Proteome alterations during clonal isolation of established human pancreatic cancer cell lines.

Clonal isolation is an integral step of numerous workflows in genome editing and cell engineering. It comprises the isolation of a single progenitor cell from a defined cell line population with subsequent expansion to obtain a monoclonal cell population. This process is associated with transient loss of cell-cell contacts and absence of a multicellular microenvironment. Previous studies have revealed transcriptomic changes upon clonal isolation with cell line specific extent. Since transcriptome alterations are only partially reflected on the proteome level, we sought to investigate the impact of clonal isolation on the cellular proteome to a depth of > 6000 proteins in three established pancreatic cancer cell lines. We show that clonal isolation does have an impact on the cellular proteome, however, with cell line specific extent, affecting different biological processes, and also depending on the isolation method. We demonstrate a different impact of clonal isolation on mesenchymal- and epithelial-derived cell lines mainly affecting cell proliferation, metabolism, cell adhesion and cellular stress. The results bear relevance to the field of genomic editing and cell engineering and highlight the need to consider the impact of clonal isolation when interpreting data stemming from experiments that include this step.

[Three-dimensional reconstruction of solid tumors : Morphological evidence for tumor heterogeneity]. / Dreidimensionale Rekonstruktion solider Tumoren : Morphologische Evidenz für Tumorheterogenität.

BACKGROUND: In histopathological routine diagnostics, three-dimensional tissue samples are analyzed histologically and/or immunohistochemically in two-dimensional sectional planes due to the high expenditure of time and the lack of digitization possibilities. AIM: Here, we demonstrate the application of three-dimensional reconstruction to solid tumors and analyze inter-/intratumoral heterogeneity with respect to epithelial-mesenchymal transition (EMT). METHODS: Tissue samples from pancreatic, lung, colorectal, and breast cancers as well as colorectal liver metastases were serially processed in 4µm sections. For individual analyses, alternating stains (cytokeratin AE1/3, zinc finger E­box-binding homeobox 1 (ZEB1), eCadherin) were performed. Subsequently, the tumor cells were analyzed for their morphology (epitheloid amoeboid, mesenchymal) and the expression of ZEB1 and eCadherin. For statistical analysis, all tumor cell aggregates were hierarchically annotated and analyzed. RESULTS: Tumor buds are predominantly associated with the main tumor mass. Furthermore, a shutteling of eCadherin could be observed within tumor cell aggregates smaller than nine cells. ZEB1 is only increasingly expressed in tumor cell groups smaller than five cells. CONCLUSIONS: The initial tumor budding and the subsequent decoupling of the tumor bud from the main tumor mass is most likely a two-part process. However, the EMT is not statistically significantly increased within the tumor bud detached from the main tumor mass. It could be shown that the currently valid and known definition of a tumor bud as a cell cluster of less than or equal to five cells cannot be completely classified in the concept of EMT represented by eCadherin and ZEB1.

Quantitative proteomic analysis of formalin-fixed, paraffin-embedded clear cell renal cell carcinoma tissue using stable isotopic dimethylation of primary amines.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness. RESULTS: In the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non-malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins. CONCLUSION: Our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC.

Cancer cell invasion and EMT marker expression: a three-dimensional study of the human cancer-host interface.

Cancer cell invasion takes place at the cancer-host interface and is a prerequisite for distant metastasis. The relationships between current biological and clinical concepts such as cell migration modes, tumour budding and epithelial-mesenchymal transition (EMT) remains unclear in several aspects, especially for the 'real' situation in human cancer. We developed a novel method that provides exact three-dimensional (3D) information on both microscopic morphology and gene expression, over a virtually unlimited spatial range, by reconstruction from serial immunostained tissue slices. Quantitative 3D assessment of tumour budding at the cancer-host interface in human pancreatic, colorectal, lung and breast adenocarcinoma suggests collective cell migration as the mechanism of cancer cell invasion, while single cancer cell migration seems to be virtually absent. Budding tumour cells display a shift towards spindle-like as well as a rounded morphology. This is associated with decreased E-cadherin staining intensity and a shift from membranous to cytoplasmic staining, as well as increased nuclear ZEB1 expression.

