Primary Teacher Identity, Commitment and Career in Performative School Cultures

ERIC Educational Resources Information Center

Troman, Geoff

2008-01-01

The research reported here maps changes in primary teachers' identity, commitment and perspectives and subjective experiences of occupational career in the context of performative primary school cultures. The research aimed to provide in-depth knowledge of performative school culture and teachers' subjective experiences in their work of teaching.…

[Characterization of epithelial primary culture from human conjunctiva].

PubMed

Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

2014-01-01

To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Isolation, Purification, and Culture of Primary Murine Sensory Neurons.

PubMed

Katzenell, Sarah; Cabrera, Jorge R; North, Brian J; Leib, David A

2017-01-01

Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection.

Establishment and characterization of Xenopus oviduct cells in primary culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marsh, J.; Tata, J.R.

1987-11-01

Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the samemore » characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.« less

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

PubMed

Mosqueda, Juan; Cossío-Bayugar, Raquel; Rodríguez, Elba; Falcón, Alfonso; Ramos, Alberto; Figueroa, Julio V; Alvarez, Antonio

2008-12-01

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

rhEPO Enhances Cellular Anti-oxidant Capacity to Protect Long-Term Cultured Aging Primary Nerve Cells.

PubMed

Wang, Huqing; Fan, Jiaxin; Chen, Mengyi; Yao, Qingling; Gao, Zhen; Zhang, Guilian; Wu, Haiqin; Yu, Xiaorui

2017-08-01

Erythropoietin (EPO) may protect the nervous system of animals against aging damage, making it a potential anti-aging drug for the nervous system. However, experimental evidence from natural aging nerve cell models is lacking, and the efficacy of EPO and underlying mechanism of this effect warrant further study. Thus, the present study used long-term cultured primary nerve cells to successfully mimic the natural aging process of nerve cells. Starting on the 11th day of culture, cells were treated with different concentrations of recombinant human erythropoietin (rhEPO). Using double immunofluorescence labeling, we found that rhEPO significantly improved the morphology of long-term cultured primary nerve cells and increased the total number of long-term cultured primary cells. However, rhEPO did not improve the ratio of nerve cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure nerve cell activity and showed that rhEPO significantly improved the activity of long-term cultured primary nerve cells. Moreover, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double immunofluorescence labeling flow cytometry revealed that rhEPO reduced the apoptotic rate of long-term cultured primary nerve cells. Senescence-associated β-galactosidase (SA-β-gal) immunohistochemistry staining showed that rhEPO significantly reduced the aging rate of long-term cultured primary nerve cells. Immunochemistry revealed that rhEPO enhanced intracellular superoxide dismutase (SOD) activity and glutathione (GSH) abundance and reduced the intracellular malondialdehyde (MDA) level. In addition, this effect depended on the dose, was maximized at a dose of 100 U/ml and was more pronounced than that of vitamin E. In summary, this study finds that rhEPO protects long-term cultured primary nerve cells from aging in a dose-dependent manner. The mechanism of this effect may be associated with the enhancement of the intracellular anti

Team structure and culture are associated with lower burnout in primary care.

PubMed

Willard-Grace, Rachel; Hessler, Danielle; Rogers, Elizabeth; Dubé, Kate; Bodenheimer, Thomas; Grumbach, Kevin

2014-01-01

Burnout is a threat to the primary care workforce. We investigated the relationship between team structure, team culture, and emotional exhaustion of clinicians and staff in primary care practices. We surveyed 231 clinicians and 280 staff members of 10 public and 6 university-run primary care clinics in San Francisco in 2012. Predictor variables included team structure, such as working in a tight teamlet, and perception of team culture. The outcome variable was the Maslach emotional exhaustion scale. Generalized estimation equation models were used to account for clustering at the clinic level. Working in a tight team structure and perceptions of a greater team culture were associated with less clinician exhaustion. Team structure and team culture interacted to predict exhaustion: among clinicians reporting low team culture, team structure seemed to have little effect on exhaustion, whereas among clinicians reporting high team culture, tighter team structure was associated with less exhaustion. Greater team culture was associated with less exhaustion among staff. However, unlike for clinicians, team structure failed to predict exhaustion among staff. Fostering team culture may be an important strategy to protect against exhaustion in primary care and enhance the benefit of tight team structures.

Mineralization and Expression of Col1a1-3.6GFP Transgene in Primary Dental Pulp Culture

PubMed Central

Balic, Anamaria; Rodgers, Barbara; Mina, Mina

2008-01-01

We have examined and compared the effects of various differentiation-inducing media on mineralization, cell morphology and expression of pOBCol3.6GFP (3.6-GFP) in primary dental pulp cultures derived from 3.6-GFP transgenic mice. Our results show that media containing ascorbic acid only could not induce mineralization in primary dental pulp cultures. On the other hand, media containing ascorbic acid and β-glycerophosphate induced formation of mineralized matrix-containing dentin. The amount of mineralized matrix was increased by addition of dexamethasone. Cells treated with ascorbic acid and β-glycerophosphate were fibroblast like and cells treated with dexamethasone were cuboidal. In all culture conditions, high levels of 3.6-GFP were expressed in areas of mineralization PMID:18781059

Examining School Culture in Flemish and Chinese Primary Schools

ERIC Educational Resources Information Center

Zhu, Chang; Devos, Geert; Tondeur, Jo

2014-01-01

The aim of this research is to gain understanding about school culture characteristics of primary schools in the Flemish and Chinese context. The study was carried out in Flanders (Belgium) and China, involving a total of 44 Flemish schools and 40 Chinese schools. The School Culture Scales were used to measure five school culture dimensions with…

Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

PubMed

Ziemińska, Elżbieta; Strużyńska, Lidia

2016-02-01

Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

Predicting Organizational Commitment from Organizational Culture in Turkish Primary Schools

ERIC Educational Resources Information Center

Ipek, Cemalettin

2010-01-01

This study aims to describe organizational culture and commitment and to predict organizational commitment from organizational culture in Turkish primary schools. Organizational Culture Scale (Ipek "1999") and Organizational Commitment Scale (Balay "2000") were used in the data gathering process. The data were collected from…

A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency

PubMed Central

Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

2015-01-01

Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose–asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes. PMID:25848252

[Isolation, purification and primary culture of rat pancreatic beta-cells].

PubMed

Liu, Yu-Pu; Lü, Qing-Guo; Tong, Nan-Wei

2009-01-01

To isolate and purify rat pancreatic beta-cells and to explore the best conditions for the primary culture of the pancreatic beta-cells in vitro. The pancreas of Norman Wistar rats were digested by collagenase V. The islets were purified by mesh sieve. The activity of the islets was stimulated by different concentrations of glucose and detected by dithizone dye. The purified islets were put into RPMI-1640 nutritive medium for culture overnight. The cultured islets were digested again with trypsin and DNAase to obtain the suspension containing single pancreatic cells. The beta-cells were separated and purified in a fluorescence-activated cell sorter (FACS) in the medium containing 2.8 mmol/L glucose. The purified beta-cells were identified by immunohistochemistry and glucose stimulating test. Ham's F-10 with different concentrations of glucose and 3-Isobutyl-1-methylxanthine (IBMX) were used as nutritive medium for the primary cell culture for 24 hours. The best conditions for the culture were identified. An average of 550 +/- 90 islets with fine activities were obtained per rat. The purification with FACS obtained about 5688 beta-cells per rat, with a recovery rate of (93.69 +/- 1.26)% and a purity of (85.5 +/- 1.24)%. A concentration of 10.0 mmol/L and 16.0 mmol/L glucose in primary culture for 24 hours produced the highest survival rates of beta-cells, but IBMX did not increase the survival rates of beta-cells. FACS is effective in purifying pancreatic beta-cells from the suspension with a medium containing 2.8 mmol/L glucose. Pancreatic beta-cells maintain relatively high activities in Ham's F-10 medium containing 10.0-16.0 mmol/L glucose in primary culture.

Improving Patient Safety Culture in Primary Care: A Systematic Review.

PubMed

Verbakel, Natasha J; Langelaan, Maaike; Verheij, Theo J M; Wagner, Cordula; Zwart, Dorien L M

2016-09-01

Patient safety culture, described as shared values, attitudes and behavior of staff in a health-care organization, gained attention as a subject of study as it is believed to be related to the impact of patient safety improvements. However, in primary care, it is yet unknown, which effect interventions have on the safety culture. To review literature on the use of interventions that effect patient safety culture in primary care. Searches were performed in PubMed, EMBASE, CINAHL, and PsychINFO on March 4, 2013. Terms defining safety culture were combined with terms identifying intervention and terms indicating primary care. Inclusion followed if the intervention effected patient safety culture, and effect measures were reported. The search yielded 214 articles from which two were eligible for inclusion. Both studies were heterogeneous in their interventions and outcome; we present a qualitative summary. One study described the implementation of an electronic medical record system in general practices as part of patient safety improvements. The other study facilitated 2 workshops for general practices, one on risk management and another on significant event audit. Results showed signs of improvement, but the level of evidence was low because of the design and methodological problems. These studies in general practice provide a first understanding of improvement strategies and their effect in primary care. As the level of evidence was low, no clear preference can be determined. Further research is needed to help practices make an informed choice for an intervention.

[A method for the primary culture of fibroblasts isolated from human airway granulation tissues].

PubMed

Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu-ling; Pei, Ying-hua

2013-04-01

To establish a feasible method to culture primary fibroblasts isolated from human airway granulation tissues, and therefore to provide experimental data for the investigation of the pathogenesis of benign airway stenosis. The granulation tissues were collected from 6 patients during routine bronchoscopy at our department of Beijing Tiantan Hospital from April to June 2011. Primary fibroblasts were obtained by culturing the explanted tissues. Cell growth was observed under inverted microscope. All of these 6 primary cultures were successful. Fibroblast-like cells were observed to migrate from the tissue pieces 3 d after inoculation. After 9-11 d of culture, cells reached to 90% confluence and could be sub-cultured. After passage, the cells were still in a typical elongated spindle-shape and grew well. The cells could be sub-cultured further when they formed a monolayer. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissues.

Grounding Signs of Culture: Primary Intersubjectivity in Social Semiosis

ERIC Educational Resources Information Center

Cowley, Stephen J.; Moodley, Sheshni; Fiori-Cowley, Agnese

2004-01-01

The article examines how infants are first permeated by culture. Building on Thibault (2000), semiogenesis is traced to the joint activity of primary intersubjectivity. Using an African example, analysis shows how--at 14 weeks--an infant already uses culturally specific indicators of "what a caregiver wants." Human predispositions and…

Generation of organotypic raft cultures from primary human keratinocytes.

PubMed

Anacker, Daniel; Moody, Cary

2012-02-22

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)(1). The life cycle of HPV is tightly linked to the differentiation of squamous epithelium(2). Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production(3,4,5). In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras(6) and modified by Kopan et al.(7), the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies(8). Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as

A Microfluidic Interface for the Culture and Sampling of Adiponectin from Primary Adipocytes

PubMed Central

Godwin, Leah A.; Brooks, Jessica C.; Hoepfner, Lauren D.; Wanders, Desiree; Judd, Robert L.; Easley, Christopher J.

2014-01-01

Secreted from adipose tissue, adiponectin is a vital endocrine hormone that acts in glucose metabolism, thereby establishing its crucial role in diabetes, obesity, and other metabolic disease states. Insulin exposure to primary adipocytes cultured in static conditions has been shown to stimulate adiponectin secretion. However, conventional, static methodology for culturing and stimulating adipocytes falls short of truly mimicking physiological environments. Along with decreases in experimental costs and sample volume, and increased temporal resolution, microfluidic platforms permit small-volume flowing cell culture systems, which more accurately represent the constant flow conditions through vasculature in vivo. Here, we have integrated a customized primary tissue culture reservoir into a passively operated microfluidic device made of polydimethylsiloxane (PDMS). Fabrication of the reservoir was accomplished through unique PDMS “landscaping” above sampling channels, with a design strategy targeted to primary adipocytes to overcome issues of positive cell buoyancy. This reservoir allowed three-dimensional culture of primary murine adipocytes, accurate control over stimulants via constant perfusion, and sampling of adipokine secretion during various treatments. As the first report of primary adipocyte culture and sampling within microfluidic systems, this work sets the stage for future studies in adipokine secretion dynamics. PMID:25423362

Inhibitory effect of mitomycin C on proliferation of primary cultured fibroblasts from human airway granulation tissues.

PubMed

Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu Ling; Pei, Ying Hua

2013-01-01

Airway granulation tissue and scar formation pose a challenge because of the high incidence of recurrence after treatment. As an emerging treatment modality, topical application of mitomycin C has potential value in delaying the recurrence of airway obstruction. Several animal and clinical studies have already proven its feasibility and efficacy. However, the ideal dosage has still not been determined. To establish a novel method for culturing primary fibroblasts isolated from human airway granulation tissue, and to investigate the dose-effect of mitomycin C on the fibroblast proliferation in vitro, so as to provide an experimental reference for clinical practitioners. Granulation tissues were collected during the routine bronchoscopy at our department. The primary fibroblasts were obtained by culturing the explanted tissues. The cells were treated with different concentrations of mitomycin C (0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml) for 5 min followed by additional 48-hour culture before an MTT assay was performed to measure cell viability. MTT assay showed that mitomycin C reduced cell viability at all tested concentrations. The inhibitory ratios were 10.26, 26.77, 32.88, 64.91 and 80.45% for cells treated with mitomycin C at 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml, respectively. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissue, and mitomycin C can inhibit proliferation of the fibroblasts in vitro. Copyright © 2013 S. Karger AG, Basel.

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

PubMed

Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

2012-12-01

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

Primary culture of human Schwann and schwannoma cells: improved and simplified protocol.

PubMed

Dilwali, Sonam; Patel, Pratik B; Roberts, Daniel S; Basinsky, Gina M; Harris, Gordon J; Emerick, Kevin S; Stankovic, Konstantina M

2014-09-01

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. Copyright © 2014 Elsevier B.V. All rights reserved.

Primary culture of human Schwann and schwannoma cells: Improved and simplified protocol

PubMed Central

Dilwali, Sonam; Patel, Pratik B.; Roberts, Daniel S.; Basinsky, Gina M.; Harris, Gordon J.; Emerick, Kevin; Stankovic, Konstantina M.

2014-01-01

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. PMID:24910344

Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

PubMed

Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

2012-08-15

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated

Neuroprotective effect of rotigotine against complex I inhibitors, MPP⁺ and rotenone, in primary mesencephalic cell culture.

PubMed

Radad, Khaled; Scheller, Dieter; Rausch, Wolf-Dieter; Reichmann, Heinz; Gille, Gabrielle

2014-01-01

Dopamine agonists are suggested to be more efficacious in treating Parkinson's disease (PD) as they have neuroprotective properties in addition to their receptor-related actions. The present study was designed to investigate the neuroprotective effects of rotigotine, a D3/D2/D1 dopamine receptor agonist, against the two powerful complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+) and rotenone, in primary mesencephalic cell culture relevant to PD. Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala at gestation day 14. Three sets of cultures were treated with rotigotine alone, rotigotine and MPP⁺, and rotigotine and rotenone to investigate the effect of rotigotine on the survival of dopaminergic neurons against age-, MPP⁺- and rotenone-induced cell death. At the end of each treatment, cultures were fixed and stained immunohistochemically against tyrosine hydroxylase (TH). The effect of rotigotine against rotenone-induced reactive oxygen species (ROS) production was measured using CH-H2DCFDA fluorescence dye. Rotigotine alone did not influence the survival of tyrosine hydroxylase immunoreactive (THir) neurons except at 10 µM, it significantly decreased the number of THir neurons by 40% compared to untreated controls. Treatment of cultures with 0.01 µM rotigotine rescued 10% of THir neurons against MPP⁺-induced cell death. Rotigotine was also found to significantly rescue 20% of THir neurons at 0.01 µM of rotenone-treated cultures. Using of CH-H2DCFDA fluorescence dye, it was found that rotigotine significantly attenuated ROS production compared to rotenone-treated cultures. Rotigotine provides minor protection against MPP⁺ and rescues a significant number of THir neurons against rotenone in primary mesencephalic cell cultures relevant to PD.

A Multi-Cultural Interaction through Video Conferencing in Primary Schools

ERIC Educational Resources Information Center

Duygu Eristi, Suzan

2012-01-01

This study investigated Turkish and Canadian primary school students' ways of expressing their perception of cultural understanding through video conferencing and that of cultural interaction through video conferencing. The qualitative research data were collected in the form of interviews. The results obtained were analyzed and interpreted based…

Clinicians' interpretations of point of care urine culture versus laboratory culture results: analysis from the four-country POETIC trial of diagnosis of uncomplicated urinary tract infection in primary care.

PubMed

Hullegie, Saskia; Wootton, Mandy; Verheij, Theo J M; Thomas-Jones, Emma; Bates, Janine; Hood, Kerenza; Gal, Micaela; Francis, Nick A; Little, Paul; Moore, Michael; Llor, Carl; Pickles, Timothy; Gillespie, David; Kirby, Nigel; Brugman, Curt; Butler, Christopher C

2017-08-01

Urine culture at the point of care minimises delay between obtaining the sample and agar inoculation in a microbiology laboratory, and quantification and sensitivity results can be available more rapidly in primary care. To identify the degree to which clinicians' interpretations of a point-of-care-test (POCT) urine culture (Flexicult™ SSI-Urinary Kit) agrees with laboratory culture in women presenting to primary care with symptoms of uncomplicated urinary tract infections (UTI). Primary care clinicians used the Flexicult™-POCT, recorded their findings and took a photograph of the result, which was interpreted by microbiology laboratory technicians. Urine samples were additionally processed in routine care laboratories. Cross tabulations were used to identify important differences in organism identification, quantification and antibiotic susceptibility between these three sources of data. The influence of various laboratory definitions for UTI on culture were assessed. Primary care clinicians identified 202/289 urine samples (69.9%) as positive for UTI using the Flexicult™-POCT, whereas laboratory culture identified 94-190 (32.5-65.7%) as positive, depending on definition thresholds. 82.9% of samples identified positive for E. coli on laboratory culture were also considered positive for E. coli using the Flexicult™ -POCT, and susceptibilities were reasonably concordant. There were major discrepancies between laboratory staff interpretation of Flexicult™ photographs, clinicians' interpretation of the Flexicult™ test, and laboratory culture results. Flexicult™-POCT overestimated the positivity rate of urine samples for UTI when laboratory culture was used as the reference standard. However, it is unclear whether point-of-care or laboratory based urine culture provides the most valid diagnostic information. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

EPA Science Inventory

Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

Ion transport by primary cultures of canine tracheal epithelium: methodology, morphology, and electrophysiology.

PubMed

Welsh, M J

1985-01-01

Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of C1 secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of C1 secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of C1 secretion by airway epithelia.

Relationship among team dynamics, care coordination and perception of safety culture in primary care.

PubMed

Blumenthal, Karen J; Chien, Alyna T; Singer, Sara J

2018-05-18

There remains a need to improve patient safety in primary care settings. Studies have demonstrated that creating high-performing teams can improve patient safety and encourage a safety culture within hospital settings, but little is known about this relationship in primary care. To examine how team dynamics relate to perceptions of safety culture in primary care and whether care coordination plays an intermediating role. This is a cross-sectional survey study with 63% response (n = 1082). The study participants were attending clinicians, resident physicians and other staff who interacted with patients from 19 primary care practices affiliated with Harvard Medical School. Three domains corresponding with our main measures: team dynamics, care coordination and safety culture. All items were measured on a 5-point Likert scale. We used linear regression clustered by practice site to assess the relationship between team dynamics and perceptions of safety culture. We also performed a mediation analysis to determine the extent to which care coordination explains the relationship between perceptions of team dynamics and of safety culture. For every 1-point increase in overall team dynamics, there was a 0.76-point increase in perception of safety culture [95% confidence interval (CI) 0.70-0.82, P < 0.001]. Care coordination mediated the relationship between team dynamics and the perception of safety culture. Our findings suggest there is a relationship between team dynamics, care coordination and perceptions of patient safety in a primary care setting. To make patients safer, we may need to pay more attention to how primary care providers work together to coordinate care.

Creativity and Performativity Policies in Primary School Cultures

ERIC Educational Resources Information Center

Troman, Geoff; Jeffrey, Bob; Raggl, Andrea

2007-01-01

Cultures of performativity in English primary schools refer to systems and relationships of: target-setting; Ofsted inspections; school league tables constructed from pupil test scores; performance management; performance related pay; threshold assessment; and advanced skills teachers. Systems which demand that teachers "perform" and in…

Measurement tools and process indicators of patient safety culture in primary care. A mixed methods study by the LINNEAUS collaboration on patient safety in primary care.

PubMed

Parker, Dianne; Wensing, Michel; Esmail, Aneez; Valderas, Jose M

2015-09-01

There is little guidance available to healthcare practitioners about what tools they might use to assess the patient safety culture. To identify useful tools for assessing patient safety culture in primary care organizations in Europe; to identify those aspects of performance that should be assessed when investigating the relationship between safety culture and performance in primary care. Two consensus-based studies were carried out, in which subject matter experts and primary healthcare professionals from several EU states rated (a) the applicability to their healthcare system of several existing safety culture assessment tools and (b) the appropriateness and usefulness of a range of potential indicators of a positive patient safety culture to primary care settings. The safety culture tools were field-tested in four countries to ascertain any challenges and issues arising when used in primary care. The two existing tools that received the most favourable ratings were the Manchester patient safety framework (MaPsAF primary care version) and the Agency for healthcare research and quality survey (medical office version). Several potential safety culture process indicators were identified. The one that emerged as offering the best combination of appropriateness and usefulness related to the collection of data on adverse patient events. Two tools, one quantitative and one qualitative, were identified as applicable and useful in assessing patient safety culture in primary care settings in Europe. Safety culture indicators in primary care should focus on the processes rather than the outcomes of care.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

PubMed

Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

2018-02-20

Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

Cultural democracy: the way forward for primary care of hard to reach New Zealanders.

PubMed

Finau, Sitaleki A; Finau, Eseta

2007-09-01

The use of cultural democracy, the freedom to practice one's culture without fear, as a framework for primary care service provision is essential for improved health service in a multi cultural society like New Zealand. It is an effective approach to attaining health equity for all. Many successful health ventures are ethnic specific and have gone past cultural competency to the practice of cultural democracy. That is, the services are freely taking on the realities of clients without and malice from those of other ethnicities. In New Zealand the scientific health service to improve the health of a multi cultural society are available but there is a need to improve access and utilization by hard to reach New Zealanders. This paper discusses cultural democracy and provide example of how successful health ventures that had embraced cultural democracy were implemented. It suggests that cultural democracy will provide the intellectual impetus and robust philosophy for moving from equality to equity in health service access and utilization. This paper would provide a way forward to improved primary care utilization, efficiency, effectiveness and equitable access especially for the hard to reach populations. use the realities of Pacificans in New Zealand illustrate the use of cultural democracy, and thus equity to address the "inverse care law" of New Zealand. The desire is for primary care providers to take cognizance and use cultural democracy and equity as the basis for the design and practice of primary health care for the hard to reach New Zealanders.

Humanized medium (h7H) allows long-term primary follicular thyroid cultures from human normal thyroid, benign neoplasm, and cancer.

PubMed

Bravo, Susana B; Garcia-Rendueles, Maria E R; Garcia-Rendueles, Angela R; Rodrigues, Joana S; Perez-Romero, Sihara; Garcia-Lavandeira, Montserrat; Suarez-Fariña, Maria; Barreiro, Francisco; Czarnocka, Barbara; Senra, Ana; Lareu, Maria V; Rodriguez-Garcia, Javier; Cameselle-Teijeiro, Jose; Alvarez, Clara V

2013-06-01

Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor. The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype. Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed. We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas. Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.

Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

PubMed Central

Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

2016-01-01

BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when

Rapid selection of mesenchymal stem and progenitor cells in primary prostate stromal cultures.

PubMed

Brennen, W Nathaniel; Kisteman, L Nelleke; Isaacs, John T

2016-05-01

Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping

Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research.

PubMed

Ray, Balmiki; Chopra, Nipun; Long, Justin M; Lahiri, Debomoy K

2014-09-16

Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.

Measurement tools and process indicators of patient safety culture in primary care. A mixed methods study by the LINNEAUS collaboration on patient safety in primary care

PubMed Central

Parker, Dianne; Wensing, Michel; Esmail, Aneez; Valderas, Jose M

2015-01-01

ABSTRACT Background: There is little guidance available to healthcare practitioners about what tools they might use to assess the patient safety culture. Objective: To identify useful tools for assessing patient safety culture in primary care organizations in Europe; to identify those aspects of performance that should be assessed when investigating the relationship between safety culture and performance in primary care. Methods: Two consensus-based studies were carried out, in which subject matter experts and primary healthcare professionals from several EU states rated (a) the applicability to their healthcare system of several existing safety culture assessment tools and (b) the appropriateness and usefulness of a range of potential indicators of a positive patient safety culture to primary care settings. The safety culture tools were field-tested in four countries to ascertain any challenges and issues arising when used in primary care. Results: The two existing tools that received the most favourable ratings were the Manchester patient safety framework (MaPsAF primary care version) and the Agency for healthcare research and quality survey (medical office version). Several potential safety culture process indicators were identified. The one that emerged as offering the best combination of appropriateness and usefulness related to the collection of data on adverse patient events. Conclusion: Two tools, one quantitative and one qualitative, were identified as applicable and useful in assessing patient safety culture in primary care settings in Europe. Safety culture indicators in primary care should focus on the processes rather than the outcomes of care. PMID:26339832

An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.

PubMed

Li, Xingnan; Ootani, Akifumi; Kuo, Calvin

2016-01-01

Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

Nurses' perceptions of workplace culture in primary health care in Finland.

PubMed

Hahtela, N; Paavilainen, E; McCormack, B; Helminen, M; Slater, P; Suominen, T

2015-12-01

This study aimed to describe nurses' perceptions of workplace culture, especially in regard to stress levels, job satisfaction and the practice environment in primary health care. Health care is facing many challenges related to its attractiveness as a place of employment and the maintenance of a sufficient workforce supply. Previous studies report increasing rates of nurse job dissatisfaction and intentions to leave their current positions both in Finland and also globally. Improving workplace culture is thus vital in meeting the challenges related to recruitment and retention. A cross-sectional descriptive design was used to describe nurses' perceptions of workplace culture. Data were collected by questionnaire from 22 units in nine primary healthcare organizations in Finland, and analysed using descriptive and inferential statistics. Most of the respondents indicated that they were not certain whether their workplace culture was either positive or negative. Profession, age and work shift characteristics had an effect on the respondents' perceptions of workplace culture. Younger licensed practical and registered nurses assessed their workplace culture more positively, whereas older registered nurses and those working rotating rosters viewed workplace culture more negatively. The findings suggest that both unit and demographic characteristics affect workplace culture. This survey highlights that a positive workplace culture is one of the key factors in retaining and recruiting nurses, and provides an essential evidence that may be considered by other healthcare organizations. Nurse managers and healthcare leaders need to address workload management and take into account the related variables that affect a unit's workplace culture. © 2015 International Council of Nurses.

Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense

NASA Astrophysics Data System (ADS)

Wang, Changliu; Zhang, Shicui; Su, Feng; Wang, Lei; Li, Hongyan

2009-02-01

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.

Organizational culture predicts job satisfaction and perceived clinical effectiveness in pediatric primary care practices.

PubMed

Brazil, Kevin; Wakefield, Dorothy B; Cloutier, Michelle M; Tennen, Howard; Hall, Charles B

2010-01-01

In recent years, there has been a growing understanding that organizational culture is related to an organization's performance. However, few studies have examined organizational culture in medical group practices. The purpose of this study was to examine the relationship of organizational culture on provider job satisfaction and perceived clinical effectiveness in primary care pediatric practices. This cross-sectional study included 36 primary care pediatric practices located in Connecticut. There were 374 participants in this study, which included 127 clinicians and 247 nonclinicians. Office managers completed a questionnaire that recorded staff and practice characteristics; all participants completed the Organizational Culture Scale, a questionnaire that assessed the practice on four cultural domains (i.e., group, developmental, rational, and hierarchical), and the Primary Care Organizational Questionnaire that evaluated perceived effectiveness and job satisfaction. Hierarchical linear models using a restricted maximum likelihood estimation method were used to evaluate whether the practice culture types predicted job satisfaction and perceived effectiveness. Group culture was positively associated with both satisfaction and perceived effectiveness. In contrast, hierarchical and rational culture were negatively associated with both job satisfaction and perceived effectiveness. These relationships were true for clinicians, nonclinicians, and the practice as a whole. Our study demonstrates that practice culture is associated with job satisfaction and perceived clinical effectiveness and that a group culture was associated with high job satisfaction and perceived effectiveness.

Decursinol and decursin protect primary cultured rat cortical cells from glutamate-induced neurotoxicity.

PubMed

Kang, So Young; Kim, Young Choong

2007-06-01

We previously reported six neuroprotective decursinol derivatives, coumarins from Angelica gigas (Umbelliferae) roots. To elucidate the action patterns of decursinol derivatives, we investigated the neuroprotective effects of decursinol and decursin, which showed highly significant activity and were major constituents of A. gigas, using primary cultures of rat cortical cells in-vitro. At concentrations of 0.1-10.0 microM, both decursinol and decursin exerted a significant neuroprotective activity pretreatment and throughout treatment. In addition, decursin had a neuroprotective impact in the post-treatment paradigm implying that decursin might possess different action mechanisms from that of decursinol in the protection of neurons against glutamate injury. Both decursinol and decursin effectively reduced the glutamate-induced increased intracellular calcium ([Ca(2+)](i)) in cortical cells, suggesting that these two coumarins may exert neuroprotection by reducing calcium influx by overactivation of glutamate receptors. This suggestion was supported by the result that decursinol and decursin protected neurons against kainic acid (KA)-induced neurotoxicity better than against that induced by N-methyl-D-aspartate (NMDA). Moreover, both decursinol and decursin significantly prevented glutamate-induced decreases in glutathione, a cellular antioxidant, and glutathione peroxidase activity. In addition, both compounds efficiently reduced the overproduction of cellular peroxide in glutamate-injured cortical cells. These results suggested that both decursinol and decursin protected primary cultured rat cortical cells against glutamate-induced oxidative stress by both reducing calcium influx and acting on the cellular antioxidative defence system. Moreover, decursin is considered to probably have a different action mechanism from that of decursinol in protecting cortical cells against glutamate injury.

Relevant principal factors affecting the reproducibility of insect primary culture.

PubMed

Ogata, Norichika; Iwabuchi, Kikuo

2017-06-01

The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

A soluble biocompatible guanidine-containing polyamidoamine as promoter of primary brain cell adhesion and in vitro cell culturing

NASA Astrophysics Data System (ADS)

Tonna, Noemi; Bianco, Fabio; Matteoli, Michela; Cagnoli, Cinzia; Antonucci, Flavia; Manfredi, Amedea; Mauro, Nicolò; Ranucci, Elisabetta; Ferruti, Paolo

2014-08-01

This paper reports on a novel application of an amphoteric water-soluble polyamidoamine named AGMA1 bearing 4-butylguanidine pendants. AGMA1 is an amphoteric, prevailingly cationic polyelectrolyte with isoelectric point of about 10. At pH 7.4 it is zwitterionic with an average of 0.55 excess positive charges per unit, notwithstanding it is highly biocompatible. In this work, it was found that AGMA1 surface-adsorbed on cell culturing coverslips exhibits excellent properties as adhesion and proliferation promoter of primary brain cells such as microglia, as well as of hippocampal neurons and astrocytes. Microglia cells cultured on AGMA1-coated coverslips substrate displayed the typical resting, ramified morphology of those cultured on poly-L-lysine and poly-L-ornithine, employed as reference substrates. Mixed cultures of primary astrocytes and neuronal cells grown on AGMA1- and poly-L-lysine coated coverslips were morphologically undistinguishable. On both substrates, neurons differentiated axon and dendrites and eventually established perfectly functional synaptic contacts. Quantitative immunocytochemical staining revealed no difference between AGMA1 and poly-L-lysine. Electrophysiological experiments allowed recording neuron spontaneous activity on AGMA1. In addition, cell cultures on both AGMA1 and PLL displayed comparable excitatory and inhibitory neurotransmission, demonstrating that the synaptic contacts formed were fully functional.

Eyewitness Culture and History: Primary Written Sources. The Iconoclast.

ERIC Educational Resources Information Center

McMurtry, John

1995-01-01

Asserts that contemporary history and historiography is "official" history that ignores the daily struggles of people for their continued survival. Argues that, while public illiteracy has nearly disappeared, individuals are ignorant of the wealth of primary-source materials of other cultures' histories. (CFR)

Long-term primary culture of mouse mammary tumor cells: production of virus.

PubMed

Young, L J; Cardiff, R D; Ashley, R L

1975-05-01

Long-term primary cultures of mouse mammary tumor cells proved an excellent source of mouse mammary tumor virus (MMTV). Virus purified from these primary cultures had the same morphologic biochemical, immunologic, and biologic characteristics as MMTV. Quantitation of MMTV-protein equivalents released into the medium was measured by the radioimmunoassay for MMTV. Peak production levels were 20-40 mug MMTV protien equivalents/75-cm-2 flask/24 hours. These cultures produced MMTV for as long as 90 days. MMTV cultivation depended on the initial cell-plating density and hormones. Maximal MMTV release was obtained at a plating density of 1 times 10-6 cells/cm-2 in the presence of insulin and hydrocortisone. Insulin alone gave basal levels of MMTV, and hydrocortisone alone increased MMTV release only three-fold, but insulin and hydrocortisone together effected an eightfold increase in MMTV release. This suggested that hydrocortisone had a primary effect on MMTV release and insulin acted synergistically with hydrocortisone to maximize MMTV release.

