts its signaling function is unknown. Here, guided by a chemical proteomics approach employing a synthetic stable Fhit–Ap3A complex, we found that the Fhit–Ap3A complex, but not Fhit or Ap3A alone, impedes translation. Our findings provide a mechanistic model in which Fhit translocates from the nucleolus into the cytosol upon stress to form an Fhit–Ap3A complex. The Fhit–Ap3A complex impedes translation both in vitro and in vivo, resulting in reduced cell viability. Overall, our findings provide a mechanistic model by which the tumor suppressor Fhit collaborates with the alarmone Ap3A to regulate cellular proliferation. published

The development of an improved kilogram-scale synthesis of the JAK1 inhibitor GDC-4379 for the treatment of asthma is described. The new process is highlighted by a step-economical construction of ...

Diadenosine polyphosphates (ApnAs) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed ApnAs for their usage as cosubstrate for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as cosubstrate is a response to ER stress. Here, we demonstrate that Ap4A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4A levels during cellular stress differs from when Ap4A is present at low concentrations allowing response to extracellular cues. published

Late-stage functionalization (LSF) is an economical approach to optimize the properties of drug candidates. However, the chemical complexity of drug molecules often makes late-stage diversification challenging. To address this problem, an LSF platform based on geometric deep learning and high-throughput reaction screening was developed. Considering borylation as a critical step in LSF, the computational model predicted reaction yields for diverse reaction conditions with a mean absolute error margin of 4–5%, while the reactivity of novel reactions with known and unknown substrates were classified with a balanced accuracy of 92% and 67%, respectively. The regioselectivity of the major products was accurately captured in up to 90% of the cases studied. When applied to 23 diverse commercial drug molecules, the platform successfully identified numerous opportunities for structural diversification. The influence of steric and quantum mechanical information on model performance was quantified and a new comprehensive simple user-friendly reaction format (SURF) is introduced which proved to be a key enabler for seamlessly integrating deep learning and high-throughput experimentation (HTE) for LSF.

Reverse transcriptases are DNA polymerases that can use RNA as a template for DNA synthesis. They thus catalyze the reverse of transcription. Although discovered in 1970, reverse transcriptases are still of great interest and are constantly being further developed for numerous modern research approaches. They are frequently used in biotechnological and molecular diagnostic applications. In this review, we describe the discovery of these fascinating enzymes and summarize research results and applications ranging from molecular cloning, direct virus detection, and modern sequencing methods to xenobiology. published

Carboxypeptidase A1 (CPA1) is a zinc metalloprotease that is produced in pancreatic acinar cells and plays a role in cleaving C-terminal branched-chain and aromatic amino acids from dietary proteins. This study assessed the utility of immunohistochemical CPA1 staining for diagnosing pancreatic acinar cell carcinoma (ACC). A total of 12,274 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types were interpretable by immunohistochemistry in a tissue microarray format. CPA1 was strongly expressed in acinar cells of all normal pancreas samples but not in any other normal tissues. CPA1 immunostaining was detected in 100% of 11 pancreatic ACCs and 1 mixed acinar endocrine carcinoma, but absent in 449 pancreatic ductal adenocarcinomas, 75 adenocarcinomas of the ampulla Vateri, and 11,739 other evaluable cancers from 128 different tumor entities. A weak to moderate diffuse staining of epithelial and stromal cells of cancer tissues immediately adjacent to non-neoplastic pancreatic acinar cells often occurred and was considered to be caused by the diffusion of the highly abundant CPA1 from normal acinar cells that may have suffered some autolytic cell damage. In conclusion, our data show that CPA1 is a highly sensitive and largely specific marker for normal and neoplastic pancreatic acinar cells. CPA1 immunohistochemistry greatly facilitates the otherwise often difficult diagnosis of pancreatic ACC.