Revisiting Mt Fuji's groundwater origins with helium, vanadium and environmental DNA tracers.

Known locally as the water mountain, for millennia Japan's iconic Mt Fuji has provided safe drinking water to millions of people via a vast network of groundwater and freshwater springs. Groundwater, which is recharged at high elevations, flows down Fuji's flanks within three basaltic aquifers, ultimately forming countless pristine freshwater springs among Fuji's foothills. Here we challenge the current conceptual model of Fuji being a simple system of laminar groundwater flow with little to no vertical exchange between its three aquifers. This model contrasts strongly with Fuji's extreme tectonic instability due to its unique location on top of the only known continental trench-trench-trench triple junction, its complex geology and its unusual microbial spring water communities. On the basis of a unique combination of microbial environmental DNA, vanadium and helium tracers, we provide evidence for prevailing deep circulation and a previously unknown deep groundwater contribution to Fuji's freshwater springs. The most substantial deep groundwater upwelling has been found along Japan's most tectonically active region, the Fujikawa-kako Fault Zone. Our findings broaden the hydrogeological understanding of Fuji and demonstrate the vast potential of combining environmental DNA, on-site noble gas and trace element analyses for groundwater science.

Validation of a turbulent Kelvin-Helmholtz shear layer model using a high-energy-density OMEGA laser experiment.

Following the successful demonstration of an OMEGA laser-driven platform for generating and studying nearly two-dimensional unstable plasma shear layers [Hurricane et al., Phys. Plasmas 16, 056305 (2009); Harding et al., Phys. Rev. Lett. 103, 045005 (2009)], this Letter reports on the first quantitative measurement of turbulent mixing in a high-energy-density plasma. As a blast wave moves parallel to an unperturbed interface between a low-density foam and a high-density plastic, baroclinic vorticity is deposited at the interface and a Kelvin-Helmholtz instability-driven turbulent mixing layer is created in the postshock flow due to surface roughness. The spatial scale and density profile of the turbulent layer are diagnosed using x-ray radiography with sufficiently small uncertainty so that the data can be used to ~0.17 µm) in the postshock plasma flow are consistent with an "inertial subrange," within which a Kolmogorov turbulent energy cascade can be active. An illustration of comparing the data set with the predictions of a two-equation turbulence model in the ares radiation hydrodynamics code is also presented.

Series "matrix metalloproteinases in lung health and disease": Biological role of matrix metalloproteinases: a critical balance.

Matrix metalloproteinases (MMPs) are members of the metzincin group of proteases which share the conserved zinc-binding motif in their catalytic active site. It was originally thought that their main function is to degrade the various components of the extracellular matrix (ECM), yet recent studies have led us to appreciate their significance as regulators of extracellular tissue signalling networks. Due to the broad spectrum of their substrate specificity, MMPs contribute to the homeostasis of many tissues and participate in several physiological processes, such as bone remodelling, angiogenesis, immunity and wound healing. MMP activity is tightly controlled at the level of transcription, pro-peptide activation and inhibition by tissue inhibitors of MMPs. Dysregulated MMP activity leads to pathological conditions such as arthritis, inflammation and cancer, thus highlighting MMPs as promising therapeutic targets. Analysis of MMP mutant mice has proved to be an essential tool for the identification of novel functions and interactions of single MMP members. Advancing our understanding of the MMP contribution to tissue homeostasis will lead us to identify causal relationships between their dysregulation and the development of disease pathologies, thus guiding us to successful MMP-directed therapies.

Automation of peptide desalting for proteomic liquid chromatography - tandem mass spectrometry by centrifugal microfluidics.

