Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1 mM sulindac over 16 h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433–451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1 mM sulindac treatment for 8 h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30 K, 3 K and 1 K). Proteins isolated in the > 30 K and 3–30 K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3 K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS–MS. Collectively, our data show that LIM1215 cells treated with 1 mM sulindac for 8 h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5 AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8 h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1 mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell–cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

Hospital Differences in Cesarean Deliveries in Massachusetts (US) 2004–2006: The Case against Case-Mix Artifact

Objective

We examined the extent to which differences in hospital-level cesarean delivery rates in Massachusetts were attributable to hospital-level, rather than maternal, characteristics.

Methods

Birth certificate and maternal in-patient hospital discharge records for 2004–06 in Massachusetts were linked. The study population was nulliparous, term, singleton, and vertex births (NTSV) (n = 80,371) in 49 hospitals. Covariates included mother''s age, race/ethnicity, education, infant birth weight, gestational age, labor induction (yes/no), hospital shift at time of birth, and preexisting health conditions. We estimated multilevel logistic regression models to assess the likelihood of a cesarean delivery

Results

Overall, among women with NTSV births, 26.5% births were cesarean, with a range of 14% to 38.3% across hospitals. In unadjusted models, the between-hospital variance was 0.103 (SE 0.022); adjusting for demographic, socioeconomic and preexisting medical conditions did not reduce any hospital-level variation 0.108 (SE 0.023).

Conclusion

Even after adjusting for both socio-demographic and clinical factors, the chance of a cesarean delivery for NTSV pregnancies varied according to hospital, suggesting the importance of hospital practices and culture in determining a hospital''s cesarean rate.     

An Agent-Based Modeling Template for a Cohort of Veterans with Diabetic Retinopathy

Background

Agent-based models are valuable for examining systems where large numbers of discrete individuals interact with each other, or with some environment. Diabetic Veterans seeking eye care at a Veterans Administration hospital represent one such cohort.

Objective

The objective of this study was to develop an agent-based template to be used as a model for a patient with diabetic retinopathy (DR). This template may be replicated arbitrarily many times in order to generate a large cohort which is representative of a real-world population, upon which in-silico experimentation may be conducted.

Methods

Agent-based template development was performed in java-based computer simulation suite AnyLogic Professional 6.6. The model was informed by medical data abstracted from 535 patient records representing a retrospective cohort of current patients of the VA St. Louis Healthcare System Eye clinic. Logistic regression was performed to determine the predictors associated with advancing stages of DR. Predicted probabilities obtained from logistic regression were used to generate the stage of DR in the simulated cohort.

Results

The simulated cohort of DR patients exhibited no significant deviation from the test population of real-world patients in proportion of stage of DR, duration of diabetes mellitus (DM), or the other abstracted predictors. Simulated patients after 10 years were significantly more likely to exhibit proliferative DR (P<0.001).

Conclusions

Agent-based modeling is an emerging platform, capable of simulating large cohorts of individuals based on manageable data abstraction efforts. The modeling method described may be useful in simulating many different conditions where course of disease is described in categorical stages.     

Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

CD4⁺ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4⁺ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4⁺ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4⁺ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.     

Development of Nascent Focal Adhesions in Spreading Cells

The eukaryotic cell develops organelles to sense and respond to the mechanical properties of its surroundings. These mechanosensing organelles aggregate into symmetry-breaking patterns to mediate cell motion and differentiation on substrate. The spreading of a cell plated onto a substrate is one of the simplest paradigms in which angular symmetry-breaking assemblies of mechanical sensors are seen to develop. We review evidence for the importance of the edge of the cell-extracellular matrix adhesion area in the aggregation of mechanosensors and develop a theoretical model for the clustering of mechanosensors into nascent focal adhesions on this contact ring. To study the spatial patterns arising on this topological feature, we use a one-dimensional lattice model with a nearest-neighbor interaction between individual integrin-mediated mechanosensors. We find the effective Ginzburg-Landau free energy for this model and determine the spectrum of spatial modes as the cell spreads and increases its contact area with the substrate. To test our model, we compare its predictions with measured distributions of paxillin in spreading fibroblasts.     

Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.     

The bryophyte flora of northern Iturup (north-west Pacific): between the circumboreal and East Asian floristic regions

Introduction. The Kuril Islands Chain demarcates the boundary between the circumpolar and East Asian floras in the amphi-Pacific and is a trans-Oceanic migration route from warm-temperate insular East Asia to north-east Asia and Beringia. This paper reviews the flora of the northern part of Iturup Island in regards to its bryogeography and taxonomic diversity, as well as its relative position among other bryophyte floras of north-east and East Asia.

Methods. The paper uses morphological methods to identify the species composition and detrended correspondence analysis to identify the position of the northern Iturup Island in the system of local floras of the eastern, extra-tropical, Pacific.

Key results. The flora is closely related to the bryophyte floras of the southern Kurils and Hokkaido Island, but has been found to be distinctly distant from the ‘true’ cool-temperate East Asian floras, as well as from the boreal and hemiarctic floras of north-east Asia. Instead, the floras of the south Kurils and northern Hokkaido Island belong to a peculiar intermediate phytogeographic province that cannot be unambiguously placed in either the East Asian or circumboreal floristic regions. The hemiboreal character of the bryophyte flora of northern Iturup Island is in contrast to the general appearance of many of the dominant vegetation communities on the island, which are more characteristic of the hemiarctic.

Conclusion. There is no robust evidence to refer the bryophyte flora of northern Iturup either to the circumboreal or East Asian Floristic Region, the flora belongs to a unique intermediate province between the two regions.