To evaluate cytokeratin 19 (CK19) expression in normal and cancerous tissues, 15,977 samples from 122 tumor types and 608 samples of 76 normal tissue types were analyzed by immunohistochemistry (IHC). In normal tissues, CK19 expression occurred in epithelial cells of most glandular organs but was strictly limited to the basal cell layer of nonkeratinizing squamous epithelium and absent in the skin. CK19 expression in ≥90% of cases was seen in 34% of the tumor entities including the adenocarcinomas of the pancreas (99.4%), colorectum (99.8%), esophagus (98.7%), and stomach (97.7%), as well as breast cancer (90.0%-100%), high-grade serous (99.1%) or endometrioid (97.8%) ovarian cancer, and urothelial carcinoma (92.6%-100%). A low CK19 positivity rate (0.1-10%) was seen in 5 of 122 tumor entities including hepatocellular carcinoma and seminoma. A comparison of tumor versus normal tissue findings demonstrated that upregulation and downregulation of CK19 can occur in cancer and that both alterations can be linked to unfavorable phenotypes. CK19 downregulation was linked to high grade (p = 0.0017) and loss of estrogen receptor- and progesterone receptor-expression (p 0.0001 each) in invasive breast carcinoma of no special type. CK19 upregulation was linked to nodal metastases in neuroendocrine tumors and papillary thyroid carcinomas (p 0.05 each) and to poor grade in clear cell renal cell carcinoma (p 0.05). CK19 upregulation was particularly common in squamous cell carcinomas. We concluded that CK19 IHC might separate primary liver cell carcinoma from liver metastases, seminoma from other testicular tumors, and helps in the detection of early neoplastic transformation in squamous epithelium.

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification mass spectrometry and show that site-specific acetylation results in overlapping but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together, our data suggest that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.

Intracellular dinucleoside polyphosphates (Npn Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap3 A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap3 A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap3 A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap3 A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap3 A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit. published

The chromatin in eukaryotic cells plays a fundamental role in all processes during a cell’s life cycle. This nucleoprotein is normally tightly packed but needs to be unpacked for expression and division. The linker histones are critical for such packaging processes and while most experimental and simulation works recognize their crucial importance, the focus is nearly always set on the nucleosome as the basic chromatin building block. Linker histones can undergo several modifications, but only few studies on their ubiquitylation have been conducted. Mono-ubiquitylated linker histones (HUb), while poorly understood, are expected to influence DNA compaction. The size of ubiquitin and the globular domain of the linker histone are comparable and one would expect an increased disorder upon ubiquitylation of the linker histone. However, the formation of higher order chromatin is not hindered and ubiquitylation of the linker histone may even promote gene expression. Structural data on chromatosomes is rare and HUb has never been modeled in a chromatosome so far. Descriptions of the chromatin complex with HUb would greatly benefit from computational structural data. In this study we generate molecular dynamics simulation data for six differently linked HUb variants with the help of a sampling scheme tailored to drive the exploration of phase space. We identify conformational sub-states of the six HUb variants using the sketch-map algorithm for dimensionality reduction and iterative HDBSCAN for clustering on the excessively sampled, shallow free energy landscapes. We present a highly efficient geometric scoring method to identify sub-states of HUb that fit into the nucleosome. We predict HUb conformations inside a nucleosome using on-dyad and off-dyad chromatosome structures as reference and show that unbiased simulations of HUb produce significantly more fitting than non-fitting HUb conformations. A tetranucleosome array is used to show that ubiquitylation can even occur in chromatin without too much steric clashes.Author summaryIn eukaryotic cells the linker histones play a crucial role in the formation of higher order nucleoprotein complex of DNA, especially for the arrangement of the nucleosomes. Histones can undergo several modifications, but modification of a linker histone with a single udiquitin (mono-ubiquitylation) remains one of the least understood epigenetic modifications. One reason is the inaccessibility of homogeneously modified linker histones for experimental methods, which are crucial for distinct studies. We combine molecular dynamics simulations with machine learning-based approaches to study the influence of mono-ubiquitylation in linker histones on DNA interaction and their ability to form higher order chromatin structures. We were able to determine the probable states in six differently linked histone-ubiquitin complexes via accelerating classical molecular dynamics simulations and using advanced state characterization techniques. As it is computationally unfeasible to simulate the whole chromatosome with different modified histones we developed efficient geometric scoring technique to select biologically relevant structures of all six mono-ubiquitylated linker histone that can bound to nucleosome.

