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.

Therapies with CCL25 require controlled release via microparticles to avoid strong inflammatory reactions.

BACKGROUND: Chemokine therapy with C-C motif chemokine ligand 25 (CCL25) is currently under investigation as a promising approach to treat articular cartilage degeneration. We developed a delayed release mechanism based on Poly (lactic-co-glycolic acid) (PLGA) microparticle encapsulation for intraarticular injections to ensure prolonged release of therapeutic dosages. However, CCL25 plays an important role in immune cell regulation and inflammatory processes like T-cell homing and chronic tissue inflammation. Therefore, the potential of CCL25 to activate immune cells must be assessed more thoroughly before further translation into clinical practice. The aim of this study was to evaluate the reaction of different immune cell subsets upon stimulation with different dosages of CCL25 in comparison to CCL25 released from PLGA particles. RESULTS: Immune cell subsets were treated for up to 5 days with CCL25 and subsequently analyzed regarding their cytokine secretion, surface marker expression, polarization, and migratory behavior. The CCL25 receptor C-C chemokine receptor type 9 (CCR9) was expressed to a different extent on all immune cell subsets. Direct stimulation of peripheral blood mononuclear cells (PBMCs) with high dosages of CCL25 resulted in strong increases in the secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-1ß (IL-1ß), tumor-necrosis-factor-α (TNF-α) and interferon-γ (IFN-γ), upregulation of human leukocyte antigen-DR (HLA-DR) on monocytes and CD4+ T-cells, as well as immune cell migration along a CCL25 gradient. Immune cell stimulation with the supernatants from CCL25 loaded PLGA microparticles caused moderate increases in MCP-1, IL-8, and IL-1ß levels, but no changes in surface marker expression or migration. Both CCL25-loaded and unloaded PLGA microparticles induced an increase in IL-8 and MCP-1 release in PBMCs and macrophages, and a slight shift of the surface marker profile towards the direction of M2-macrophage polarization. CONCLUSIONS: While supernatants of CCL25 loaded PLGA microparticles did not provoke strong inflammatory reactions, direct stimulation with CCL25 shows the critical potential to induce global inflammatory activation of human leukocytes at certain concentrations. These findings underline the importance of a safe and reliable release system in a therapeutic setup. Failure of the delivery system could result in strong local and systemic inflammatory reactions that could potentially negate the benefits of chemokine therapy.

Socio-spatial inequalities in smoking among young adults: What a 'go-along' study says about local smoking practices.

Smoking prevalence in Canada has declined; yet, not among all segments of the population. Residents of high-deprivation neighbourhoods and young adults are over-represented among current smokers. There is a dearth of theoretically informed research focused on better understanding the connection between smoking, social inequalities, and place among young adults. To contribute to this understudied area, we undertook a qualitative study drawing upon the collective lifestyles framework. We conducted walking interviews (go-alongs) with 27 young adults (smokers and non-smokers) living in five diverse urban neighbourhoods in Montreal, Canada. We present the findings as neighbourhood portraits wherein participants' accounts revealed how local smoking practices shaped and were shaped by rules and regulations and the meaning and use of neighbourhood resources. In the discussion we reflect on the opportunities and constraints to smoking with a focus on young adults. We also consider the possible implications for socio-spatial inequalities in smoking and the relationship to tobacco control strategies.

Can reflexivity be learned? An experience with tobacco control practitioners in Canada.

Summary: To explore an example of a reflexive intervention with health professionals working in tobacco control (TC). This study reports the perceived intervention effects regarding: (i) participants' understanding of reflexivity and personal learning and (ii) conditions needed in order to integrate reflexivity into professional and organizational practices. This is a qualitative study using an interpretative evaluation framework to assess the perceived effects of a reflexive intervention in Montréal, Québec. Semi-structured qualitative interviews (n = 8) gathered data. Data analysis began deductively, guided by the broad categories found in research questions. Sub-categories to populate these broad categories captured the inhibitors and facilitators through an inductive thematic analysis. Our study reveals that, following the intervention, most participants had a generally good understanding of reflexivity and described concrete learning in association with the intervention. Main facilitators and inhibitors to conducting a reflexive workshop pertained to the organizational context as well as to the professional and individual characteristics of the participants. Some participants implemented sustainable changes as a result of the intervention, such as creating a tool, reviewing work plans and developing new mechanisms to integrate the voice of their clientele in the planning process. The need and interest for dialogue among health professionals about how TC intervention activities may inadvertently contribute to social inequalities in smoking is apparent. While there appears to be potential for reflexive practice, the integration of reflexivity into practice is reliant upon the organizational context (financial and time constraints, culture, support, and climate) and the reflexivity concept itself (intangibility, complexity and fuzziness).

