The long way from raw data to NAM-based information: Overview on data layers and processing steps.

Toxicological test methods generate raw data and provide instructions on how to use these to determine a final outcome such as a classification of test compounds as hits or non-hits. The data processing pipeline provided in the test method description is often highly complex. Usually, multiple layers of data, ranging from a machine-generated output to the final hit definition, are considered.

A New Severe Congenital Neutropenia Syndrome Associated with Autosomal Recessive COPZ1 Mutations.

We have identified a new inherited bone marrow (BM) failure syndrome with severe congenital neutropenia (CN) caused by autosomal recessive mutations in the coatomer protein complex I (COPI) subunit zeta 1 (COPZ1) gene. A stop-codon COPZ1 mutation and a missense mutation were found in three patients from two unrelated families. While two affected siblings with a stop-codon COPZ1 mutation suffered from congenital neutropenia (CN) that involves other hematological lineages, and non-hematological tissues, the patient with a missense COPZ1 mutation had isolated neutropenia.

Tissue Factor and Its Cerebrospinal Fluid Protein Profiles in Parkinson's Disease.

Background: Prior investigations have elucidated pathophysiological interactions involving blood coagulation and neurodegenerative diseases. These interactions pertain to age-related effects and a mild platelet antiaggregant function of exogenous α-Synuclein.

Objective: Our study sought to explore whether cerebrospinal fluid (CSF) levels of tissue factor (TF), the initiator of the extrinsic pathway of hemostasis, differ between controls (CON) compared to patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), considering that these conditions represent a spectrum of α-Synuclein pathology.

Dicloxacillin is an inducer of intestinal P-glycoprotein but neither dicloxacillin nor flucloxacillin increases the risk of stroke/systemic embolism in direct oral anticoagulant users.

Aim: We aimed to assess if dicloxacillin/flucloxacillin reduces the therapeutic efficacy of direct oral anticoagulants (DOACs) and the underlying molecular mechanism.

Methods: In a randomized, crossover study, we assessed whether dicloxacillin reduces oral absorption of drugs through P-glycoprotein (P-gp) during 10 and 28 days of treatment. To study the impact of dicloxacillin/flucloxacillin on intestinal and hepatic expression of P-gp in vitro, we usd LS174T cells and 3D spheroids of primary human hepatocytes.

Nature as blueprint: Global phenotype engineering of CHO production cells based on a multi-omics comparison with plasma cells.

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits.

Immunoaffinity proteomics for kidney injury biomarkers in male beagle dogs.

Drug-induced kidney injury (DIKI) is a cause of drug development failure. Dogs represent a common non-rodent animal model in pre-clinical safety studies; however, biomarker assays for detecting nephrotoxicity in dogs are limited. To identify novel proteins and gain insight into the molecular mechanisms involved in DIKI, we developed an assay to evaluate proteomic changes associated with DIKI in male beagle dogs that received nephrotoxic doses of tobramycin for 10 consecutive days.

The molecular interaction pattern of lenvatinib enables inhibition of wild-type or kinase-mutated FGFR2-driven cholangiocarcinoma.

Fibroblast growth factor receptor (FGFR)-2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA.

Elements and development processes for test methods in toxicology and human health-relevant life science research.

Many laboratory procedures generate data on properties of chemicals, but they cannot be equated with toxicological "test methods". This apparent discrepancy is not limited to in vitro testing, using animal-free new approach methods (NAM), but also applies to animal-based testing approaches. Here, we give a brief overview of the differences between data generation and the setup or use of a complete test method.

Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment.

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs).

Correlation between Clinical Characteristics and Antibody Levels in COVID-19 Convalescent Plasma Donor Candidates.

COVID-19 convalescent plasma (CCP) with high neutralizing antibodies has been suggested in preventing disease progression in COVID-19. In this study, we investigated the relationship between clinical donor characteristics and neutralizing anti-SARS-CoV-2 antibodies in CCP donors. COVID-19 convalescent plasma donors were included into the study.

Flucloxacillin Is a Weak Inducer of CYP3A4 in Healthy Adults and 3D Spheroid of Primary Human Hepatocytes.

