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Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, often diagnosed at stages that dis‐qualify for surgical resection. Neoadjuvant therapies offer potential tumor regression and improved resectability. Although features of the tumor biology (e.g., molecular markers) may guide adjuvant therapy, biological alterations after neoadjuvant therapy remain largely unexplored. We performed mass spectrometry to characterize the proteomes of 67 PDAC resection specimens of patients who received either neoadjuvant chemo (NCT) or chemo‐radiation (NCRT) therapy. We employed data‐independent acquisition (DIA), yielding a proteome coverage in excess of 3500 proteins. Moreover, we successfully integrated two publicly available proteome datasets of treatment‐naïve PDAC to unravel proteome alterations in response to neoadjuvant therapy, highlighting the feasibility of this approach. We found highly distinguishable proteome profiles. Treatment‐naïve PDAC was characterized by enrichment of immunoglobulins, complement and extracellular matrix (ECM) proteins. Post‐NCT and post‐NCRT PDAC presented high abundance of ribosomal and metabolic proteins as compared to treatment‐naïve PDAC. Further analyses on patient survival and protein expression identified treatment‐specific prognostic candidates. We present the first proteomic characterization of the residual PDAC mass after NCT and NCRT, and potential protein candidate markers associated with overall survival. We conclude that residual PDAC exhibits fundamentally different proteome profiles as compared to treatment‐naïve PDAC, influenced by the type of neoadjuvant treatment. These findings may impact adjuvant or targeted therapy options. [ABSTRACT FROM AUTHOR]

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.

Introduction: Breast cancer is globally the leading cancer in women, and despite the high 5-year survival rate the most frequent cause of cancer related deaths. Surgery, systemic therapy and radiotherapy are the three pillars of curative breast cancer treatment. However, locoregional recurrences frequently occur after initial treatment and are often challenging to treat, amongst others due to high doses of previous radiotherapy treatments. Radiotherapy can be combined with local hyperthermia to sensitize tumor cells to radiation and thereby significantly reduce the required radiation dose. Therefore, the combination treatment of mild local hyperthermia, i.e. locally heating of the tissue to 39-43°C, and reirradiation with a reduced total dose is a relevant treatment option for previously irradiated patients. The mechanisms of this effect in the course of the therapy are to date not well understood and will be investigated in the HISTOTHERM study. Methods and analyses: Patients with local or (loco)regional recurrent breast cancer with macroscopic tumors are included in the study. Local tumor control is evaluated clinically and histologically during the course of a combination treatment of 60 minutes mild superficial hyperthermia (39 - 43°C) using water- filtered infrared A (wIRA) irradiation, immediately followed by hypofractionated re-irradiation with a total dose of 20-24 Gy, administered in weekly doses of 4 Gy. Tumor and tumor stroma biopsies as well as blood samples will be collected prior to treatment, during therapy (at a dose of 12 Gy) and in the follow-up to monitor therapy response. The treatment represents the standard operating procedure for hyperthermia plus re-irradiation. Various tissue and blood-based markers are analyzed. We aim at pinpointing key mechanisms and markers for therapy response which may help guiding treatment decisions in future. In addition, quality of life in the course of treatment will be assessed and survival data will be evaluated. [ABSTRACT FROM AUTHOR]

This article describes a modular ensemble-based data assimilation (DA) system which is developed for an integrated surface–subsurface hydrological model. The software environment for DA is the Parallel Data Assimilation Framework (PDAF), which provides various assimilation algorithms like the ensemble Kalman filters, non-linear filters, 3D-Var and combinations among them. The integrated surface–subsurface hydrological model is HydroGeoSphere (HGS), a physically based modelling software for the simulation of surface and variably saturated subsurface flow, as well as heat and mass transport. The coupling and capabilities of the modular DA system are described and demonstrated using an idealised model of a geologically heterogeneous alluvial river–aquifer system with drinking water production via riverbank filtration. To demonstrate its modularity and adaptability, both single and multivariate assimilations of hydraulic head and soil moisture observations are demonstrated in combination with individual and joint updating of multiple simulated states (i.e. hydraulic heads and water saturation) and model parameters (i.e. hydraulic conductivity). With the integrated model and this modular DA framework, we have essentially developed the hydrologically and DA-wise robust toolbox for developing the basic model for operational management of coupled surface water–groundwater resources. [ABSTRACT FROM AUTHOR]

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche. [ABSTRACT FROM AUTHOR]

The present study addresses older adults' developmental regulation when faced with progressive and irreversible vision loss. We used the motivational theory of life span development as a conceptual framework and examined changes in older adults' striving for control over everyday goal achievement, and their association with affective well-being, in a sample of 364 older adults diagnosed with age-related macular degeneration. Using longitudinal data from 5 occasions at 6-month intervals, we examined intraindividual change in control strategies, and how it was related to change in affective well-being, in terms of self-rated happiness and depressive symptoms. Mixed model analyses confirmed our hypotheses that (a) intraindividual change, particularly in selective primary control and in compensatory secondary control (CSC), predict change toward higher happiness ratings and lower depression; and (b) as functional abilities (instrumental activities of daily living) declined, CSC became increasingly predictive of better affective well-being. Overall, the findings suggest that CSC strategies are essential for maintaining affective well-being when physical functioning declines. Intensified selective primary control striving may be effective to achieve goals that have become difficult to reach but are not associated with affective well-being, possibly because struggling with difficulties undermines the experience of enjoyable mastery. In contrast, goal adjustments and self-protective thinking may help to find pleasure even from restricted daily activities.