For large-scale analysis of complex protein mixtures, liquid chromatography - tandem mass spectrometry (LC-MS/MS) has been proven to be one of the most versatile tools due to its high sensitivity and ability to both identify and quantify thousands of proteins in a single measurement. Sample preparation typically comprises site-specific cleavage of proteins into peptides, followed by desalting and concomitant peptide enrichment, which is commonly performed by solid phase extraction. Desalting workflows may include multiple liquid handling steps and are thus error prone and labour intensive. To improve the reproducibility of sample preparation for low amounts of protein, we present a centrifugal microfluidic disk that automates all liquid handling steps required for peptide desalting by solid phase extraction (DesaltingDisk). Microfluidic implementation was enabled by a novel centrifugal microfluidic dosing on demand structure that enabled mapping multiple washing steps onto a microfluidic disk. Evaluation of the microfluidic disk was performed by LC-MS/MS analysis of tryptic HEK-293 eukaryotic cell peptide mixtures desalted either using the microfluidic disk or a manual workflow. A comparable number of peptides were identified in the disk and manual set with 19 775 and 20 212 identifications, respectively. For a core set of 10 444 peptides that could be quantified in all injections, intensity coefficients of variation were calculated based on label-free quantitation intensities. The disk set featured smaller variability with a median CV of 9.3% compared to the median CV of 12.6% for the manual approach. Intensity CVs on protein level were lowered from 5.8% to 4.2% when using the LabDisk. Interday reproducibility for both workflows was assessed by LC-SRM/MS analysis of samples that were spiked with 11 synthetic peptides of varying hydrophobicity. Except for the most hydrophilic and hydrophobic peptides, the average CV was lowered to 3.6% for the samples processed with the disk compared to 7.2% for the manual workflow. The presented centrifugal microfluidic DesaltingDisk demonstrates the potential to improve reproducibility in the sample preparation workflow for proteomic mass spectrometry, especially for application with limited amount of sample material.

Topsoil structure stability in a restored floodplain: Impacts of fluctuating water levels, soil parameters and ecosystem engineers.

Ecosystem services provided by floodplains are strongly controlled by the structural stability of soils. The development of a stable structure in floodplain soils is affected by a complex and poorly understood interplay of hydrological, physico-chemical and biological processes. This paper aims at analysing relations between fluctuating groundwater levels, soil physico-chemical and biological parameters on soil structure stability in a restored floodplain. Water level fluctuations in the soil are modelled using a numerical surface-water-groundwater flow model and correlated to soil physico-chemical parameters and abundances of plants and earthworms. Causal relations and multiple interactions between the investigated parameters are tested through structural equation modelling (SEM). Fluctuating water levels in the soil did not directly affect the topsoil structure stability, but indirectly through affecting plant roots and soil parameters that in turn determine topsoil structure stability. These relations remain significant for mean annual days of complete and partial (>25%) water saturation. Ecosystem functioning of a restored floodplain might already be affected by the fluctuation of groundwater levels alone, and not only through complete flooding by surface water during a flood period. Surprisingly, abundances of earthworms did not show any relation to other variables in the SEM. These findings emphasise that earthworms have efficiently adapted to periodic stress and harsh environmental conditions. Variability of the topsoil structure stability is thus stronger driven by the influence of fluctuating water levels on plants than by the abundance of earthworms. This knowledge about the functional network of soil engineering organisms, soil parameters and fluctuating water levels and how they affect soil structural stability is of fundamental importance to define management strategies of near-natural or restored floodplains in the future.

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance.

The WD40-domain containing protein CORO2B is specifically enriched in glomerular podocytes and regulates the ventral actin cytoskeleton.