The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus.

PubMed

Zhou, Jingxiang; Wang, Hao; Zhu, Xia; Li, Xingwei; Lv, Wenliang; Zhang, Dongming

2013-10-01

The explosive Koi herpesvirus (KHV) epidemic has caused the deaths of a large number of carp and carp variants and has produced serious economic losses. The mirror carp (Cyprinus carpio var. specularis) exhibits strong environmental adaptability and its primary cells can be used to isolate KHV. This study utilized the tissue explant method to systematically investigate primary cell culture conditions for mirror carp snout and caudal fin tissues. We demonstrated that cells from these two tissue types had strong adaptability, and when cultured in Medium 199 (M199) containing 20% serum at 26 to 30°C, the cells from the snout and caudal fin tissues exhibited the fastest egress and proliferation. Inoculation of these two cell types with KHV-infected fish kidney tissues produced typical cytopathic effects; additionally, identification by electron microscopy, and PCR indicated that KHV could be isolated from both cell types.

Long-enduring primary hepatocyte-based co-cultures improve prediction of hepatotoxicity.

PubMed

Novik, Eric I; Dwyer, Jacquelyn; Morelli, James K; Parekh, Amit; Cho, Cheul; Pludwinski, Eitan; Shrirao, Anil; Freedman, Robert M; MacDonald, James S; Jayyosi, Zaid

2017-12-01

The failure of drug candidates during clinical trials and post-marketing withdrawal due to Drug Induced Liver Injury (DILI), results in significant late-stage attrition in the pharmaceutical industry. Animal studies have proven insufficient to definitively predict DILI in the clinic, therefore a variety of in vitro models are being tested in an effort to improve prediction of human hepatotoxicity. The model system described here consists of cryopreserved primary rat, dog or human hepatocytes co-cultured together with a fibroblast cell line, which aids in the hepatocytes' maintenance of more in vivo-like characteristics compared to traditional hepatic mono-cultures, including long term viability and retention of activity of cytochrome P450 isozymes. Cell viability was assessed by measurement of ATP following treatment with 29 compounds having known hepatotoxic liabilities. Hμrelrat™, Hμreldog™, and Hμrelhuman™ hepatic co-cultures were treated for 24h, or under repeat-dosing for 7 or 13days, and compared to rat and human hepatic mono-cultures following single-dose exposure for 24h. The results allowed for a comparison of cytotoxicity, species-specific responses and the effect of repeat compound exposure on the prediction of hepatotoxic potential in each model. Results show that the co-culture model had greater sensitivity compared to that of the hepatic mono-cultures. In addition, "time-based ratios" were determined by dividing the compounds' 24-hour TC 50 /C max values by TC 50 /C max values measured after dosing for either 7 or 13days. The results suggest that this approach may serve as a useful adjunct to traditional measurements of hepatotoxicity, improving the predictive value of early screening studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

PubMed

Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

2016-09-06

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

LIVER REGENERATION STUDIES WITH RAT HEPATOCYTES IN PRIMARY CULTURE

EPA Science Inventory

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr...

Preparation of Human Primary Colon Tissue-Derived Organoid Using Air Liquid Interface Culture.

PubMed

Usui, Tatsuya; Sakurai, Masashi; Umata, Koji; Yamawaki, Hideyuki; Ohama, Takashi; Sato, Koichi

2018-02-21

In vitro analysis of intestinal epithelium has been hindered by a lack of suitable culture systems useful for gastrointestinal research. To overcome the problem, an air liquid interface (ALI) method using a collagen gel was established to culture three-dimensional primary cells containing both primary epithelial and mesenchymal components from mouse gastrointestinal tissues. ALI organoids accurately recapitulate organ structures, multilineage differentiation, and physiology. Since ALI organoids from human tissues have not been produced, we modified the previous protocol for mouse ALI organoid culture to establish the culture system of ALI organoids from normal and tumor colorectal tissues of human patients. The current unit presents a protocol for preparation of the ALI organoid culture from normal and tumor colorectal tissues of human patients. ALI organoid culture from human tissues might be useful for examining not only resistance to chemotherapy in a tumor microenvironment but also toxic effects on organoids. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

PubMed

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-05-10

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats

PubMed Central

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-01-01

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function. PMID:28489060

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level.

PubMed

Ventura, P; Toullec, G; Fricano, C; Chapron, L; Meunier, V; Röttinger, E; Furla, P; Barnay-Verdier, S

2018-04-01

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5 and + 8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a + 8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.

Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

PubMed Central

Kierszenbaum, A L; Spruill, W A; White, M G; Tres, L L; Perkins, J P

1985-01-01

Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule. Images PMID:2984678

Gelatin for purification and proliferation of primary keratinocyte culture for use in chronic wounds and burns.

PubMed

Rahsaz, Marjan; Geramizadeh, Bita; Kaviani, Maryam; Marzban, Saeed

2015-04-01

Human epidermal keratinocytes are currently established as a treatment for burns and wounds and have laboratory applications. Keratinocyte culture contamination by unwanted cells and inhibition of cell proliferation are barriers in primary keratinocyte culture. According to the recent literature, these cells are hard to culture. The present study was conducted to evaluate the efficacy of gelatin-coated surfaces in keratinocyte cultures. After enzymatic isolation of keratinocytes from normal epidermis by trypsin, the cells were cultured on gelatin-coated flasks in serum-free medium. Another group of cells were cultured as a control group without gelatin coating. We showed positive effects of surface coating with gelatin on the primary culture of keratinocytes. Culture of these cells on a gelatincoated surface showed better proliferation with suitable morphology. By using gelatin, adhesion of these cells to the surface was more efficient and without contamination by small round cells. Successful primary culture of keratinocytes on a gelatin-coated surface may provide better yield and optimal number of cells for research and clinical applications.

Measuring safety culture in Dutch primary care: psychometric characteristics of the SCOPE-PC questionnaire.

PubMed

Verbakel, Natasha J; Zwart, Dorien L M; Langelaan, Maaike; Verheij, Theo J M; Wagner, Cordula

2013-09-17

Patient safety has been a priority in primary healthcare in the last years. The prevailing culture is seen as an important condition for patient safety in practice and several tools to measure patient safety culture have therefore been developed. Although Dutch primary care consists of different professions, such as general practice, dental care, dietetics, physiotherapy and midwifery, a safety culture questionnaire was only available for general practices. The purpose of this study was to modify and validate this existing questionnaire to a generic questionnaire for all professions in Dutch primary care. A validated Dutch questionnaire for general practices was modified to make it usable for all Dutch primary care professions. Subsequently, this questionnaire was administered to a random sample of 2400 practices from eleven primary care professions. The instrument's factor structure, reliability and validity were examined using confirmatory and explorative factor analyses. 921 questionnaires were returned. Of these, 615 were eligible for factor analysis. The resulting SCOPE-PC questionnaire consisted of seven dimensions: 'open communication and learning from errors', 'handover and teamwork', 'adequate procedures and working conditions', 'patient safety management', 'support and fellowship', 'intention to report events' and 'organisational learning' with a total of 41 items. All dimensions had good reliability with Cronbach's alphas ranging from 0.70-0.90, and the questionnaire had a good construct validity. The SCOPE-PC questionnaire has sound psychometric characteristics for use by the different professions in Dutch primary care to gain insight in their safety culture.

Rotigotine protects against glutamate toxicity in primary dopaminergic cell culture.

PubMed

Oster, Sandra; Radad, Khaled; Scheller, Dieter; Hesse, Marlen; Balanzew, Wladimir; Reichmann, Heinz; Gille, Gabriele

2014-02-05

In Parkinson disease the degeneration of dopaminergic neurones is believed to lead to a disinhibition of the subthalamic nucleus thus increasing the firing rate of the glutamatergic excitatory projections to the substantia nigra. In consequence, excessive glutamatergic activity will cause excitotoxicity and oxidative stress. In the present study we investigated mechanisms of glutamate toxicity and the neuroprotective potential of the dopamine agonist rotigotine towards dopaminergic neurones in mouse mesencephalic primary culture. Glutamate toxicity was mediated by the N-methyl-d-aspartic acid (NMDA) receptor and accompanied by a strong calcium influx into dopaminergic neurones for which the L-type voltage-sensitive calcium channels play an important role. The rate of superoxide production in the culture was highly increased. Deleterious nitric oxide production did not participate in glutamate-mediated excitotoxicity. Pretreatment of cultures with rotigotine significantly increased the survival of dopaminergic neurones exposed to glutamate. Rotigotine exerted its protective effects via dopamine receptor stimulation (presumably via dopamine D3 receptor) and decreased significantly the production of superoxide radicals. When cultures were preincubated with Phosphoinositol 3-Kinase (PI3K) inhibitors the protective effect of rotigotine was abolished suggesting a decisive role of the PI3K/Akt pathway in rotigotine-mediated neuroprotection. Consistently, exposure to rotigotine induced the activation of Akt by phosphorylation followed by phosphorylation, and thus inactivation, of the pro-apoptotic factor glycogen synthase kinase-3-beta (GSK-3-β). Taken together, our work contributed to elucidating the mechanisms of glutamate toxicity in mesencephalic culture and unravelled the signalling pathways associated with rotigotine-induced neuroprotection against glutamate toxicity in primary dopaminergic cultures. Copyright © 2013 Elsevier B.V. All rights reserved.

The terminator mouse: salvation for primary cell culture.

PubMed

Kabgani, Nazanin; Moeller, Marcus J

2013-11-01

The Terminator had to come back from the future already several times in an effort to bring salvation to mankind. In the present issue of Kidney International, Guo et al. brought us a novel transgenic mouse model: the terminator mouse. This highly elegant mouse may facilitate significantly the derivation of primary cultures of a specific cell type from a tissue containing multiple cell populations.

Creative Partnerships? Cultural Policy and Inclusive Arts Practice in One Primary School

ERIC Educational Resources Information Center

Hall, Christine; Thomson, Pat

2007-01-01

This article traces the "cultural turn" in UK educational policy through an analysis of the Creative Partnerships policy (New Labour's "flagship programme in the cultural education field") and a consideration of an arts project funded under this initiative in one primary school. It argues that current educational policy…

Culture and Sampling of Primary Adipose Tissue in Practical Microfluidic Systems.

PubMed

Brooks, Jessica C; Judd, Robert L; Easley, Christopher J

2017-01-01

Microfluidic culture of primary adipose tissue allows for reduced sample and reagent volumes as well as constant media perfusion of the cells. By continuously flowing media over the tissue, microfluidic sampling systems can more accurately mimic vascular flow in vivo. Quantitative measurements can be performed on or off chip to provide time-resolved secretion data, furthering insight into the dynamics of the function of adipose tissue. Buoyancy resulting from the large lipid storage capacity in this tissue presents a unique challenge for culture, and it is important to account for this buoyancy during microdevice design. Herein, we describe approaches for microfluidic device fabrication that utilize 3D-printed interface templating to help counteract cell buoyancy. We apply such methods to the culture of both isolated, dispersed primary adipocytes and epididymal adipose explants. To facilitate more widespread adoption of the methodology, the devices presented here are designed for user-friendly operation. Only handheld syringes are needed to control flow, and devices are inexpensive and disposable.

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

PubMed

Yamazaki, Taisuke; Wakai, Mariko; Enosawa, Shin; Tokiwa, Takayoshi

2017-06-01

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Hydrocortisone effect of arylsulfatase A in primary mouse brain cell cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcelo, A.; Pieringer, R.A.

The primary goal of this study was to study the mechanism of action of hydrocortisone (HC) on arylsulfatase A (ASA) in primary cultures of cells that were dissociated from the brains of embryonic mice. Cells were cultured in a defined medium in the absence or in the presence of 3 ..mu..M HC. The specific activity of ASA in nontreated cells was 1.297 U/mg (U = ..mu..mol/hr) while the value for the HC-treated cells was 0.783 U/mg. The authors data shows that HC inhibits ASA activity in these cultures cells (p < 0.001). The determination of the ASA enzyme activity wasmore » assayed primarily with the artificial substrate p-nitrocatechol sulfate. However, the natural substrate (cerebroside /sup 35/S-sulfate) also as active and correlated linearly with the activity of p-nitrocatechol sulfate. Purified ASA was isolated from calf brains and used to generate an antibody (Ab) against ASA. The specificity of the Ab for the ASA protein of cell cultures was tested in Ouchterlony double immunodiffusion studies. The Ab was used in a competitive enzyme-linked immunosorbent assay to quantify the number of ASA molecules in the cell extracts from the embryonic mouse cell cultures. Preliminary data suggest that HC decreases the number of ASA molecules.« less

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

PubMed Central

Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

1998-01-01

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

PubMed Central

2011-01-01

Background Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast. Results Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (kLa) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium

Health system factors affecting communication with pediatricians: gendered work culture in primary care.

PubMed

Lynch, Sean

2011-01-01

This qualitative study examined the roles that practice setting, education level, and gender may play in social workers' communication satisfaction with pediatricians. Taking an ethnographic approach, the researcher interviewed social workers and pediatricians who worked together to provide mental health services in primary care. The results suggested that gender at the health system level may be an issue and that gendered work culture in primary care was a factor in communication. In particular, reimbursement, an aspect of the gendered work culture, was a substantial communication barrier, and the implications for Medicaid billing are discussed.

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

PubMed Central

Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

2014-01-01

ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

[Primary culture of human malignant meningioma cells and its intracranial orthotopic transplantation in nude mice].

PubMed

Hu, Mei-Xin; Liu, Jia-le; Chen, Xuan-Bo; Xu, An-Qi; Shu, Song-Ren; Wang, Chao-Hu; Liu, Yi

2018-03-20

To obtain stable primary cultures of human malignant meningioma cells and establish an intracranial in-situ tumor model in nude mice. Ten surgical specimens of highly suspected malignant meningioma were obtained with postoperative pathological confirmation. Primary malignant meningioma cells were cultured from the tissues using a modified method and passaged. After identification with cell immunofluorescence, the cultured cells were inoculated into the right parietal lobe of 6 nude mice using stereotaxic apparatus and also transplanted subcutaneously in another 6 nude mice. The nude mice were executed after 6 weeks, and HE staining and immunohistochmistry were used to detect tumor growth and the invasion of the adjacent brain tissues. The primary malignant meningioma cells were cultured successfully, and postoperative pathology reported anaplastic malignant meningioma. Cell immunofluorescence revealed positivity for vimentin and EMA in the cells, which showed a S-shaped growth curve in culture. Flow cytometry revealed a cell percentage in the Q3 area of (95.99∓2.58)%. Six weeks after transplantation, tumor nodules occurred in the subcutaneous tumor group, and the nude mice bearing the in situ tumor showed obvious body weight loss. The xenografts in both groups contained a mean of (36∓5.35)% cells expressing Ki-67, and the intracranial in situ tumor showed obvious invasion of the adjacent peripheral brain tissues. We obtained stable primary cultures of malignant meningioma cells and successfully established a nude mouse model bearing in situ human malignant meningioma.

Traditional Indigenous Games promoting physical activity and cultural connectedness in primary schools--cluster randomised control trial.

PubMed

Kiran, Asha; Knights, Janice

2010-08-01

This study investigated the effectiveness of Traditional Indigenous Games (TIG) to improve physical activity and cultural connectedness among primary school students in the community renewal areas of Townsville in North Queensland. A cluster randomised control trial was conducted in four primary schools in 2007. Baseline and post implementation surveys were conducted in two intervention and two control schools and the results were compared. TIG delivered in primary schools every week over period of three months did not contribute to any statistically significant improvement in intervention and control groups in physical activity levels or cultural connectedness. Further research specifically in terms of intensity and duration of TIG may inform whether physical activity may be improved. Enhancing the Indigenous cultural features of the existing TIG kit might positively influence Indigenous cultural connectedness.

Primary care resident perceived preparedness to deliver cross-cultural care: an examination of training and specialty differences.

PubMed

Greer, Joseph A; Park, Elyse R; Green, Alexander R; Betancourt, Joseph R; Weissman, Joel S

2007-08-01

Previous research has shown that resident physicians report differences in training across primary care specialties, although limited data exist on education in delivering cross-cultural care. The goals of this study were to identify factors that relate to primary care residents' perceived preparedness to provide cross-cultural care and to explore the extent to which these perceptions vary across primary care specialties. Cross-sectional, national mail survey of resident physicians in their last year of training. Eleven hundred fifty primary care residents specializing in family medicine (27%), internal medicine (23%), pediatrics (26%), and obstetrics/gynecology (OB/GYN) (24%). Male residents as well as those who reported having graduated from U.S. medical schools, access to role models, and a greater cross-cultural case mix during residency felt more prepared to deliver cross-cultural care. Adjusting for these demographic and clinical factors, family practice residents were significantly more likely to feel prepared to deliver cross-cultural care compared to internal medicine, pediatric, and OB/GYN residents. Yet, when the quantity of instruction residents reported receiving to deliver cross-cultural care was added as a predictor, specialty differences became nonsignificant, suggesting that training opportunities better account for the variability in perceived preparedness than specialty. Across primary care specialties, residents reported different perceptions of preparedness to deliver cross-cultural care. However, this variation was more strongly related to training factors, such as the amount of instruction physicians received to deliver such care, rather than specialty affiliation. These findings underscore the importance of formal education to enhance residents' preparedness to provide cross-cultural care.

Changes in Inward Rectifier K+ Channels in Hepatic Stellate Cells During Primary Culture

PubMed Central

Lee, Dong Hyeon; Kong, In Deok; Lee, Joong-Woo

2008-01-01

Purpose This study examined the expression and function of inward rectifier K+ channels in cultured rat hepatic stellate cells (HSC). Materials and Methods The expression of inward rectifier K+ channels was measured using real-time RT-PCR, and electrophysiological properties were determined using the gramicidin-perforated patch-clamp technique. Results The dominant inward rectifier K+ channel subtypes were Kir2.1 and Kir6.1. These dominant K+ channel subtypes decreased significantly during the primary culture throughout activation process. HSC can be classified into two subgroups: one with an inward-rectifying K+ current (type 1) and the other without (type 2). The inward current was blocked by Ba2+ (100 µM) and enhanced by high K+ (140 mM), more prominently in type 1 HSC. There was a correlation between the amplitude of the Ba2+-sensitive current and the membrane potential. In addition, Ba2+ (300 µM) depolarized the membrane potential. After the culture period, the amplitude of the inward current decreased and the membrane potential became depolarized. Conclusion HSC express inward rectifier K+ channels, which physiologically regulate membrane potential and decrease during the activation process. These results will potentially help determine properties of the inward rectifier K+ channels in HSC as well as their roles in the activation process. PMID:18581597

Designing a primary science curriculum in a globalizing world: How do social constructivism and Vietnamese culture meet?

NASA Astrophysics Data System (ADS)

Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

2017-09-01

The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that is, the description of the design of a formal, written curriculum for primary science education in which features of social constructivist approaches to learning are synthesized with essential aspects of Vietnamese culture. The written design comprises learning aims, a framework that is the synthesis of learning functions, learning settings and educational expectations for learning phases, and exemplary curriculum units. Learning aims are formulated to comprehensively develop scientific knowledge, skills, and attitudes toward science for primary students. Derived from these learning aims, the designed framework consists of four learning phases respectively labeled as Engagement, Experience, Exchange, and Follow-up. The designed framework refers to knowledge of the "nature of science" education and characteristics of Vietnamese culture as an example of Confucian heritage culture. The curriculum design aims to serve as an educational product that addresses previously analyzed problems of primary science education in the Vietnamese culture in a globalizing world.

Where Cultural Games Count: The Voices of Primary Classroom Teachers

ERIC Educational Resources Information Center

Nabie, Michael Johnson

2015-01-01

This study explored Ghanaian primary school teachers' values and challenges of integrating cultural games in teaching mathematics. Using an In-depth conversational interview, ten (10) certificated teachers' voices on the values and challenges of integrating games were examined. Thematic data analysis was applied to the qualitative data from the…

Preventive effect of piracetam and vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture.

PubMed

Solanki, P; Prasad, D; Muthuraju, S; Sharma, A K; Singh, S B; Ilavzhagan, G

2011-04-01

The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N(2), 5% CO(2)) for 3h and followed by 1h of reoxygenation (21% O(2) and 5% CO(2)) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia+Piracetam, Hypoxia+Vinpocetine, Normoxia + Piracetam and Normoxia+Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cultural aspects of primary healthcare in india: A case- based analysis.

PubMed

Worthington, Roger P; Gogne, Anupriya

2011-06-16

Delivering quality primary care to large populations is always challenging, and that is certainly the case in India. While the sheer magnitude of patients can create difficulties, not all challenges are about logistics. Sometimes patient health-seeking behaviour leads to delays in obtaining medical help for reasons that have more to do with culture, social practice and religious belief. When primary care is accessed via busy state-run outpatient departments there is often little time for the physician to investigate causes behind a patient's condition, and these factors can adversely affect patient outcomes. We consider the case of a woman with somatic symptoms seemingly triggered by psychological stresses associated with social norms and familial cultural expectations. These expectations conflict with her personal and professional aspirations, and although she eventually receives psychiatric help and her problems are addressed, initially, psycho-social factors underlying her condition posed a hurdle in terms of accessing appropriate medical care. While for many people culture, belief and social norms exert a stabilising, positive influence, in situations where someone's personal expectations differ significantly from accepted social norms, individual autonomy can be directly challenged, and in which case, something has to give. The result of such challenges can negatively impact on health and well-being, and for patients with immature defence mechanisms for dealing with inner conflict, such an experience can be damaging and ensuing somatic disturbances are often difficult to treat. Patients with culture-bound symptoms are not uncommon within primary care in India or in other Asian countries and communities. We argue that such cases need to be properly understood if satisfactory patient outcomes are to be achieved. While some causes are structural, having to do with how healthcare is accessed and delivered, others are about cultural values, social practices and

Cultural aspects of primary healthcare in india: A case- based analysis

PubMed Central

2011-01-01

Delivering quality primary care to large populations is always challenging, and that is certainly the case in India. While the sheer magnitude of patients can create difficulties, not all challenges are about logistics. Sometimes patient health-seeking behaviour leads to delays in obtaining medical help for reasons that have more to do with culture, social practice and religious belief. When primary care is accessed via busy state-run outpatient departments there is often little time for the physician to investigate causes behind a patient's condition, and these factors can adversely affect patient outcomes. We consider the case of a woman with somatic symptoms seemingly triggered by psychological stresses associated with social norms and familial cultural expectations. These expectations conflict with her personal and professional aspirations, and although she eventually receives psychiatric help and her problems are addressed, initially, psycho-social factors underlying her condition posed a hurdle in terms of accessing appropriate medical care. While for many people culture, belief and social norms exert a stabilising, positive influence, in situations where someone's personal expectations differ significantly from accepted social norms, individual autonomy can be directly challenged, and in which case, something has to give. The result of such challenges can negatively impact on health and well-being, and for patients with immature defence mechanisms for dealing with inner conflict, such an experience can be damaging and ensuing somatic disturbances are often difficult to treat. Patients with culture-bound symptoms are not uncommon within primary care in India or in other Asian countries and communities. We argue that such cases need to be properly understood if satisfactory patient outcomes are to be achieved. While some causes are structural, having to do with how healthcare is accessed and delivered, others are about cultural values, social practices and

Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

PubMed Central

Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E.

2007-01-01

The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Upregulation occurred in both “on” and “stand-by” modes in neurons, but only in “on” mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons and astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes. PMID:17187929

Isolation and functional studies of human fetal gastric epithelium in primary culture.

PubMed

Chailler, Pierre; Beaulieu, Jean-François; Ménard, Daniel

2012-01-01

Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistence of viable chief cells able to synthesize and secrete gastric digestive enzymes, i.e., pepsinogen and gastric lipase. In wounding experiments, epithelial restitution occurred in serum-reduced conditions and was modulated by exogenous agents. This culture system is thus representative of the foveolus-gland axis and offers new perspectives to establish the influence of individual growth factors and extracellular matrix components as well as their combinatory effects on gastric epithelium homeostasis.

Primary Care Resident Perceived Preparedness to Deliver Cross-cultural Care: An Examination of Training and Specialty Differences

PubMed Central

Park, Elyse R.; Green, Alexander R.; Betancourt, Joseph R.; Weissman, Joel S.

2007-01-01

Objective Previous research has shown that resident physicians report differences in training across primary care specialties, although limited data exist on education in delivering cross-cultural care. The goals of this study were to identify factors that relate to primary care residents’ perceived preparedness to provide cross-cultural care and to explore the extent to which these perceptions vary across primary care specialties. Design Cross-sectional, national mail survey of resident physicians in their last year of training. Participants Eleven hundred fifty primary care residents specializing in family medicine (27%), internal medicine (23%), pediatrics (26%), and obstetrics/gynecology (OB/GYN) (24%). Results Male residents as well as those who reported having graduated from U.S. medical schools, access to role models, and a greater cross-cultural case mix during residency felt more prepared to deliver cross-cultural care. Adjusting for these demographic and clinical factors, family practice residents were significantly more likely to feel prepared to deliver cross-cultural care compared to internal medicine, pediatric, and OB/GYN residents. Yet, when the quantity of instruction residents reported receiving to deliver cross-cultural care was added as a predictor, specialty differences became nonsignificant, suggesting that training opportunities better account for the variability in perceived preparedness than specialty. Conclusions Across primary care specialties, residents reported different perceptions of preparedness to deliver cross-cultural care. However, this variation was more strongly related to training factors, such as the amount of instruction physicians received to deliver such care, rather than specialty affiliation. These findings underscore the importance of formal education to enhance residents’ preparedness to provide cross-cultural care. PMID:17516107

Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

EPA Science Inventory

Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

Evaluation of the metabolic capability of primary human hepatocytes in three-dimensional cultures on microstructural plates.

PubMed

Koyama, Satoshi; Arakawa, Hiroshi; Itoh, Manabu; Masuda, Norio; Yano, Kentaro; Kojima, Hajime; Ogihara, Takuo

2018-04-01

The NanoCulture Plate (NCP) is a novel microstructural plate designed as a base for the three-dimensional culture of cells/tissues. This study examined whether or not the metabolic capability of human primary hepatocytes is well maintained during culture on NCPs. The hepatocytes formed aggregates after seeding and their ATP content was well maintained during culture for 21 days. Expression of CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 mRNAs was detected throughout the 21-day culture period. Addition of CYP substrate drugs (midazolam, diclofenac, lamotrigine and acetaminophen) resulted in the formation of multiple metabolites with a corresponding decrease in the amounts of the unchanged compounds. The inducers omeprazole, phenobarbital and rifampicin increased the levels of CYP1A2, 2B6 and 3A4 mRNAs by 110-fold, 12.5-fold and 5.4-fold, respectively, at day 2, compared with control human hepatocytes. CYP activities were also increased at 2 days after inducer treatment (CYP1A2, 2.2-fold; CYP2B6, 20.6-fold; CYP3A4, 3.3-fold). The results indicate that the hepatocyte spheroids on NCP have detectable and inducible metabolic abilities during the 7-day culture period. Copyright © 2018 John Wiley & Sons, Ltd.

Multi-Cellular 3D Human Primary Liver Cell Cultures Elevate Metabolic Activity Under Fluidic Flow

PubMed Central

Esch, Mandy B.; Prot, Jean-Matthieu; Wang, Ying I.; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A.; Applegate, Dawn R.

2015-01-01

Predicting drug-induced liver injury with in vitro cell culture models more accurately would be of significant value to the pharmaceutical industry. To this end we have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop and bidirectional fluid flow (average flow rate of 650 μL/min, and a maximum shear stress of 0.64 dyne/cm2). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures derived from human tissues increase their metabolic activity in response to bidirectional fluidic flow. Since bidirectional flow drastically changes the behavior of other cells types that are shear sensitive, the finding that bidirectional flow increases the metabolic activity of primary liver cells also supports the theory that this increase in metabolic activity is likely caused by increased levels of gas and metabolite exchange or by the accumulation of soluble growth factors rather than by shear sensing. Our results indicate that device operation with bi-directional gravity-driven medium

Rapid communication between neurons and astrocytes in primary cortical cultures.

PubMed

Murphy, T H; Blatter, L A; Wier, W G; Baraban, J M

1993-06-01

The identification of neurotransmitter receptors and voltage-sensitive ion channels on astrocytes (reviewed by Barres, 1991) has renewed interest in how these cells respond to neuronal activity. To investigate the physiology of neuron astrocyte signaling, we have employed primary cortical cultures that contain both neuronal and glial cells. As the neurons in these cultures exhibit synchronous spontaneous synaptic activity, we have used both calcium imaging and whole-cell recording techniques to identify physiological activity in astrocytes related to neuronal activity. Whole-cell voltage-clamp records from astrocytes revealed rapid inward currents that coincide with bursts of electrical activity in neighboring neurons. Calcium imaging studies demonstrate that these currents in astrocytes are not always associated with slowly propagating calcium waves. Inclusion of the dye Lucifer yellow within patch pipettes confirmed that astrocytes are extensively coupled to each other but not to adjacent neurons, indicating that the currents observed are not due to gap junction connections between these cell types. These currents do not reflect widespread diffusion of glutamate or potassium released during neuronal activity since a population of small, round, multipolar presumed glial cells that are not dye coupled to adjacent cells did not display electrical currents coincident with neuronal firing, even though they respond to locally applied glutamate and potassium. These findings indicate that, in addition to the relatively slow signaling conveyed by calcium waves, astrocytes also display rapid electrical responses to neuronal activity.

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

PubMed Central

Tricoli, Lucas; Berry, Deborah L.; Albanese, Chris

2018-01-01

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive “living biobanks” of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research. PMID:28287583

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

PubMed

Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

2015-02-01

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. Copyright © 2014 Elsevier Inc. All rights reserved.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

PubMed

Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

2015-08-07

Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes.

PubMed

Hamel, Francine; Grondin, Mélanie; Denizeau, Francine; Averill-Bates, Diana A; Sarhan, Fathey

2006-11-05

Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent. (c) 2006 Wiley Periodicals, Inc.

Cultural significance of primary teeth for caregivers in Northeast Brazil.

PubMed

Nations, Marilyn K; Calvasina, Paola Gondim; Martin, Michele N; Dias, Hilma Fontenele

2008-04-01

This anthropological study critically evaluates Brazilian caregivers' symbolic production and significance of their malnourished offspring's primary teeth, as well as their own, and describes popular dental practices. From January to June 2004, ethnographic interviews of 27 poor, low-literacy mothers were conducted at a public Malnutrition Treatment Center in Fortaleza, Ceará State. Participant observation of clinical pathways and home environments supplemented the data. Content analysis was performed. Results confirm that primary teeth are imbued with cultural significance in Northeast Brazil. Mothers examine mouths, perceive signs of decay, associate primary with permanent tooth healthiness, identify ethnodental illnesses, seek assistance, and perform rituals with exfoliated teeth. The mother's motivation to care for primary teeth is sparked by her memories of past toothache and attempts to avoid stigma and discrimination. Social determinants, not mothers' beliefs or behaviors, are the most critical obstacles to effective dental care. Legitimizing lay knowledge and empowering caregivers and children can improve oral health in Northeast Brazil.

Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

PubMed Central

Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

2009-01-01

Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

Cationic amino acid based lipids as effective nonviral gene delivery vectors for primary cultured neurons.

PubMed

Aoshima, Yumiko; Hokama, Ryosuke; Sou, Keitaro; Sarker, Satya Ranjan; Iida, Kabuto; Nakamura, Hideki; Inoue, Takafumi; Takeoka, Shinji

2013-12-18

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

Cationic Amino Acid Based Lipids as Effective Nonviral Gene Delivery Vectors for Primary Cultured Neurons

PubMed Central

2013-01-01

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl–) and trifluoroacetic acid (TFA–), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA– induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA– showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA– is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity. PMID:24087930

Exploring the Effects of Classroom Culture on Primary Pre-Service Teachers' Professional Development

ERIC Educational Resources Information Center

Altun, Taner

2013-01-01

This study aims to examine primary student teachers' (PSTs) perceptions about the effects of pre-formed classroom culture on their professional development. In the study, a mixed method approach was used. The study group consisted of 4th year student teachers who attend a primary teacher education program leading to a B.Ed. degree at the Fatih…

Long-term primary culture of neurons taken from chick embryo brain: A model to study neural cell biology, synaptogenesis and its dynamic properties.

PubMed

Kumar, Awanish; Mallick, Birendra Nath

2016-04-01

Studying neuronal growth, development and synaptogenesis are among the hot research topics. However, it is faced with various challenges and technical limitations that include but not limited to donor's species and health, threat to life, age of embryo, glial contamination, real-time tracking, and follow-up. We have successfully standardized a method for long-term primary culture of neurons collected from post-fertilized 9 day incubated chicken embryo brain overcoming the limitations mentioned above. Fertilized eggs were incubated in the laboratory and neurons from the embryonic brain were collected and low-density culture, apparently without glial contamination, was studied at least for 35 days in vitro (DIV). Neurons were characterized by double immunostaining using stringent neuronal and glial markers. Neuronal differentiation, cytomorphology, neurite and axon formation, development and maturation, spine formation and synaptogenesis were tracked in real-time in a stage and time dependent manner. The neurons were transfected with Synaptophysin-RFP to label synaptic vesicles, which were followed in real-time under live-cell imaging. Every step was carried out under controlled laboratory conditions. Eggs are easily available, easy to handle, neurons from desired day of incubation could be conveniently studied for long period in apparently glia-free condition. In addition to common factors affecting primary culture, selection of culture media and cover glass coating are other key factors affecting neuronal cultures. We describe an inexpensive, simpler pure primary neuronal culture method for studying neuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayered brain in vitro. Copyright © 2016 Elsevier B.V. All rights reserved.