Back pain prevalence in adolescent athletes.

The research aimed to investigate back pain (BP) prevalence in a large cohort of young athletes with respect to age, gender, and sport discipline. BP (within the last 7 days) was assessed with a face scale (face 1-2 = no pain; face 3-5 = pain) in 2116 athletes (m/f 61%/39%; 13.3 ± 1.7 years; 163.0 ± 11.8 cm; 52.6 ± 13.9 kg; 4.9 ± 2.7 training years; 8.4 ± 5.7 training h/week). Four different sports categories were devised (a: combat sports, b: game sports; c: explosive strength sport; d: endurance sport). Analysis was described descriptively, regarding age, gender, and sport. In addition, 95% confidence intervals (CI) were calculated. About 168 (8%) athletes were allocated into the BP group. About 9% of females and 7% of males reported BP. Athletes, 11-13 years, showed a prevalence of 2-4%; while prevalence increased to 12-20% in 14- to 17-year olds. Considering sport discipline, prevalence ranged from 3% (soccer) to 14% (canoeing). Prevalences in weight lifting, judo, wrestling, rowing, and shooting were ≥10%; in boxing, soccer, handball, cycling, and horse riding, ≤6%. 95% CI ranged between 0.08-0.11. BP exists in adolescent athletes, but is uncommon and shows no gender differences. A prevalence increase after age 14 is obvious. Differentiated prevention programs in daily training routines might address sport discipline-specific BP prevalence.

Is Sonographic Assessment of Intratendinous Blood Flow in Achilles Tendinopathy Patients Reliable?: Consistency of Doppler Ultrasound Modes and Intra- and Inter-observer Reliability.

PURPOSE: The purpose of this study was to investigate the consistency between different Doppler ultrasound (DU) modes as well as the intra- and inter-observer reliability of investigators with different experience level in assessing intratendinous blood flow (IBF) in Achilles tendinopathy patients. MATERIAL AND METHODS: 18 participants (36 Achilles tendons, AT) with Achilles tendinopathy (24 AT) were examined with power Doppler ultrasound (PDU), colour Doppler ultrasound (CDU) and "Advanced Dynamic Flow" (ADF) (Toshiba Xario SSA-660 A; 14MHz transducer) by 2 investigators (experienced, EI; inexperienced, II) in a test-retest design (M1/M2). A modified Öhberg score was used to quantify IBF. Data was analysed descriptively (absolute and relative). Consistency of the 3 modes was presented by Kendall's Coefficient of Concordance (Kendall's W). Intra- and inter-observer reliability were calculated by use of Kendall's tau b correlation coefficient. RESULTS: IBF was detected in 79-92% of symptomatic AT and in 33-50% of contralateral asymptomatic AT. Comparing the 3 modes, Kendall's W ranged from 0.97-0.98. Analysis of intra-observer reliability resulted in Kendall's tau 0.90-0.92 for EI and 0.84-0.87 for II. Inter-observer reliability resulted in Kendall's tau 0.64-0.69 in M1 and 0.68-0.70 in M2. CONCLUSION: The very good consistency between PDU, CDU and ADF indicates a comparable applicability for assessing IBF in ATs. Intra-observer reliability was high for both investigators, independent of experience. The moderate inter-observer reliability reflects the challenge in sonographic detection of intratendinous blood flow (IBF) amount.

Tendon Adaptation to Sport-specific Loading in Adolescent Athletes.

Tendon adaptation due to mechanical loading is controversially discussed. However, data concerning the development of tendon thickness in adolescent athletes is sparse. The purpose of this study was to examine possible differences in Achilles (AT) and patellar tendon (PT) thickness in adolescent athletes while considering age, gender and sport-specific loading. In 500 adolescent competitive athletes of 16 different sports and 40 recreational controls both ATs and PTs were sonographically measured. Subjects were divided into 2 age groups (< 13; ≥ 13 years) and 6 sport type categories (ball, combat, and water sports, combined disciplines, cycling, controls). In addition, 3 risk groups (low, moderate, high) were created according to the athlete's risk of developing tendinopathy. AT and PT thickness did not significantly differ between age groups (AT/PT:<13: 5.4±0.7 mm/3.6±0.5 mm;≥13: 5.3±0.7 mm/3.6±0.5 mm). In both age groups males presented higher tendon thickness than females (p<0.001). AT thickness was highest in ball sports/cyclists and lowest in controls (p≤0.002). PT thickness was greatest in water sports and lowest in controls (p=0.02). High risk athletes presented slightly higher AT thickness compared to the low risk group (p=0.03). Increased AT and PT thickness in certain sport types compared to controls supports the hypothesis of structural tendon adaptation due to sport-specific loading.