Podocytes are highly specialized epithelial cells essentially required to establish and maintain the kidney filtration barrier. Due to their complex cellular architecture these cells rely on an elaborated cytoskeletal apparatus providing plasticity as well as adaptive adhesion properties to withstand significant physical filtration forces. However, our knowledge about podocyte specific components of the cytoskeletal machinery is still incomplete. Employing cross-analysis of various quantitative omics-data sets we identify the WD40-domain containing protein CORO2B as a podocyte enriched protein. Furthermore, we demonstrate the distinct localization pattern of CORO2B to the ventral actin cytoskeleton serving as a physical linkage module to cell-matrix adhesion sites. Analysis of a novel Coro2b knockout mouse revealed that CORO2B modulates stress response of podocytes in an experimental nephropathy model. Using quantitative focal adhesome proteomics we identify the recruitment of CFL1 via CORO2B to focal adhesions as an underlying mechanism. Thus, we describe CORO2B as a novel podocyte enriched protein influencing cytoskeletal plasticity and stress adaptation.

Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations.

Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) α is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAPα, we investigate how FAPα shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAPα activity has a strong impact on the secreted CAF proteome ("secretome"), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) ß, and an impact on matrix processing enzymes. Functionally, FAPα mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAPα leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGFß. In collagen contraction assays, FAPα induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAPα, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By "terminal amine labeling of substrates" (TAILS) we explored FAPα-dependent cleavage events. Although FAPα acts predominantly as an amino-dipeptidase, putative FAPα cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAPα cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAPα with distinct positional profiles. Generally, our findings link FAPα to key aspects of CAF biology and attribute an important role in tumor-stroma interaction to FAPα.

Taspase1: a 'misunderstood' protease with translational cancer relevance.

Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a 'non-oncogene addiction' protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1's pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1's structure-function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field.

Psychosocial consequences of age-related visual impairment: comparison with mobility-impaired older adults and long-term outcome.

Indices of behavioral competence (activities of daily living [ADLs], instrumental activities of daily living [IADLs], use of outdoor resources, leisure activity level) as well as emotional adaptation (subjective well-being, future orientation) were used to investigate the psychosocial consequences of age-related vision impairment in a threefold manner: (a) comparison of visually impaired and unimpaired elders, (b) comparison of visually impaired and mobility-impaired elders, and (c) long-term adaptation across 5 years. The research design used (a) 42 severely visually impaired elders, (b) 42 blind elders, (c) 42 mobility-impaired elders, and (d) 42 unimpaired elders. Compared with the mobility impaired, the visually impaired demonstrated lower IADL competence but no difference in emotional adaptation. The long-term adjustment of the visually impaired remained relatively stable in the behavioral domain, although lower compared with the unimpaired elders. Emotional adaptation decreased over the 5-year longitudinal interval in the visually impaired and the unimpaired group, but the decrease was generally higher in the visually impaired group. Conceptual ideas from environmental gerontology as well as psychological resilience are used to interpret these results.

The Work-Health-Check (WHC): a brief new tool for assessing psychosocial stress in the workplace.

OBJECTIVE: Brief, psychometrically robust questionnaires assessing work-related psychosocial stressors are lacking. The purpose of the study is to evaluate the psychometric properties of a brief new questionnaire for assessing sources of work-related psychosocial stress. PARTICIPANTS: Managers, blue- and white-collar workers (n= 628 at measurement point one, n=459 at measurement point two), sampled from an online panel of a German marketing research institute. METHODS: We either developed or identified appropriate items from existing questionnaires for ten scales, which are conceptually based in work stress models and reflected either work-related demands or resources. Factorial structure was evaluated by confirmatory factor analyses (CFA). Scale reliability was assessed by Cronbach's Alpha, and test-retest; correlations with work-related efforts demonstrated convergent and discriminant validity for the demand and resource scales, respectively. Scale correlations with health indicators tested criterion validity. RESULTS: All scales had satisfactory reliability (Cronbach's Alpha: 0.74-0.93, retest reliabilities: 0.66-0.81). CFA supported the anticipated factorial structure. Significant correlations between job-related efforts and demand scales (mean r=0.44) and non-significant correlations with the resource scales (mean r=0.07) suggested good convergent and discriminant validity, respectively. Scale correlations with health indicators demonstrated good criterion validity. CONCLUSION: The WHC appears to be a brief, psychometrically robust instrument for assessing work-related psychosocial stressors.