Organizational culture in the primary healthcare setting of Cyprus.

PubMed

Zachariadou, Theodora; Zannetos, Savvas; Pavlakis, Andreas

2013-03-24

The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization's characteristic cultural values orientation along a five-point Likert scale ranging from "Very Much = 1" to "Not at all= 5". Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension "performance orientation" was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). "Supportiveness" and "social responsibility" were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). This was the first study performed in Cyprus assessing organizational culture in the PHC setting. In the forthcoming health system reform

Organizational culture in the primary healthcare setting of Cyprus

PubMed Central

2013-01-01

Background The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. Methods The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization’s characteristic cultural values orientation along a five-point Likert scale ranging from “Very Much = 1” to “Not at all= 5”. Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. Results From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension “performance orientation” was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). “Supportiveness” and “social responsibility” were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). Conclusions This was the first study performed in Cyprus assessing organizational culture in

[Improving Primary Culture of Pulmonary Microvascular Endothelial Cells of Rats].

PubMed

Jiang, Ling; Hu, Yuan-Dong; Xu, Fei-Fei; Wang, Ting-Hua

2016-09-01

To improve the culturing method of pulmonary microvascular endothelial cells (PMEVCs) of SD rats. The culturing processes in regard to obtaining peripheral lung tissue, attaching tissue block,preparing medium and subculturing were modified.These included an injection of heparin sodium before anesthesia, abdominal bleeding, opening of chest when breathing stopped, improvement of operational details, reduction of pollution by adding penicillin and streptomycin, discard of tissues after 48 h of primary culturing, remove of fibroblasts by a second digestion, and identification of cells using a fluorescence microscope for binding with lectin from BSI (FITC-BSI).An inverted microscope was used to observe the morphological characteristics of PMEVCs. Purified PMEVCs were obtained,which displayed a polygon or short fusiform, exhibiting a typical cobblestone-like morphology. The morphology of PMVECs turned into swirling or long fusiform following subculture or changes in culture conditions. The results of FITC-BSI assay showed that more than 90% cells were stained with green fluorescence. Purified PMEVCs with a good growth state and subculture stability can be obtained using the modified method.

Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

PubMed

Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

2012-01-01

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples. Copyright © 2011 Elsevier Inc. All rights reserved.

Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzawa, Tetsuo; Itoh, Nao; Department of Clinical Pharmacy, School of Pharmaceutical Science, Showa University

2006-07-07

To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was establishedmore » to analyze the promoter activity of the amelogenin gene. The region between -1102 bp and -261 bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.« less

Alginate foam-based three-dimensional culture to investigate drug sensitivity in primary leukaemia cells.

PubMed

Karimpoor, Mahroo; Yebra-Fernandez, Eva; Parhizkar, Maryam; Orlu, Mine; Craig, Duncan; Khorashad, Jamshid S; Edirisinghe, Mohan

2018-04-01

The development of assays for evaluating the sensitivity of leukaemia cells to anti-cancer agents is becoming an important aspect of personalized medicine. Conventional cell cultures lack the three-dimensional (3D) structure of the bone marrow (BM), the extracellular matrix and stromal components which are crucial for the growth and survival of leukaemia stem cells. To accurately predict the sensitivity of the leukaemia cells in an in vitro assay a culturing system containing the essential components of BM is required. In this study, we developed a porous calcium alginate foam-based scaffold to be used for 3D culture. The new 3D culture was shown to be cell compatible as it supported the proliferation of both normal haematopoietic and leukaemia cells. Our cell differential assay for myeloid markers showed that the porous foam-based 3D culture enhanced myeloid differentiation in both leukaemia and normal haematopoietic cells compared to two-dimensional culture. The foam-based scaffold reduced the sensitivity of the leukaemia cells to the tested antileukaemia agents in K562 and HL60 leukaemia cell line model and also primary myeloid leukaemia cells. This observation supports the application of calcium alginate foams as scaffold components of the 3D cultures for investigation of sensitivity to antileukaemia agents in primary myeloid cells. © 2018 The Author(s).

Lipoprotein degradation and cholesterol esterification in primary cell cultures of rabbit atherosclerotic lesions.

PubMed Central

Jaakkola, O.; Nikkari, T.

1990-01-01

Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding. The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions. The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture. The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures. DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells. The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL. The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively. Incubation with unlabeled acetyl-LDL enhanced the incorporation of [3H]oleate into cholesteryl esters and increased the cellular cholesteryl ester content. These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL. Images Figure 2 PMID:2201201

Culturally Relevant Literature: What Matters Most to Primary-Age Urban Learners

ERIC Educational Resources Information Center

Cartledge, Gwendolyn; Keesey, Susan; Bennett, Jessica G.; Ramnath, Rajiv; Council, Morris R., III.

2016-01-01

The ratings and rationales primary-age urban learners gave culturally relevant reading passages was the focus of this descriptive study. First- and second-grade students each read 30 researcher-developed passages reflecting the students' immediate and historical backgrounds. The students rated the passages and gave a reason for their ratings. A…

Hanging Drop, A Best Three-Dimensional (3D) Culture Method for Primary Buffalo and Sheep Hepatocytes.

PubMed

Shri, Meena; Agrawal, Himanshu; Rani, Payal; Singh, Dheer; Onteru, Suneel Kumar

2017-04-26

Livestock, having close resemblance to humans, could be a better source of primary hepatocytes than rodents. Herein, we successfully developed three-dimensional (3D) culturing system for primary sheep and buffalo hepatocytes. The 3D-structures of sheep hepatocytes were formed on the fifth-day and maintained until the tenth-day on polyHEMA-coated plates and in hanging drops with William's E media (HDW). Between the cultured and fresh cells, we observed a similar expression of GAPDH, HNF4α, ALB, CYP1A1, CK8 and CK18. Interestingly, a statistically significant increase was noted in the TAT, CPS, AFP, AAT, GSP and PCNA expression. In buffalo hepatocytes culture, 3D-like structures were formed on the third-day and maintained until the sixth-day on polyHEMA and HDW. The expression of HNF4α, GSP, CPS, AFP, AAT, PCNA and CK18 was similar between cultured and fresh cells. Further, a statistically significant increase in the TAT and CK8 expression, and a decrease in the GAPDH, CYP1A1 and ALB expression were noted. Among the culture systems, HDW maintained the liver transcript markers more or less similar to the fresh hepatocytes of the sheep and buffalo for ten and six days, respectively. Taken together, hanging drop is an efficient method for 3D culturing of primary sheep and buffalo hepatocytes.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

PubMed

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Sugammadex, a Neuromuscular Blockade Reversal Agent, Causes Neuronal Apoptosis in Primary Cultures

PubMed Central

Palanca, José M.; Aguirre-Rueda, Diana; Granell, Manuel V.; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L.

2013-01-01

Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we determine toxicity of Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types. PMID:23983586

Establishment of primary cell cultures from fish calcified tissues

PubMed Central

Marques, Cátia L.; Rafael, Marta S.; Cancela, M. Leonor

2007-01-01

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case. PMID:19002990

Knowing in Primary Physical Education in the UK: Negotiating Movement Culture

ERIC Educational Resources Information Center

Ward, Gavin; Quennerstedt, Mikael

2015-01-01

This paper aims to understand how pupils and teachers actions-in-context constitute being-a-pupil and being-a-teacher within a primary school physical education (PE) movement culture. Dewey and Bentley's theory of transaction, which views organism-in-environment-as-a-whole, enables the researcher to explore how actions-in-ongoing activities…

Effects of lactic acid on astrocytes in primary culture.

PubMed

Norenberg, M D; Mozes, L W; Gregorios, J B; Norenberg, L O

1987-03-01

Excessive tissue lactic acidosis is considered to be detrimental to the central nervous system (CNS) and may adversely affect recovery from anoxia, ischemia, trauma and epilepsy. Since astrocytes are believed to play a role in pH regulation in the CNS, we studied the effect of this acid on primary astrocyte cultures. Cells exposed to lactic acid showed chromatin clumping, an increase of lipid and dense bodies, a loss of polyribosomal clusters, slightly increased cytoplasmic lucency, swollen mitochondria and tangled intermediate filaments. These alterations progressed with lower pH and longer exposure. Irreversible changes occurred one to two hours after exposure at pH 6; after 30 to 60 minutes (min) at pH 5.5 and after ten to 30 min at pH 5. Comparable results were obtained with the use of other weak acids indicating that the observed changes were due to increased hydrogen ion concentration rather than secondary to lactate per se. Additionally, various concentrations of lactic acid adjusted to identical pH produced similar morphologic alterations. Thus, while lactic acid caused marked and at times irreversible alterations in astrocytes, severe and prolonged acidosis was required to produce such injurious effects. This relative resistance of astrocytes to acidosis is in keeping with their potential role in pH regulation in brain.

Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

PubMed Central

Popova, Daria; Stonier, Adam; Pain, David; Titchener‐Hooker, Nigel J.

2016-01-01

Abstract Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost‐effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale‐down (USD) mimics requiring 25–110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost‐effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario. PMID:27067803

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

NASA Technical Reports Server (NTRS)

Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

1998-01-01

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists

Modeling Fetal Alcohol Spectrum Disorder: validating an ex vivo primary hippocampal cell culture system

PubMed Central

Tunc-Ozcan, Elif; Ferreira, Adriana B.; Redei, Eva E.

2016-01-01

Background Fetal Alcohol Spectrum Disorder (FASD) is the leading non-genetic cause of mental retardation. There are no treatments for FASD to date. Preclinical in vivo and in vitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an ex vivo model that combines the physiological advantages of prenatal ethanol (E) exposure in vivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal E. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (in vivo) and primary hippocampal culture (ex vivo). The similarity of prenatal E effects in these two paradigms would deem the ex vivo culture acceptable to screen possible treatments for FASD. Methods Pregnant Sprague-Dawley rats received one of three diets: ad libitum standard lab chow (control-C), isocaloric pair-fed (PF, nutritional control), and E containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (in vivo) or on GD18 for primary culture (ex vivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1 and Zac1 were measured by RT-qPCR. Results Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal E on the hippocampal expression of the insulin pathway genes was parallel in the in vivo and the ex vivo conditions. Conclusions The similarity of gene expression changes in response to prenatal E between the in vivo and the ex vivo conditions ascertain that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the ex vivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD. PMID:27162054

Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyoshi, Ko, E-mail: miyoshi@cc.okayama-u.ac.jp; Kasahara, Kyosuke; Miyazaki, Ikuko

2009-10-30

The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed withmore » Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.« less

Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

PubMed Central

Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

2016-01-01

Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

[Protective effects of Fufangdengzhanhua dripping pill on apoptosis induced by glutamate in cultured primary hippocampal neurons of rats].

PubMed

Wang, Lijun; Wan, Lei

2010-03-01

To explore the protective effects and the inhibited mechanism of Fufangdengzhanhua dripping pill (FDD) on the apoptosis induced by glutamate (Glu) of cultured primary hippocampal neurons of rats. By the seropharmacological method, we obtained the drug-contained serum. The primary hippocampal neurons of rat cerebrum were cultured for 10 days, then exposed to 500 micromol x L(-1) glutamate acid (Glu) for 20 minutes to build the model. The 5% drug-contained sera which included normal, model, 0.05 g x kg(-1) nimodipine (Nim), 5.00 g x kg(-1) FDD and 1.25 g x kg(-1) FDD were added to the nutrient solution of cultured neurons. In this study, we observed the following indexes: the viability of cultured primary hippocampal neurons by MTT assay, the injured cell morphological changes with fluorescence microscope by using Hoechst 33342 & Propicium Iodide (PI) staining, intracellular Ca2+ concentration and the percentage of apoptosis by flow cytometry. When the hippocampal neurons were exposed to Glu, the cells were seriously damaged: nuclei were shrunken and cloven and the apoptosis body and the viability of cultured primary hippocampal neurons were decreased dramatically compared with the control. The FDD (5.00, 1.25 g x kg(-1)) and Nim could prevent the above changes Glu-induced. The necrosis rates and the percentage of cellular apoptosis of cultured hippocampal neurons pretreated with the serum of containing FDD decreased significantly and the number of surviving cells was increased significantly compared with model. Intracellular Ca2+ concentration Glu-induced were increased markedly compared with the control and the FDD (5.00, 1.25 g x kg(-1)) could prevent the above changes . FDD has protective effects on the apoptosis induced by glutamate (Glu) of cultured primary hippocampal neurons of rats, which possibly is related to reducing the intracellular Ca2+.

The Effect of Organizational Trust on the Culture of Teacher Leadership in Primary Schools

ERIC Educational Resources Information Center

Demir, Kamile

2015-01-01

The purpose of this research is to examine the effect of the level of trust of primary school teachers towards their organization in relation to their perceptions of the school having a culture of teacher leadership. Participants of the study consisted of 378 teachers working in Burdur public primary schools. The data collection tool used two…

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

PubMed Central

Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

PubMed Central

Dohmen, Amy J. C.; Swartz, Justin E.; Van Den Brekel, Michiel W. M.; Willems, Stefan M.; Spijker, René; Neefjes, Jacques; Zuur, Charlotte L.

2015-01-01

Primary human tumor culture models allow for individualized drug sensitivity testing and are therefore a promising technique to achieve personalized treatment for cancer patients. This would especially be of interest for patients with advanced stage head and neck cancer. They are extensively treated with surgery, usually in combination with high-dose cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to the intended treatment regimen and to test novel drugs. One of such models is the technique of culturing primary human tumor tissue. This review discusses the feasibility and success rate of existing primary head and neck tumor culturing techniques and their corresponding chemo- and radiosensitivity assays. A comprehensive literature search was performed and success factors for culturing in vitro are debated, together with the actual value of these models as preclinical prediction assay for individual patients. With this review, we aim to fill a gap in the understanding of primary culture models from head and neck tumors, with potential importance for other tumor types as well. PMID:26343729

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

PubMed

Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa; Ibrahim, Toni

2017-02-15

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. © 2016. Published by The Company of Biologists Ltd.

Meeting and treating cultural difference in primary care: a qualitative interview study.

PubMed

Wachtler, Caroline; Brorsson, Annika; Troein, Margareta

2006-02-01

Primary care doctors see patients from diverse cultural backgrounds and communication plays an important role in diagnosis and treatment. Communication problems can arise when patient and doctor do not share the same cultural background. The aim of this study was to examine how consultations with immigrant patients are understood by GPs and how GPs manage these consultations. Semi-structured interviews with GPs about their experiences with immigrant patients were recorded on audio-tape, transcribed and analysed using a qualitative thematic analysis methodology. A constructivist approach was taken to analysis and interpretation. Culture is not in focus when GPs meet immigrant patients. The consultation is seen as a meeting between individuals, where cultural difference is just one of many individual factors that influence how well doctor and patient understand each other. However, when mutual understanding is poor and the consultation not successful, cultural differences are central. The GPs try to conduct their consultations with immigrant patients in the same way that they conduct all their consultations. There is no specific focus on culture, instead, GPs tend to avoid addressing even pronounced cultural differences. This study indicates that cultural difference is not treated in GPs consultation with immigrant patients. Learning about cultural difference's effect on mutual understanding between doctor and patient could improve GPs cross-cultural communication. Increased awareness of the culture the doctor brings to the consultation could facilitate management of cross-cultural consultations.

Identification of cultural determinants of antibiotic use cited in primary care in Europe: a mixed research synthesis study of integrated design "Culture is all around us".

PubMed

Touboul-Lundgren, Pia; Jensen, Siri; Drai, Johann; Lindbæk, Morten

2015-09-17

Inappropriate antibiotic prescribing, particularly for respiratory tract infections (RTI) in ambulatory care, has become a worldwide public health threat due to resulting antibiotic resistance. In spite of various interventions and campaigns, wide variations in antibiotic use persist between European countries. Cultural determinants are often referred to as a potential cause, but are rarely defined. To our knowledge, so far no systematic literature review has focused on cultural determinants of antibiotic use. The aim of this study was to identify cultural determinants, on a country-specific level in ambulatory care in Europe, and to describe the influence of culture on antibiotic use, using a framework of cultural dimensions. A computer-based systematic literature review was conducted by two research teams, in France and in Norway. Eligible publications included studies exploring antibiotic use in primary care in at least two European countries based on primary study results, featuring a description of cultural determinants, and published between 1997 and 2015. Quality assessment was conducted independently by two researchers, one in each team, using appropriate checklists according to study design. Each included paper was characterized according to method, countries involved, sampling and main results, and cultural determinants mentioned in each selected paper were extracted, described and categorized. Finally, the influence of Hofstede's cultural dimensions associated with antibiotic consumption within a primary care setting was described. Among 24 eligible papers, 11 were rejected according to exclusion criteria. Overall, 13 papers meeting the quality assessment criteria were included, of which 11 used quantitative methods and two qualitative or mixed methods. The study participants were patients (nine studies) and general practitioners (two studies). This literature review identified various cultural determinants either patient-related (illness perception

Organization Complexity and Primary Care Providers' Perceptions of Quality Improvement Culture Within the Veterans Health Administration.

PubMed

Korom-Djakovic, Danijela; Canamucio, Anne; Lempa, Michele; Yano, Elizabeth M; Long, Judith A

2016-01-01

This study examined how aspects of quality improvement (QI) culture changed during the introduction of the Veterans Health Administration (VHA) patient-centered medical home initiative and how they were influenced by existing organizational factors, including VHA facility complexity and practice location. A voluntary survey, measuring primary care providers' (PCPs') perspectives on QI culture at their primary care clinics, was administered in 2010 and 2012. Participants were 320 PCPs from hospital- and community-based primary care practices in Pennsylvania, West Virginia, Delaware, New Jersey, New York, and Ohio. PCPs in community-based outpatient clinics reported an improvement in established processes for QI, and communication and cooperation from 2010 to 2012. However, their peers in hospital-based clinics did not report any significant improvements in QI culture. In both years, compared with high-complexity facilities, medium- and low-complexity facilities had better scores on the scales assessing established processes for QI, and communication and cooperation. © The Author(s) 2014.

Addition of Carbon to the Culture Medium Improves the Detection Efficiency of Aflatoxin Synthetic Fungi

PubMed Central

Suzuki, Tadahiro; Iwahashi, Yumiko

2016-01-01

Aflatoxin (AF) is a harmful secondary metabolite that is synthesized by the Aspergillus species. Although AF detection techniques have been developed, techniques for detection of AF synthetic fungi are still required. Techniques such as plate culture methods are continually being modified for this purpose. However, plate culture methods require refinement because they suffer from several issues. In this study, activated charcoal powder (carbon) was added to a culture medium containing cyclodextrin (CD) to enhance the contrast of fluorescence and improve the detection efficiency for AF synthetic fungi. Two culture media, potato dextrose agar and yeast extract sucrose agar, were investigated using both plate and liquid cultures. The final concentrations of CD and carbon in the media were 3 mg/mL and 0.3 mg/mL, respectively. Addition of carbon improved the visibility of fluorescence by attenuating approximately 30% of light scattering. Several fungi that could not be detected with only CD in the medium were detected with carbon addition. The carbon also facilitated fungal growth in the potato dextrose liquid medium. The results suggest that addition of carbon to media can enhance the observation of AF-derived fluorescence. PMID:27854283

Insulin and IGF-I effects on the proliferation of an osteoblast primary culture from sea bream (Sparus aurata).

PubMed

Capilla, Encarnación; Teles-García, Agueda; Acerete, Laura; Navarro, Isabel; Gutiérrez, Joaquim

2011-05-15

Bone deformities in several fish species, like gilthead sea bream (Sparus aurata), are currently a major problem in aquaculture. To gain knowledge of fish skeletal development, a primary cell culture has been established from sea bream vertebra. The initial fibroblastic phenotype of the cells changed to a polygonal shape during the culture, and the addition of an osteogenic medium promoted the deposition of minerals in the extracellular matrix. Cell proliferation was analyzed using the MTT assay in control and mineralizing conditions at different culture days, up to day 20. The capacity of the cells to differentiate into osteoblasts was evaluated using Alizarin red stain. The cells showed slightly increased proliferation and differentiation in the presence of osteogenic medium. Furthermore, pluripotentiality of these cells was demonstrated by inducing them to differentiate into adipocytes, and the accumulation of lipids into the cells was detected with Oil Red O staining. Subsequently, the effects of insulin (1, 10, 100 and 1000 nM) and IGF-I (0.1, 1 and 10nM) on cell proliferation were evaluated with the MTT assay at day 3. Both peptides significantly stimulated the proliferation of the cells in a dose-dependent manner after either 24 or 48 h of incubation, with IGF-I apparently being more potent than insulin. In summary, a primary culture of sea bream osteoblasts has been characterized. This cellular system can be a good model to study the process of osteoblastogenesis in fish and its endocrine regulation, which may help to improve the quality of the product in aquaculture. Copyright © 2011 Elsevier Inc. All rights reserved.

Features of morfological changes in primary thyroid gland CTLL cultures of rats descendants prenatally exposed by radioisotopes of iodine-131.

PubMed

Boiko, O A; Lavrenchuk, H Yo; Lypska, A I; Talko, V V; Asmolkov, V S

2017-12-01

to investigate morphological changes in the primary thyroid cell culture of rat infants whose parents were prenatally exposed by radioisotope iodine 131. obtaining and culturing of thyroid tissue primary cell cultures of newborn rats, cytological (receipt and analysis of cell cultures agents for optical microscopy), biophysical (flow cytometry), statistics. It was shown that cells in thyroid primary culture of offspring rats prenatally exposed by radioisotopes of iodine 131 signs of destructive degenerative changes were observed mostly when animals of both sexes were irra diated. Increased number of two and three nuclear cells and induction of ring like cells is an evidence of signifi cant genotoxic violation and points to the genome instability in offspring of animals exposed by radioisotope iodine 131. Analysis and quantitative morphological parameters of cells in thyroid primary culture of newborn rats whose parents were exposed prenatally by radioisotopes of iodine 131 showed that upon exposure to radiation thy roid undergoes destructive changes at the cellular level and, even in the second generation of offspring, leads to disruption of its functions. O. A. Boiko, H. Yo. Lavrenchuk, A. I. Lypska, V. V. Talko, V. S. Asmolkov.

Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface.

PubMed

Chang-Lin, Joan-En; Kim, Kwang-Jin; Lee, Vincent H L

2005-06-01

Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits. Primary cultured RCrECL were grown at an air-interface on Clear-Snapwells precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na(+) and Cl(-) transport to overall short-circuit current (I(sc)) was evaluated by removing Na(+) and Cl(-), respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na(+) and Cl(-) using (22)Na and (36)Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na(+)-channel activities to net Na(+) fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl(2) and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl(-)-channel, and basolateral K(+)-channel and Na(+)(K(+))2Cl(-)-cotransporter activities, respectively, in active ion transport across RCrECL. I(sc) of RCrECL derived from pigmented rabbits was comprised of 64+/-2% and 44+/-5% for active Na(+) and Cl(-) transport, respectively, consistent with net Na(+) absorption and Cl(-) secretion of 0.062+/-0.006 and 0.046+/-0.008 muEq/cm(2)/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited I(sc) by up to approximately 50% with an IC(50) of 1 and 0.1 microm, respectively, consistent with participation of apical epithelial Na(+)-channels to net Na(+) absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl(2) to the basolateral and bumetanide to basolateral fluid decreased I(sc) by 86+/-1.5%, 53+/-3%, 18+/-1.8% and 13+/-1.9% in RCr

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

PubMed

Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

2018-06-26

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Effects of Trichostatin A on drug uptake transporters in primary rat hepatocyte cultures

PubMed Central

Ramboer, Eva; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

2015-01-01

The present study was set up to investigate the effects of Trichostatin A (TSA), a prototypical epigenetic modifier, on the expression and activity of hepatic drug uptake transporters in primary cultured rat hepatocytes. To this end, the expression of the sinusoidal transporters sodium-dependent taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 4 (Oatp4) was monitored by real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblotting. The activity of the uptake transporters was analyzed using radiolabeled substrates and chemical inhibitors. Downregulation of the expression and activity of Oatp4 and Ntcp was observed as a function of the cultivation time and could not be counteracted by TSA. In conclusion, the epigenetic modifier TSA does not seem to exert a positive effect on the expression and activity of the investigated uptake transporters in primary rat hepatocyte cultures. PMID:26648816

Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

NASA Astrophysics Data System (ADS)

Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

NASA Astrophysics Data System (ADS)

Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

Morphology and function of lacrimal gland acinar cells in primary culture.

PubMed

Hann, L E; Tatro, J B; Sullivan, D A

1989-01-01

The objectives of the current investigation were fourfold: (1) to establish an effective procedure for the isolation of acinar cells from the rat lacrimal gland; (2) to evaluate the functional capacity of freshly isolated cells; (3) to determine defined culture conditions which permit maintenance of viable, differentiated cells, as well as secretory component (SC) production, during long-term culture; and (4) to characterize the morphological features of cultured cells. Acinar cells were isolated by serial incubation of gland fragments in chelating and enzymatic solutions, followed by centrifugation through a Ficoll gradient. The yield of viable cells/gland appeared to be age-dependent: cell recovery was inversely proportional to the age of the animals. Immunofluorescence analysis of freshly isolated cells showed the presence of SC, the IgA antibody receptor, within isolated cells. In addition, experiments with a labeled analog (Nle4-D-Phe7-alpha MSH) of alpha-melanocyte-stimulating hormone (alpha-MSH) demonstrated specific binding sites on freshly isolated cells; alpha-MSH is a known modulator of acinar protein secretion. Maximum binding of the alpha-MSH analog occurred within 30 min, was dependent upon cell density and was reduced by coincubation with unlabeled alpha-MSH. To determine the culture requirements of acinar cells, cells were cultured on a variety of substrates (plastic or modified plastic [Primaria], coated with or without extracellular matrix [Matrigel]) in the presence or absence of various supplements and/or fetal calf serum (FCS) for 0.7 to 3.5 weeks. Cell attachment, function and long-term viability required an extracellular matrix. Moreover, in long term cultures (25 days), acinar cell attachment was enhanced by the inclusion of supplements to media containing 10% FCS. Replacement of serum with fibroblast growth factor, high-density lipoprotein and an increased concentration of epidermal growth factor resulted in a distinct "cobblestone

Use of primary cultures of Kenyon cells from bumblebee brains to assess pesticide side effects.

PubMed

Wilson, Daniel E; Velarde, Rodrigo A; Fahrbach, Susan E; Mommaerts, Veerle; Smagghe, Guy

2013-09-01

Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain. © 2013 Wiley Periodicals, Inc.

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants

PubMed Central

Vagasi, Alexandra S.; Rahman, Mohammad M.; Chaudhari, Snehal N.; Kipreos, Edward T.

2017-01-01

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016. PMID:28868332

Primary care units in Emilia-Romagna, Italy: an assessment of organizational culture.

PubMed

Pracilio, Valerie P; Keith, Scott W; McAna, John; Rossi, Giuseppina; Brianti, Ettore; Fabi, Massimo; Maio, Vittorio

2014-01-01

This study investigates the organizational culture and associated characteristics of the newly established primary care units (PCUs)-collaborative teams of general practitioners (GPs) who provide patients with integrated health care services-in the Emilia-Romagna Region (RER), Italy. A survey instrument covering 6 cultural dimensions was administered to all 301 GPs in 21 PCUs in the Local Health Authority (LHA) of Parma, RER; the response rate was 79.1%. Management style, organizational trust, and collegiality proved to be more important aspects of PCU organizational culture than information sharing, quality, and cohesiveness. Cultural dimension scores were positively associated with certain characteristics of the PCUs including larger PCU size and greater proportion of older GPs. The presence of female GPs in the PCUs had a negative impact on collegiality, organizational trust, and quality. Feedback collected through this assessment will be useful to the RER and LHAs for evaluating and guiding improvements in the PCUs. © 2013 by the American College of Medical Quality.

Primary Retinal Cultures as a Tool for Modeling Diabetic Retinopathy: An Overview

PubMed Central

Varano, Monica; Mallozzi, Cinzia; Gaddini, Lucia; Formisano, Giuseppe; Pricci, Flavia

2015-01-01

Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight. PMID:25688355

High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

PubMed

Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

2012-07-01

In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Primary-Grade Students' Knowledge and Thinking about Shelter as a Cultural Universal.

ERIC Educational Resources Information Center

Brophy, Jere; Alleman, Janet

The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. A study was designed to provide information with respect to the topic of shelter, and in the process, to assess claims that primary grade students do not need instruction in the topic because they…

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

PubMed

Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

2018-02-01

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

[Cultural dimensions of the childhood primary health care delivery from the mothers' perceptions in the Araucania-Chile].

PubMed

Alarcón-Muñoz, Ana Maria; Vidal-Herrera, Aldo Conrado

2005-01-01

To explore the cultural dimensions of the childhood primary health care delivery process from the mothers' perceptions in the Araucania region of Chile. Qualitative study performed in the year 2003 within the zone with the highest ethnicity rate of the country. Ninety four Mapuche and non-Mapuche mothers agreed to be in depth interviewed. The analysis drew three cultural dimensions: a) Explanatory models of disease were associated with cultural, political-economy, and environmental factors; b) The therapeutic itinerary blends indigenous, popular, and biomedical resources and; c) Health care delivery process lacks of cultural competence. The mothers explain their children diseases articulating religious, magic, and natural (hot, cold, humidity) causes. The main challenge of the primary healthcare delivery process is to overcome the communicational barriers due to the social and linguistic differences between mothers and health care providers.

Contribution to Cultural Organization, Working Motivation and Job Satisfaction on the Performance of Primary School Teacher

ERIC Educational Resources Information Center

Murtedjo; Suharningsih

2016-01-01

The purposes of this study are: (1) describes the performance of the teacher, organizational culture, work motivation and job satisfaction; (2) determine whether there is a significant direct relationship between organizational culture, work motivation and job satisfaction on the performance of primary school teachers. Through the study of the…

Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

EPA Science Inventory

Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

EPA Science Inventory

Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

The potential of silk sericin protein as a serum substitute or an additive in cell culture and cryopreservation.

PubMed

Cao, Ting-Ting; Zhang, Yu-Qing

2017-06-01

Cell culture and cryopreservation are necessary for clinical therapy and cells storage. The addition of 10% (v/v) foetal bovine serum (FBS) to basal culture media has been common practice and is one of the most widely used methods. FBS media added with 10% DMSO (dimethyl sulfoxide) have also been used for cryopreservation cells. Ideally, FBS should be avoided because of high cost and bio-safety. Silk sericin has been used as a serum substitute and an additive due to its good hydrophilicity and biological safety. This article summarizes a few details about the processing of sericin and its application as a serum substitute or an additive for cell culture and cryopreservation media. Sericin can be a potential novel serum substitute or an additive for cell culture and cryopreservation media.

[Application of the cultural competence model in the experience of care in nursing professionals Primary Care].

PubMed

Gil Estevan, María Dolores; Solano Ruíz, María Del Carmen

2017-11-01

To know the experiences and perceptions of nurses in providing care and health promotion, women belonging to groups at risk of social vulnerability, applying the model of cultural competence Purnell. Phenomenological qualitative study. Department of Health Elda. A total of 22 primary care professional volunteers. Semi-structured interviews and focus groups with recording and content analysis, according to the theory model of cultural competence. Socio-cultural factors influence the relationship between professionals and users of the system. The subtle racism and historical prejudices create uncomfortable situations and mistrust. The language barrier makes it difficult not only communication, but also the monitoring and control of the health-disease process. The physical appearance and stereotypes are determining factors for primary care professionals. Although perceived misuse of health services are also talking about changes. The spiritual aspects of religious beliefs alone are taken into account in the case of Muslim women, not being considered as important in the case of Gypsy women and Romanian women. To provide quality care, consistent and culturally competent, it is necessary to develop training programs for professionals in cultural competence, to know the culture of other, and work without preconceived ideas, and ethnocentric; since the greater the knowledge of the cultural group being served, the better the quality of care provided. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Neurotensin and neuromedin N undergo distinct catabolic processes in murine astrocytes and primary cultured neurons.

PubMed

Vincent, B; Vincent, J P; Checler, F

1994-04-01

We examined the occurrence of various endopeptidases and exopeptidases and their subcellular partition within soluble and membrane-associated compartments of 15-day-old astrocytes and 4-day-old primary cultured neurons. Peptidases were monitored with chromogenic or fluorimetric substrates and identified by means of specific inhibitors. We assessed the contribution of these peptidases in the catabolism of two related neuropeptides, neurotensin and neuromedin N. Metabolites were separated by HPLC and the identity of the proteolytic activities involved in their formation was established using specific inhibitors. Neuromedin N and neurotensin undergo both quantitative and qualitative differential proteolysis. Initial maximal rates of neuromedin N degradation were higher than those of neurotensin in both cell types. Furthermore, the two peptides were inactivated much more rapidly by the soluble than by the membrane-associated fractions prepared from both cell cultures. Neuromedin N was rapidly broken down by an aminopeptidase M/leucine aminopeptidase attack, leading to the functionally silent Des-Lys1-neuromedin N metabolite. In the astrocytic membrane-associated fraction, neuromedin N underwent an additional minor endoproteolytic cleavage at the Pro3-Tyr4 bond elicited by endopeptidase 24.11, as suggested by the protective effect of its blocking agent phosphoramidon. Unlike neuromedin N, neurotensin totally resisted hydrolysis by aminopeptidases. Primary inactivating cleavages detected in both cell types appeared mainly located at the Arg8-Arg9 and Pro10-Tyr11 bonds, leading to the formations of neurotensin-(1-8) and neurotensin-(1-10) as the major biologically inactive neurotensin catabolites. Endopeptidase 24.15 appeared mainly responsible for neurotensin-(1-8) formation by the soluble fraction of neurons and astrocytes. In contrast, endopeptidase 24.16 was involved in neurotensin-(1-10) formation by both soluble and membrane-associated fractions of the two cell

Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis.