Flucloxacillin is a widely used antibiotic. It is an agonist to the nuclear receptor PXR that regulates the expression of cytochrome P450 (CYP) enzymes. Treatment with flucloxacillin reduces warfarin efficacy and plasma concentrations of tacrolimus, voriconazole, and repaglinide.

Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 preparations.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive Proarrhythmia Assay (CiPA).

Light Stimulation of Neurons on Organic Photocapacitors Induces Action Potentials with Millisecond Precision.

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. This research has benchmarked the performance of organic electrolytic photocapacitor (OEPC) optoelectronic stimulators at the level of single mammalian cells: human embryonic kidney (HEK) cells with heterologously expressed voltage-gated K channels and hippocampal primary neurons. OEPCs act as extracellular stimulation electrodes driven by deep red light.

Transcription Factors STAT3 and MYC Are Key Players of Human Platelet Lysate-Induced Cell Proliferation.

Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL's growth-enhancing effect.

Generation and characterization of three induced pluripotent stem cells lines from an 86-year old female individual diagnosed with an invasive lobular mammary carcinoma.

Invasive lobular carcinoma (ILC) is a distinct type of breast cancer and is accounting up to 10-15 % of all mammary carcinomas showing a pronounced increase in incidence rates over the last two decades. We generated three induced pluripotent stem cell (iPSC) lines from CD34+ progenitor cells isolated from a mammary carcinoma patient diagnosed with ILC. Here, we describe the characterization of the iPSCs by array-based comparative genomic hybridization (array CGH), immunocytochemistry, flow cytometry, reverse transcriptase polymerase chain reaction and directed in vitro differentiation.

Plasma biomarkers TAP, CPA1, and CPA2 for the detection of pancreatic injury in rat: the development of a novel multiplex IA-LC-MS/MS assay and biomarker performance evaluation.

Drug-induced pancreatic injury (DIPI) is an issue seen in drug development both in nonclinical and clinical contexts. DIPI is typically monitored by measurement of lipase and/or amylase, however, both enzymes lack sensitivity and specificity. Although candidate protein biomarkers specific to pancreas exist, antibody-based assay development is difficult due to their small size or the rapid cleavage by proteolytic enzymes released during pancreatic injury.

Protein Profiling of Breast Carcinomas Reveals Expression of Immune-Suppressive Factors and Signatures Relevant for Patient Outcome.

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined.

Establishment of Four Induced Pluripotent Stem Cell Lines from CD34+ Hematopoietic Stem and Progenitor Cells from a Patient Diagnosed with an Invasive Lobular Mammary Carcinoma.

CD34+ cells were isolated from peripheral blood of a breast cancer patient. By the introduction of five integration-free episomal vectors, the CD34+ cells were successfully reprogrammed and resulted in four iPSC clones. Flow Cytometry, reverse transcriptase PCR and immunocytochemistry confirm a robust expression of pluripotency factors and the concomitant loss of exogenous reprogramming plasmids.

Spatial Proteomics Reveals Differences in the Cellular Architecture of Antibody-Producing CHO and Plasma Cell-Derived Cells.

Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels.

Proteomic analysis of hepatic effects of phenobarbital in mice with humanized liver.

Activation of the constitutive androstane receptor (CAR) may induce adaptive but also adverse effects in rodent liver, including the induction of drug-metabolizing enzymes, transient hepatocellular proliferation, and promotion of liver tumor growth. Human relevance of CAR-related adverse hepatic effects is controversially debated. Here, we used the chimeric FRG-KO mouse model with livers largely repopulated by human hepatocytes, in order to study human hepatocytes and their response to treatment with the model CAR activator phenobarbital (PB) in vivo.

A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions.

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide -glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

Background: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.

Nicotinamide-N-methyltransferase is a promising metabolic drug target for primary and metastatic clear cell renal cell carcinoma.

Background: The metabolic enzyme nicotinamide-N-methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour-promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target.

Methods: NNMT expression was assessed in primary ccRCC (n = 134), non-tumour tissue and ccRCC-derived metastases (n = 145) by microarray analysis and/or immunohistochemistry.