Ultrasound Applied to Subcutaneous Fat Tissue Measurements in International Elite Canoeists.

Subcutaneous adipose tissue (SAT) measurements with ultrasound have recently been introduced to assess body fat in elite athletes. However, appropriate protocols and data on various groups of athletes are missing. We investigated intra-rater reliability of SAT measurements using ultrasound in elite canoe athletes. 25 international level canoeists (18 male, 7 female; 23±4 years; 81±11 kg; 1.83±0.09 m; 20±3 training h/wk) were measured on 2 consecutive days. SAT was assessed with B-mode ultrasound at 8 sites (ISAK): triceps, subscapular, biceps, iliac crest, supraspinal, abdominal, front thigh, medial calf, and quantified using image analysis software. Data was analyzed descriptively (mean±SD, [range]). Coefficient of variation (CV%), intraclass correlation coefficient (ICC, 2.1) and absolute (LoA) and ratio limits of agreement (RLoA) were calculated for day-to-day reliability. Mean sum of SAT thickness was 30.0±19.4 mm [8.0, 80.1 mm], with 3.9±1.8 mm [1.2 mm subscapular, 8.0 mm abdominal] for individual sites. CV for the sum of sites was 4.7%, ICC 0.99, LoA 1.7±3.6 mm, RLoA 0.940 ( * /÷1.155). Measuring SAT with ultrasound has proved to have excellent day-to-day reliability in elite canoe athletes. Recommendations for standardization of the method will further increase accuracy and reproducibility.

Prevalence of Achilles and patellar tendinopathy and their association to intratendinous changes in adolescent athletes.

Achilles (AT) and patellar tendons (PT) are commonly affected by tendinopathy in adult athletes but prevalence of symptoms and morphological changes in adolescents is unclear. The study aimed to determine prevalence of tendinopathy and intratendinous changes in ATs and PTs of adolescent athletes. A total of 760 adolescent athletes (13.0 ± 1.9 years; 160 ± 13 cm; 50 ± 14 kg) were examined. History, local clinical examination, and longitudinal Doppler ultrasound analysis for both ATs and PTs were performed including identification of intratendinous echoic changes and vascularization. Diagnosis of tendinopathy was complied clinically in case of positive history of tendon pain and tendon pain on palpation. Achilles tendinopathy was diagnosed in 1.8% and patellar tendinopathy in 5.8%. Vascularizations were visible in 3.0% of ATs and 11.4% of PTs, hypoechogenicities in 0.7% and 3.2% as well as hyperechogenicities in 0% and 0.3%, respectively. Vascularizations and hypoechogenicities were statistically significantly more often in males than in females (P ≤ 0.02). Subjects with patellar tendinopathy had higher prevalence of structural intratendinous changes than those without PT symptoms (P ≤ 0.001). In adolescent athletes, patellar tendinopathy is three times more frequent compared with Achilles tendinopathy. Longitudinal studies are necessary to investigate physiological or pathological origin of vascularizations and its predictive value in development of tendinopathy.

Influence of intensive care treatment on the protein binding of sufentanil and hydromorphone during pain therapy in postoperative cardiac surgery patients.

BACKGROUND: Our objective was to evaluate the effect of intensive care treatment on the protein binding of sufentanil and hydromorphone in cardiac surgery patients during postoperative analgesia using a target-controlled infusion (TCI) and patient-controlled analgesia (PCA). METHODS: Fifty adult patients were enrolled in this prospective randomized study; of which, 49 completed the study (age range 40-81 yr). Sufentanil was administered as an analgesic intraoperatively, and hydromorphone was dosed after operation with TCI and PCA until 8 a.m. on the first postoperative day. Arterial plasma samples were collected for drug and protein concentration measurements up to 24 h after cardiac surgery. Corresponding patient data were collected from the electronic patient data system. After explorative data analysis with principal component analysis, multivariate regression analysis and non-linear mixed effects modelling was used to study the effect of treatment on protein binding. RESULTS: Data of 35 patients were analysed. The median protein binding of sufentanil and hydromorphone was 88.4% (IQ range 85.7-90.5%) and 11.6% (IQ range 9.5-14.3%), respectively. Free fraction of sufentanil increased towards the end of the study period, whereas hydromorphone free fraction remained nearly constant. The total sufentanil concentration and volume balance were identified as significant covariates for the protein binding of sufentanil. For the protein binding of hydromorphone, no significant covariate effects were found. CONCLUSIONS: Sufentanil protein binding was significantly dependent on changes in the total drug concentration and volume balance addressing the importance of adequate dosing and fluid-guided therapy. Hydromorphone protein binding was nearly constant throughout the study period. CLINICAL TRIAL REGISTRATION: EudraCT 2011-003648-31 and ClinicalTrials.gov: NCT01490268.