PubMed

Pifferi, Massimo; Bush, Andrew; Montemurro, Francesca; Pioggia, Giovanni; Piras, Martina; Tartarisco, Gennaro; Di Cicco, Maria; Chinellato, Iolanda; Cangiotti, Angela M; Boner, Attilio L

2013-04-01

Diagnosis of primary ciliary dyskinesia (PCD) sometimes requires repeated nasal brushing to exclude secondary ciliary alterations. Our aim was to evaluate whether the use of a new method of nasal epithelial cell culture can speed PCD diagnosis in doubtful cases and to identify which are the most informative parameters by means of a multilayer artificial neural network (ANN). A cross-sectional study was performed in patients with suspected PCD. All patients underwent nasal brushing for ciliary motion analysis, ultrastructural assessment and evaluation of ciliary function after ciliogenesis in culture by ANN. 151 subjects were studied. A diagnostic suspension cell culture was obtained in 117 nasal brushings. A diagnosis of PCD was made in 36 subjects (29 of whom were children). In nine out of the 36 patients the diagnosis was made only after a second brushing, because of equivocal results of both tests at first examination. In each of these subjects diagnosis of PCD was confirmed by cell culture results. Cell culture in suspension evaluated by means of ANN allows the separation of PCD from secondary ciliary dyskinesia patients after only 5 days of culture and allows diagnosis to be reached in doubtful cases, thus avoiding the necessity of a second sample.

Mechanism of ion transport by avian salt gland primary cell cultures.

PubMed

Lowy, R J; Dawson, D C; Ernst, S A

1989-06-01

Confluent sheets formed from primary culture of avian salt gland secretory cells exhibit a short-circuit current (Isc) in response to cholinergic and beta-adrenergic stimulation [Lowy, R. J., D. C. Dawson, and S. A. Ernst. Am J. Physiol. 249 (Cell Physiol. 18): C41-C47, 1985]. To establish the ionic basis for the Isc, transmural fluxes of 22Na and 36Cl were measured. Under short-circuit conditions there was little net flux of either ion in the absence of agonists. Addition of carbachol elevated net serosal-to-mucosal Cl flux to 1.71 mu eq.h-1.cm-2, whereas a smaller increase to 0.85 mu eq.h-1.cm-2 occurred with isoproterenol. Neither agonist altered net Na flux. The stimulated Isc accounted for 70% of the net Cl flux induced by carbachol and nearly 100% of that induced by isoproterenol. Replacement of Cl by gluconate or Na by choline abolished (carbachol) or greatly reduced (isoproterenol) the Isc, which could be restored in a dose-dependent fashion by ion restitution. Active ion transport was preferentially inhibited by basal (vs. apical) addition of ouabain, furosemide, or barium. The results provide evidence that cholinergic and beta-adrenergic agonists elicit active transmural Cl secretion. They further suggest that transport is dependent on the Na+-K+-adenosine-triphosphatase, a Na-Cl cotransport process, and a basal K conductance, all features of a secondary active Cl secretory mechanism.

Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action.

PubMed Central

Thompson, D B; Pratley, R; Ossowski, V

1996-01-01

Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans. It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia. To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities. These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN. Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures. The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58). This defect in insulin action is stable in a uniform culture environment and is retained over time. The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle. PMID:8941652

A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

PubMed

Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

2015-08-01

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

Differential stimulation of neurotrophin release by the biocompatible nano-material (carbon nanotube) in primary cultured neurons.

PubMed

Kim, Yun Gi; Kim, Jong Wan; Pyeon, Hee Jang; Hyun, Jung Keun; Hwang, Ji-Young; Choi, Seong-Jun; Lee, Ja-Yeon; Deák, Ferenc; Kim, Hae-Won; Lee, Young Il

2014-01-01

In order to develop novel, effective therapies for central nervous system regeneration, it is essential to better understand the role of neurotrophic factors and to design, accordingly, better artificial scaffolds to support both neurite outgrowth and synapse formation. Both nerve growth factor and brain-derived neurotrophic factor are major factors in neural survival, development, synaptogenesis, and synaptic connectivity of primary cultured neurons. As a prime candidate coating material for such neural cultures, carbon nanotubes offer unique structural, mechanical, and electrical properties. In this study, carbon nanotubes coated glass-coverslips were used as the matrix of a primary neural culture system used to investigate the effects of carbon nanotubes on neurite outgrowth and nerve growth factor/brain-derived neurotrophic factor release and expression. For these purposes, we performed comparative analyses of primary cultured neurons on carbon nanotubes coated, non-coated, and Matrigel-coated coverslips. The morphological findings showed definite carbon nanotubes effects on the neurite outgrowths and synaptogenic figures in both cortical and hippocampal neurons when compared with the non-coated negative control. Although the carbon nanotubes did not change neurotrophin expression levels, it stimulated brain-derived neurotrophic factor release into the media from both types of neurons. Accordingly, we suggest a different mechanism of action between carbon nanotubes and Matrigel in relation to the specific neurotrophic factors. Since carbon nanotubes supply long-term extracellular molecular cues for the survival and neurite outgrowths of cultured neurons, the results from this study will contribute to an understanding of carbon nanotubes biological effects and provide new insight into their role in the secretion of neurotrophic factors.

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

PubMed Central

Degrassi, A; Hilbert, D M; Rudikoff, S; Anderson, A O; Potter, M; Coon, H G

1993-01-01

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion. Images Fig. 1 Fig. 2 PMID:8446628

Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

PubMed

Napoli, Alessandro; Obeid, Iyad

2016-03-01

Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans. © 2015 Wiley Periodicals, Inc.

Rapamycin (sirolimus) protects against hypoxic damage in primary heart cultures via Na+/Ca2+ exchanger activation.

PubMed

El-Ani, Dalia; Stav, Hagit; Guetta, Victor; Arad, Michael; Shainberg, Asher

2011-07-04

Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic-reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca(2+) homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage. We demonstrate here that rapamycin protects rat heart cultures from hypoxic-reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) measurements, and desmin immunostaining. As a result of hypoxia, intracellular calcium levels ([Ca(2+)](i)) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of [Ca(2+)](i). Rapamycin also attenuated (45)Ca(2+) uptake into the sarcoplasmic reticulum (SR) of skinned heart cultures in a dose- and time-dependent manner. KB-R7943, which inhibits the "reverse" mode of Na(+)/Ca(2+) exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased [Ca(2+)](i) following its elevation by extracellular Ca(2+) ([Ca(2+)](o)) influx, thapsigargin treatment, or depolarization with KCl. We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca(2+) outside the cardiomyocytes. According to our findings, rapamycin preserves Ca(2+) homeostasis and prevents Ca(2+) overload via extrusion of Ca(2+) surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress. Copyright © 2011 Elsevier Inc. All rights reserved.

Recapitulating cortical development with organoid culture in vitro and modeling abnormal spindle-like (ASPM related primary) microcephaly disease.

PubMed

Li, Rui; Sun, Le; Fang, Ai; Li, Peng; Wu, Qian; Wang, Xiaoqun

2017-11-01

The development of a cerebral organoid culture in vitro offers an opportunity to generate human brain-like organs to investigate mechanisms of human disease that are specific to the neurogenesis of radial glial (RG) and outer radial glial (oRG) cells in the ventricular zone (VZ) and subventricular zone (SVZ) of the developing neocortex. Modeling neuronal progenitors and the organization that produces mature subcortical neuron subtypes during early stages of development is essential for studying human brain developmental diseases. Several previous efforts have shown to grow neural organoid in culture dishes successfully, however we demonstrate a new paradigm that recapitulates neocortical development process with VZ, OSVZ formation and the lamination organization of cortical layer structure. In addition, using patient-specific induced pluripotent stem cells (iPSCs) with dysfunction of the Aspm gene from a primary microcephaly patient, we demonstrate neurogenesis defects result in defective neuronal activity in patient organoids, suggesting a new strategy to study human developmental diseases in central nerve system.

Proapoptotic effect of a micropollutant (tris-(2-chloroethyl)-phosphate) at environmental level in primary cultured renal proximal tubule cells.

PubMed

Ren, Xianghao; Han, Ho Jae; Lee, Yu Jin; Lee, Sang Hun; Ng, How Yong; Chae, Kyu-Jung; Kim, In S

2012-12-01

Being a typical micropollutant, tris-(2-chloroethyl)-phosphate (TCEP) is often found in aquatic environments. However, the potential effects of TCEP at environmental concentrations on apoptotic mechanisms are mostly unknown. Thus, the purpose of this study is to investigate the apoptotic regulatory protein expression of TCEP at environmental concentration in primary cultured renal proximal tubule cells (PTCs). The results show that TCEP at 0.01 and 1 mg L(-1) significantly increased the phosphorylation of c-Jun-NH2-terminal kinase (JNK) (135.5 and 138.0% of the control, respectively), and significantly decreased the expression of Bcl-2 and cIAP-2 at all tested concentrations, except for a slight decrease of Bcl-2 at 0.01 mg L(-1). In addition, TCEP significantly increased the expression of caspase-3 at all three concentrations (132.6, 172.6 and 167.9% of the control, respectively) and caspase-9 at 1 and 10 mg L(-1) (128.3 and 144.5% of the control, respectively). Furthermore, TCEP increased the apoptotic cell population in a flow cytometry analysis. In conclusion, environmental TCEP might have a dose-dependent proapoptotic effect with a decrease of DNA synthesis and cell number in primary cultured renal PTCs.

U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

PubMed Central

Liu, Xu; Chen, Ben; Chen, Lulan; Ren, Wan-Ting; Liu, Juan; Wang, Guoxiang; Fan, Wei; Wang, Xin; Wang, Yun

2013-01-01

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media. PMID:23560076

Misconception on Addition and Subtraction of Fraction at Primary School Students in Fifth-Grade

NASA Astrophysics Data System (ADS)

Trivena, V.; Ningsih, A. R.; Jupri, A.

2017-09-01

This study aims to investigate the mastery concept of the student in mathematics learning especially in addition and subtraction of fraction at primary school level. By using qualitative research method, the data were collected from 23 grade five students (10-11-year-old). Instruments included a test, that is accompanied by Certainty Response Index (CRI) and interview with students and teacher. The result of the test has been obtained, then processed by analyzing the student’s answers for each item and then grouped by the CRI categories that combined with the results of the interview with students and teacher. The results showed that student’s mastery-concept on additional and subtraction dominated by category ‘misconception’. So, we can say that mastery-concept on addition and subtraction of fraction at fifth-grade students is still low. Finally, the impact can make most of primary student think that learning addition and subtraction of fraction in mathematics is difficult.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

PubMed

Fiorentino, Ilaria; Gualtieri, Roberto; Barbato, Vincenza; Mollo, Valentina; Braun, Sabrina; Angrisani, Alberto; Turano, Mimmo; Furia, Maria; Netti, Paolo A; Guarnieri, Daniela; Fusco, Sabato; Talevi, Riccardo

2015-01-15

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Aboriginal Community Engagement in Primary Schooling: Promoting Learning through a Cross-Cultural Lens

ERIC Educational Resources Information Center

Turner, Angela; Wilson, Katie; Wilks, Judith L.

2017-01-01

This article reports on action research conducted at a primary school in rural New South Wales, Australia. The research responded to an expressed school aspiration to foster greater understanding of local Aboriginal culture, historical perspectives and knowledge systems within the school. An exploratory model was developed using a mixed methods…

Promoting Cultural Understandings through Song across the Tasman: Pre-Service Primary Teacher Education

ERIC Educational Resources Information Center

Joseph, Dawn; Trinick, Robyn

2016-01-01

As tertiary music educators across the Tasman we argue that music, particularly song, is an effective medium for teaching and learning about non-western music when preparing generalist primary Pre-Service Teachers (PSTs). Using "voice" as a portable and accessible vehicle to transmit cultural understandings, we draw on the Zimbabwean…

Cultures of Engagement in Challenging Circumstances: Four Lebanese Primary Schools in Urban Beirut

ERIC Educational Resources Information Center

Nabhani, Mona; Busher, Hugh; Bahous, Rima

2012-01-01

This study of four private primary schools in Beirut, Lebanon, investigated why the children in the schools appeared to out-perform their peers in other schools. The study investigated the cultures that teachers and principals constructed in schools with children and their parents, wondering whether they would exhibit characteristics said to…

Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells

PubMed Central

Raab, Monika; Kappel, Sven; Krämer, Andrea; Sanhaji, Mourad; Matthess, Yves; Kurunci-Csacsko, Elisabeth; Calzada-Wack, Julia; Rathkolb, Birgit; Rozman, Jan; Adler, Thure; Busch, Dirk H.; Esposito, Irene; Fuchs, Helmut; Gailus-Durner, Valérie; Klingenspor, Martin; Wolf, Eckhard; Sänger, Nicole; Prinz, Florian; Angelis, Martin Hrabě de; Seibler, Jost; Yuan, Juping; Bergmann, Martin; Knecht, Rainald; Kreft, Bertolt; Strebhardt, Klaus

2011-01-01

High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions. PMID:21772266

Heme Derived from Corynebacterium glutamicum: A Potential Iron Additive for Swine and an Electron Carrier Additive for Lactic Acid Bacterial Culture.

PubMed

Choi, Su-In; Park, Jihoon; Kim, Pil

2017-03-28

To investigate the potential applications of bacterial heme, aminolevulinic acid synthase (HemA) was expressed in a Corynebacterium glutamicum HA strain that had been adaptively evolved against oxidative stress. The red pigment from the constructed strain was extracted and it exhibited the typical heme absorbance at 408 nm from the spectrum. To investigate the potential of this strain as an iron additive for swine, a prototype feed additive was manufactured in pilot scale by culturing the strain in a 5 ton fermenter followed by spray-drying the biomass with flour as an excipient (biomass: flour = 1:10 (w/w)). The 10% prototype additive along with regular feed was supplied to a pig, resulting in a 1.1 kg greater increase in weight gain with no diarrhea in 3 weeks as compared with that in a control pig that was fed an additive containing only flour. To verify if C. glutamicum -synthesized heme is a potential electron carrier, lactic acid bacteria were cultured under aerobic conditions with the extracted heme. The biomasses of the aerobically grown Lactococcus lactis , Lactobacillus rhamosus , and Lactobacillus casei were 97%, 15%, and 4% greater, respectively, than those under fermentative growth conditions. As a potential preservative, cultures of the four strains of lactic acid bacteria were stored at 4°C with the extracted heme and living lactic acid bacterial cells were counted. There were more L. lactis and L. plantarum live cells when stored with heme, whereas L. rhamosus and L. casei showed no significant differences in live-cell numbers. The potential uses of the heme from C. glutamicum are further discussed.

Habitus and Flow in Primary School Musical Practice: Relations between Family Musical Cultural Capital, Optimal Experience and Music Participation

ERIC Educational Resources Information Center

Valenzuela, Rafael; Codina, Nuria

2014-01-01

Based on Bourdieu's idea that cultural capital is strongly related to family context, we describe the relations between family musical cultural capital and optimal experience during compulsory primary school musical practice. We analyse whether children from families with higher levels of musical cultural capital, and specifically with regard to…

Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

PubMed

Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

2015-10-01

Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Designing a Primary Science Curriculum in a Globalizing World: How Do Social Constructivism and Vietnamese Culture Meet?

ERIC Educational Resources Information Center

H?ng, Ngô Vu Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

2017-01-01

The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that is, the description of the design of a formal, written…

The Development of Students' Use of Additive and Proportional Methods along Primary and Secondary School

ERIC Educational Resources Information Center

Fernandez, Ceneida; Llinares, Salvador; Van Dooren, Wim; De Bock, Dirk; Verschaffel, Lieven

2012-01-01

This study investigates the development of proportional and additive methods along primary and secondary school. In particular, it simultaneously investigates the use of additive methods in proportional word problems and the use of proportional methods in additive word problems. We have also studied the role played by integer and non-integer…

Low-molecular-weight lignin-rich fraction in the extract of cultured Lentinula edodes mycelia attenuates carbon tetrachloride-induced toxicity in primary cultures of rat hepatocytes.

PubMed

Yoshioka, Yasuko; Kojima, H; Tamura, A; Tsuji, K; Tamesada, M; Yagi, K; Murakami, N

2012-01-01

The extract of cultured Lentinula edodes mycelia (LEM) is a medicinal food ingredient that has hepatoprotective effects. In this study, we fractionated the LEM extract to explore novel active compounds related to hepatoprotection by using primary cultures of rat hepatocytes exposed to carbon tetrachloride (CCl(4)). The LEM extract and the fractions markedly inhibited the release of alanine aminotransferase (ALT) from hepatocytes damaged by CCl(4) into the culture medium. The strongest hepatocyte-protective activity was seen in a fraction (Fr. 2) in which a 50% ethanol extract was further eluted with 50% methanol and separated using reverse-phase HPLC. Fr. 2 had an average molecular weight of 2753, and the main components are lignin (49%) and saccharides (36%, of which xylose comprises 41%). Therefore, Fr. 2 was presumed to be a low-molecular-weight compound consisting mainly of lignin and xylan-like polysaccharides. The hepatocyte-protective activity was observed even after digestion of xylan-like polysaccharides in Fr.2 and confirmed with low-molecular-weight lignin (LM-lignin) alone. In addition, Fr. 2, the xylan-digested Fr. 2 and LM-lignin showed higher superoxide dismutase (SOD)-like activity than the LEM extract. These results suggested that the effective fraction in the LEM extract related to hepatocyte protection consisted mainly of LM-lignin, and its antioxidant activity partially contributes to the hepatocyte-protective activity of the LEM extract.

The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs

PubMed Central

Bohan, Amy E; Purvis, Katelyn N; Bartosh, Julia L; Brandebourg, Terry D

2014-01-01

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig. PMID:26317057

Neuroglobin overexpression inhibits oxygen-glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons.

PubMed

Yu, Zhanyang; Liu, Ning; Li, Yadan; Xu, Jianfeng; Wang, Xiaoying

2013-08-01

Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb-VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD(+) release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD(+) release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD(+) release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngb's neuroprotection. Copyright © 2013 Elsevier Inc. All rights reserved.

Blood culture-PCR to optimise typhoid fever diagnosis after controlled human infection identifies frequent asymptomatic cases and evidence of primary bacteraemia.

PubMed

Darton, Thomas C; Zhou, Liqing; Blohmke, Christoph J; Jones, Claire; Waddington, Claire S; Baker, Stephen; Pollard, Andrew J

2017-04-01

Improved diagnostics for typhoid are needed; a typhoid controlled human infection model may accelerate their development and translation. Here, we evaluated a blood culture-PCR assay for detecting infection after controlled human infection with S. Typhi and compared test performance with optimally performed blood cultures. Culture-PCR amplification of blood samples was performed alongside daily blood culture in 41 participants undergoing typhoid challenge. Study endpoints for typhoid diagnosis (TD) were fever and/or bacteraemia. Overall, 24/41 (59%) participants reached TD, of whom 21/24 (86%) had ≥1 positive blood culture (53/674, 7.9% of all cultures) or 18/24 (75%) had ≥1 positive culture-PCR assay result (57/684, 8.3%). A further five non-bacteraemic participants produced culture-PCR amplicons indicating infection; overall sensitivity/specificity of the assay compared to the study endpoints were 70%/65%. We found no significant difference between blood culture and culture-PCR methods in ability to identify cases (12 mismatching pairs, p = 0.77, binomial test). Clinical and stool culture metadata demonstrated that additional culture-PCR amplification positive individuals likely represented true cases missed by blood culture, suggesting the overall attack rate may be 30/41 (73%) rather than 24/41 (59%). Several participants had positive culture-PCR results soon after ingesting challenge providing new evidence for occurrence of an early primary bacteraemia. Overall the culture-PCR assay performed well, identifying extra typhoid cases compared with routine blood culture alone. Despite limitations to widespread field-use, the benefits of increased diagnostic yield, reduced blood volume and faster turn-around-time, suggest that this assay could enhance laboratory typhoid diagnostics in research applications and high-incidence settings. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

PubMed

Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

2015-01-01

The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

Effect of Ga Addition on Morphology and Recovery of Primary Si During Al-Si Alloy Solidification Refining

NASA Astrophysics Data System (ADS)

Li, Jingwei; Bai, Xiaolong; Li, Yanlei; Ban, Boyuan; Chen, Jian

2015-12-01

The effect of Ga addition on alloy macrostructure, morphology and recovery rate of primary Si during the Al-Si-Ga alloy solvent refining process of silicon was studied in this work. The addition of Ga to Al-Si alloy could change the morphology of the primary Si. The average plate thickness of the primary Si increases with increase of Ga content. With the increase of Ga content, the average plate length of the primary Si crystals becomes larger when the Ga content is less than 5% in the Al-30%Si-xGa alloy, but becomes smaller when the Ga content exceeds 5%. Al-Si-Ga alloys consist of three types, primary Si, GaxAl1-x, (α-Al+Si+β-Ga) eutectic. (111) is the preferred growth surface of the plate-like primary Si. The recovery rate of the primary Si increases with the increase of Ga content. When the Ga content increased to 20% in Al-30%Si-xGa alloy, the relative recovery rate of the primary Si increased to 50.41% than that in Al-30%Si alloy.

Hepatoprotective effects of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] on alcohol-damaged primary rat hepatocyte culture in vitro.

PubMed

Jiang, Wenhua; Bian, Yuzhu; Wang, Zhenghui; Chang, Thomas Ming Swi

2017-02-01

We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

Progesterone and synthetic progestin, dienogest, induce apoptosis of human primary cultures of adenomyotic stromal cells.

PubMed

Yamanaka, Akiyoshi; Kimura, Fuminori; Kishi, Yohei; Takahashi, Kentaro; Suginami, Hiroshi; Shimizu, Yutaka; Murakami, Takashi

2014-08-01

To investigate the direct effects of progesterone receptor (PR) agonists on proliferation and apoptosis of human adenomyotic cells. Human primary cultures of adenomyotic stromal cells (ASCs) from 24 patients with adenomyosis were co-treated with estradiol (E2) plus the PR agonists, endogenous progesterone (P) or the synthetic progestin dienogest (DNG), which is used to treat endometriosis. In ASCs, anti-proliferative effects and induction of apoptosis were evaluated in the presence or absence of P (10(-8)-10(-6)M) or DNG (10(-8)-10(-6)M) in culture medium containing E2. Cellular proliferation was analyzed with bromodeoxyuridine incorporation and flow cytometry. Apoptosis was detected with annexin V/7-amino-actinomycin D (7-AAD) staining with flow cytometry and cellular caspase 3/7 activity. P and DNG significantly decreased the proportion of cells in the S phase. In addition, both P and DNG increased apoptosis as measured by annexin V-positive/7-AAD -negative cells and caspase 3/7 activity. Both endogenous P and synthetic progestin directly inhibited cellular proliferation and induced apoptosis in human ASCs. These pharmacological features of progestational compounds provide insight into the therapeutic strategy for the treatment of adenomyosis. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression.

PubMed

Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; Lombardi, Francesca; La Torre, Cristina; Dehcordi, Soheila Raysi; Galzio, Renato; Cimini, Annamaria; Giordano, Antonio; Cifone, Maria Grazia

2017-04-11

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Adverse events analysis as an educational tool to improve patient safety culture in primary care: a randomized trial.

PubMed

González-Formoso, Clara; Martín-Miguel, María Victoria; Fernández-Domínguez, Ma José; Rial, Antonio; Lago-Deibe, Fernando Isidro; Ramil-Hermida, Luis; Pérez-García, Margarita; Clavería, Ana

2011-06-14

Patient safety is a leading item on the policy agenda of both major international health organizations and advanced countries generally. The quantitative description of the phenomena has given rise to intense concern with the issue in institutions and organizations, leading to a number of initiatives and research projects and the promotion of patient safety culture, with training becoming a priority both in Spain and internationally. To date, most studies have been conducted in a hospital setting, even though primary care is the type most commonly used by the public, in our experience. Our study aims to achieve the following:--Assess the registry of adverse events as an education tool to improve patient safety culture in the Family and Community Teaching Units of Galicia.--Find and analyze educational tools to improve patient safety culture in primary care.--Evaluate the applicability of the Hospital Survey on Patient Safety Culture by the Agency for Healthcare Research and Quality, Spanish version, in the context of primary health care. Experimental unifactorial study of two groups, control and intervention. Tutors and residents in Family and Community Medicine in last year of studies in Galicia, Spain. From the population universe through voluntary participation. Twenty-seven tutor-resident units in each group required, randomly assigned. Residents and their respective tutor (tutor-resident pair) in teaching units on Family and Community Medicine from throughout Galicia will be invited to participate. Tutor-resident pair that agrees to participate will be sent the Hospital Survey on Patient Safety Culture. Then, tutor-resident pair will be assigned to each group--either intervention or control--through simple random sampling. The intervention group will receive specific training to record the adverse effects found in patients under their care, with subsequent feedback, after receiving instruction on the process. No action will be taken in the control group. After

Cranberry Products Inhibit Adherence of P-Fimbriated Escherichia Coli to Primary Cultured Bladder and Vaginal Epithelial Cells

PubMed Central

Gupta, K.; Chou, M. Y.; Howell, A.; Wobbe, C.; Grady, R.; Stapleton, A. E.

2011-01-01

Purpose Cranberry proanthocyanidins have been identified as possible inhibitors of Escherichia coli adherence to uroepithelial cells. However, little is known about the dose range of this effect. Furthermore, it has not been studied directly in the urogenital system. To address these issues we tested the effect of a cranberry powder and proanthocyanidin extract on adherence of a P-fimbriated uropathogenic E. coli isolate to 2 new urogenital model systems, namely primary cultured bladder epithelial cells and vaginal epithelial cells. Materials and Methods E. coli IA2 was pre-incubated with a commercially available cranberry powder (9 mg proanthocyanidin per gm) or with increasing concentrations of proanthocyanidin extract. Adherence of E. coli IA2 to primary cultured bladder epithelial cells or vaginal epithelial cells was measured before and after exposure to these products. Results Cranberry powder decreased mean adherence of E. coli IA2 to vaginal epithelial cells from 18.6 to 1.8 bacteria per cell (p <0.001). Mean adherence of E. coli to primary cultured bladder epithelial cells was decreased by exposure to 50 μg/ml proanthocyanidin extract from 6.9 to 1.6 bacteria per cell (p <0.001). Inhibition of adherence of E. coli by proanthocyanidin extract occurred in linear, dose dependent fashion over a proanthocyanidin concentration range of 75 to 5 μg/ml. Conclusions Cranberry products can inhibit E. coli adherence to biologically relevant model systems of primary cultured bladder and vaginal epithelial cells. This effect occurs in a dose dependent relationship. These findings provide further mechanistic evidence and biological plausibility for the role of cranberry products for preventing urinary tract infection. PMID:17509358

Are Mammographically Occult Additional Tumors Identified More Than 2 Cm Away From the Primary Breast Cancer on MRI Clinically Significant?

PubMed

Goodman, Sarah; Mango, Victoria; Friedlander, Lauren; Desperito, Elise; Wynn, Ralph; Ha, Richard

2018-06-08

To evaluate the clinical significance of mammographically occult additional tumors identified more than 2cm away from the primary breast cancer on preoperative magnetic resonance imaging (MRI). An Institutional Review Board approved review of consecutive preoperative breast MRIs performed from 1/1/08 to 12/31/14, yielded 667 patients with breast cancer. These patients underwent further assessment to identify biopsy proven mammographically occult breast tumors located more than 2cm away from the edge of the primary tumor. Additional MRI characteristics of the primary and secondary tumors and pathology were reviewed. Statistical analysis was performed using SPSS (v. 24). Of 667 patients with breast cancer, 129 patients had 150 additional ipsilateral mammographically occult tumors that were more than 2cm away from the edge of the primary tumor. One hundred twelve of 129 (86.8%) patients had one additional tumor and 17/129 (13.2%) had two or more additional tumors. In 71/129 (55.0%), additional tumors were located in a different quadrant and in 58/129 (45.0%) additional tumors were in the same quadrant but ≥2cm away. Overall, primary tumor size was significantly larger (mean 1.87± 1.25 cm) than the additional tumors (mean 0.79 ± 0.61cm, p < 0.001). However, in 20/129 (15.5%) the additional tumor was larger and in 26/129 (20.2%) the additional tumor was ≥1cm. The primary tumor was significantly more likely to be invasive (81.4%, 105/129) compared to additional tumors (70%, 105/150, p = 0.03). In 9/129 (7.0%) patients, additional tumors yielded unsuspected invasive cancer orhigher tumor grade. The additional tumor was more likely to be nonmass lesion type (37.3% vs 24% p = 0.02) and focus lesion type (10% vs 0.08%, p < 0.001) compared to primary tumor. Mammographically occult additional tumors identified more than 2cm away from the primary breast tumor on MRI are unlikely to be surgically treated if undiagnosed and may be clinically significant. Copyright �

Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

PubMed

Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

2015-05-21

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation

Cultural responses to pain in UK children of primary school age: a mixed-methods study.

PubMed

Azize, Pary M; Endacott, Ruth; Cattani, Allegra; Humphreys, Ann

2014-06-01

Pain-measurement tools are often criticized for not addressing the influence of culture and ethnicity on pain. This study examined how children who speak English as a primary or additional language discuss pain. Two methods were used in six focus group interviews with 34 children aged 4-7 years: (i) use of drawings from the Pediatric Pain Inventory to capture the language used by children to describe pain; and (ii) observation of the children's placing of pain drawings on red/amber/green paper to denote perceived severity of pain. The findings demonstrated that children with English as an additional language used less elaborate language when talking about pain, but tended to talk about the pictures prior to deciding where they should be placed. For these children, there was a positive significant relationship between language, age, and length of stay in the UK. The children's placement of pain drawings varied according to language background, sex, and age. The findings emphasize the need for sufficient time to assess pain adequately in children who do not speak English as a first language. © 2013 Wiley Publishing Asia Pty Ltd.

Leptin inhibits amyloid β-protein degradation through decrease of neprilysin expression in primary cultured astrocytes.

PubMed

Yamamoto, Naoki; Tanida, Mamoru; Ono, Yoko; Kasahara, Rika; Fujii, Yuko; Ohora, Kentaro; Suzuki, Kenji; Sobue, Kazuya

2014-02-28

Pathogenesis of Alzheimer's disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK. Copyright © 2014 Elsevier Inc. All rights reserved.

Importance of perceived naturalness for acceptance of food additives and cultured meat.

PubMed

Siegrist, Michael; Sütterlin, Bernadette

2017-06-01

Four experiments examined some factors influencing the perceived naturalness of food products and their biasing effect on risk perception. The results of Experiment 1a showed that three food additives displaying their respective E-numbers (i.e., codes for food additives in the European Union and Switzerland) decreased perceived naturalness. Experiment 1b demonstrated that mentioning possible health effects decreased the perceived naturalness of a plant-based food additive. This experiment further showed that it would not matter for perceived naturalness whether the food was synthetic or nature-identical. Moreover, the results of Experiments 2 and 3 suggested that the same risk associated with meat consumption was much more acceptable for traditionally produced meat compared with in-vitro meat. Experiment 3 further indicated that the perceived naturalness of the meat (i.e., traditional or cultured meat) had a full mediation effect on participants' evaluation of the acceptability of the risk of colon cancer associated with the meat consumption. Even if the new production method (i.e., cultured meat) was more environmentally friendly and less harmful to animals, the perceived lack of naturalness might reduce the acceptability of the risk associated with such a product. The present study provides evidence that consumers rely on symbolic information when evaluating foods, which may lead to biased judgments and decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Process cost and facility considerations in the selection of primary cell culture clarification technology.

PubMed

Felo, Michael; Christensen, Brandon; Higgins, John

2013-01-01

The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined. © 2013 American Institute of Chemical Engineers.

Establishment of a long-term primary culture of striatal neurons.

PubMed

Sebben, M; Gabrion, J; Manzoni, O; Sladeczek, F; Gril, C; Bockaert, J; Dumuis, A

1990-03-01

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.

The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures.

PubMed

Dringen, R; Hamprecht, B; Bröer, S

1998-07-01

The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.