Cytokeratin 10 (CK10) is a type I acidic low molecular weight cytokeratin which is mainly expressed in keratinizing squamous epithelium of the skin. Variable levels of CK10 protein have been described in squamous carcinomas of different sites and in some other epithelial neoplasms. To comprehensively determine the prevalence of CK10 expression in normal and neoplastic tissues, a tissue microarray containing 11,021 samples from 131 different tumor types and subtypes was analyzed by immunohistochemistry. CK10 immunostaining was detectable in 41 (31.3 %) of 131 tumor categories, including 18 (13.7 %) tumor types with at least one strongly positive case. The highest rate of positive staining was found in squamous cell carcinomas from various sites of origin (positive in 18.6 %-66.1 %) and in Warthin tumors of salivary glands (47.8 %), followed by various tumor entities known to potentially exhibit areas with squamous cell differentiation such as teratomas (33.3 %), basal cell carcinomas of the skin (14.3 %), adenosquamous carcinomas of the cervix (11.1 %), and several categories of urothelial neoplasms (3.1 %-16.8 %). In a combined analysis of 956 squamous cell carcinomas from 11 different sites of origin, reduced CK10 staining was linked to high grade (p 0.0001) and advanced stage (p = 0.0015) but unrelated to HPV infection. However, CK10 staining was not statistically related to grade (p = 0.1509) and recurrence-free (p = 0.5247) or overall survival (p = 0.5082) in 176 cervical squamous cell carcinomas. In the urinary bladder, CK10 staining occurred more commonly in muscle-invasive (17.7 %) than in non-invasive urothelial carcinomas (4.0 %-6.0 %; p 0.0001). In summary, our data corroborate a role of CK10 as a suitable marker for mature, keratinizing squamous cell differentiation in epithelial tissues. CK10 immunohistochemistry may thus be instrumental for a more objective evaluation of the clinical significance of focal squamous differentiation in cancer.

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.

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.

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 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 tumor suppressor protein

Objective: Anoctamin 7 (ANO7) is a calcium2+-dependent chloride ion channel protein. Its expression is restricted to prostate epithelial cells. The exact function is unknown. This study aimed to analyze ANO7 expression and its clinical significance in prostate cancer (PCa). Methods: ANO7 expression was assessed by immunohistochemistry in 17,747 clinical PCa specimens. Results: ANO7 was strongly expressed in normal prostate glandular cells but often less abundant in cancer cells. ANO7 staining was interpretable in 13,594 cancer tissues and considered strong in 34.4%, moderate in 48.7%, weak in 9.3%, and negative in 7.6%. Reduced staining was tightly linked to adverse tumor features [high classical and quantitative Gleason grade, lymph node metastasis, advanced tumor stage, high Ki67 labeling index, positive surgical margin, and early biochemical recurrence (P < 0.0001 each)]. The univariate Cox hazard ratio for prostate-specific antigen (PSA) recurrence after prostatectomy in patients with negative vs. strong ANO7 expression was 2.98 (95% confidence interval 2.61–3.38). The prognostic impact was independent of established pre- or postoperatively available parameters (P < 0.0001). Analysis of annotated molecular data showed that low ANO7 expression was linked to TMPRSS2:ERG fusions (P < 0.0001), elevated androgen receptor expression (P < 0.0001), as well as presence of 9 of 11 chromosomal deletions (P < 0.05 each). A particularly strong association of low ANO7 expression with phosphatase and tensin homolog (PTEN) deletion may indicate a functional relationship with the PTEN/AKT pathway. Conclusions: These data identify reduced ANO7 protein expression as a strong and independent predictor of poor prognosis in PCa. ANO7 measurement, either alone or in combination, might provide clinically useful prognostic information in PCa.

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 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

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

Special AT-rich sequence-binding protein 2 (SATB2) induces local chromatin loops to facilitate transcription. SATB2 immunostaining is commonly used as a marker for colorectal adenocarcinoma and osteosarcoma.To extend our knowledge on the diagnostic value of SATB2 analysis in a comprehensive set of human tumors.Tissue microarrays with 15 012 samples from 120 tumor types and 608 samples of 76 different normal tissues were analyzed.SATB2 positivity was found in 89 of 120 different tumor types (74%), including 59 of 120 (49%) with at least 1 moderately positive tumor and 38 of 120 tumor types (32%) with at least 1 strongly positive tumor. Expression was frequent in adenomas (44/42-47/44; 94%-96% positive), adenocarcinomas (1747 of 2023; 86%) and various subtypes of neuroendocrine neoplasms (3/7-12/12; 43%-100%) of the colorectum and appendix, Merkel cell carcinoma (25 of 34, 74%), osteosarcomas (15 of 25; 60%), and papillary renal cell carcinoma (RCC) (121 of 235; 52%). Associations to clinicopathologic tumor features were assessed in colorectal and kidney cancers. In colorectal cancer, weak SATB2 expression was linked to high pT (P.001), nodal metastasis (P.001), right-sided tumor location (P.001), microsatellite instability (P.001), and BRAF mutations (P = .02). In papillary RCC, low SATB2 expression was associated with high pT (P = .02), distant metastasis (P = .04), and reduced tumor-specific survival (P = .04). In clear cell RCC, low SATB2 expression was linked to high pT (P.001), high Union for International Cancer Control stage (P.001), high Thoenes grade (P = .02), and reduced recurrence-free survival (P = .02).Strong SATB2 expression argues for a colorectal origin within adenocarcinomas and neuroendocrine neoplasms. Weak SATB2 expression reflects progression and poor prognosis in colorectal and kidney cancer.

