Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining ...gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes.
The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the ...general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.
Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these ...various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.
Background and purpose
The presentation of Parkinson's disease patients with mutations in the LRRK2 gene (PDLRRK2) is highly variable, suggesting a strong influence of modifying factors. In this ...context, inflammation is a potential candidate inducing clinical subtypes.
Methods
An extensive battery of peripheral inflammatory markers was measured in human serum in a multicentre cohort of 142 PDLRRK2 patients from the MJFF LRRK2 Consortium, stratified by three different subtypes as recently proposed for idiopathic Parkinson's disease: diffuse/malignant, intermediate and mainly pure motor.
Results
Patients classified as diffuse/malignant presented with the highest levels of the pro‐inflammatory proteins interleukin 8 (IL‐8), monocyte chemotactic protein 1 (MCP‐1) and macrophage inflammatory protein 1‐β (MIP‐1‐β) paralleled by high levels of the neurotrophic protein brain‐derived neurotrophic factor (BDNF). It was also possible to distinguish the clinical subtypes based on their inflammatory profile by using discriminant and area under the receiver operating characteristic curve analysis.
Conclusions
Inflammation seems to be associated with the presence of a specific clinical subtype in PDLRRK2 that is characterized by a broad and more severely affected spectrum of motor and non‐motor symptoms. The pro‐inflammatory metabolites IL‐8, MCP‐1 and MIP‐1‐β as well as BDNF are interesting candidates to be included in biomarker panels that aim to differentiate subtypes in PDLRRK2 and predict progression.
The application of human pluripotent stem cells (hPSCs) in vitro is of high value for therapeutic and industrial applications. Perfusion was shown to result in a more homogeneous culture environment ...and enabled 47% higher cell yields compared with conventional repeated batch culture. Furthermore, results show the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development.
The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high‐quality cells by defined processes. Culturing hPSCs as cell‐only aggregates in (three‐dimensional 3D) suspension has the potential to overcome numerous limitations of conventional surface‐adherent (two‐dimensional 2D) cultivation. Utilizing single‐use instrumented stirred‐tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse‐transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix‐free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture‐induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP‐compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture.
Significance
Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single‐use, clinically compliant bioreactor technology to develop advanced, matrix‐free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.
An involvement of the central-nervous and peripheral, innate and adaptive immune system in the pathogenesis of Parkinson's disease (PD) is nowadays well established.
We face several open questions in ...preparation of clinical trials aiming at disease-modification by targeting the immune system: Do peripheral (blood) inflammatory profiles reflect central (CSF) inflammatory processes? Are blood/CSF inflammatory markers associated with CSF levels of neurodegenerative/PD-specific biomarkers?
Using a multiplex assay we assessed 41 inflammatory markers in CSF/serum pairs in 453 sporadic PD patients. We analyzed CSF/serum correlation as well as associations of inflammatory markers with clinical outcome measures (UPDRS-III, H&Y, MoCA) and with CSF levels of α-synuclein, Aβ
,
Tau, p181-Tau and NFL. All analyses were stratified by sex as the immune system shows relevant sex-specific differences.
Correlations between CSF and serum were sparse and detected in only 25% (9 out of 36) of the analysable inflammatory markers in male PD patients and in only 38% (12 out of 32) of female PD patients. The most important pro-inflammatory mediators associated with motor and cognitive decline as well as with neurodegenerative/PD-specific biomarkers were FABP, ICAM-1, IL-8, MCP-1, MIP-1-beta, and SCF. Results were more robust for CSF than for serum.
Levels of central-nervous and peripheral inflammatory markers might be regulated independently of each other with CSF inflammatory markers reflecting CNS pathology more accurately than peripheral markers. These findings along with sex-specific characteristics have to be considered when designing clinical trials aiming at disease-modification by targeting the immune system.
There is evidence for a relevant role of inflammation in the pathogenesis of Parkinson's disease (PD). Mutations in the LRRK2 gene represent the most frequent genetic cause for autosomal dominant PD. ...LRRK2 is highly expressed in macrophages and microglia suggesting an involvement in inflammatory pathways. The objectives are to test (1) whether idiopathic PD and LRRK2-associated PD share common inflammatory pathways or present distinct profiles and (2) whether non-manifesting LRRK2 mutation carriers present with similar aspects of inflammatory profiles as seen in PD-affected patients.
We assessed serum profiles of 23 immune-associated markers and the brain-derived neurotrophic factor in 534 individuals from the MJFF LRRK2 consortium.
A large proportion of inflammatory markers were gender-dependent. Both PD-affected cohorts showed increased levels of the pro-inflammatory marker fatty-acid-binding protein. Additionally, idiopathic PD but not LRRK2-associated PD patients showed increased levels of the pro-inflammatory marker interleukin-12-p40 as well as the anti-inflammatory species interleukin-10, brain-derived neurotrophic factor, and stem cell factor. Non-manifesting LRRK2 mutation carriers including those with prodromal characteristics of PD presented with control-like inflammatory profiles.
Concomitant inflammation seems to be associated with idiopathic and LRRK2-associated PD. Identifying PD patients in whom inflammatory processes play a major role in their pathophysiology might offer a new therapeutic window at least for a subgroup of patients. Since non-manifesting LRRK2 mutation carriers with symptoms of the prodromal phase of PD did not show inflammatory profiles, activation of the immune system seems not an early event in the disease cascade.
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. Label-free quantitative discovery proteomics analysis on representative kidney cortex tissues collected on Day 11 showed that the tobramycin-induced kidney injury led to a significant differential regulation of 94 proteins mostly associated with mechanisms of nephrotoxicity such as oxidative stress and proteasome degradation. For verification of the proteomic results, we developed a multiplex peptide-centric immunoaffinity liquid chromatography tandem mass spectrometry assay (IA LC-MS/MS) to evaluate the association of eight DIKI protein biomarker candidates using kidney cortices collected on Day 11 and urine samples collected on Days -4, 1, 3, 7 and 10. The results showed that most biomarkers evaluated were detected in the kidney cortices and their expression profile in tissue aligned with the label-free data. Cystatin C was the most consistent marker regardless of the magnitude of the renal injury while fatty acid-binding protein-4 (FABP4) and kidney injury molecule-1 (KIM-1) were the most affected biomarkers in response to moderate proximal tubular injury in absence of changes in serum-based concentrations of blood urea nitrogen or creatinine. In the urine, clusterin is considered the most consistent biomarker regardless of the magnitude and time of the renal injury. To our knowledge, this is the most comprehensive multiplex assay for the quantitative analysis of mechanism-based proximal tubular injury biomarkers in dogs.
During various inflammatory processes circulating cytokines including IL-6, IL-1β, and TNFα elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the ...simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXRα playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRα gene silencing versus IL-6 induced negative gene regulation (rs = 0.79; P<0.0001). These results concur with RXRα functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism.
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