Adverse events analysis as an educational tool to improve patient safety culture in primary care: A randomized trial

PubMed Central

2011-01-01

Background Patient safety is a leading item on the policy agenda of both major international health organizations and advanced countries generally. The quantitative description of the phenomena has given rise to intense concern with the issue in institutions and organizations, leading to a number of initiatives and research projects and the promotion of patient safety culture, with training becoming a priority both in Spain and internationally. To date, most studies have been conducted in a hospital setting, even though primary care is the type most commonly used by the public, in our experience. Our study aims to achieve the following: - Assess the registry of adverse events as an education tool to improve patient safety culture in the Family and Community Teaching Units of Galicia. - Find and analyze educational tools to improve patient safety culture in primary care. - Evaluate the applicability of the Hospital Survey on Patient Safety Culture by the Agency for Healthcare Research and Quality, Spanish version, in the context of primary health care. Design and methods Design Experimental unifactorial study of two groups, control and intervention. Study population Tutors and residents in Family and Community Medicine in last year of studies in Galicia, Spain. Sample From the population universe through voluntary participation. Twenty-seven tutor-resident units in each group required, randomly assigned. Intervention Residents and their respective tutor (tutor-resident pair) in teaching units on Family and Community Medicine from throughout Galicia will be invited to participate. Tutor-resident pair that agrees to participate will be sent the Hospital Survey on Patient Safety Culture. Then, tutor-resident pair will be assigned to each group-either intervention or control-through simple random sampling. The intervention group will receive specific training to record the adverse effects found in patients under their care, with subsequent feedback, after receiving

Cognition, emotion, and arithmetic in primary school: A cross-cultural investigation.

PubMed

Rodic, Maja; Cui, Jiaxin; Malykh, Sergey; Zhou, Xinlin; Gynku, Elena I; Bogdanova, Elena L; Zueva, Dina Y; Y Bogdanova, Olga; Kovas, Yulia

2018-06-01

The study investigated cross-cultural differences in variability and average performance in arithmetic, mathematical reasoning, symbolic and non-symbolic magnitude processing, intelligence, spatial ability, and mathematical anxiety in 890 6- to 9-year-old children from the United Kingdom, Russia, and China. Cross-cultural differences explained 28% of the variance in arithmetic and 17.3% of the variance in mathematical reasoning, with Chinese children outperforming the other two groups. No cross-cultural differences were observed for spatial ability and mathematical anxiety. In all samples, symbolic magnitude processing and mathematical reasoning were independently related to early arithmetic. Other factors, such as non-symbolic magnitude processing, mental rotation, intelligence, and mathematical anxiety, produced differential patterns across the populations. The results are discussed in relation to potential influences of parental practice, school readiness, and linguistic factors on individual differences in early mathematics. Statement of contribution What is already known on this subject? Cross-cultural differences in mathematical ability are present in preschool children. Similar mechanisms of mathematical development operate in preschool children from the United Kingdom, Russia, and China. Tasks that require understanding of numbers are best predictors of arithmetic in preschool children. What does this study add? Cross-cultural differences in mathematical ability become greater with age/years of formal education. Similar mechanisms of mathematical development operate in early primary school children from the United Kingdom, Russia, and China. Symbolic number magnitude and mathematical reasoning are the main predictors of arithmetic in all three populations. © 2018 The Authors British Journal of Developmental Psychology published by John Wiley & Sons Ltd on behalf of British Psychological Society.

Short communication: Tryptic β-casein hydrolysate modulates enteric nervous system development in primary culture.

PubMed

Cossais, F; Clawin-Rädecker, I; Lorenzen, P C; Klempt, M

2017-05-01

The intestinal tract of the newborn is particularly sensitive to gastrointestinal disorders, such as infantile diarrhea or necrotizing colitis. Perinatal development of the gut also encompasses the maturation of the enteric nervous system (ENS), a main regulator of intestinal motility and barrier functions. It was recently shown that ENS maturation can be enhanced by nutritional factors to improve intestinal maturation. Bioactivity of milk proteins is often latent, requiring the release of bioactive peptides from inactive native proteins. Several casein-derived hydrolysates presenting immunomodulatory properties have been described recently. Furthermore, accumulating data indicate that milk-derived hydrolysate can enhance gut maturation and enrichment of milk formula with such hydrolysates has recently been proposed. However, the capability of milk-derived bioactive hydrolysate to target ENS maturation has not been analyzed so far. We, therefore, investigated the potential of a recently described tryptic β-casein hydrolysate to modulate ENS growth parameters in an in vitro model of rat primary culture of ENS. Rat primary cultures of ENS were incubated with a bioactive tryptic β-casein hydrolysate and compared with untreated controls or to cultures treated with native β-casein or a Prolyve β-casein hydrolysate (Lyven, Colombelles, France). Differentiation of enteric neurons and enteric glial cells, and establishment of enteric neural network were analyzed using immunohistochemistry and quantitative PCR. Effect of tryptic β-casein hydrolysate on bone morphogenetic proteins (BMP)/Smad pathway, an essential regulator of ENS development, was further assessed using quantitative PCR and immunochemistry. Tryptic β-casein hydrolysate stimulated neurite outgrowth and simultaneously modulated the formation of enteric ganglia-like structures, whereas native β-casein or Prolyve β-casein hydrolysate did not. Additionally, treatment with tryptic bioactive �

Influence of workplace culture on nursing-sensitive nurse outcomes in municipal primary health care.

PubMed

Hahtela, Nina; Paavilainen, Eija; McCormack, Brendan; Slater, Paul; Helminen, Mika; Suominen, Tarja

2015-10-01

To explore the influence of workplace culture on sickness absences, overtime work and occupational injuries in municipal primary health care. The need to improve nursing sensitive outcomes has been highlighted. Therefore, an adequate understanding of the influence of workplace culture on nursing-sensitive nurse outcomes is essential for nurse managers to meet the requirements of improving nursing outcomes. A cross-sectional survey design was used to incorporating the data from 21 inpatient acute care units of nine organisations at the Finnish municipal primary health care system from 2011 to 2012. Findings emphasise in particular the importance of the practice environment as being an interpretative factor for nurses' absences owing to sickness, overtime work and occupational injuries. To ensure favourable nursing sensitive outcomes it is essential that there is a shared interest in the unit to invest in the creation of a supportive practice environment. Outcome improvements require a special focus on issues related to nursing management, adequate staffing and resources and intention to leave. © 2014 John Wiley & Sons Ltd.

Studying Mucin Secretion from Human Bronchial Epithelial Cell Primary Cultures

PubMed Central

Abdullah, Lubna H.; Wolber, Cédric; Kesimer, Mehmet; Sheehan, John K.; Davis, C. William

2016-01-01

Mucin secretion is regulated by extracellular signaling molecules emanating from local, neuronal, or endocrine sources. Quantifying the rate of this secretion is important to understanding how the exocytic process is regulated, and also how goblet/mucous cells synthesize and release mucins under control and pathological conditions. Consequently, measuring mucins in a quantitatively accurate manner is the key to many experiments addressing these issues. This paper describes procedures used to determine agonist-induced mucin secretion from goblet cells in human bronchial epithelial (HBE) cell cultures. It begins with primary epithelial cell culture, offers methods for purifying MUC5AC and MUC5B mucins for standards, and describes five different microtiter plate binding assays which use various probes for mucins. A polymeric mucin-specific antibody is used in standard and sandwich ELISA formats for two assays while the others target the extensive glycosylated domains of mucins with lectin, periodate oxidation, and antibody-based probes. Comparing the data derived from the different assays applied to the same set of samples of HBE cell cultures indicates a qualitative agreement between baseline and agonist stimulated mucin release; however, the polymeric mucin-specific assays yield substantially lower values than the assays using nonspecific molecular reporters. These results indicate that the more non-specific assays are suitable to assess overall secretory responses by goblet cells, but are likely unsuited for specific measurements of polymeric mucins, per se. PMID:22259142

Inorganic carbon addition stimulates snow algae primary productivity

NASA Astrophysics Data System (ADS)

Hamilton, T. L.; Havig, J. R.

2017-12-01

Earth has experienced glacial/interglacial oscillations throughout its history. Today over 15 million square kilometers (5.8 million square miles) of Earth's land surface is covered in ice including glaciers, ice caps, and the ice sheets of Greenland and Antarctica, most of which are retreating as a consequence of increased atmospheric CO2. Glaciers are teeming with life and supraglacial snow and ice surfaces are often red due to blooms of photoautotrophic algae. Recent evidence suggests the red pigmentation, secondary carotenoids produced in part to thrive under high irradiation, lowers albedo and accelerates melt. However, there are relatively few studies that report the productivity of snow algae communities and the parameters that constrain their growth on snow and ice surfaces. Here, we demonstrate that snow algae primary productivity can be stimulated by the addition of inorganic carbon. We found an increase in light-dependent carbon assimilation in snow algae microcosms amended with increasing amounts of inorganic carbon. Our snow algae communities were dominated by typical cosmopolitan snow algae species recovered from Alpine and Arctic environments. The climate feedbacks necessary to enter and exit glacial/interglacial oscillations are poorly understood. Evidence and models agree that global Snowball events are accompanied by changes in atmospheric CO2 with increasing CO2 necessary for entering periods of interglacial time. Our results demonstrate a positive feedback between increased CO2 and snow algal productivity and presumably growth. With the recent call for bio-albedo effects to be considered in climate models, our results underscore the need for robust climate models to include feedbacks between supraglacial primary productivity, albedo, and atmospheric CO2.

Moclobemide exerts anti-inflammatory effect in lipopolysaccharide-activated primary mixed glial cell culture.

PubMed

Bielecka, A M; Paul-Samojedny, M; Obuchowicz, E

2010-12-01

An increasing body of evidence indicates that glial activation and neuroinflammation play an important role in the pathogenesis of psychiatric and neurodegenerative diseases. Activated glial cells secrete various cytokines that influence neurotransmission, hypothalamus-pituitary-adrenal axis activity, neuronal plasticity and neurogenesis. It has been suggested that alterations in cytokine networks are involved in the mechanism of action of antidepressant drugs. Until now, only a few studies demonstrated that some tricyclic antidepressants and selective serotonin reuptake inhibitors reduced production of pro-inflammatory cytokines in brain glia cells. We have investigated for the first time whether the antidepressant, moclobemide (a reversible selective inhibitor of monoamine oxidase-A) has an influence on pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and anti-inflammatory cytokine (IL-10) in primary rat mixed glial cell cultures stimulated by lipopolysaccharide (LPS). Our results showed that moclobemide used in a wide range of concentrations diminished LPS-stimulated IL-1β and TNF-α mRNAs expression in cellular extracts and remarkably reduced the levels of both pro-inflammatory cytokines in culture medium. In opposite to this, the drug had no influence on IL-10 mRNA and slightly reduced IL-10 concentration. Moreover, moclobemide decreased LPS-stimulated translocation of NFκB p65 subunit into cellular nuclei. These results suggest that moclobemide exerts anti-inflammatory effect in the central nervous system because it affects the balance between pro- and anti-inflammatory cytokines (IL-1β, TNF-α/IL-10) in primary mixed glial cell cultures.

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts.

PubMed

Bratka-Robia, Christine B; Mitteregger, Gerda; Aichinger, Amanda; Egerbacher, Monika; Helmreich, Magdalena; Bamberg, Elmar

2002-02-01

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.

Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells.

PubMed

Pizzi, M; Fallacara, C; Arrighi, V; Memo, M; Spano, P F

1993-08-01

Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration-dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans-1-aminocyclopentane-1,3-dicarboxylate or by quisqualate counteracts both glutamate- and kainate-induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR-evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration-dependently counteracted glutamate-, kainate-, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans-1-aminocyclopentane-1,3-dicarboxylate. In addition, aniracetam potentiated the mGluR-coupled stimulation of phospholipase C, as revealed by the measurement of 3H-inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.

Osmotic regulation of myo-inositol uptake in primary astrocyte cultures.

PubMed

Isaacks, R E; Bender, A S; Kim, C Y; Prieto, N M; Norenberg, M D

1994-03-01

Uptake of myo-inositol by astrocytes in hypertonic medium (440 mosm/kg H2O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein). myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H2O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation of myo-inositol uptake. A 30 to 40 mosm/kg H2O deviation from physiological osmolality can influence myo-inositol homeostasis. The intracellular content of myo-inositol in astrocytes in isotonic medium was 25.6 +/- 1.3 micrograms/mg protein (28 mM). This level of myo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.

Effects of phenobarbital on aniline metabolism in primary liver cell culture of rats with ethionine-induced liver disorder.

PubMed

Noguchi, M; Nitoh, S; Mabuchi, M; Kawai, Y

1996-04-01

In experiment 1, the amount of aniline (AN) metabolites in the primary cell culture medium of the liver cells obtained from ethionine (ET)-treated rats was compared with that of the control (normal) rats. Although the metabolites detected in both groups were p-aminophenol (p-AP), N-acetyl-p-AP (AAP), acetoanilide (AAN), AAP-glucuronide (AAPG), phenylhydroxylamine sulfate (PHAS) and p-AP-glucuronide (p-APG), the amount of AAP was lower and that of p-APG was markedly higher in the ET-treated rats than in the control rats. In experiment 2, phenobarbital (PB) was orally administered to the ET-treated and control rats at a dose of 100 mg/kg. The time course changes in AN metabolites in the primary cell culture medium of liver cells obtained at 2 or 48 hr after PB treatment were compared with those without PB treatment. In the ET-treated rats, the amount of PHAS was slightly higher at 2 hr after PB treatment, and that of AAP was lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. In the control rats, the amounts of AAP, AAN, p-AP and p-APG at 2 hr after PB treatment remained lower than those without PB treatment, and that of AAP was markedly lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. These findings indicated greater detoxication in the primary liver cell culture in the ET-treated rats than in the control rats. Furthermore, detoxication was greater in the primary cell culture of liver cell obtained from the ET-treated rats after PB treatment than from those without PB treatment, because the production of acetylates (AAP) decreased and p-APG increased (induction of conjugated enzyme) in the PB treatment group.

Advances in primary recovery: centrifugation and membrane technology.

PubMed

Roush, David J; Lu, Yuefeng

2008-01-01

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification. Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers. Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead-end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary

Computational modeling of colorimetric primary transducer for metrological assurance in additive manufacturing

NASA Astrophysics Data System (ADS)

Skliarov, Volodymyr

2018-03-01

Many additive manufacturing (AM) systems are based on laser technology. The advantage of laser technology is that it provides a high-intensity and high-collimation energy beam that can be controlled. Since AM requires that the material on each layer has to be solid or connected to the previous one, the energy of laser radiation is exactly the needed technical tool for the processing of the material. AM uses two types of laser processing: cutting and heating. One of the most popular (common) types of measurements in the field of laser metrology is the control of the energy parameters of the sources of laser radiation. At present, calorimeters provide the highest accuracy of absolute measurements of laser radiation in the power range from several watts to tens of kilowatts. The main elements that determine the accuracy of reproduction, maintenance and transfer of the unit of laser power are the primary measuring converters (PMCs), which are the part of the equipment of the national primary measurement standards of Ukraine. A significant contribution to the uncertainty budget of the primary measuring calorimetric converter is the unbalanced replacement of laser radiation by the heat flux that calibrates this converter. The heterogeneous internal structure of the calorimetric primary converter, the nonlinearity of processes occurring in it, and the multifactorial process of its calibration substantially complicate the development of primary measuring converters. The purpose of this paper is to simulate the thermal field of the primary converter for maximum reduction of the uncertainty of calibration. The presented research is a part of the scientific work that NSC "Institute of Metrology" carries out under COOMET and EMPIRE projects. The modeling was performed in the academic version of ANSYS.

Political, cultural and economic foundations of primary care in Europe.

PubMed

Kringos, Dionne S; Boerma, Wienke G W; van der Zee, Jouke; Groenewegen, Peter P

2013-12-01

This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda, economy, prevailing values, and type of healthcare system are all important factors that influence the development of strong PC. Wealthier countries are associated with a weaker PC structure and lower PC accessibility, while Eastern European countries seemed to have used their growth in national income to strengthen the accessibility and continuity of PC. Countries governed by left-wing governments are associated with a stronger PC structure, accessibility and coordination of PC. Countries with a social-security based system are associated with a lower accessibility and continuity of PC; the opposite is true for transitional systems. Cultural values seemed to affect all aspects of PC. It can be concluded that strengthening PC means mobilising multiple leverage points, policy options, and political will in line with prevailing values in a country. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells.

PubMed

Márquez, Laura B; Araoz, Alicia; Repetto, Horacio A; Ibarra, Fernando R; Silberstein, Claudia

2016-10-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes post-diarrheal Hemolytic Uremic Syndrome (HUS), which is one of the most common causes of acute renal failure in children in Argentine. The aim of the present work was to study the effects of Shiga toxin type 2 (Stx2) on regenerative mechanisms of primary cultures of human cortical renal tubular epithelial cells (HRTEC) and three-dimensional (3D) cultures of HRTEC. Primary cultures of HRTEC were able to develop tubular structures when grown in matrigel, which showed epithelial cells surrounding a central lumen resembling the original renal tubules. Exposure to Stx2 inhibited tubulogenesis in 3D-HRTEC cultures. Moreover, a significant increase in apoptosis, and decrease in cell proliferation was observed in tubular structures of 3D-HRTEC exposed to Stx2. A significant reduction in cell migration and vimentin expression levels was observed in HRTEC primary cultures exposed to Stx2, demonstrating that the holotoxin affected HRTEC dedifferentiation. Furthermore, a decreased number of cells expressing CD133 progenitor marker was found in HRTEC cultures treated with Stx2. The CD133 positive cells also expressed the Stx receptor globotriaosylceramide, which may explain their sensitivity to Stx2. In conclusion, Stx2 affects the regenerative processes of human renal tubular epithelial cells in vitro, by inhibiting cell dedifferentiation mechanisms, as well as tubules restoration. The development of 3D-HRTEC cultures that resemble original human renal proximal tubules is a novel in vitro model to study renal epithelial repair mechanisms after injury. Copyright © 2016 Elsevier Ltd. All rights reserved.

Culture Care Theory: a proposed practice theory guide for nurse practitioners in primary care settings.

PubMed

McFarland, Marilyn M; Eipperle, Marilyn K

2008-04-01

Leininger's Theory of Culture Care Diversity and Universality is presented as a foundational basis for the educational preparation, primary care contextual practice, and outcomes-focused research endeavours of advanced practice nursing. Discussion emphasises the value of care and caring as the essence of advanced practice nursing through the use of three modes of care, use of the Sunrise and other enablers, and the ethnonursing method. Education, research, practice, and key concepts of the theory are connected as essential components toward the provision of culturally congruent care to meet the healthcare needs of diverse individuals, families, groups, and communities by family nurse practitioners.

[Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

PubMed

Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

2017-04-12

Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

PubMed Central

Jeppesen, Maria; Hagel, Grith; Glenthoj, Anders; Vainer, Ben; Ibsen, Per; Harling, Henrik; Thastrup, Ole; Jørgensen, Lars N.

2017-01-01

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient’s tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine. PMID:28877221

Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kern, P.A.; Marshall, S.; Eckel, R.H.

1985-01-01

To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific /sup 125/I-insulin binding. In addition, chloroquine induced an increase in cell-associated /sup 125/I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measuredmore » as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation. In addition, HR was found to correlate logarithmically and inversely with body mass index. Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology.« less

Patient safety culture in out-of-hours primary care services in the Netherlands: a cross-sectional survey.

PubMed

Smits, Marleen; Keizer, Ellen; Giesen, Paul; Deilkås, Ellen Catharina Tveter; Hofoss, Dag; Bondevik, Gunnar Tschudi

2018-03-01

To examine patient safety culture in Dutch out-of-hours primary care using the safety attitudes questionnaire (SAQ) which includes five factors: teamwork climate, safety climate, job satisfaction, perceptions of management and communication openness. Cross-sectional observational study using an anonymous web-survey. Setting Sixteen out-of-hours general practitioner (GP) cooperatives and two call centers in the Netherlands. Subjects Primary healthcare providers in out-of-hours services. Main outcome measures Mean scores on patient safety culture factors; association between patient safety culture and profession, gender, age, and working experience. Overall response rate was 43%. A total of 784 respondents were included; mainly GPs (N = 470) and triage nurses (N = 189). The healthcare providers were most positive about teamwork climate and job satisfaction, and less about communication openness and safety climate. The largest variation between clinics was found on safety climate; the lowest on teamwork climate. Triage nurses scored significantly higher than GPs on each of the five patient safety factors. Older healthcare providers scored significantly higher than younger on safety climate and perceptions of management. More working experience was positively related to higher teamwork climate and communication openness. Gender was not associated with any of the patient safety factors. Our study showed that healthcare providers perceive patient safety culture in Dutch GP cooperatives positively, but there are differences related to the respondents' profession, age and working experience. Recommendations for future studies are to examine reasons for these differences, to examine the effects of interventions to improve safety culture and to make international comparisons of safety culture. Key Points Creating a positive patient safety culture is assumed to be a prerequisite for quality and safety. We found that: • healthcare providers in Dutch GP cooperatives

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

PubMed

Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D

2016-04-01

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation. © 2015 International Federation for Cell Biology.

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture.

PubMed

Nakanishi, Takeo; Fukushi, Akimasa; Sato, Masanobu; Yoshifuji, Mayuko; Gose, Tomoka; Shirasaka, Yoshiyuki; Ohe, Kazuyo; Kobayashi, Masato; Kawai, Keiichi; Tamai, Ikumi

2011-12-05

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.

Teaching Cultural History from Primary Events

ERIC Educational Resources Information Center

Carson, Robert N.

2004-01-01

This article explores the relationship between specific cultural events such as Galileo's work with the pendulum and a curriculum design that seeks to establish in skeletal form a comprehensive epic narrative about the co-evolution of cultural systems and human consciousness. The article explores some of the challenges and some of the strategies…

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2.

PubMed

Hong, Fashui; Sheng, Lei; Ze, Yuguan; Hong, Jie; Zhou, Yingjun; Wang, Ling; Liu, Dong; Yu, Xiaohong; Xu, Bingqing; Zhao, Xiaoyang; Ze, Xiao

2015-06-01

Numerous studies have indicated that nano-titanium dioxide (TiO2) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO2 affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO2 for 24 h, and nano-TiO2 internalization, dendritic growth, glutamate metabolism, expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (INa) and potassium current (IK) were examined. Our findings demonstrated that nano-TiO2 crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO2 induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca(2+)]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the INa and IK. In addition, nano-TiO2 increased nitric oxide and nitrice synthase, attenuated the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO2 inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Revealing Additional Dimensions of Globalisation and Cultural Hegemony: A Response to Roland S. Persson's Call for Cultural Sensitivity in Gifted Studies

ERIC Educational Resources Information Center

Ambrose, Don

2012-01-01

In this commentary, the author finds the interdisciplinary approach of Roland S. Persson's (2012a) target article refreshing. Persson's (2012a) additional emphases on ethnocentricity, cultural bias and strong threads of influence from the global economy also are helpful. They shed light on some strong contextual influences that shape the…

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level

PubMed Central

Meißner, Joachim D; Kubis, Hans-Peter; Scheibe, Renate J; Gros, Gerolf

2000-01-01

The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression. PMID:10673542

Impact of the organisational culture on primary care staff members' intention to engage in research and development.

PubMed

Morténius, Helena; Baigi, Amir; Palm, Lars; Fridlund, Bengt; Björkelund, Cecilia; Hedberg, Berith

2015-01-01

The purpose of this paper is to understand how organisational culture influences the intentions of primary care staff members (PCSM) to engage in research and development (R&D). The participants (n=30) were PCSM employed in a care centre in south-western Sweden. The study had an observational design with an ethnographic approach. The data were collected by means of observations, interviews and analysis of documents. The results revealed the perceptions of PCSM in two domains, research and clinical practice, both of which existed at three different cultural levels: visible (structures and policy), semi-visible (norms and values) and invisible (taken-for-granted attitudes). It is difficult to conduct a purely objective ethnographic study because the investigation is controlled by its context. However, it is necessary to highlight and discuss the invisible level to improve understanding of negative attitudes and preconceptions related to the implementation of R&D in the clinical setting. By highlighting the invisible level of culture, the management of an organisation has the opportunity to initiate discussion of issues related to concealed norms and values as well as attitudes towards new thinking and change in the primary health context. This paper is one of the very few studies to investigate the influence of organisational culture on the intentions of PCSM to engage in R&D.

Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture

PubMed Central

Kubis, Hans-Peter; Haller, Ernst-August; Wetzel, Petra; Gros, Gerolf

1997-01-01

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs. PMID:9108130

Cultural differences in complex addition: efficient Chinese versus adaptive Belgians and Canadians.

PubMed

Imbo, Ineke; LeFevre, Jo-Anne

2009-11-01

In the present study, the authors tested the effects of working-memory load on math problem solving in 3 different cultures: Flemish-speaking Belgians, English-speaking Canadians, and Chinese-speaking Chinese currently living in Canada. Participants solved complex addition problems (e.g., 58 + 76) in no-load and working-memory load conditions, in which either the central executive or the phonological loop was loaded. The authors used the choice/no-choice method to obtain unbiased measures of strategy selection and strategy efficiency. The Chinese participants were faster than the Belgians, who were faster and more accurate than the Canadians. The Chinese also required fewer working-memory resources than did the Belgians and Canadians. However, the Chinese chose less adaptively from the available strategies than did the Belgians and Canadians. These cultural differences in math problem solving are likely the result of different instructional approaches during elementary school (practice and training in Asian countries vs. exploration and flexibility in non-Asian countries), differences in the number language, and informal cultural norms and standards. The relevance of being adaptive is discussed as well as the implications of the results in regards to the strategy choice and discovery simulation model of strategy selection (J. Shrager & R. S. Siegler, 1998).

Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine.

PubMed

Lobo, Nazleen C; Gedye, Craig; Apostoli, Anthony J; Brown, Kevin R; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A S; Ailles, Laurie

2016-07-16

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The

A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

PubMed

Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

2016-01-01

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance

PubMed Central

Breslin, Susan; O'Driscoll, Lorraine

2016-01-01

Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs. PMID:27304190

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons.

PubMed

Rubiolo, Juan Andrés; Vale, Carmen; Boente-Juncal, Andrea; Hirama, Masahiro; Yamashita, Shuji; Camiña, Mercedes; Vieytes, Mercedes R; Botana, Luis M

2018-05-10

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.

The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

PubMed Central

Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

2015-01-01

To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

Ergothioneine Contents in Fruiting Bodies and Their Enhancement in Mycelial Cultures by the Addition of Methionine

PubMed Central

Lee, Wi Young; Ahn, Jin Kwon; Ka, Kang-Hyeon

2009-01-01

The levels of ergothioneine (ERG), which have been shown to act as an excellent antioxidant, were determined in both fruiting bodies and mycelia of various mushroom species. We found that ERG accumulated at different levels in fruiting bodies of mushrooms and showed up to a 92.3-fold difference between mushrooms. We also found that ERG accumulated at higher levels in mycelia than in fruiting bodies of economically important mushroom species such as Ganoderma neo-japonicum, G. applanatum and Paecilomyces tenuipes. The addition of 2 mM methionine (Met) to mycelial culture medium increased the ERG contents in most mushroom species tested, indicating that Met is a good additive to enhance the ERG levels in a variety of mushroom species. Taking these results into consideration, we suggest that the addition of Met to the mycelial culture medium is an efficient way to enhance the antioxidant properties in economically important mushroom species. PMID:23983506

Designing and Using an Organisational Culture Inquiry Tool to Glimpse the Relational Nature of Leadership and Organisational Culture within a South Australian Primary School

ERIC Educational Resources Information Center

Giles, David; Bills, Andrew

2017-01-01

This case study research found that the relational leadership and organisational culture at a public primary school situated in a high poverty location in South Australia was built upon the strength of the inter-relationships between the teachers, teachers and leadership, and between teachers and students. Supported by what we called "dynamic…

p53 participates in the protective effects of ischemic post-conditioning against OGD-reperfusion injury in primary cultured spinal cord neurons.

PubMed

Li, Jinquan; Chen, Gong; Gao, Xinjie; Shen, Chao; Zhou, Ping; Wu, Xing; Che, Xiaoming; Xie, Rong

2017-01-18

Spinal cord ischemia-reperfusion (I/R) injury is a severe clinical condition, while the mechanism is still not clarified and the therapeutic approach is limited. Ischemia post-conditioning (PC) has been found to have the protective effects against I/R injury in brain. Recently p53 has been reported to take part in the regulation and protection of I/R injury. We hypothesize that PC has the protective effects in primary cultured spinal cord neurons against ischemia-reperfusion injury, and MDM2-p53 signaling pathway may involve in its protective mechanism. In this study, we used an OGD (oxygen and glucose deprivation)-reperfusion model in primary cultured spinal cord neurons to simulate the I/R injury of spinal cord in vitro, and PC was conducted by 3 cycles of 15min restoration of glucose and oxygen with 15min OGD, followed by 6h fully restoration as reperfusion. Lentiviral vectors were used to knock down MDM2 or over-express p53 genes in primary cultured spinal cord neurons. The results showed that 3 cycles of 15min PC generated the most significant protective effects in primary cultured spinal cord neurons against OGD-reperfusion injury. The levels of MDM2 were decreased while p53, Bax, and cleaved Caspase 3 were increased under OGD-reperfusion condition. PC could significantly reverse the down-regulation of MDM2 and up-regulation of p53, Bax, and cleaved Caspase 3 by OGD-reperfusion injury. Moreover, MDM2 knockdown or p53 over-expression could induce the cleaved Caspase 3 expression and blocked the protective effects of PC in primary cultured spinal cord neurons against OGD-reperfusion injury. In conclusion, our work demonstrated that MDM2-p53 pathway plays a pivotal role in the protective effect of PC against OGD-reperfusion injury and PC may be a feasible therapy strategy in the treatment for spinal cord I/R injury. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Strategies of Pre-Service Primary School Teachers for Solving Addition Problems with Negative Numbers

ERIC Educational Resources Information Center

Almeida, Rut; Bruno, Alicia

2014-01-01

This paper analyses the strategies used by pre-service primary school teachers for solving simple addition problems involving negative numbers. The findings reveal six different strategies that depend on the difficulty of the problem and, in particular, on the unknown quantity. We note that students use negative numbers in those problems they find…

Exposing primary rat retina cell cultures to γ-rays: An in vitro model for evaluating radiation responses.

PubMed

Gaddini, Lucia; Balduzzi, Maria; Campa, Alessandro; Esposito, Giuseppe; Malchiodi-Albedi, Fiorella; Patrono, Clarice; Matteucci, Andrea

2018-01-01

Retinal tissue can receive incidental γ-rays exposure during radiotherapy either of tumors of the eye and optic nerve or of head-and-neck tumors, and during medical diagnostic procedures. Healthy retina is therefore at risk of suffering radiation-related side effects and the knowledge of pathophysiological response of retinal cells to ionizing radiations could be useful to design possible strategies of prevention and management of radiotoxicity. In this study, we have exploited an in vitro model (primary rat retinal cell culture) to study an array of biological effects induced on retinal neurons by γ-rays. Most of the different cell types present in retinal tissue - either of the neuronal or glial lineages - are preserved in primary rat retinal cultures. Similar to the retina in situ, neuronal cells undergo in vitro a maturational development shown by the formation of polarized neuritic trees and operating synapses. Since 2 Gy is the incidental dose received by the healthy retina per fraction when the standard treatment is delivered to the brain, retina cell cultures have been exposed to 1 or 2 Gy of γ-rays at different level of neuronal differentiation in vitro: days in vitro (DIV)2 or DIV8. At DIV9, retinal cultures were analyzed in terms of viability, apoptosis and characterized by immunocytochemistry to identify alterations in neuronal differentiation. After irradiation at DIV2, MTT assay revealed an evident loss of cell viability and βIII-tubulin immunostaining highlighted a marked neuritic damage, indicating that survived neurons showed an impaired differentiation. Differentiated cultures (DIV8) appeared to be more resistant with respect to undifferentiated, DIV2 cultures, both in terms of cell viability and differentiation. Apoptosis evaluated with TUNEL assay showed that irradiation at both DIV2 and DIV8 induced a significant increase in the apoptotic rate. To further investigate the effects of γ-rays on retinal neurons, we evaluated the

Primary cultures of human colon cancer as a model to study cancer stem cells.

PubMed

Koshkin, Sergey; Danilova, Anna; Raskin, Grigory; Petrov, Nikolai; Bajenova, Olga; O'Brien, Stephen J; Tomilin, Alexey; Tolkunova, Elena

2016-09-01

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Adaptation and cross-cultural validation of the United States Primary Care Assessment Tool (expanded version) for use in South Africa.

PubMed

Bresick, Graham; Sayed, Abdul-Rauf; le Grange, Cynthia; Bhagwan, Susheela; Manga, Nayna

2015-06-19

Measuring primary care is important for health sector reform. The Primary Care Assessment Tool (PCAT) measures performance of elements essential for cost-effective care. Following minor adaptations prior to use in Cape Town in 2011, a few findings indicated a need to improve the content and cross-cultural validity for wider use in South Africa (SA). This study aimed to validate the United States of America-developed PCAT before being used in a baseline measure of primary care performance prior to major reform. Public sector primary care clinics, users, practitioners and managers in urban and rural districts in the Western Cape Province. Face value evaluation of item phrasing and a combination of Delphi and Nominal Group Technique (NGT) methods with an expert panel and user focus group were used to obtain consensus on content relevant to SA. Original and new domains and items with > = 70% agreement were included in the South African version--ZA PCAT. All original PCAT domains achieved consensus on inclusion. One new domain, the primary healthcare (PHC) team, was added. Three of 95 original items achieved < 70% agreement, that is consensus to exclude as not relevant to SA; 19 new items were added. A few items needed minor rephrasing with local healthcare jargon. The demographic section was adapted to local socio-economic conditions. The adult PCAT was translated into isiXhosa and Afrikaans. The PCAT is a valid measure of primary care performance in SA. The PHC team domain is an important addition, given its emphasis in PHC re-engineering. A combination of Delphi and NGT methods succeeded in obtaining consensus on a multi-domain, multi-item instrument in a resource-constrained environment.

