A Syngeneic Orthotopic Murine Model of Pancreatic Adenocarcinoma in the C57/BL6 Mouse Using the Panc02 and 6606PDA Cell Lines

Background/Aims: To develop a clinically relevant immunocompetent murine model to study pancreatic cancer using two different syngeneic pancreatic cancer cell lines and to assess MRI for its applicability in this model. Methods: Two cell lines, 6606PDA and Panc02, were employed for the experiments. Cell proliferation and migration were monitored in vitro. Matrigel™ was tested for its role in tumor induction. Tumor cell growth was assessed after orthotopic injection of tumor cells into the pancreatic head of C57/BL6 mice by MRI and histology. Results: Proliferation and migration of Panc02 were significantly faster than those of 6606PDA. Matrigel did not affect tumor growth/migration but prevented tumor cell spread after injection thus avoiding undesired peritoneal tumor growth. MRI could reliably monitor longitudinal tumor growth in both cell lines: Panc02 had a more irregular finger-like growth, and 6606PDA grew more spherically. Both tumors showed local invasiveness. Histologically, Panc02 showed a sarcoma-like undifferentiated growth pattern, whereas 6606PDA displayed a moderately differentiated glandular tumor growth. Panc02 mice had a significantly shorter (28 days) survival than 6606PDA mice (50 days). Conclusion: This model closely mimics human pancreatic cancer. MRI was invaluable for longitudinal monitoring of tumor growth thus reducing the number of mice required. Employing two different cell lines, this model can be used for various treatment and imaging studies

Prediction of huge X-ray Faraday rotation at the Gd N_4,5 threshold

X-ray absorption spectra in a wide energy range around the 4d-4f excitation threshold of Gd were recorded by total electron yield from in-plane magnetized Gd metal films. Matching the experimental spectra to tabulated absorption data reveals unprecedented short light absorption lengths down to 3 nm. The associated real parts of the refractive index for circularly polarized light propagating parallel or antiparallel to the Gd magnetization, determined through the Kramers-Kronig transformation, correspond to a magneto-optical Faraday rotation of 0.7 degrees per atomic layer. This finding shall allow the study of magnetic structure and magnetization dynamics of lanthanide elements in nanosize systems and dilute alloys.Comment: 4 pages, 2 figures, final version resubmitted to Phys. Rev. B, Brief Reports. Minor change

Whole-exome sequencing identifies SLC52A1 and ZNF106 variants as novel genetic risk factors for (early) multiple-organ failure in acute pancreatitis

Objective: The aim of this study was to identify genetic variants associated with early multiple organ failure (MOF) in acute pancreatitis.Summary background data: MOF is a life-threatening complication of acute pancreatitis, and risk factors are largely unknown, especially in early persistent MOF. Genetic risk factors are thought to enhance severity in complex diseases such as acute pancreatitis.Methods: A 2-phase study design was conducted. First, we exome sequenced 9 acute pancreatitis patients with early persistent MOF and 9 case-matched patients with mild edematous pancreatitis (phenotypic extremes) from our initial Dutch cohort of 387 patients. Secondly, 48 candidate variants that were overrepresented in MOF patients and 10 additional variants known from literature were genotyped in a replication cohort of 286 Dutch and German patients.Results: Exome sequencing resulted in 161,696 genetic variants, of which the 38,333 nonsynonymous variants were selected for downstream analyses. Of these, 153 variants were overrepresented in patients with multiple-organ failure, as compared with patients with mild acute pancreatitis. In total, 58 candidate variants were genotyped in the joined Dutch and German replication cohort. We found the rs12440118 variant of ZNF106 to be overrepresented in patients with MOF (minor allele frequency 20.4% vs 11.6%, Padj = 0.026). Additionally, SLC52A1 rs346821 was found to be overrepresented (minor allele frequency 48.0% vs 42.4%, Padj = 0.003) in early MOF. None of the variants known from literature were associated.Conclusions: This study indicates that SLC52A1, a riboflavin plasma membrane transporter, and ZNF106, a zinc finger protein, may be involved in disease progression toward (early) MOF in acute pancreatitis.Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.Genetics of disease, diagnosis and treatmen

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high resolution X ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state of the art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results. Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening CFS at the Helmholtz Zentrum Berlin HZB are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two day procedure, a 96 membered CFS focused library provided as dried ready to use plates is employed to soak 192 crystals, which are then flash cooled individually. The final diffraction experiments can be performed within one day at the robot mounting supported beamlines BL14.1 and BL14.2 at the BESSY II electron storage ring operated by the HZB in Berlin Adlershof Germany . Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input. Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical application

Substrate binding modes of purine and pyrimidine nucleotides to human ecto 5 nucleotidase CD73 and inhibition by their bisphosphonic acid derivatives

Human ecto 5 nucleotidase CD73 is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small molecule inhibitors to date being non hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5 amp; 8242; nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological propertie

The receptor tyrosine kinase p185HER2 is expressed on a subset of B-lymphoid blasts from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia

The class I receptor tyrosine kinase (RTK) HER2 is an oncoprotein that is frequently involved in the pathogenesis of tumors of epithelial origin. Here we report mRNA expression in peripheral blood and bone marrow cells from healthy donors in hematopoietic cell lines and leukemic blasts from patients with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and chronic myeloid leukemia (CML). However, cell surface expression of HER2 protein (p185HER2) was found exclusively on a subset of leukemic cells of the B-lymphoblastic lineage. p185HER2 expression was found on blasts in 2 of 15 samples from infants, 9 of 19 samples from adult patients with C-ALL (CD19+CD10+), and 1 of 2 samples from patients with pro-B ALL (CD19+CD10-), whereas none of the leukemic cells from patients with AML (0/30), T-ALL (0/7), CLL (0/5) (CD19+CD5+), or CML in chronic and accelerated phase (0/5) or in blast crisis with myeloid differentiation (0/14) were positive for p185HER2. However, cells from 3 of 4 patients with CML in B-lymphoid blast crisis (CD19+CD10+) expressed high levels of p185HER2, which was also found on the surface of the CML-derived B-cell lines BV-173 and Nalm-1. Our study shows p185HER2 expression on malignant cells of hematopoietic origin for the first time. Aberrant expression of this oncogenic receptor tyrosine kinase in hematopoietic cell types may be an oncogenic event contributing to the development of a subset of B-lymphoblastic leukemias

Structures of endothiapepsin fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96 compound fragment library

Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein-ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin-fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity

The macromolecular crystallography beamlines at BESSY II of the Helmholtz Zentrum Berlin Current status and perspectives

For a little over a decade now, the Macromolecular Crystallography MX group at the Helmholtz Zentrum Berlin HZB has been operating three state of the art synchrotron beamlines for MX at the BESSY II storage ring in Berlin. The three HZB MX beamlines, BL14.1, BL14.2 and BL14.3, serve a stable and growing user community of currently more than 100 independent research groups from Berlin, Germany and Europe. Every year, the beamlines provide close to 200 days of MX beamtime. Over time, the HZB MX beamlines and endstations, in particular BL14.1, have been continually developed and upgraded and, since 2010, they operate as the most productive MX beamlines in Germany. The environment of the beamlines includes various ancillary equipment as well as additional facilities, such as office space adjacent to the beamlines, a sample preparation laboratory, a safety level 1 biology laboratory HZB MX BioLab and all necessary computing resources. In this paper, the current status of the beamlines as well as the ongoing developments are describe