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.     

Amygdala Reward Neurons Form and Store Fear Extinction Memory

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A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

Cell lines from imaginal discs ofDrosophila melanogaster

Summary New cell lines, designated as ML-DmDl≈10, were established from dissociated imaginal discs ofDrosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang’s M3(BF) medium, supplemented with 10% heat inactivated fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary culture supernatan, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums, imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to 10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae, they differentiated at high frequency into adult cuticular strutures in the early phase of the primary culture. This differentiation of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in vitro.     

c-fos expression in the amygdala:In vivo antisense modulation and role in anxiety

Summary 1. The amygdaloid complex is a key structure in mechanisms of fear and anxiety. Expression of the immediate-early gene c-fos has been reported in the central nucleus of the amygdala following various stressors, but the functional role of this phenomenon has remained unknown.2. c-fos expression was observed in the central nucleus when rats were subjected to a pharmacologically validated animal model of anxiety, the Vogel conflict test, but not after mere exposure to the test apparatus. Bilateral amygdala injection of a 15-mer phosphorothioate c-fos antisense oligodeoxynucleotide prior to testing blocked conflict-induced c-fos expression and had behavioral effects similar to those of established antianxiety drugs.3. Separate experiments determined that antisense treatment did not affect conflict behavior by acting on shock thresholds or drinking motivation.4. These findings provide evidence that neuronal activation and c-fos induction in the amygdala may be of importance for mechanisms of fear and anxiety.     

Lack of expression on cultured human T-lymphocytes of a T-cell antigen shared by the molt-4 cell line and normal human thymocytes

Summary Four hematopoietic cell lines (CCRF-CEM, HSB-2, MOLT-4, and RPMI-8402), derived from acute lymphoblastic leukemia and expressing T-cell surface markers (T-HCL), were studied with two specific anti-T-cell sera. The sera were raised in rabbits against human thymocytes (anti-HTY) and against T-cells cultured in the presence of conditioned medium derived from lymphocytes stimulated with PHA (anti-CTC). Both sera were absorbed to obtain a T-cell specific pattern of reaction and were further absorbed with normal peripheral blood lymphocytes or with each of the four T-HCL. The anti-HTY sera absorbed with CEM, 8402, and HSB-2 still reacted with MOLT-4. A similar pattern of reactivity was found only with the anti-CTC absorbed with 8402, whereas, after absorptions with the other cell lines, this antiserum was unreactive against MOLT-4. After absorption with normal peripheral blood lymphocytes, anti-HTY still reacted with thymocytes and MOLT-4 but was negative on CTC. In contrast, anti-CTC absorbed with peripheral blood lymphocytes (PBL) was negative on thymocytes and MOLT-4 but still reacted against CTC. Our data confirm the existence of a T-cell antigen (probably an early T-cell differentiation antigen) shared between thymus and MOLT-4. This antigen is not expressed on CTC, although these cells express an antigenic pattern more complex than PBL. Antisera to CTC represents a source of anti-T-cell sera free of contamination with antibodies to early thymus-related antigens but containing other T-cell-related specificities. Supported in part by Naval Medical Research and Development Command, Research Task No. ZF51.524.013.1025, and National Cancer Institute Contract No. Y01-CB-00319. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council.     

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans of other products of smooth muscle cell metabolism.     

Mesenchymal stromal cell‐derived factors promote the colonization of collagen 3D scaffolds with human skin cells

The development of stem cell technology in combination with advances in biomaterials has opened new ways of producing engineered tissue substitutes. In this study, we investigated whether the therapeutic potential of an acellular porous scaffold made of type I collagen can be improved by the addition of a powerful trophic agent in the form of mesenchymal stromal cells conditioned medium (MSC‐CM) in order to be used as an acellular scaffold for skin wound healing treatment. Our experiments showed that MSC‐CM sustained the adherence of keratinocytes and fibroblasts as well as the proliferation of keratinocytes. Moreover, MSC‐CM had chemoattractant properties for keratinocytes and endothelial cells, attributable to the content of trophic and pro‐angiogenic factors. Also, for the dermal fibroblasts cultured on collagen scaffold in the presence of MSC‐CM versus serum control, the ratio between collagen III and I mRNAs increased by 2‐fold. Furthermore, the gene expression for α‐smooth muscle actin, tissue inhibitor of metalloproteinase‐1 and 2 and matrix metalloproteinase‐14 was significantly increased by approximately 2‐fold. In conclusion, factors existing in MSC‐CM improve the colonization of collagen 3D scaffolds, by sustaining the adherence and proliferation of keratinocytes and by inducing a pro‐healing phenotype in fibroblasts.