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 nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells., Diadenosine polyphosphates (ApAs) are involved in cellular stress signaling but only a few molecular targets have been characterized so far. Here, the authors develop ApnA-based photoaffinity-labeling probes and use them to identify Ap3A and Ap4A binding proteins in human cell lysates.

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

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

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

Placental alkaline phosphatase (PLAP) is commonly expressed at high levels in testicular germ cell tumors. PLAP immunohistochemistry (IHC) is thus often used to confirm this diagnosis, especially in cases of putative metastasis. However, other tumors can also express PLAP. To comprehensively determine PLAP expression in normal and tumor tissue, a tissue microarray containing 16,166 samples from 131 different tumor types and subtypes as well as 608 samples from 76 different normal tissue types was analyzed by IHC. Moderate to strong PLAP positivity was found in 27 (21%) of 131 different tumor types including seminoma (96%), embryonal carcinoma (85%), and yolk sac tumors of the testis (56%); endometrioid carcinoma of the endometrium (28%) and the ovary (20%); gastric adenocarcinoma (22%); serous carcinoma (not otherwise specified) of the ovary (17%) and the uterus (11%); adenocarcinoma of the ampulla of Vater (15%); carcinosarcoma of the ovary (11%) and the uterus (8%); esophageal adenocarcinoma (10%); invasive urothelial carcinoma (4%); cholangiocarcinoma (2%); and adenocarcinoma of the lung (1%). Low‐level PLAP immunostaining, often involving only a small fraction of tumor cells, was seen in 21 additional tumor entities. The clinical significance of PLAP expression may vary between tumor types as high PLAP expression was linked to advanced pathological tumor stage (p = 0.0086), nodal metastasis (p = 0.0085), and lymphatic (p = 0.0007) and blood vessel invasion (p = 0.0222) in colorectal cancer, but to low pathological tumor stage in endometrial cancer (p = 0.0043). In conclusion, our data identify several tumor entities that can show PLAP expression at comparable levels to testicular germ cell tumors. These tumor entities need to be considered in cases of PLAP‐positive metastasis. Low‐level PLAP expression can be found in various other tumor entities and should generally not be viewed as a strong argument for germ cell neoplasia.

Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1., While the role of specific posttranslational modifications (PTMs) is increasingly well understood for core histones, this is not the case for linker histone H1. Here the authors show that site-specific ubiquitylation of H1 results in distinct interactomes, regulates phase separation, and modulates assembly of chromatosomes.

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.

p16 (CDKN2A) is a member of the INK4 class of cell cycle inhibitors, which is often dysregulated in cancer. However, the prevalence of p16 expression in different cancer types is controversial. 15,783 samples from 124 different tumor types and 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. p16 was detectable in 5,292 (45.0%) of 11,759 interpretable tumors. Except from adenohypophysis in islets of Langerhans, p16 staining was largely absent in normal tissues. In cancer, highest positivity rates were observed in uterine cervix squamous cell carcinomas (94.4%), non-invasive papillary urothelial carcinoma, pTaG2 (100%), Merkel cell carcinoma (97.7%), and small cell carcinomas of various sites of origin (54.5%-100%). All 124 tumor categories showed at least occasional p16 immunostaining. Comparison with clinico-pathological data in 128 vulvar, 149 endometrial, 295 serous ovarian, 396 pancreatic, 1365 colorectal, 284 gastric, and 1245 urinary bladder cancers, 910 breast carcinomas, 620 clear cell renal cell carcinomas, and 414 testicular germ cell tumors revealed only few statistically significant associations. Comparison of human papilloma virus (HPV) status and p16 in 497 squamous cell carcinomas of different organs revealed HPV in 80.4% of p16 positive and in 20.6% of p16 negative cancers (p

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.