Incorporating Cultural and Linguistic Diversity into Policy and Practice: Case Studies from an English Primary School

ERIC Educational Resources Information Center

Dakin, Justine

2017-01-01

Global migration has increased the number of non-English speaking pupils in UK schools, challenging a system which is politically and ideologically monolingual. This article examines how staff at a UK primary school positioned newly arrived pupils and their families, both culturally and linguistically, in terms of Cummins' educator role…

A Primary Xenograft Model of Small Cell Lung Cancer Reveals Irreversible Changes in Gene Expression Imposed by Culture In-Vitro

PubMed Central

Daniel, Vincent C.; Marchionni, Luigi; Hierman, Jared S.; Rhodes, Jonathan T.; Devereux, Wendy L.; Rudin, Charles M.; Yung, Rex; Parmigani, Giovanni; Dorsch, Marion; Peacock, Craig D.; Watkins, D. Neil

2009-01-01

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media. This is particularly true of small cell lung cancer (SCLC), where surgically resected tissue is rarely available. Recent attention has focused on the need for better models that preserve the integrity of cancer stem cell populations, as well as three-dimensional tumor-stromal interactions. Here we describe a primary xenograft model of SCLC in which endobronchial tumor specimens obtained from chemo-naive patients are serially propagated in vivo in immunodeficient mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then re-implanted to generate secondary xenografts. Using the Affymetrix platform, we analyzed gene expression in primary xenograft, xenograft-derived cell line, and secondary xenograft, and compared these data to similar analyses of unrelated primary SCLC samples and laboratory models. When compared to normal lung, primary tumors, xenografts and cell lines displayed a gene expression signature specific for SCLC. Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to tissue culture, and that was not regained when the tumors were re-established as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development. PMID:19351829

Patient safety culture in out-of-hours primary care services in the Netherlands: a cross-sectional survey

PubMed Central

Smits, Marleen; Keizer, Ellen; Giesen, Paul; Deilkås, Ellen Catharina Tveter; Hofoss, Dag; Bondevik, Gunnar Tschudi

2018-01-01

Objective To examine patient safety culture in Dutch out-of-hours primary care using the safety attitudes questionnaire (SAQ) which includes five factors: teamwork climate, safety climate, job satisfaction, perceptions of management and communication openness. Design Cross-sectional observational study using an anonymous web-survey. Setting Sixteen out-of-hours general practitioner (GP) cooperatives and two call centers in the Netherlands. Subjects Primary healthcare providers in out-of-hours services. Main outcome measures Mean scores on patient safety culture factors; association between patient safety culture and profession, gender, age, and working experience. Results Overall response rate was 43%. A total of 784 respondents were included; mainly GPs (N = 470) and triage nurses (N = 189). The healthcare providers were most positive about teamwork climate and job satisfaction, and less about communication openness and safety climate. The largest variation between clinics was found on safety climate; the lowest on teamwork climate. Triage nurses scored significantly higher than GPs on each of the five patient safety factors. Older healthcare providers scored significantly higher than younger on safety climate and perceptions of management. More working experience was positively related to higher teamwork climate and communication openness. Gender was not associated with any of the patient safety factors. Conclusions Our study showed that healthcare providers perceive patient safety culture in Dutch GP cooperatives positively, but there are differences related to the respondents’ profession, age and working experience. Recommendations for future studies are to examine reasons for these differences, to examine the effects of interventions to improve safety culture and to make international comparisons of safety culture. Key Points Creating a positive patient safety culture is assumed to be a prerequisite for quality and safety. We found that: �

Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

PubMed

de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

2010-12-15

Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

Social Cultural Factors Influencing Appointment of Headteachers in Primary Schools in Eldoret East Sub-County, Kenya

ERIC Educational Resources Information Center

Suter, Esther J.

2017-01-01

Headteachers' position is at the critical point of which all the mechanism of Education system: planning, delivery and management rest. The purpose of the study was to investigate social cultural factors influencing appointment of headteachers in primary schools in Eldoret East Sub-County, Uasin Gishu County. The target population comprised of 275…

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

PubMed

Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

2016-05-01

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

Enrichment of prostate cancer stem cells from primary prostate cancer cultures of biopsy samples

PubMed Central

Wang, Shunqi; Huang, Shengsong; Zhao, Xin; Zhang, Qimin; Wu, Min; Sun, Feng; Han, Gang; Wu, Denglong

2014-01-01

This study was to enrich prostate cancer stem cells (PrCSC) from primary prostate cancer cultures (PPrCC). Primary prostate cancer cells were amplified in keratinocyte serum-free medium with epidermal growth factor (EGF) and bovine pituitary extract (BPE), supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF) and cholera toxin. After amplification, cells were transferred into ultra-low attachment dishes with serum-free DMEM/F12 medium, supplemented with EGF, basic fibroblast growth factor (bFGF), bovine serum albumin (BSA), insulin, and N2 nutrition. Expression of cell-type-specific markers was determined by RT-qPCR and immunostaining. Tumorigenicity of enriched PrCSC was determined by soft agar assay and xenograft assay in NOD/SCID mice. Biopsy samples from 19 confirmed prostate cancer patients were used for establishing PPrCC, and 18 cases (95%) succeeded. Both basal marker (CK5) and luminal markers (androgen receptor and CK8) strongly co-expressed in most of PPrCC, indicating their basal epithelial origin. After amplification under adherent culture condition in vitro, transient amplifying cells were the dominant cells. Sphere formation efficiency (SFE) of passaged PPrCC was about 0.5%, which was 27 times lower than SFE of LNCaP (13.67%) in the same condition. Compared with adherent cells from PPrCC, prostasphere from PPrCC showed up regulated stem cell markers and increased tumorigenic potential in soft-agar assay. However, spheroid cells from PPrCC prostasphere failed to initiate tumor in xenograft assay in 6 months. Thus, PPrCC can be established and amplified from prostate cancer biopsy samples. Our modified sphere culture system can enrich PrCSC from PPrCC. PMID:24427338

Hyaline Cartilage Tissue Is Formed through the Co-culture of Passaged Human Chondrocytes and Primary Bovine Chondrocytes

PubMed Central

Taylor, Drew W.; Ahmed, Nazish; Hayes, Anthony J.; Ferguson, Peter; Gross, Allan E.; Caterson, Bruce

2012-01-01

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks. PMID:22610463

Communication in cross-cultural consultations in primary care in Europe: the case for improvement. The rationale for the RESTORE FP 7 project.

PubMed

van den Muijsenbergh, Maria; van Weel-Baumgarten, Evelyn; Burns, Nicola; O'Donnell, Catherine; Mair, Frances; Spiegel, Wolfgang; Lionis, Christos; Dowrick, Chris; O'Reilly-de Brún, Mary; de Brun, Tomas; MacFarlane, Anne

2014-04-01

The purpose of this paper is to substantiate the importance of research about barriers and levers to the implementation of supports for cross-cultural communication in primary care settings in Europe. After an overview of migrant health issues, with the focus on communication in cross-cultural consultations in primary care and the importance of language barriers, we highlight the fact that there are serious problems in routine practice that persist over time and across different European settings. Language and cultural barriers hamper communication in consultations between doctors and migrants, with a range of negative effects including poorer compliance and a greater propensity to access emergency services. It is well established that there is a need for skilled interpreters and for professionals who are culturally competent to address this problem. A range of professional guidelines and training initiatives exist that support the communication in cross-cultural consultations in primary care. However, these are commonly not implemented in daily practice. It is as yet unknown why professionals do not accept or implement these guidelines and interventions, or under what circumstances they would do so. A new study involving six European countries, RESTORE (REsearch into implementation STrategies to support patients of different ORigins and language background in a variety of European primary care settings), aims to address these gaps in knowledge. It uses a unique combination of a contemporary social theory, normalisation process theory (NPT) and participatory learning and action (PLA) research. This should enhance understanding of the levers and barriers to implementation, as well as providing stakeholders, with the opportunity to generate creative solutions to problems experienced with the implementation of such interventions.

Culture of porcine embryonic germ cells in serum-supplemented and serum-free conditions: the effects of serum and growth factors on primary and long-term culture.

PubMed

Petkov, Stoyan G; Anderson, Gary B

2008-06-01

Fetal bovine serum (FBS) is a commonly used medium supplement with variable and undefined composition, which presents problems in culture of pluripotent stem cells. The purpose of this study was to determine if FBS can be replaced with Knockout Serum Replacement (KSR), a defined medium supplement, and to examine the effects of FBS and growth factors on short- and long-term culture of pig embryonic germ cells (EGC). No significant differences were observed in total and mean colony areas in primary cultures between FBS- and KSR-supplemented medium (421 x 10(3) mum(2) vs. 395 x 10(3) microm(2), p = 0.68, n = 11, and 6375 microm(2) vs. 6407 microm(2), p = 0.885, respectively). Total and mean colony areas were significantly larger in KSR-supplemented medium compared with medium supplemented with KSR and growth factors (505 x 10(3) microm(2) vs. 396 x 10(3) microm(2), p = 0.016, n = 12, and 8769 microm(2) vs. 6513 microm(2), p = 0.003, respectively). The cultures proliferated for significantly higher numbers of passages in FBS-supplemented medium and in medium supplemented with KSR and growth factors compared with medium containing KSR alone (31.1 vs. 21.9, p = 0.004, n = 10, and 35.5 vs. 21.6, p = 002, n = 10, respectively). Porcine EGC maintained in serum-free conditions were positive for pluripotent stem cell markers, maintained stable karyotypes for up to 54 passages, and were capable of differentiating in vitro into cells from the three primary germ layers. These results will help improve and standardize culture of pluripotent stem cells in the pig.

Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells.

PubMed

Hong, Jong Kwang; Lee, Gyun Min; Yoon, Sung Kwan

2011-09-10

The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q(Ab). Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q(Ab) and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q(Glc), q(Lac) and Y(Lac/Glc), and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q(Ab) and extend culture longevity. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of Quality Assurance System in Culture and Nation Character Education in Primary Education in Indonesia

ERIC Educational Resources Information Center

Susilana, Rudi; Asra

2013-01-01

The purpose of national education is to develop skills and build dignified national character and civilization in educating nation life (Act No. 20, 2003). The paper describes a system of quality assurance in culture and character education in primary education. This study employs the six sigma model which consists of the formula DMAIC (Define,…

Implementing guidelines and training initiatives to improve cross-cultural communication in primary care consultations: a qualitative participatory European study.

PubMed

Teunissen, E; Gravenhorst, K; Dowrick, C; Van Weel-Baumgarten, E; Van den Driessen Mareeuw, F; de Brún, T; Burns, N; Lionis, C; Mair, F S; O'Donnell, C; O'Reilly-de Brún, M; Papadakaki, M; Saridaki, A; Spiegel, W; Van Weel, C; Van den Muijsenbergh, M; MacFarlane, A

2017-02-10

Cross-cultural communication in primary care is often difficult, leading to unsatisfactory, substandard care. Supportive evidence-based guidelines and training initiatives (G/TIs) exist to enhance cross cultural communication but their use in practice is sporadic. The objective of this paper is to elucidate how migrants and other stakeholders can adapt, introduce and evaluate such G/TIs in daily clinical practice. We undertook linked qualitative case studies to implement G/TIs focused on enhancing cross cultural communication in primary care, in five European countries. We combined Normalisation Process Theory (NPT) as an analytical framework, with Participatory Learning and Action (PLA) as the research method to engage migrants, primary healthcare providers and other stakeholders. Across all five sites, 66 stakeholders participated in 62 PLA-style focus groups over a 19 month period, and took part in activities to adapt, introduce, and evaluate the G/TIs. Data, including transcripts of group meetings and researchers' fieldwork reports, were coded and thematically analysed by each team using NPT. In all settings, engaging migrants and other stakeholders was challenging but feasible. Stakeholders made significant adaptations to the G/TIs to fit their local context, for example, changing the focus of a G/TI from palliative care to mental health; or altering the target audience from General Practitioners (GPs) to the wider multidisciplinary team. They also progressed plans to deliver them in routine practice, for example liaising with GP practices regarding timing and location of training sessions and to evaluate their impact. All stakeholders reported benefits of the implemented G/TIs in daily practice. Training primary care teams (clinicians and administrators) resulted in a more tolerant attitude and more effective communication, with better focus on migrants' needs. Implementation of interpreter services was difficult mainly because of financial and other

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons

PubMed Central

Rubiolo, Juan Andrés; Boente-Juncal, Andrea; Hirama, Masahiro; Yamashita, Shuji; Camiña, Mercedes; Vieytes, Mercedes R.

2018-01-01

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin. PMID:29748486

Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

PubMed

Martyniszyn, L; Szulc-Dąbrowska, L; Boratyńska-Jasińska, A; Niemiałtowski, M

2013-01-01

Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

[Culture and health services: studying the participation of cultural traits of Brazilian society in the work process of primary healthcare services].

PubMed

Pinto, Alessandra Maria Silva; Najar, Alberto Lopes

2011-11-01

The analysis of institutions is a widely researched area of health. The culture of organizations is understood as a symbolic possibility contained in a larger dimension, called "national culture". This premise justifies the incorporation of the social anthropological approach to the study of organizational culture. This study sought to establish the perceptions of employees of two primary healthcare services in Niterói, State of Rio de Janeiro, regarding commonly used social navigation strategies from the theory developed by Roberto DaMatta. The results showed the relational character associated with the stereotype of the Brazilian people manifested by conflicts arising from the existence of values based on the `individual' and the `person'. Among them are the distortions observed between discourse and practice, and the mobilization strategies of social navigation like "making do" - to establish a mediation between the person and the impersonal law. The organization of the services of the Niterói Family Medical Program apparently sets its employees the concrete challenge of balancing the egalitarian principle that underpins the Unified Health System (SSU) with the set of values upon which personal relations are based in Brazilian society.

Adaptation and cross-cultural validation of the United States Primary Care Assessment Tool (expanded version) for use in South Africa

PubMed Central

Sayed, Abdul-Rauf; le Grange, Cynthia; Bhagwan, Susheela; Manga, Nayna

2015-01-01

Background Measuring primary care is important for health sector reform. The Primary Care Assessment Tool (PCAT) measures performance of elements essential for cost-effective care. Following minor adaptations prior to use in Cape Town in 2011, a few findings indicated a need to improve the content and cross-cultural validity for wider use in South Africa (SA). Aim This study aimed to validate the United States of America-developed PCAT before being used in a baseline measure of primary care performance prior to major reform. Setting Public sector primary care clinics, users, practitioners and managers in urban and rural districts in the Western Cape Province. Methods Face value evaluation of item phrasing and a combination of Delphi and Nominal Group Technique (NGT) methods with an expert panel and user focus group were used to obtain consensus on content relevant to SA. Original and new domains and items with > = 70% agreement were included in the South African version – ZA PCAT. Results All original PCAT domains achieved consensus on inclusion. One new domain, the primary healthcare (PHC) team, was added. Three of 95 original items achieved < 70% agreement, that is consensus to exclude as not relevant to SA; 19 new items were added. A few items needed minor rephrasing with local healthcare jargon. The demographic section was adapted to local socio-economic conditions. The adult PCAT was translated into isiXhosa and Afrikaans. Conclusion The PCAT is a valid measure of primary care performance in SA. The PHC team domain is an important addition, given its emphasis in PHC re-engineering. A combination of Delphi and NGT methods succeeded in obtaining consensus on a multi-domain, multi-item instrument in a resource- constrained environment. PMID:26245610

Establishment of primary cultures of craniopharyngioma cells★

PubMed Central

Liu, Hao; Liu, Liang; Liu, Zhiyong; Li, Qiang; You, Chao; Xu, Jianguo

2012-01-01

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research. PMID:25745451

Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue.

PubMed

Horai, Sawako; Yanagi, Kumiko; Kaname, Tadashi; Yamamoto, Masatatsu; Watanabe, Izumi; Ogura, Go; Abe, Shintaro; Tanabe, Shinsuke; Furukawa, Tatsuhiko

2014-11-01

The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity.

Cultural Dimensions of Feedback at an Australian University: A Study of International Students with English as an Additional Language

ERIC Educational Resources Information Center

Warner, Richard; Miller, Julia

2015-01-01

International students with English as an additional language face transitional challenges when entering a new academic culture. One such challenge involves optimising feedback to help foster their academic development, bearing in mind that feedback is not a culturally neutral entity (Nazif, A., Biswas, D., & Hilbig, R. (2004-2005). Towards an…

Additively Manufactured Device for Dynamic Culture of Large Arrays of 3D Tissue Engineered Constructs.

PubMed

Costa, Pedro F; Hutmacher, Dietmar W; Theodoropoulos, Christina; Gomes, Manuela E; Reis, Rui L; Vaquette, Cédryck

2015-04-22

The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.

2010-05-15

Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAsmore » methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also

Culture and art: Importance of art practice, not aesthetics, to early human culture.

PubMed

Zaidel, Dahlia W

2018-01-01

Art is expressed in multiple formats in today's human cultures. Physical traces of stone tools and other archaeological landmarks suggest early nonart cultural behavior and symbolic cognition in the early Homo sapiens (HS) who emerged ~300,000-200,000 years ago in Africa. Fundamental to art expression is the neural underpinning for symbolic cognition, and material art is considered its prime example. However, prior to producing material art, HS could have exploited symbolically through art-rooted biological neural pathways for social purpose, namely, those controlling interpersonal motoric coordination and sound codependence. Aesthetics would not have been the primary purpose; arguments for group dance and rhythmical musical sounds are offered here. In addition, triggers for symbolic body painting are discussed. These cultural art formats could well have preceded material art and would have enhanced unity, inclusiveness, and cooperative behavior, contributing significantly to already existing nonart cultural practices. © 2018 Elsevier B.V. All rights reserved.

Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

PubMed

Strobel, Oliver; Dadabaeva, Nigora; Felix, Klaus; Hackert, Thilo; Giese, Nathalia A; Jesenofsky, Ralf; Werner, Jens

2016-02-01

Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

Conducting Museum Education Activities within the Context of Developing a Nature Culture in Primary School Students: MTA Natural History Museum Example

ERIC Educational Resources Information Center

Dilli, Rukiye

2016-01-01

The present study, aiming to develop nature culture in primary school students and to help them to become acquainted with their close environment, is a quasi-experimental study. Museum education activities were conducted with the study group which consisted of 128 fourth-grade primary school students. At the end of the study, the students gained…

Quantitative Nuclease Protection Assays (qNPA) as Windows into Chemical-Induced Adaptive Response in Cultures of Primary Human Hepatocytes (Concentration and Time-Response)

EPA Science Inventory

Cultures of primary human hepatocytes have been shown to be dynamic in vitro model systems that retain liver-like functionality (e.g. metabolism, transport, induction). We have utilized these culture models to interrogate 309 ToxCast chemicals. The study design characterized both...

The influence of inquiry learning model on additives theme with ethnoscience content to cultural awareness of students

NASA Astrophysics Data System (ADS)

Sudarmin, S.; Selia, E.; Taufiq, M.

2018-03-01

The purpose of this research is to determine the influence of inquiry learning model on additives theme with ethnoscience content to cultural awareness of students and how the students’ responses to learning. The method applied in this research is a quasi-experimental with non-equivalent control group design. The sampling technique applied in this research is the technique of random sampling. The samples were eight grade students of one of junior high schools in Semarang. The results of this research were (1) thestudents’ cultural awareness of the experiment class is better than the control class (2) inquiry learning model with ethnoscience content strongly influencing the cultural awareness of students by 78% and (3) students gave positive responses to inquiry learning model with ethnoscience content. The conclusions of this research are inquiry-learning model with ethnoscience content has positive influence on students’ cultural awareness.

3D cell culture systems--towards primary drug discovery platforms: an interview with Heinz Ruffner (Novartis) and Jan Lichtenberg (InSphero).

PubMed

Ruffner, Heinz; Lichtenberg, Jan

2012-07-01

Advanced cell culture systems for regenerative medicine, drug efficacy and toxicity testing, enabling technologies to create and analyze 3D cell culture systems were the topics of the 3D cell culture meeting taking place in March 14-16, 2012 at the Technopark in Zurich, Switzerland. At this meeting Biotechnology Journal had the pleasure to talk to Dr. Heinz Ruffner, Novartis AG, and Dr. Jan Lichtenberg, co-founder and CEO of InSphero AG, about challenges and perspectives in using 3D cell culture systems as primary drug discovery platforms. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Influence of Language and Culture in the Primary Care of Spanish-Speaking Latino Adults with Poorly Controlled Diabetes: A Qualitative Study.

PubMed

Zamudio, Cindy D; Sanchez, Gabriela; Altschuler, Andrea; Grant, Richard W

2017-01-01

We examined the role of language and culture in the interactions between Spanish-speaking Latino patients with poorly controlled diabetes - a fast-growing population in the United States - and their primary care providers. We conducted four focus groups with 36 non-US born Spanish-speaking patients with elevated HbA1c. Participants were insured health plan members with either English-speaking (2 groups) or Spanish-speaking (2 groups) primary care providers. Moderated discussions focused on visit preparation, communication during visit, and role of other care team members. Key themes derived from these discussions were then linked to corresponding Latino cultural constructs. Patients had a mean age of 57.9 (±11.2) years and last measured HbA1c was 8.6% (1.5%). Two communication-related themes (reluctance to switch providers and use of intermediaries) and two visit-related themes (provider-driven visit agendas and problem-based visits) emerged from our analyses. These themes reflected the cultural constructs of confianza (trust), familismo (family), respeto (deference), and simpatía (harmonious relationship). Trust in the patient-provider relationship led many participants to remain with English-speaking providers who treated them well. Patients with either language concordant and discordant providers reported reliance on family or other intermediaries to close communication gaps. Deference to physician expertise and authority led to visit expectations that it is the doctor's job to know what to ask and that visits were intended to address specific, often symptom-driven problems. Spanish-speaking Latino patients' cultural expectations play an important role in framing their primary care interactions. Recognizing culturally influenced visit expectations is an important step toward improving patient-provider communication.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

DOE PAGES

Pilgrim, Matthew G.; Lengyel, Imre; Lanzirotti, Antonio; ...

2017-02-01

Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-lm-thickmore » inserts with 0.4-lm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. In conclusion, the data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch’s membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.« less

Cross cultural training in primary mental health care consultations in Moldova - The tEACH perspective.

PubMed

Møller, Jane Ege; van Weel-Baumgarten, Evelyn

2017-09-01

This article reports experiences and challenges encountered in a cross-cultural training project in Moldova that was undertaken by tEACH, the teaching subcommittee of EACH: International Association for Communication in Healthcare, in cooperation with local and international stakeholders. As part of a major health policy reform, the aim was to equip a group of trainers with the skills to train Moldovan professionals in skills for primary mental health care, including communication skills. The project consisted of 3 weeks of training using mainly experiential teaching methods to allow participants to practice content and methods, including interactive lecturing, roleplay, feedback and video. A majority of the participants reported that they acquired key facilitation skills. They valued the opportunity to practice and receive feedback. However, some reported that there was too much focus on communication skills, which was thought to be less relevant in a Moldovan context. Furthermore our learner-centered approach was occasionally experienced as a lack of structure CONCLUSION: The tEACH expertise plays an important role in supporting trainers in cross-cultural contexts with effective communication skills methods. Teaching in a cross-cultural context is only successful through continuous dialogue with stakeholders and demands attention to cultural differences. Copyright © 2017. Published by Elsevier B.V.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

PubMed

Pilgrim, Matthew G; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C; Messinger, Jeffrey D; Read, Russell W; Guidry, Clyde; Curcio, Christine A

2017-02-01

Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

PubMed Central

Pilgrim, Matthew G.; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C.; Messinger, Jeffrey D.; Read, Russell W.; Guidry, Clyde; Curcio, Christine A.

2017-01-01

Purpose Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss. PMID:28146236

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pilgrim, Matthew G.; Lengyel, Imre; Lanzirotti, Antonio

Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-lm-thickmore » inserts with 0.4-lm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. In conclusion, the data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch’s membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.« less

Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

PubMed Central

Coles, Janice L.; Williams, Gwyneth; Rutman, Andrew; Goggin, Patricia M.; Adam, Elizabeth C.; Page, Anthony; Evans, Hazel J.; Lackie, Peter M.; O’Callaghan, Christopher; Lucas, Jane S.

2014-01-01

Background The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. Methods We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Results Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. Conclusions The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia. PMID:24586956

The potential use of primary human upper urinary tract urothelial cell carcinoma (UUT-UCC) cultured cells for prognostic indicators and chemosensitivity test.

PubMed

Hsieh, Teng-Fu; Chen, Chi-Cheng; Chang, Chao-Hsiang; Yu, Ai-Lin; Ma, Wen-Lung; Shyr, Chih-Rong

2013-07-01

Upper urinary tract urothelial cell carcinoma (UUT-UCC) is a rare yet aggressive urologic tumor with a high rate of recurrence and metastasis, resulting in high mortality. Chemotherapy has been used to prevent recurrence and treat metastatic UUT-UCCs. Although UUT-UCC is sensitive to chemotherapy, the patients' responses to therapy vary and the therapy outcome is unpredictable. Therefore, the identification of patients who are at high risk of failure in chemotherapy is important for accurate prognostication, patient counseling, and better therapy. We have obtained cells from UUT-UCC tumor tissues after surgery and established individual primary cultured cell lines, which were used to evaluate E-cadherin and Ki-67 proliferation marker expression and their chemosensitivity to chemotherapeutic drugs. Differential Ki-67 expression and chemosensitivity were observed in these primary cultured cell lines, suggesting these types of UUT-UCC cell lines could provide a platform for determining prognostic makers and evaluating the efficacy of chemotherapy. In conclusion, primary cultured cell lines from individual patients will be a great tool for evaluating and determining each individual's personalized chemotherapy course and for testing and screening new chemotherapeutic agents against UUT-UCCs. Copyright © 2012 Elsevier GmbH. All rights reserved.

[Primary culture and characteristics of colorectal cancer-associated fibroblasts].

PubMed

Wen, Huan; Nie, Qianqian; Jiang, Zhinong; Deng, Hong

2015-10-01

To compare the biological characteristics of colorectal cancer associated fibroblasts (CAFs) with normal fibroblasts (NFs). CAFs and NFs were isolated from fresh specimens of colorectal cancer and their paired normal colon tissue and cultured by tissue explant method. Light microscopy, quantitative polymerase chain reaction (qPCR), Western blot, immunofluorescence microscopy, electron microscopy and flow cytometry were used to identify isolated fibroblasts and to explore their characteristics of activation and growth. Primary colorectal CAFs and NFs were isolated and cultured successfully. NFs showed spindled morphology and were arranged in interlacing or spiral bundles. CAFs were polygonal or spindle, but were fatter than NFs. They were distributed randomly and arranged irregularly, and had obvious actin expression. CAFs and NFs both expressed fibronectin, but not E-cadherin, CD31 and caldesmon. qPCR showed that CAFs expressed more fibroblast activation protein (FAP) and less fibroblast specific protein 1 (FSP1) than that of NFs. There was no difference in the expression of α-SMA between NFs and CAFs by Western blot. α-SMA was bundled in parallel to the long axis of the cell by immunofluorescence. By electron microscopy, CAFs but not NFs showed dense myofilament that was arranged regularly. Flow cytometry showed that the percentage of S- and G2-phase in CAFs were significantly lower than that in NFs. mRNA expression of transforming growth factor β1, stromal derived factor 1 (SDF-1) and platelet derived growth factor (PDGF)-D in CAFs were lower while that for PDGFC was higher than that in NFs. That indicated the proliferation of CAFs was inhibited and the secretion of some cytokines was different when compared with NFs. CAFs show differences with NFs in morphology, characteristics of activation and secretion of some cytokines. The proliferation of CAFs is down regulated as compared with NFs.

Development and validation of primary human myometrial cell culture models to study pregnancy and labour.

PubMed

Mosher, Andrea A; Rainey, Kelly J; Bolstad, Seunghwa S; Lye, Stephen J; Mitchell, Bryan F; Olson, David M; Wood, Stephen L; Slater, Donna M

2013-01-01

The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni's multiple comparisons test was performed. We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10. Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain

Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.

PubMed

McKeever, P E; Wahl, R L; Shakui, P; Jackson, G A; Letica, L H; Liebert, M; Taren, J A; Beierwaltes, W H; Hoff, J T

1990-06-01

To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.

Assessing the prevalence of depression in Punjabi and English primary care attenders: the role of culture, physical illness and somatic symptoms.

PubMed

Bhui, Kamaldeep; Bhugra, Dinesh; Goldberg, David; Sauer, Justin; Tylee, Andre

2004-09-01

Previous studies exploring the prevalence of depression among South Asians reported inconsistent findings. Research artefacts due to sampling bias, measurements errors and a failure to include ethnographic methods may all explain this. We estimated the prevalence of depression, and variations of prevalence with culture, cultural adaptation, somatic symptoms and physical disability in a cross-sectional primary care survey of Punjabi and English attendees. We included a culture specific screening instrument, culturally adapted the instruments and offered bilingual interviews. We found that, compared with their English counterparts, depressive diagnoses were more common among Punjabis, Punjabi women, Punjabis with physical complaints and, contrary to expectation, even Punjabis with low scores for somatic symptoms.

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.

PubMed

Heslop, James A; Rowe, Cliff; Walsh, Joanne; Sison-Young, Rowena; Jenkins, Roz; Kamalian, Laleh; Kia, Richard; Hay, David; Jones, Robert P; Malik, Hassan Z; Fenwick, Stephen; Chadwick, Amy E; Mills, John; Kitteringham, Neil R; Goldring, Chris E P; Kevin Park, B

2017-01-01

The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems.

Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow.

PubMed

Costa, Pedro F; Vaquette, Cédryck; Baldwin, Jeremy; Chhaya, Mohit; Gomes, Manuela E; Reis, Rui L; Theodoropoulos, Christina; Hutmacher, Dietmar W

2014-09-01

This study reports on an original concept of additive manufacturing for the fabrication of tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept towards the development of anatomically relevant TECs, this concept was utilized for the design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold device was investigated by micro-computed tomography and scanning electron microscopy confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as the inner architecture of the device can significantly impact the perfusion of fluid within the scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant importance for controlling the nutrition flow pattern within the scaffold and the bioreactor chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue development. The bioreactor/scaffold device was dynamically seeded with human primary osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human osteoblasts were observed homogenously distributed throughout the scaffold, and were viable for the six week culture period. This work demonstrates a novel application for additive manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility of the additive manufacturing technology platform developed, more complex culture systems can be fabricated which would contribute to the advances in customized and patient-specific tissue engineering strategies for a wide range of applications.

Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

PubMed Central

Neeper, I D; Patton, D L; Kuo, C C

1990-01-01

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

Resveratrol protects primary cortical neuron cultures from transient oxygen-glucose deprivation by inhibiting MMP-9.

PubMed

Gao, Dakuan; Huang, Tao; Jiang, Xiaofan; Hu, Shijie; Zhang, Lei; Fei, Zhou

2014-06-01

It was recently shown that resveratrol exerts neuroprotective effects against cerebral ischemia in mice. The aim of the present study was to further confirm these effects in in vitro primary cortical neuron cultures with transient oxygen-glucose deprivation (OGD), and to investigate whether these effects are due to the inhibition of matrix metalloproteinase-9 (MMP-9) and of cell apoptosis. Neuronal primary cultures of cerebral cortex were prepared from BALB/c mice embryos (13-15 days). Cells from 14- to 16-day cultures were subjected to OGD for 3 h, followed by 21 h of reoxygenation to simulate transient ischemia. Different doses of resveratrol were added into the culture medium during the simulation of transient ischemia. The effect of the extracellular signal-regulated kinase (ERK) inhibitor U0126 was studied by adding U0126 (5 µg/µl, 4 µl) into the culture medium during transient ischemia; as a control, we used treatment of cells with 50 µM of resveratrol. Cell viability was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Cell apoptosis was assessed by flow cytometry. The effects of resveratrol on the expression of MMP-9 were analyzed by western blotting and reverse transcription-polymerase chain reaction (RT-PCR), while the levels of ERK, phosphorylated (p)-ERK, cleaved caspase-3, Bax and Bcl-2 were measured by western blotting. The results of the MTT assay showed that cell viability is significantly reduced by transient OGD. OGD induced cell apoptosis, the expression of Bax and the activation of caspase-3 and ERK, inhibited the expression of Bcl-2 and increased the expression of MMP-9, while these effects were reversed by treatment with resveratrol. The therapeutic efficacy of resveratrol was shown to be dose-dependent, with the most suitable dose range determined at 50-100 µM. Treatment with U0126 inhibited MMP-9 and Bax expression and caspase-3 activation, while it further promoted the

Effects of dimethyl sulfoxide on the morphology and viability of primary cultured neurons and astrocytes.

