A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM flow cytometry - FITC fluorescein-iso-thiocyanate - LB Luria broth - MM minimal salt medium - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

The major light-harvesting complex of Photosystem II: aspects of its molecular and cell biology

The light-harvesting complex of photosystem II (LHC II) contains one major (LHC IIb) and at least three minor chlorophyll-protein components. The apoproteins of LHC IIb (LHCP) are encoded by nuclear genes and synthesized in the cytoplasm as a higher molecular weight precursor(s) (pLHCP). Several genes coding for pLHCP have been cloned from various higher plant species. The expression of these genes is dependent upon a variety of factors such as light, the developmental stage of the plastids and the plant. After its synthesis in the cytoplasm, pLHCP is imported into plastids, inserted into thylakoids, processed to its mature form, and assembled into LHC IIb. The pathway of assembly of LHC IIb in the thylakoid membranes is currently being investigated in several laboratories. We present a model that gives some details of the steps in the assembly process. Many of the steps involved in the synthesis and assembly are dependent on light and the stage of plastid development.Abbreviations PS Photosystem - LHC II Light-harvesting complex of PS II - LHCP Apoproteins of LHC IIb - pLHCP Precursor of LHCP - PAGE Polyacrylamide gel electrophoresis     

Predator driven changes in community structure

Summary The zooplankton community of a small pond changed markedly with temporal variation in predation pressure. Long term changes in zooplankton community structure occurred following the replacement of planktivorous fish by phantom midge (Chaoborus americanus) larvae as the predominant predator of zooplankton. The interannual changes following the establishment of Chaoborus included the apparent or near extinction, of species ill adapted to the new predation pressure and the successful colonization of well adapted species. Seasonal changes in the species composition and size distribution of the zooplankton community correlate with temporal variation in predation intensity associated with temperature-activity patterns of the predator or changes in the stage structure of the predator population.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.     

Twenty years of numerical syntaxonomy

The development of numerical syntaxonomy during its first 20 yr is reviewed. The use of methods of numerical classification and ordination is the dominating feature of the development. National and local phytosociological data banks were established, large data sets handled and many important vegetation monographs were methodically based on multivariate data analysis. Particularly the development in Italy, the Netherlands, Czechoslovakia, and Sweden contributed to new theoretical elements of numerical syntaxonomy. Ordination became a common tool of searching for reticulate synsystematic relations between community types. The most popular ordination techniques have been Principal Components Analysis and Detrended Correspondence Analysis. Hierarchical agglomerative techniques of clustering still prevail in classification, although the divisive strategy of TWINSPAN has also become an effective tool for phytosociological clustering and table sorting. Extensive program packages, also for personal computers have now become standard equipment for many vegetation scientists.