Cannabinoid receptor subtypes 1 and 2 mediate long-lasting neuroprotection and improve motor behavior deficits after transient focal cerebral ischemia.

The endocannabinoid system is crucially involved in the regulation of brain activity and inflammation. We have investigated the localization of cannabinoid CB1 and CB2 receptors in adult rat brains before and after focal cerebral ischemia due to endothelin-induced transient occlusion of the middle cerebral artery (eMCAO). Using immunohistochemistry, both receptor subtypes were identified in cortical neurons. After eMCAO, neuronal cell death was accompanied by reduced neuronal CB1 and CB2 receptor-linked immunofluorescence. In parallel, CB1 receptor was found in activated microglia/macrophages 3 days post eMCAO and in astroglia cells at days 3 and 7. CB2 receptor labeling was identified in activated microglia/macrophages or astroglia 3 and 7d ays post ischemia, respectively. In addition, immune competent CD45-positive cells were characterized by pronounced CB2 receptor staining 3 and 7 days post eMCAO. KN38-72717, a potent and selective CB1 and CB2 receptor agonist, revealed a significant, dose-dependent and long-lasting reduction of cortical lesion sizes due to eMCAO, when applied consecutively before, during and after eMCAO. In addition, severe motor deficits of animals suffering from eMCAO were significantly improved by KN38-7271. KN38-7271 remained effective, even if its application was delayed up to 6h post eMCAO. Finally, we show that the endocannabinoid system assembles a comprehensive machinery to defend the brain against the devastating consequences of cerebral ischemia. In summary, this study underlines the therapeutic potential of CB1 and/or CB2 receptor agonists against neurodegenerative diseases or injuries involving acute or chronic imbalances of cerebral blood flow and energy consumption.

The propeptide of yeast cathepsin D inhibits programmed necrosis.

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast.

The social context of smoking: the next frontier in tobacco control?

A better understanding of the social context of smoking may help to enhance tobacco control research and practice.

Apoptosis in yeast: a new model for aging research.

Apoptosis is a form of programmed cell death with a central role in development and homeostasis of metazoan organisms. Recent research indicates the presence of an apoptotic cell death program in unicellular eukaryotes. Yeast can be killed by expression of mammalian proapoptotic genes or in response to oxygen stress, which is an inducer of mammalian apoptosis. The dying yeast cells show morphological alterations typical for apoptosis. Yeast provides a simple model for cellular aging. The observation that old yeast cells produce oxygen radicals and die apoptotically may provide clues to a similar sequence of events in mammalian aging.

The proteasomal substrate Stm1 participates in apoptosis-like cell death in yeast.

We have identified the yeast gene STM1 in an overexpression screen for new proteasomal substrates. Stm1 is unstable in wild-type cells and stabilized in cells with defective proteasomal activity and thus a bona fide substrate of the proteasome. It is localized in the perinuclear region and is required for growth in the presence of mutagens. Overexpression in cells with impaired proteasomal degradation leads to cell death accompanied with cytological markers of apoptosis: loss of plasma membrane asymmetry, chromatin condensation, and DNA cleavage. Cells lacking Stm1 display deficiency in the apoptosis-like cell death process induced by treatment with low concentrations of H(2)O(2). We suggest that Stm1 is involved in the control of the apoptosis-like cell death in yeast. Survival is increased when Stm1 is completely missing from the cells or when inhibition of Stm1 synthesis permits proteasomal degradation to decrease its amount in the cell. Conversely, Stm1 accumulation induces cell death. In addition we identified five other genes whose overexpression in proteasomal mutants caused similar apoptotic phenotypes.

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis.

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

Alcohol enhances oxysterol-induced apoptosis in human endothelial cells by a calcium-dependent mechanism.

Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with physiologically occurring oxysterols on atherogenesis.