PubMed

Zhang, Chen; Deng, Yuanying; Dai, Hongmei; Zhou, Wenjuan; Tian, Jing; Bing, Guoying; Zhao, Lingling

2017-01-01

Dimethyl sulfoxide (DMSO) is a widely used solvent and vehicle for in vivo and in vitro administration of test compounds. Effects of DMSO independent of the test compound, such as in studies examining morphological plasticity or neurotoxic responses, may lead to spurious results. To investigate effects of DMSO concentration ([DMSO]) on morphology and survival of primary cultured neurons and astrocytes. Primary cultured neurons and astrocytes were treated with 0.25%-10.00% [DMSO] for 12-48h. Viable cell number and morphology were compared to untreated cultures using the CCK-8 assay and phase-contrast microscopy. Expression levels of the neuronal marker NeuN and astrocyte marker glial fibrillary acidic protein (GFAP) were determined by immunofluorescence and western blotting. A [DMSO]≤0.50% had no effect on neuronal number or NeuN expression up to 24h, while ≥1.00% induced a progressive and dramatic loss of both viability and NeuN expression even after 12h. Brief (12h) exposure to ≤1.00% DMSO had no effect on astrocytes survival or GFAP expression, while ≥5.00% significantly reduced both at all exposure durations. In contrast to neurons, exposure to 0.50% and 1.00% DMSO for 24 or 48h enhanced astrocytes proliferation and GFAP expression. Astrocytic processes were maintained at 0.50% and 1.00% DMSO, while neurons exhibited marked neurite retraction at ≥0.50%. A [DMSO]≥0.5% markedly disrupts neuronal morphology and reduces viability, even after brief exposure. In astrocytes, 0.50% and 1.00% DMSO appear to induce reactive gliosis. For treatment of neural cells, [DMSO] should be ≤0.25% to obviate spurious vehicle effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

PubMed

Chen, Wan-Chun; Lin, Hsi-Hui; Tang, Ming-Jer

2014-09-15

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis. Copyright © 2014 the American Physiological Society.

Palmitoylethanolamide Blunts Amyloid-β42-Induced Astrocyte Activation and Improves Neuronal Survival in Primary Mouse Cortical Astrocyte-Neuron Co-Cultures.

PubMed

Beggiato, Sarah; Borelli, Andrea Celeste; Ferraro, Luca; Tanganelli, Sergio; Antonelli, Tiziana; Tomasini, Maria Cristina

2018-01-01

Based on the pivotal role of astrocytes in brain homeostasis and the strong metabolic cooperation existing between neurons and astrocytes, it has been suggested that astrocytic dysfunctions might cause and/or contribute to neuroinflammation and neurodegenerative processes. Therapeutic approaches aimed at both neuroprotection and neuroinflammation reduction may prove particularly effective in slowing the progression of these diseases. The endogenous lipid mediator palmitoylethanolamide (PEA) displayed neuroprotective and anti(neuro)inflammatory properties, and demonstrated interesting potential as a novel treatment for Alzheimer's disease. We firstly evaluated whether astrocytes could participate in regulating the Aβ42-induced neuronal damage, by using primary mouse astrocytes cell cultures and mixed astrocytes-neurons cultures. Furthermore, the possible protective effects of PEA against Aβ42-induced neuronal toxicity have also been investigated by evaluating neuronal viability, apoptosis, and morphometric parameters. The presence of astrocytes pre-exposed to Aβ42 (0.5μM; 24 h) induced a reduction of neuronal viability in primary mouse astrocytes-neurons co-cultures. Furthermore, under these experimental conditions, an increase in the number of neuronal apoptotic nuclei and a decrease in the number of MAP-2 positive neurons were observed. Finally, astrocytic Aβ42 pre-exposure induced an increase in the number of neurite aggregations/100μm as compared to control (i.e., untreated) astrocytes-neurons co-cultures. These effects were not observed in neurons cultured in the presence of astrocytes pre-exposed to PEA (0.1μM), applied 1 h before and maintained during Aβ42 treatment. Astrocytes contribute to Aβ42-induced neurotoxicity and PEA, by blunting Aβ42-induced astrocyte activation, improved neuronal survival in mouse astrocyte-neuron co-cultures.

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures.

PubMed

Petrenko, Volodymyr; Saini, Camille; Perrin, Laurent; Dibner, Charna

2016-11-11

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

INCIDENCE AND DETECTION OF PLEUROPNEUMONIA-LIKE ORGANISMS IN CELL CULTURES BY FLUORESCENT ANTIBODY AND CULTURAL PROCEDURES1

PubMed Central

Barile, Michael F.; Malizia, Walter F.; Riggs, Donald B.

1962-01-01

Barile, Michael F. (National Institutes of Health, Bethesda, Md.), Walter F. Malizia, and Donald B. Riggs. Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol. 84:130–136. 1962—A total of 102 tissue-cell cultures from 17 separate laboratories was examined for pleuropneumonia-like organisms (PPLO) by the fluorescent antibody and cultural procedures. PPLO were isolated from 48 of the 49 tissue-cell cultures found positive for PPLO by the fluorescent antibody procedure, and results of the two procedures agreed in 101 of the 102 (99%) cases. PPLO were isolated from none of 10 primary-cell cultures prepared from six animal species and from 48 of 92 (52%) continuous-cell cultures prepared from eight animal species. Cells grown in media containing antibiotics were more frequently contaminated with PPLO (72%) than cells grown in antibiotic-free media (7%). Cultures (91%) from tissue-culture-producing laboratories and cultures (76%) used for propagation of microorganisms were contaminated with PPLO, although none used for tissue-culture metabolic studies was contaminated. In addition, our findings support the view that PPLO contamination of cell cultures is probably owing to bacterial contaminants which revert to L forms in the presence of antibiotics. Images PMID:13865001

Live imaging of dense-core vesicles in primary cultured hippocampal neurons.

PubMed

Kwinter, David M; Silverman, Michael A; Kwinter, David; Michael, Silverman

2009-05-29

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is

Developmentally and Culturally Appropriate Screening in Primary Care: Development of the Behavioral Health Checklist

PubMed Central

Koshy, Anson J.; Watkins, Marley W.; Cassano, Michael C.; Wahlberg, Andrea C.; Mautone, Jennifer A.; Blum, Nathan J.

2013-01-01

Objective To evaluate the construct validity of the Behavioral Health Checklist (BHCL) for children aged from 4 to 12 years from diverse backgrounds. Method The parents of 4–12-year-old children completed the BHCL in urban and suburban primary care practices affiliated with a tertiary-care children’s hospital. Across practices, 1,702 were eligible and 1,406 (82.6%) provided consent. Children of participating parents were primarily non-Hispanic black/African American and white/Caucasian from low- to middle-income groups. Confirmatory factor analyses examined model fit for the total sample and subsamples defined by demographic characteristics. Results The findings supported the hypothesized 3-factor structure: Internalizing Problems, Externalizing Problems, and Inattention/Hyperactivity. The model demonstrated adequate to good fit across age-groups, gender, races, income groups, and suburban versus urban practices. Conclusion The findings provide strong evidence of the construct validity, developmental appropriateness, and cultural sensitivity of the BHCL when used for screening in primary care. PMID:23978505

[Regulation of age-dependent phenomena. Influence of C6-substituted purines on cell aggregation and cell migration in primary cultures of lense epithelial cells].

PubMed

Glässer, D; Iwig, M; Weber, E

1975-01-01

The existence of an age dependent latent period of cell emigration has been proved in the primary culture of epithelial cells of bovine lenses. The previously described aggregation phenomenon as well as the latent period of the cell emigration increase with the age of the sponsor animals. Extracellular adenine and other C6-substituted purines, isolated from the cells themselves and added to the medium, act the same way on the lens cells in the primary culture as the increasing age of the sponsor animals. Adenine stimulates cell aggregation and inhibits the adhesion of the cells to the substratum, the cell flattening and the cell migration. The adenine action has been proved down to a concentration of 3 X 10(-6) M. During the primary culture, the lens cells gradually los the adenine sensitivity. The adenine action also occurs on single cells, isolated by trypsination, it differs from the reaction of ouabain and can be removed at low concentration by washing procedures. The results favour the suggestion C6-substituted purines to be involved in cell ageing.

Neuroprotective effect of schizandrin A on oxygen and glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons.

PubMed

Wang, Cai-Ping; Li, Gui-Cai; Shi, Yun-Wei; Zhang, Xiao-Chuan; Li, Jian-Long; Wang, Zhi-Wei; Ding, Fei; Liang, Xin-Miao

2014-09-01

Brain ischemia appears to be associated with innate immunity. Recent reports showed that C3a and C5a, as potent targets, might protect against ischemia induced cell death. In traditional Chinese medicine, the fruit of Schizandra chinesis Baill (Fructus schizandrae) has been widely used as a tonic. In the present study, we sought to evaluate the neuroprotective effects of schizandrin A, a composition of S. chinesis Baill, against oxygen and glucose deprivation followed by reperfusion (OGD/R)-induced cell death in primary culture of rat cortical neurons, and to test whether C3a and C5a affected cortical neuron recovery from ischemic injury after schizandrin A treatment. The results showed that schizandrin A significantly reduced cell apoptosis and necrosis, increased cell survival, and decreased intracellular calcium concentration ([Ca(2+)]i) and lactate dehydrogenase (LDH) release in primary culture of rat cortical neurons after OGD/R. Mechanism studies suggested that the modulation of extracellular-regulated kinase (ERK), c-Jun NH2-terminal kinases (JNK), and p38, as well as caspase-3 activity played an important role on the progress of neuronal apoptosis. C5aR participated in the neuroprotective effect of schizandrin A in primary culture of rat cortical neurons after OGD/R. Our findings suggested that schizandrin A might act as a candidate therapeutic target drug used for brain ischemia and related diseases.

Blood culture contamination with Enterococci and skin organisms: implications for surveillance definitions of primary bloodstream infections.

PubMed

Freeman, Joshua T; Chen, Luke Francis; Sexton, Daniel J; Anderson, Deverick J

2011-06-01

Enterococci are a common cause of bacteremia but are also common contaminants. In our institution, approximately 17% of positive blood cultures with enterococci are mixed with skin organisms. Such isolates are probable contaminants. The specificity of the current definition of primary bloodstream infection could be increased by excluding enterococci mixed with skin organisms. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

PubMed Central

Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

2013-01-01

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

Pericellular oxygen concentration of cultured primary human trophoblasts

PubMed Central

Chen, Baosheng; Longtine, Mark S.; Nelson, D. Michael

2012-01-01

Introduction Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. Methods We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. Results The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤ 0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. Conclusions We conclude that culture conditions of 21% oxygen are unable to replicate the pO2 of 40–60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions. PMID:23211472

Effect of primary-zone equivalence ratio and hydrogen addition on exhaust emission in a hydrocarbon-fueled combustor

NASA Technical Reports Server (NTRS)

Norgren, C. T.; Ingebo, R. D.

1974-01-01

The effects of reducing the primary-zone equivalence ratio on the exhaust emission levels of oxides of nitrogen, carbon monoxide, and unburned hydrocarbons in experimental hydrocarbon-fueled combustor segments at simulated supersonic cruise and idle conditions were investigated. In addition, the effects of the injection of hydrogen fuel (up to 4 percent of the total weight of fuel) on the stability of the hydrocarbon flame and exhaust emissions were studied and compared with results obtained without hydrogen addition.

Functional characterization of Na(+)/H(+) exchangers in primary cultures of prairie dog gallbladder.

PubMed

Narins, S C; Park, E H; Ramakrishnan, R; Garcia, F U; Diven, J N; Balin, B J; Hammond, C J; Sodam, B R; Smith, P R; Abedin, M Z

2004-01-15

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

The Implementation of a Social Constructivist Approach in Primary Science Education in Confucian Heritage Culture: The Case of Vietnam

ERIC Educational Resources Information Center

H?ng, Ngô Vu Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

2015-01-01

Social constructivism has been increasingly studied and implemented in science school education. Nevertheless, there is a lack of holistic studies on the implementation of social constructivist approach in primary science education in Confucian heritage culture. This study aims to determine to what extent a social constructivist approach is…

Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

PubMed

Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

2015-09-01

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions. Copyright © 2012 John Wiley & Sons, Ltd.

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

PubMed Central

Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

2016-01-01

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

PubMed

Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

2016-03-04

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

PubMed Central

Gentile, Luciana B.; Nagamine, Marcia K.; Biondi, Luiz R.; Sanches, Daniel S.; Toyota, Fábio; Giovani, Tatiane M.; de Jesus, Isis P.; da Fonseca, Ivone I. M.; Queiroz-Hazarbassanov, Nicolle; Diaz, Bruno L.; Salles Gomes, Cristina de O. Massoco

2017-01-01

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development. PMID:28945747

Attitudes of Arab and Jewish patients toward integration of complementary medicine in primary care clinics in Israel: a cross-cultural study.

PubMed

Ben-Arye, Eran; Karkabi, Khaled; Karkabi, Sonia; Keshet, Yael; Haddad, Maria; Frenkel, Moshe

2009-01-01

The purpose of this cross-cultural study was to evaluate patient perspectives on complementary and alternative medicine (CAM) integration within primary care clinics. It is one of the first multiethnic studies to explore patients' perspectives on the best model for integrating CAM into the conventional care setting. We developed a 13-item questionnaire that addresses issues of CAM use, expectations from the primary care physicians concerning CAM, and attitudes toward CAM integration within a patient's primary care clinic. We constructed the questionnaire with cross-cultural sensitivity concerning the core concepts of CAM and traditional medicine in both the Arab and Jewish communities in northern Israel. Data for statistical analysis were obtained from 3840 patients attending seven primary care clinics. Of the 3713 respondents who were willing to identify their religion, 2184 defined themselves as Muslims, Christians, or Druze and 1529 as Jews. Respondents in the two groups were equally distributed by sex but differed significantly by age, education, self-rated religiosity, and self-reported chronic diseases in their medical background. Respondents in the two groups reported comparable overall CAM use during the previous year, but the Arab respondents reported more use of herbs and traditional medicine. Respondents in both groups stated that their primary expectation from a family physician concerning CAM was to refer them appropriately and safely to a CAM practitioner. Respondents in both groups greatly supported a theoretical scenario of CAM integration into primary medical care. However, Arab respondents were more supportive of the option that non-physician CAM practitioners would provide CAM rather than physicians.

Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

PubMed

Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

2014-01-01

Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

PubMed

Márquez, Laura B; Velázquez, Natalia; Repetto, Horacio A; Paton, Adrienne W; Paton, James C; Ibarra, Cristina; Silberstein, Claudia

2014-01-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

The Structure of Primary and Secondary Teachers' Attributions for Pupils' Misbehaviour: A Preliminary Cross-Phase and Cross-Cultural Investigation

ERIC Educational Resources Information Center

Gibbs, Simon; Gardiner, Marianne

2008-01-01

The purpose of this study was to see if systematic contrasts in educational culture and curricular emphases might affect the underlying structure of teachers' attributions for children's behaviour. Thus, responses to a questionnaire developed from earlier work by Miller and colleagues (2000, 2002) were gathered from primary and secondary school…

Emotional expression in music: contribution, linearity, and additivity of primary musical cues.

PubMed

Eerola, Tuomas; Friberg, Anders; Bresin, Roberto

2013-01-01

The aim of this study is to manipulate musical cues systematically to determine the aspects of music that contribute to emotional expression, and whether these cues operate in additive or interactive fashion, and whether the cue levels can be characterized as linear or non-linear. An optimized factorial design was used with six primary musical cues (mode, tempo, dynamics, articulation, timbre, and register) across four different music examples. Listeners rated 200 musical examples according to four perceived emotional characters (happy, sad, peaceful, and scary). The results exhibited robust effects for all cues and the ranked importance of these was established by multiple regression. The most important cue was mode followed by tempo, register, dynamics, articulation, and timbre, although the ranking varied across the emotions. The second main result suggested that most cue levels contributed to the emotions in a linear fashion, explaining 77-89% of variance in ratings. Quadratic encoding of cues did lead to minor but significant increases of the models (0-8%). Finally, the interactions between the cues were non-existent suggesting that the cues operate mostly in an additive fashion, corroborating recent findings on emotional expression in music (Juslin and Lindström, 2010).

The use of propidium iodide to assess excitotoxic neuronal death in primary mixed cortical cultures.

PubMed

Lau, Anthony C; Cui, Hong; Tymianski, Michael

2007-01-01

Neurodegenerative disorders are subjects of intense scrutiny in biomedical research because of their often-debilitating effects. Currently, many laboratories are engaged in developing or testing drugs to prevent neuronal loss in a variety of these pathologies. A key to testing such drugs is the use of a fast, reliable, and easily reproducible model of neurodegeneration and neuroprotection. Our laboratory has previously used propidium iodide (PI) to assess the degree of neurodegeneration and neuroprotection under a variety of conditions. Ultimately, efforts are underway in the laboratory to prevent delayed neuronal loss following acute ischemic insults using drug therapies. It is now believed that a key mechanism of neurodegeneration following acute ischemia or anoxia is a result of excitotoxicity via N-methyl-D-aspartate receptors (NMDARs) and subsequent overproduction of nitric oxide via neuronal nitric oxide synthase (nNOS). Thus, for the purposes of this chapter, the insult used to induce cell death will be various concentrations of NMDA and the compound used to demonstrate neuroprotection will be the nonspecific NOS inhibitor No-nitro-L-arginine methyl ester (L-NAME). Assessment of neuronal death is accomplished by measuring changes in PI fluorescence using a fluorescent plate reader. This chapter will outline the necessary steps required to (1) produce primary mixed cortical cultures, (2) apply PT and NMDA to these cultures, (3) quantify the results obtained from these cultures, and (4) image these cultures in conjunction with Hoechst 33342 and immunocytochemistry using fluorescence microscopy.

Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

NASA Astrophysics Data System (ADS)

Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

2016-02-01

Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

Panel management, team culture, and worklife experience.

PubMed

Willard-Grace, Rachel; Dubé, Kate; Hessler, Danielle; O'Brien, Bridget; Earnest, Gillian; Gupta, Reena; Shunk, Rebecca; Grumbach, Kevin

2015-09-01

Burnout and professional dissatisfaction are threats to the primary care workforce. We investigated the relationship between panel management capability, team culture, cynicism, and perceived "do-ability" of primary care among primary care providers (PCPs) and staff in primary care practices. We surveyed 326 PCPs and 142 staff members in 10 county-administered, 6 university-run, and 3 Veterans Affairs primary care clinics in a large urban area in 2013. Predictor variables included capability for performing panel management and perception of team culture. Outcome variables included 2 work experience measures--the Maslach Burnout Inventory cynicism scale and a 1-item measure of the "do-ability" of primary care this year compared with last year. Generalized Estimation Equation (GEE) models were used to account for clustering at the clinic level. Greater panel management capability and higher team culture were associated with lower cynicism among PCPs and staff and higher reported "do-ability" of primary care among PCPs. Panel management capability and team culture interacted to predict the 2 work experience outcomes. Among PCPs and staff reporting high team culture, there was little association between panel management capability and the outcomes, which were uniformly positive. However, there was a strong relationship between greater panel management capability and improved work experience outcomes for PCPs and staff reporting low team culture. Team-based processes of care such as panel management may be an important strategy to protect against cynicism and dissatisfaction in primary care, particularly in settings that are still working to improve their team culture. (c) 2015 APA, all rights reserved).

Socio-cultural differences in Australian primary school children's weight and weight-related behaviours.

PubMed

Hardy, Louise L; King, Lesley; Hector, Debra; Baur, Louise A

2013-08-01

To examine whether there are socio-cultural differences in overweight/obesity and weight-related behaviours of Australian primary school children. Secondary data analysis of the 2010 NSW Schools Physical Activity and Nutrition Survey (n = 4898). Height and weight were measured. Parents of Year K, 2, 4 children and Year 6 students completed a questionnaire on demographics and weight-related behaviours. Cardio-respiratory fitness was assessed by the 20-meter shuttle run test and categorised as adequately fit or unfit. Children were categorised according to the main cultural/ethnic backgrounds (Middle-Eastern, Asian and English-speaking backgrounds) and socio-economic status (SES) tertile. Within ethnic groups, SES was associated with weight-related behaviours, but not consistently, and not with body mass index status. Within ethnic groups, the odds were higher among low SES, compared with high SES to be inactive, unfit and have poorer dietary habits. Weight-related behaviours among each ethnic group also differed by gender. Compared with low SES children from English-speaking backgrounds, ethnic boys were two times as likely to be overweight/obese, and the odds were significantly higher among ethnic children to be inactive, unfit and have poor dietary patterns. The results indicate the need for obesity prevention initiatives to specifically target and reach children from low SES and Asian and Middle-Eastern backgrounds, and the importance of ensuring that such initiatives are culturally appropriate and address relevant issues. © 2013 The Authors. Journal of Paediatrics and Child Health © 2013 Paediatrics and Child Health Division (Royal Australasian College of Physicians).

Isolated primary blast alters neuronal function with minimal cell death in organotypic hippocampal slice cultures.

PubMed

Effgen, Gwen B; Vogel, Edward W; Lynch, Kimberly A; Lobel, Ayelet; Hue, Christopher D; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

2014-07-01

An increasing number of U.S. soldiers are diagnosed with traumatic brain injury (TBI) subsequent to exposure to blast. In the field, blast injury biomechanics are highly complex and multi-phasic. The pathobiology caused by exposure to some of these phases in isolation, such as penetrating or inertially driven injuries, has been investigated extensively. However, it is unclear whether the primary component of blast, a shock wave, is capable of causing pathology on its own. Previous in vivo studies in the rodent and pig have demonstrated that it is difficult to deliver a primary blast (i.e., shock wave only) without rapid head accelerations and potentially confounding effects of inertially driven TBI. We have previously developed a well-characterized shock tube and custom in vitro receiver for exposing organotypic hippocampal slice cultures to pure primary blast. In this study, isolated primary blast induced minimal hippocampal cell death (on average, below 14% in any region of interest), even for the most severe blasts tested (424 kPa peak pressure, 2.3 ms overpressure duration, and 248 kPa*ms impulse). In contrast, measures of neuronal function were significantly altered at much lower exposures (336 kPa, 0.84 ms, and 86.5 kPa*ms), indicating that functional changes occur at exposures below the threshold for cell death. This is the first study to investigate a tolerance for primary blast-induced brain cell death in response to a range of blast parameters and demonstrate functional deficits at subthreshold exposures for cell death.

Effects of endocrine disrupting chemicals on estrogen receptor alpha and heat shock protein 60 gene expression in primary cultures of loggerhead sea turtle (Caretta caretta) erythrocytes.

PubMed

Cocci, Paolo; Capriotti, Martina; Mosconi, Gilberto; Palermo, Francesco Alessandro

2017-10-01

The loggerhead turtle (Caretta caretta) can be considered a good indicator species for studying the ecological impact of endocrine disrupting chemicals (EDCs) on wildlife. However, the effect of these environmental pollutants on nuclear steroid hormone signaling has not yet been addressed in sea turtles mainly due to the legal constraints of their endangered status. Here we describe the use of primary erythrocyte cell cultures as in vitro models for evaluating the effects of different EDCs on the expression of estrogen receptor α (ERα). In addition, we evaluated erythrocyte toxicity caused by EDCs using Alamar Blue assay and heat shock proteins 60 (HSP60) expression. Primary cultures of erythrocytes were exposed to increasing concentrations of 4-nonylphenol (4NP), Diisodecyl phthalate (DiDP), Tri-m-cresyl phosphate (TMCP) and Tributyltin (TBT) for 48h. Alamar Blue demonstrated that exposure of erythrocytes to each contaminant for up to 48h led to a significant impairment of cellular metabolic activity at 100μM, with the exception of TBT. Moreover, our data indicate that loggerhead erythrocytes constitutively express ERα and HSP60 at the transcript level and respond to EDCs by up-regulating their expression. In this regard, ERα was up-regulated in a dose-dependent manner after 48h exposure to both 4NP and TMCP. Interestingly, the dosage-dependent effects of DiDP on ERα expression were opposite in comparison to that obtained following exposure to the other tested compounds. This work provides the first indication regarding the potential of primary erythrocytes as study models for evaluating the effects of EDCs on sea turtles. Copyright © 2017 Elsevier Inc. All rights reserved.

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

PubMed

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Fragile X-associated primary ovarian insufficiency: evidence for additional genetic contributions to severity.

PubMed

Hunter, Jessica Ezzell; Epstein, Michael P; Tinker, Stuart W; Charen, Krista H; Sherman, Stephanie L

2008-09-01

The fragile X mental retardation gene (FMR1) contains a CGG repeat sequence in its 5' untranslated region that can become unstable and expand in length from generation to generation. Alleles with expanded repeats in the range of approximately 55-199, termed premutation alleles, are associated with an increased risk for fragile-X-associated primary ovarian insufficiency (FXPOI). However, not all women who carry the premutation develop FXPOI. To determine if additional genes could explain variability in onset and severity, we used a random-effects Cox proportional hazards model to analyze age at menopause on 680 women from 225 families who have a history of fragile X syndrome and 321 women from 219 families from the general population. We tested for the presence of a residual additive genetic effect after adjustment for FMR1 repeat length, race, smoking, body mass index, and method of ascertainment. Results showed significant familial aggregation of age at menopause with an estimated additive genetic variance of 0.55-0.96 depending on the parameterization of FMR1 repeat size and definition of age at menopause (P-values ranging between 0.0002 and 0.0027). This is the first study to analyze familial aggregation of FXPOI. This result is important for proper counseling of women who carry FMR1 premutation alleles and for guidance of future studies to identify additional genes that influence ovarian insufficiency. (c) 2008 Wiley-Liss, Inc.

Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the mitten crab, Eriocheir sinensis.

PubMed

Hong, Yuhang; Yang, Xiaozhen; Cheng, Yongxu; Liang, Pan; Zhang, Jinbiao; Li, Meng; Shen, Cheng; Yang, Zhigang; Wang, Chun

2013-10-01

Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg(-1)) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5 × 10(7) cells ml(-1), and three subpopulations of hemocytes were distinguished as granulocytes (43.46 ± 4.98%), semigranulocytes (31.04 ± 1.95%), and hyalinocytes (25.50 ± 4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg(-1), pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Select putative neurodevelopmental toxins modify SNAP-25 expression in primary cultures of rat cerebellar granule cells.

PubMed

Zieminska, Elzbieta; Lenart, Jacek; Lazarewicz, Jerzy W

2016-08-31

A presynaptic protein SNAP-25 belonging to SNARE complex which is instrumental in intracellular vesicular trafficking and exocytosis, has been implicated in hyperactivity and cognitive abilities in some neuropsychiatric disorders. The unclear etiology of the behavior disrupting neurodevelopmental disabilities in addition to genetic causes most likely involves environmental factors. The aim of this in vitro study was to test if various suspected developmental neurotoxins can alter SNAP-25 mRNA and protein expression in neurons. Real-time PCR and Western blotting analyses were used to assess SNAP-25 mRNA and protein levels in primary cultures of rat cerebellar granule cells (CGCs). The test substances: tetrabromobisphenol-A (TBBPA), thimerosal (TH), silver nanoparticles (NAg), valproic acid (VPA) and thalidomide (THAL), were administered to CGC cultures at subtoxic concentrations for 24h. The results demonstrated that SNAP-25 mRNA levels were increased by 49 and 66% by TBBPA and THAL, respectively, whereas VPA and NAg reduced these levels to 48 and 64% of the control, respectively. The SNAP-25 protein content in CGCs was increased by 79% by TBBPA, 25% by THAL and 21% by NAg; VPA and TH reduced these levels to 73 and 69% of the control, respectively. The variety of changes in SNAP-25 expression on mRNA and protein level suggests the diversity of the mechanism of action of the test substances. This initial study provided no data on concentration-effect relations and on functional changes in CGCs. However it is the first to demonstrate the effect of different compounds that are suspected of causing neurodevelopmental disabilities on SNAP-25 expression. These results suggest that this protein may be a common target for not only inherited but also environmental modifications linked to behavioral deficits in neurodevelopmental disabilities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture.

PubMed

Tollefsen, K-E; Eikvar, Sissel; Finne, Eivind Farmen; Fogelberg, Oscar; Gregersen, Inger Katharina

2008-10-01

Alkylphenols act as estrogen mimics by binding to and transactivating estrogen receptors (ERs) in fish. In the present study, activation of ER-mediated production of the estrogenic biomarker vitellogenin (vtg) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes was used to construct a structure-activity relationship for this ubiquitous group of aquatic pollutants. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure was assessed. The results showed that most alkylphenols were estrogenic, although with 3-300 thousand times lower affinity than the endogenous estrogen 17beta-estradiol. Mono-substituted tertiary alkylphenols with moderate (C4-C5) and long alkyl chain length (C8-C9) in the para position exhibited the highest estrogenic potency. Substitution with multiple alkyl groups, presence of substituents in the ortho- and meta-position and lack of a hydroxyl group on the benzene ring reduced the estrogenic activity, although several estrogenic alkylated non-phenolics were identified. Co-exposures with the natural estrogen 17beta-estradiol led to identification of additional estrogenic compounds as well as some anti-estrogens. A combination of low affinity for the ER and cytotoxicity was identified as factors rendering some of the alkylphenols non-estrogenic in the bioassay when tested alone.

The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes.

PubMed

Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S; Dringen, Ralf

2017-11-01

Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U- 13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

PubMed Central

Gebhardt, R; Mecke, D

1983-01-01

The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells. Images Fig. 1. PMID:6138251

[Donor age affects on the «behavior» and the sensibility bone marrow cells in on copper ion of the primary culture].

PubMed

Bozhkov, A I; Ohiienko, S L; Kuznetsova, Yu A; Bondar', A Yu; Marchenko, V P; Gumennaya, M S

2017-01-01

The changes of bone marrow cells (BMC) number in the primary culture from 0 to 96 hours, the pattern (the distribution of cells) of cells morphotypes and «lifespan» (the time of cell life after isolation) of myelocytes, metamyelocytes, band and segmented neutrophils, isolated of the young (3 months) and old (20months) animals, were investigated. The number of the BMC obtained from intact old animals increased faster in primary culture, than from young animals. The Cu induced fibrosis had different influence on the rate of BMC culture growth of old and young animals. The adding of 4 mM and 8 mM CuSO4x5H2O in the BMC culture of young and old animals resulted in a dose-dependent inhibition of growth rate of young animal cells. If copper ions were added into the culture of BMC of old animals, the decreased of the BMC number was described less than for cells of young animals. The adding of 8 mM CuSO4x5H2O inhibited proliferation less, than the adding of 4 mM CuSO4x5H2O. The Cu-induced liver fibrosis had accelerated the BMC rate death of both old and young animals. However, this effect was more pronounced in young animals. It is suggested, that during the ontogenesis the BMC undergo such epigenetic changes, which change functional properties.

Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

PubMed Central

Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

1996-01-01

Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atienzar, Franck A., E-mail: franck.atienzar@ucb.com; Novik, Eric I.; Gerets, Helga H.

Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine modelmore » along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.« less

"You Get to Be Yourself": Visual Arts Programs, Identity Construction and Learners of English as an Additional Language

ERIC Educational Resources Information Center

Wielgosz, Meg; Molyneux, Paul

2015-01-01

Students learning English as an additional language (EAL) in Australian schools frequently struggle with the cultural and linguistic demands of the classroom while concurrently grappling with issues of identity and belonging. This article reports on an investigation of the role primary school visual arts programs, distinct programs with a…

Emotional expression in music: contribution, linearity, and additivity of primary musical cues

PubMed Central

Eerola, Tuomas; Friberg, Anders; Bresin, Roberto

2013-01-01

The aim of this study is to manipulate musical cues systematically to determine the aspects of music that contribute to emotional expression, and whether these cues operate in additive or interactive fashion, and whether the cue levels can be characterized as linear or non-linear. An optimized factorial design was used with six primary musical cues (mode, tempo, dynamics, articulation, timbre, and register) across four different music examples. Listeners rated 200 musical examples according to four perceived emotional characters (happy, sad, peaceful, and scary). The results exhibited robust effects for all cues and the ranked importance of these was established by multiple regression. The most important cue was mode followed by tempo, register, dynamics, articulation, and timbre, although the ranking varied across the emotions. The second main result suggested that most cue levels contributed to the emotions in a linear fashion, explaining 77–89% of variance in ratings. Quadratic encoding of cues did lead to minor but significant increases of the models (0–8%). Finally, the interactions between the cues were non-existent suggesting that the cues operate mostly in an additive fashion, corroborating recent findings on emotional expression in music (Juslin and Lindström, 2010). PMID:23908642

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators.

PubMed

Fukuda, Tomokazu; Iino, Yuuka; Eitsuka, Takahiro; Onuma, Manabu; Katayama, Masafumi; Murata, Koichi; Inoue-Murayama, Miho; Hara, Kumiko; Isogai, Emiko; Kiyono, Tohru

2016-10-01

Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

A research agenda on patient safety in primary care. Recommendations by the LINNEAUS collaboration on patient safety in primary care.

PubMed

Verstappen, Wim; Gaal, Sander; Bowie, Paul; Parker, Diane; Lainer, Miriam; Valderas, Jose M; Wensing, Michel; Esmail, Aneez

2015-09-01

Healthcare can cause avoidable serious harm to patients. Primary care is not an exception, and the relative lack of research in this area lends urgency to a better understanding of patient safety, the future research agenda and the development of primary care oriented safety programmes. To outline a research agenda for patient safety improvement in primary care in Europe and beyond. The LINNEAUS collaboration partners analysed existing research on epidemiology and classification of errors, diagnostic and medication errors, safety culture, and learning for and improving patient safety. We discussed ideas for future research in several meetings, workshops and congresses with LINNEAUS collaboration partners, practising GPs, researchers in this field, and policy makers. This paper summarizes and integrates the outcomes of the LINNEAUS collaboration on patient safety in primary care. It proposes a research agenda on improvement strategies for patient safety in primary care. In addition, it provides background information to help to connect research in this field with practicing GPs and other healthcare workers in primary care. Future research studies should target specific primary care domains, using prospective methods and innovative methods such as patient involvement.

