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.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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High-Throughput Drug Screening Identifies a Potent Wnt Inhibitor that Promotes Airway Basal Stem Cell Homeostasis

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iPSC technology-Powerful hand for disease modeling and therapeutic screen

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]     

Stylet penetration by two aphid species on susceptible and resistant lettuce

The probing behavior of two aphid species, Myzus persicae (Sulz.) and Nasonovia ribisnigri (Mosley), was electronically monitored on susceptible and resistant lettuce lines using a DC amplifier. A waveform pattern associated with extracellular stylet pathway activities, pattern C, occurred for longer periods when either aphid species probed resistant plants. This pattern is usually regularly interrupted by drops in electrical potential lasting a few seconds, reflecting cell membrane punctures followed by rapid withdrawal of the stylet tips. For M. persicae on resistant lettuce a large increase in pattern C without these potential drops accounted for the increased duration of this pattern. For N. ribisnigri the increase in pathway activity on resistant plants was due to an increase in the more typical pattern C with potential drops, as well as to an increased duration of pattern F, associated with a curious type of stylet penetration within cell walls. Both aphids made more but shorter probes on resistant than on susceptible plants, and these probes led less frequently to periods of sieve element contact and ingestion. The effects of resistance appear to involve both mesophyll and phloem factors. The underlying mechanisms, however, remain unclear. The results indicate which stylet penetration activities or waveform patterns are of interest for further investigation of resistance mechanisms.

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Résumé Le comportement de sondage de variétés sensibles et résistantes de laitues par deux espèces de pucerons, Myzus persicae (Sulz.) et Nasonovia ribisnigri (Mosley), a été enregistré électroniquement en utilisant un amplificateur DC. Les deux espèces de pucerons ont présenté une amplification de l'onde de type C associée au cheminement extracellulaire des stylets lors du sondage des variétés résistantes. Cette onde est normalement interrompue par des chutes de quelques secondes du potentiel électrique, traduisant des ponctions de la membrane cellulaire suivies par un rapide retrait de l'extrémité des stylets. La forte augmentation de l'onde C mais sans chutes de potentiel rend compte de la plus longue durée de cette onde chez M. persicae sur laitue résistante. L'accroissement du cheminement des stylets chez N. ribisnigri sur plantes résistantes est dû à une augmentation de l'onde C typique avec chutes de potentiel, ainsi qu'à une prolongation de l'onde F liée à la pénétration des stylets dans les parois cellulaires. Les deux espèces font des sondages plus brefs et plus nombreux sur variétés résistantes, et ces sondages entraînent des contacts moins fréquents avec les éléments criblés et débouchent moins souvent sur de l'ingestion. Les effects de la résistance semblent impliquer des facteurs liés à la fois au mésophylle et au phloème. Les mécanismes sous-jacents, cependant, ne sont pas encore clairs. Ces résultats Montrent que l'examen des ondes liées à la pénétration des stylets est important pour des études ultérieures sur les mécanismes de résistance.

     

Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: Comparison of different substrata and tissue culture media formulations

Summary Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells. This research was supported in part by grant 1R01-AM-26520 from the National Institute of Arthritis, Diabetes and Digestive Kidney Diseases, NIH, Bethesda, Maryland.