A research agenda on patient safety in primary care. Recommendations by the LINNEAUS collaboration on patient safety in primary care

PubMed Central

Verstappen, Wim; Gaal, Sander; Bowie, Paul; Parker, Diane; Lainer, Miriam; Valderas, Jose M.; Wensing, Michel; Esmail, Aneez

2015-01-01

ABSTRACT Background: Healthcare can cause avoidable serious harm to patients. Primary care is not an exception, and the relative lack of research in this area lends urgency to a better understanding of patient safety, the future research agenda and the development of primary care oriented safety programmes. Objective: To outline a research agenda for patient safety improvement in primary care in Europe and beyond. Methods: The LINNEAUS collaboration partners analysed existing research on epidemiology and classification of errors, diagnostic and medication errors, safety culture, and learning for and improving patient safety. We discussed ideas for future research in several meetings, workshops and congresses with LINNEAUS collaboration partners, practising GPs, researchers in this field, and policy makers. Results: This paper summarizes and integrates the outcomes of the LINNEAUS collaboration on patient safety in primary care. It proposes a research agenda on improvement strategies for patient safety in primary care. In addition, it provides background information to help to connect research in this field with practicing GPs and other healthcare workers in primary care. Conclusion: Future research studies should target specific primary care domains, using prospective methods and innovative methods such as patient involvement. PMID:26339841

Cytoprotective Effect of Peptide Sedatin, an Agonist of μ/δ-Opioid Receptors, on Primary Culture of Pulmonary Fibroblasts of Albino Rats under Conditions of Oxidative Stress.

PubMed

Sazonova, E N; Samarina, E Yu; Lebed'ko, O A; Maltseva, I M; Timoshin, S S

2016-05-01

We studied the effects of a synthetic analogue of dermorphin peptide sedatin on DNA synthesis, nucleolar apparatus, and parameters of free radical oxidation in the primary culture of pulmonary fibroblasts under conditions of oxidative stress. Oxidative stress significantly enhanced production of superoxide anion radical in the culture, sufficiently inhibited DNA synthesis in fibroblasts, and reduced the size of cell nuclei and parameters of the nucleolar apparatus. Sedatin prevented accumulation of free radical oxidation products and changes in karyometry parameters induced by oxidative stress. The peptide completely eliminated changes in the parameters of fibroblast nucleolar apparatus and abolished the inhibitory effect of oxidative stress on the number of DNA-synthesizing cells. Pretreatment with non-selective opioid receptor antagonist naloxone hydrochloride partially abolished the effects of sedatin in the primary culture of pulmonary fibroblasts.

Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

PubMed

Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

2015-07-01

The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates.

PubMed

Biffi, Emilia; Menegon, Andrea; Piraino, Francesco; Pedrocchi, Alessandra; Fiore, Gianfranco B; Rasponi, Marco

2012-01-01

In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 µm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies. Copyright © 2011 Wiley Periodicals, Inc.

Effects of Single and Repeated Exposure to a 50-Hz 2-mT Electromagnetic Field on Primary Cultured Hippocampal Neurons.

PubMed

Zeng, Ying; Shen, Yunyun; Hong, Ling; Chen, Yanfeng; Shi, Xiaofang; Zeng, Qunli; Yu, Peilin

2017-06-01

The prevalence of domestic and industrial electrical appliances has raised concerns about the health risk of extremely low-frequency magnetic fields (ELF-MFs). At present, the effects of ELF-MFs on the central nervous system are still highly controversial, and few studies have investigated its effects on cultured neurons. Here, we evaluated the biological effects of different patterns of ELF-MF exposure on primary cultured hippocampal neurons in terms of viability, apoptosis, genomic instability, and oxidative stress. The results showed that repeated exposure to 50-Hz 2-mT ELF-MF for 8 h per day after different times in culture decreased the viability and increased the production of intracellular reactive oxidative species in hippocampal neurons. The mechanism was potentially related to the up-regulation of Nox2 expression. Moreover, none of the repeated exposure patterns had significant effects on DNA damage, apoptosis, or autophagy, which suggested that ELF-MF exposure has no severe biological consequences in cultured hippocampal neurons.

Poly(ethylene glycol)-containing hydrogels promote the release of primary granules from human blood-derived polymorphonuclear leukocytes

PubMed Central

Cohen, Hannah Caitlin; Lieberthal, Tyler Jacob; Kao, W. John

2014-01-01

Polymorphonuclear leukocytes (PMNs) are recruited to sites of injury and biomaterial implants. Once activated, PMNs can exocytose their granule subsets to recruit monocytes (MCs) and mediate MC/macrophage activation. We investigated the release of myeloperoxidase (MPO), a primary granule marker, and matrix metalloproteinase-9 (MMP-9), a tertiary granule marker, from human blood-derived PMNs cultured on poly(ethylene glycol) (PEG) hydrogels, polydimethylsiloxane (PDMS), tissue culture polystyrene (TCPS) and gelatin-PEG (GP) hydrogels, with and without the presence of the bacterial peptide formyl-Met-Leu-Phe. Supernatants from PMN cultures on PEG-containing hydrogels (i.e., PEG and GP hydrogels) had higher concentrations of MPO than those from PMN cultures on PDMS or TCPS at 2 hours. PMNs on all biomaterials released comparable levels of MMP-9 at 2 hours, indicating that PMNs cultured on PEG-containing hydrogels have different mechanisms of release for primary and tertiary granules. Src family kinases were involved in the release of MPO from PMNs cultured on PEG hydrogels, TCPS and GP hydrogels and in the release of MMP-9 from PMNs cultured on all four materials. The increased release of primary granules from PMNs on PEG-containing hydrogels did not significantly increase MC chemotaxis, indicating that additional co-effectors in the dynamic inflammatory milieu in vivo modulate PMN-mediated MC recruitment. PMID:24497370

Nerve-independent and ectopically additional induction of taste buds in organ culture of fetal tongues.

PubMed

Honda, Kotaro; Tomooka, Yasuhiro

2016-10-01

An improved organ culture system allowed to observe morphogenesis of mouse lingual papillae and taste buds relatively for longer period, in which fetal tongues were analyzed for 6 d. Taste cells were defined as eosinophobic epithelial cells expressing CK8 and Sox2 within lingual epithelium. Addition of glycogen synthase kinase 3 beta inhibitor CHIR99021 induced many taste cells and buds in non-gustatory and gustatory stratified lingual epithelium. The present study clearly demonstrated induction of taste cells and buds ectopically and without innervation.

The Sustainable Impact of a Short Comparative Teaching Placement Abroad on Primary School Language Teachers' Professional, Linguistic and Cultural Skills

ERIC Educational Resources Information Center

Driscoll, Patricia; Rowe, Janet Elizabeth; Thomae, Manuela

2014-01-01

Recent research shows an increase in the number of trained teachers teaching foreign languages to learners aged 7-11 in English primary schools. Part of this increase stems from a government-funded four-week teaching placement abroad as part of a languages programme in initial teacher education (ITE). Student teachers' cultural, linguistic and…

Maintenance of primary cell cultures of immunocytes from Cacopsylla sp. psyllids: a new in vitrio tool for the study of pest insects

USDA-ARS?s Scientific Manuscript database

Psyllid species are major vectors of plant pathogens, such as phytoplasmas and Liberibacter bacteria, which threaten economic stability of fruit tee crops and vegetable production worldwide. Primary cell cultures of immunocytes have been developed from the three psyllid species, Cacopsylla melanone...

Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia.

PubMed

Wilson, P D; Anderson, R J; Breckon, R D; Nathrath, W; Schrier, R W

1987-02-01

Rabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane-like material adjacent to the dish. Differentiated properties characteristic of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK-ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT-specific anti-keratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

PubMed Central

Dong, Jia; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K.N.; Knobeloch, Daniel; Gerlach, Jörg C.; Zeilinger, Katrin

2008-01-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes. PMID:19003182

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

PubMed

Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

2008-07-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Asymmetric synthesis of α-alkenyl homoallylic primary amines via 1,2-addition of Grignard reagent to α,β-unsaturated phosphonyl imines.

PubMed

Xiong, Yiwen; Mei, Haibo; Xie, Chen; Han, Jianlin; Li, Guigen; Pan, Yi

2013-01-01

A series of chiral N -phosphonyl protected α-alkenyl homoallylic primary amines were synthesized by asymmetric addition of allylmagnesium bromide Grignard reagent towards chiral α,β-unsaturated imines. Only 1,2-adduct was obtained for all the imines with good yields and excellent diastereoselectivities. The chiral auxiliary could be easily removed under simple conditions, giving free multiple functionalized primary amines.

Teaching Language, Teaching Culture.

ERIC Educational Resources Information Center

Liddicoat, Anthony J., Ed.; Crozet, Chantal, Ed.

1997-01-01

Essays and research reports on the relationship between teaching second languages and teaching culture include: "Teaching Culture as an Integrated Part of Language Teaching: An Introduction" (Chantal Crozet, Anthony J. Liddicoat); "Primary Socialization and Cultural Factors in Second Language Learning: Wending Our Way through Semi-Charted…

Cultural similarity, cultural competence, and nurse workforce diversity.

PubMed

McGinnis, Sandra L; Brush, Barbara L; Moore, Jean

2010-11-01

Proponents of health workforce diversity argue that increasing the number of minority health care providers will enhance cultural similarity between patients and providers as well as the health system's capacity to provide culturally competent care. Measuring cultural similarity has been difficult, however, given that current benchmarks of workforce diversity categorize health workers by major racial/ethnic classifications rather than by cultural measures. This study examined the use of national racial/ethnic categories in both patient and registered nurse (RN) populations and found them to be a poor indicator of cultural similarity. Rather, we found that cultural similarity between RN and patient populations needs to be established at the level of local labor markets and broadened to include other cultural parameters such as country of origin, primary language, and self-identified ancestry. Only then can the relationship between cultural similarity and cultural competence be accurately determined and its outcomes measured.

Regulation of Iodide Uptake in Placental Primary Cultures

PubMed Central

Burns, R.; O'Herlihy, C.; Smyth, P.P.A.

2013-01-01

Background Maintenance of adequate iodide supply to the developing fetus is dependent not only on maternal dietary iodine intake but also on placental iodide transport. The objective of this study was to examine the effects of different pregnancy-associated hormones on the uptake of radioiodide by the placenta and to determine if iodide transporter expression is affected by hormone incubation. Methods Primary cultures of placental trophoblast cells were established from placentas obtained at term from pre-labor caesarean sections. They were pre-incubated with 17β-estradiol, prolactin, oxytocin, human chorionic gonadotropin (hCG) and progesterone either singly or in combination over 12 h with 125I uptake being measured after 6 h. RNA was isolated from placental trophoblasts and real-time RT-PCR performed using sodium iodide symporter (NIS) and pendrin (PDS) probes. Results Significant dose response increments in 125I uptake by trophoblast cells (p < 0.01) were observed following incubation with hCG (60% increase), oxytocin (45% increase) and prolactin (32% increase). Although progesterone (50-200 ng/ml) and 17β-estradiol (1,000-15,000 pg/ml) alone produced no significant differences in uptake, they facilitated increased uptake when combined with prolactin or oxytocin, with a combination of all four hormones producing the greatest increase (82%). Increased 125I uptake was accompanied by corresponding increments in NIS mRNA (ratio 1.52) compared to untreated control cells. No significantly increased expression levels of PDS were observed. Conclusions Pregnancy-associated hormones, particularly oxytocin and hCG, have a role in promoting placental iodide uptake which may protect the fetus against iodine deficiency. PMID:24783055

Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-γ in polymorphonuclear leukocyte primary and tertiary granule release

PubMed Central

Cohen, Hannah Caitlin; Frost, Dustin C.; Lieberthal, Tyler Jacob; Li, Lingjun; Kao, W. John

2018-01-01

In the foreign body response, infiltrating PMNs exocytose granule subsets to influence subsequent downstream inflammatory and wound healing events. In previous studies, we found that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG and gelatin + PEG hydrogels) had enhanced primary granule release, yet similar tertiary granule release compared with PMNs cultured on polydimethylsiloxane or tissue culture polystyrene. PMN primary granules contain microbicidal proteins and proteases, which can potentially injure bystander cells, degrade the extracellular matrix, and promote inflammation. Here, we sought to understand the mechanism of the enhanced primary granule release from PMNs on PEG hydrogels. We found that primary granule release from PMNs on PEG hydrogels was adhesion mediated and involved Src family kinases and PI3K-γ. The addition of gelatin to PEG hydrogels did not further enhance PMN primary granule release. Using stable-isotope dimethyl labeling-based shotgun proteomics, we identified many serum proteins – including Ig gamma constant chain region proteins and alpha-1-acid glycoprotein 1 – that were absorbed/adsorbed in higher quantities on PEG hydrogels than on TCPS, and may be involved in mediating PMN primary granule release. Ultimately, this mechanistic knowledge can be used to direct inflammation and wound healing following biomaterial implantation to promote a more favorable healing response. PMID:25736495

Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

PubMed

Hu, Hongtao; Li, Mo

2016-09-09

Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture.

PubMed

Faedmaleki, Firouz; Shirazi, Farshad H; Ejtemaeimehr, Shahram; Anjarani, Soghra; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

2016-02-01

.7 ppm or µg/ml). Then the hepatoprotective effect of silymarin and vitamin E were experimented on silver nanoparticle toxicity on mice liver primary cell culture. The results showed that silymarin at 600 µg/ml and vitamin E at 2500 µmol/l have protective effects on silver nanoparticle toxicity on mice liver primary cell culture. Viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles at 121.7 ppm and co-treatment of silymarin, and vitamin E is more than viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles and silymarin or silver nanoparticles and vitamin E.

[Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

PubMed

Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

2017-01-01

Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

Metal-mediated aminocatalysis provides mild conditions: Enantioselective Michael addition mediated by primary amino catalysts and alkali-metal ions

PubMed Central

Leven, Matthias; Neudörfl, Jörg M

2013-01-01

Summary Four catalysts based on new amides of chiral 1,2-diamines and 2-sulfobenzoic acid have been developed. The alkali-metal salts of these betaine-like amides are able to form imines with enones, which are activated by Lewis acid interaction for nucleophilic attack by 4-hydroxycoumarin. The addition of 4-hydroxycoumarin to enones gives ee’s up to 83% and almost quantitative yields in many cases. This novel type of catalysis provides an effective alternative to conventional primary amino catalysis were strong acid additives are essential components. PMID:23400419

Primary language and cultural background as factors in resident burnout in medical specialties: a study in a bilingual US city.

PubMed

Afzal, Khalid I; Khan, Farhan M; Mulla, Zuber; Akins, Ralista; Ledger, Elizabeth; Giordano, Frank L

2010-07-01

The aim of this study was to identify the degree of burnout among resident physicians enrolled in seven postgraduate training programs at Texas Tech University Health Sciences Center (TTUHSC), Paul L. Foster School of Medicine, El Paso, Texas, as it related to residents' age, gender, marital status, number of hours worked per week, primary language, race/ethnicity, and cultural background. : The Maslach Burnout Inventory Human Service Survey (MBI) was administered to measure the level of burnout according to the prevalence of emotional exhaustion (EE), depersonalization (DP), and reduced personal accomplishment (PA). : Eighty-one percent of the residents at TTUHSC participated in the study. Residents raised in the United States or Canada comprised 28% and 35% of the study, and all reported English as their primary language. The EE scale was significant for obstetrics/gynecology (OB/GYN) residents (prevalence odds ratio [POR] = 13.55, P = 0.02) and psychiatry (PSY) residents (POR = 6.50, P = 0.03). Emergency medicine (EM) residents (POR = 23.35, P = 0.002), OB/GYN (POR = 10.89, P = 0.02), and general surgery (GS) (POR = 6.24, P = 0.03) residents had high DP. Internal medicine (IM) residents (primarily Spanish-speaking) reported significantly low EE (POR = 0.22, P = 0.03) and PA (POR = 0.09, P = 0.001) scores. Residents from the United States or Canada who reported English as their primary language and noted their race as white, had high EE (POR = 3.06, P = 0.03; POR = 5.61, P = 0.0001; POR = 2.91, P = 0.004), DP (POR = 3.19, P = 0.02; POR = 8.34, P < or = 0.0001; POR = 4.70, P < or = 0.0001) and PA (POR = 2.61, P = 0.02; POR = 2.35, P = 0.05, POR 0.29, P = 0.3) scores. Using valid measures, this pilot study identified a statistically significant relationship between burnout and residents' race/ethnicity, primary language, and cultural background. Larger studies with similar focus would be necessary to generalize these findings. At-risk residents in bilingual

Investigating the cell death mechanisms in primary prostate cancer cells using low-temperature plasma treatment

NASA Astrophysics Data System (ADS)

O'Connell, Deborah; Hirst, A. M.; Packer, J. R.; Simms, M. S.; Mann, V. M.; Frame, F. M.; Maitland, N. J.

2016-09-01

Atmospheric pressure plasmas have shown considerable promise as a potential cancer therapy. An atmospheric pressure plasma driven with kHz kV excitation, operated with helium and oxygen admixtures is used to investigate the interaction with prostate cancer cells. The cytopathic effect was verified first in two commonly used prostate cancer cell lines (BPH-1 and PC-3 cells) and further extended to examine the effects in paired normal and tumour prostate epithelial cells cultured directly from patient tissues. Through the formation of reactive species in cell culture media, and potentially other plasma components, we observed high levels of DNA damage, together with reduced cell viability and colony-forming ability. We observed differences in response between the prostate cell lines and primary cells, particularly in terms of the mechanism of cell death. The primary cells ultimately undergo necrotic cell death in both the normal and tumour samples, in the complete absence of apoptosis. In addition, we provide the first evidence of an autophagic response in primary cells. This work highlights the importance of studying primary cultures in order to gain a more realistic insight into patient efficacy. EPSRC EP/H003797/1 & EP/K018388/1, Yorkshire Cancer Research: YCR Y257PA.

Effects of wound dressings on cultured primary keratinocytes.

PubMed

Esteban-Vives, Roger; Young, Matthew T; Ziembicki, Jenny; Corcos, Alain; Gerlach, Jörg C

2016-02-01

Autologous cell-spray grafting of non-cultured epidermal cells is an innovative approach for the treatment of severe second-degree burns. After treatment, wounds are covered with dressings that are widely used in wound care management; however, little is known about the effects of wound dressings on individually isolated cells. The sprayed cells have to actively attach, spread, proliferate, and migrate in the wound for successful re-epithelialization, during the healing process. It is expected that exposure to wound dressing material might interfere with cell survival, attachment, and expansion. Two experiments were performed to determine whether some dressing materials have a negative impact during the early phases of wound healing. In one experiment, freshly isolated cells were seeded and cultured for one week in combination with eight different wound dressings used during burn care. Cells, which were seeded and cultured with samples of Adaptic(®), Xeroform(®), EZ Derm(®), and Mepilex(®) did not attach, nor did they survive during the first week. Mepitel(®), N-Terface(®), Polyskin(®), and Biobrane(®) dressing samples had no negative effect on cell attachment and cell growth when compared to the controls. In a second experiment, the same dressings were exposed to pre-cultured cells in order to exclude the effects of attachment and spreading. The results confirm the above findings. This study could be of interest for establishing skin cell grafting therapies in burn medicine and also for wound care in general. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture.

PubMed

Schaffer, K; Herrmuth, H; Mueller, J; Coy, D H; Wong, H C; Walsh, J H; Classen, M; Schusdziarra, V; Schepp, W

1997-09-01

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for canine fundic D cells. Bombesin (EC50 6 X 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 X 10(-10) M, for both) were almost equally potent stimuli of somatostatin release, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2

Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction.

PubMed

Choi, Moonju; Park, Minkyung; Lee, Suhyon; Lee, Jeong Woo; Cho, Min Chul; Noh, Minsoo; Lee, Choongho

2017-05-01

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.

Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction

PubMed Central

Choi, Moonju; Park, Minkyung; Lee, Suhyon; Lee, Jeong Woo; Cho, Min Chul; Noh, Minsoo; Lee, Choongho

2017-01-01

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals. PMID:28365978

Deaf Culture. PEPNet Tipsheet

ERIC Educational Resources Information Center

Siple, Linda; Greer, Leslie; Holcomb, Barbra Ray

2004-01-01

It often comes as a surprise to people that many deaf people refer to themselves as being members of Deaf culture. The American Deaf culture is a unique linguistic minority that uses American Sign Language (ASL) as its primary mode of communication. This tipsheet provides a description of Deaf culture and suggestions for effective communication.

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katano, Takahito; Ootani, Akifumi; Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501

2013-03-22

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system withinmore » the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.« less

Influence of organizational culture on provider adherence to the diabetic clinical practice guideline: using the competing values framework in Palestinian Primary Healthcare Centers.

PubMed

Radwan, Mahmoud; Akbari Sari, Ali; Rashidian, Arash; Takian, Amirhossein; Abou-Dagga, Sanaa; Elsous, Aymen

2017-01-01

Diabetes mellitus (DM) is a serious chronic disease and an important public health issue. This study aimed to identify the predominant culture within the Palestinian Primary Healthcare Centers of the Ministry of Health (PHC-MoH) and the Primary Healthcare Centers of the United Nations Relief and Works Agency for Palestine Refugees (PHC-UNRWA) by using the competing values framework (CVF) and examining its influence on the adherence to the Clinical Practice Guideline (CPG) for DM. A cross-sectional design was employed with a census sample of all the Palestinian family doctors and nurses (n=323) who work within 71 PHC clinic. A cross-cultural adaptation framework was followed to develop the Arabic version of the CVF questionnaire. The overall adherence level to the diabetic guideline was disappointingly suboptimal (51.5%, p <0.001; 47.3% in the PHC-MoH and 55.5% in the PHC-UNRWA). In the PHC-MoH, the clan/group culture was the most predominant (mean =41.13; standard deviation [SD] =8.92), followed by hierarchical (mean =33.14; SD=5.96), while in the PHC-UNRWA, hierarchical was the prevailing culture (mean =48.43; SD =12.51), followed by clan/group (mean =29.73; SD =8.37). Although a positively significant association between the adherence to CPG and the rational culture and a negatively significant association with the developmental archetype were detected in the PHC-MoH, no significant associations were found in the PHC-UNRWA. Our study demonstrates that the organizational culture has a marginal influence on the adherence to the diabetic guideline. Future research should preferably mix quantitative and qualitative approaches and explore the use of more sensitive instruments to measure such a complex construct and its effects on guideline adherence in small-sized clinics.

Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

PubMed

Bao, Kai; Akguel, Baki; Bostanci, Nagihan

2014-01-01

In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. © 2014 S. Karger AG, Basel.

The neuroprotective effects of Berberine against amyloid β-protein-induced apoptosis in primary cultured hippocampal neurons via mitochondria-related caspase pathway.

PubMed

Liang, Yubin; Huang, Min; Jiang, Xin; Liu, Qiong; Chang, Xin; Guo, Yi

2017-08-10

Neuronal cell apoptosis is an important pathological change in Alzheimer's disease (AD). Berberine, an isoquinoline alkaloid isolated and extracted from Coptidis and rhizome and Cortex phellodendri, has a wide range of pharmacological effects. In this study, we investigated the neuroprotective effects of Berberine against neuronal insults induced by Aβ25-35 in primary cultured hippocampal neurons. Primary neuron cells have been isolated from hippocampus of C57BL/6 newborn mice. We investigated effect of Berberine against neuronal insults induced by Aβ25-35 in primary cultured hippocampal neurons. TdT-mediated dUTP nick-end labeling, MTT, Propidium iodide, MMP, Caspase activity measurement, Western blot. These neurons explosure to the β25-35 protein resulted in a loss in cell viability and a surge in apoptosis. However, the presence of Berberine significantly reversed the effects induced by Aβ25-35. Through decreasing viability and caspase activity in neurons, the pretreatment with Berberine attenuated the cytotoxic effect of the Aβ25-35. Furthermore, it's found that expression of cytochrome C, as well as the restoration of Bcl-2/Bax and Bcl-xl/Bax ratio in the presence of Berberine, led to a decline in the apoptotic rate. The neuroprotective effects of Berberine against Aβ25-35-induced neuronal apoptosis, suggesting that this may be a promising therapeutics against AD. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

PubMed

Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

2017-08-01

CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia

PubMed Central

Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun

2017-01-01

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308–331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner. PMID:28832643

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia.

PubMed

Chen, Qiang; Wu, Hui; Tao, Jia; Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun; Xu, Changshui

2017-01-01

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308-331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner.

Sponge-supported cultures of primary head and neck tumors for an optimized preclinical model.

PubMed

Dohmen, Amy J C; Sanders, Joyce; Canisius, Sander; Jordanova, Ekaterina S; Aalbersberg, Else A; van den Brekel, Michiel W M; Neefjes, Jacques; Zuur, Charlotte L

2018-05-18

Treatment of advanced head and neck cancer is associated with low survival, high toxicity and a widely divergent individual response. The sponge-gel-supported histoculture model was previously developed to serve as a preclinical model for predicting individual treatment responses. We aimed to optimize the sponge-gel-supported histoculture model and provide more insight in cell specific behaviour by evaluating the tumor and its microenvironment using immunohistochemistry. We collected fresh tumor biopsies from 72 untreated patients and cultured them for 7 days. Biopsies from 57 patients (79%) were successfully cultured and 1451 tumor fragments (95.4%) were evaluated. Fragments were scored for percentage of tumor, tumor viability and proliferation, EGF-receptor expression and presence of T-cells and macrophages. Median tumor percentage increased from 53% at day 0 to 80% at day 7. Viability and proliferation decreased after 7 days, from 90% to 30% and from 30% to 10%, respectively. Addition of EGF, folic acid and hydrocortisone can lead to improved viability and proliferation, however this was not systematically observed. No patient subgroup could be identified with higher culture success rates. Immune cells were still present at day 7, illustrating that the tumor microenvironment is sustained. EGF supplementation did not increase viability and proliferation in patients overexpressing EGF-Receptor.

Co-culture of primary human tumor hepatocytes from patients with hepatocellular carcinoma with autologous peripheral blood mononuclear cells: study of their in vitro immunological interactions

PubMed Central

2013-01-01

Background Many studies have suggested that the immune response may play a crucial role in the progression of hepatocellular carcinoma (HCC). Therefore, our aim was to establish a (i) functional culture of primary human tumor hepatocytes and non-tumor from patients with hepatocellular carcinoma (HCC) and (ii) a co-culture system of HCC and non-HCC hepatocytes with autologous peripheral blood mononuclear cells (PBMCs) in order to study in vitro cell-to-cell interactions. Methods Tumor (HCC) and non-tumor (non-HCC) hepatocytes were isolated from the liver resection specimens of 11 patients operated for HCC, while PBMCs were retrieved immediately prior to surgery. Four biopsies were obtained from patients with no liver disease who had surgery for non malignant tumor (normal hepatocytes). Hepatocytes were either cultured alone (monoculture) or co-cultured with PBMCs. Flow cytometry measurements for MHC class II expression, apoptosis, necrosis and viability (7AAD) were performed 24 h, 48 h and 72 h in co-culture and monocultures. Results HCC and non-HCC hepatocytes exhibited increased MHC-II expression at 48h and 72h in co-culture with PBMCs as compared to monoculture, with MHC II-expressing HCC hepatocytes showing increased viability at 72 h. PBMCs showed increased MHC-II expression (activation) in co-culture with HCC as compared to non-HCC hepatocytes at all time points. Moreover, CD8+ T cells had significantly increased apoptosis and necrosis at 48h in co-culture with HCC hepatocytes as compared to monocultures. Interestingly, MHC-II expression on both HCC and non-HCC hepatocytes in co-culture was positively correlated with the respective activated CD8+ T cells. Conclusions We have established an in vitro co-culture model to study interactions between autologous PBMCs and primary HCC and non-HCC hepatocytes. This direct interaction leads to increased antigen presenting ability of HCC hepatocytes, activation of PBMCs with a concomitant apoptosis of activated CD8+ T

Bacterial biodegradation of melamine-contaminated aged soil: influence of different pre-culture media or addition of activation material.

PubMed

Hatakeyama, Takashi; Takagi, Kazuhiro

2016-08-01

This study aimed to investigate the biodegrading potential of Arthrobacter sp. MCO, Arthrobacter sp. CSP, and Nocardioides sp. ATD6 in melamine-contaminated upland soil (melamine: approx. 10.5 mg/kg dry weight) after 30 days of incubation. The soil sample used in this study had undergone annual treatment of lime nitrogen, which included melamine; it was aged for more than 10 years in field. When R2A broth was used as the pre-culture medium, Arthrobacter sp. MCO could degrade 55 % of melamine after 30 days of incubation, but the other strains could hardly degrade melamine (approximately 25 %). The addition of trimethylglycine (betaine) in soil as an activation material enhanced the degradation rate of melamine by each strain; more than 50 % of melamine was degraded by all strains after 30 days of incubation. In particular, strain MCO could degrade 72 % of melamine. When the strains were pre-cultured in R2A broth containing melamine, the degradation rate of melamine in soil increased remarkably. The highest (72 %) melamine degradation rate was noted when strain MCO was used with betaine addition.

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

PubMed Central

Vorrink, Sabine U.; Ullah, Shahid; Schmidt, Staffan; Nandania, Jatin; Velagapudi, Vidya; Beck, Olof; Ingelman-Sundberg, Magnus; Lauschke, Volker M.

2017-01-01

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.—Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. PMID:28264975

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

PubMed

Vorrink, Sabine U; Ullah, Shahid; Schmidt, Staffan; Nandania, Jatin; Velagapudi, Vidya; Beck, Olof; Ingelman-Sundberg, Magnus; Lauschke, Volker M

2017-06-01

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.-Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. © The Author(s).

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

PubMed

Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

2017-03-01

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

PubMed

Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

2018-04-13

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

The myotoxic effect of bupivacaine and ropivacaine on myotubes in primary mouse cell culture and an immortalized cell line.

PubMed

Hofmann, Petra; Metterlein, Thomas; Bollwein, Gabriele; Gruber, Michael; Plank, Christoph; Graf, Bernhard M; Zink, Wolfgang

2013-09-01

The 2 local anesthetics (LAs) bupivacaine and ropivacaine have acute cytotoxic effects on different tissues. In this respect, LA-induced myotoxicity has been subject to various studies; however, the exact mechanisms are still not fully understood. Most in vitro studies use immortalized cell lines because of feasibility. Thus, establishing a primary cell line might result in more accurate results. In this study, we examined the effects of immortalization on bupivacaine- and ropivacaine-induced myotoxicity in vitro. An immortalized (N = 6) and a primary cell line (N = 8) of the same tissue and species were established, and differentiation in myotubes was induced. Cells were exposed to increasing concentrations of bupivacaine and ropivacaine for 1 or 2 hours, respectively. Twenty-four and 48 hours after treatment, the fractions of dead and vital cells were measured using flow cytometry. Significance was tested through 1-way analysis of variance with post hoc Dunnett T3 test. Medians of dataset pairs were compared by T test. In both cell lines, increasing concentrations of both LAs resulted in decreased cell survival (e.g., P < 0.001 for 5000 ppm bupivacaine, 1 or 2 hours of incubation, and 24 hours recovery in both cell lines). For the same LA concentrations, survival was significantly higher in the immortalized cell culture (e.g., P < 0.001 for 2500 ppm ropivacaine, 1 hour of incubation, and 24 hours recovery). In addition, equal concentrations of bupivacaine resulted in significantly fewer vital cells compared with ropivacaine (e.g., P = 0.032 for 2500 ppm ropivacaine, 1 hour of incubation, and 24 hours recovery). Two hours of incubation resulted in a significantly higher rate of dead cells compared with 1 hour of incubation (e.g., P = 0.004 for C2C12 cells, 2500 ppm bupivacaine, and 24 hours recovery). Primary skeletal muscle cells are more vulnerable to LAs than immortalized cells. The higher myotoxic potential of bupivacaine compared with ropivacaine in vivo can

Colocalization of neurotensin receptors and of the neurotensin-degrading enzyme endopeptidase 24-16 in primary cultures of neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chabry, J.; Checler, F.; Vincent, J.P.

1990-12-01

This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with {sup 125}I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific {sup 125}I-Tyr3-neurotensin labeling is maximal after 4 d of culture and ismore » located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.« less

Colocalization of neurotensin receptors and of the neurotensin-degrading enzyme endopeptidase 24-16 in primary cultures of neurons.

PubMed

Chabry, J; Checler, F; Vincent, J P; Mazella, J

1990-12-01

This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125I-Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.

Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO₂, and pH.

PubMed

Hodne, Kjetil; von Krogh, Kristine; Weltzien, Finn-Arne; Sand, Olav; Haug, Trude M

2012-09-01

Protocols for primary cultures of teleost cells are commonly only moderately adjusted from similar protocols for mammalian cells, the main adjustment often being of temperature. Because aquatic habitats are in general colder than mammalian body temperatures and teleosts have gills in direct contact with water, pH and buffer capacity of blood and extracellular fluid are different in fish and mammals. Plasma osmolality is generally higher in marine teleosts than in mammals. Using Atlantic cod (Gadus morhua) as a model, we have optimized these physiological parameters to maintain primary pituitary cells in culture for an extended period without loosing key properties. L-15 medium with adjusted osmolality, adapted to low pCO(2) (3.8mm Hg) and temperature (12°C), and with pH 7.85, maintained the cells in a physiologically sounder state than traditional culture medium, significantly improving cell viability compared to the initial protocol. In the optimized culture medium, resting membrane potential and response to releasing hormone were stable for at least two weeks, and the proportion of cells firing action potentials during spawning season was about seven times higher than in the original culture medium. The cells were moderately more viable when the modified medium was supplemented with newborn calf serum or artificial serum substitute. Compared to serum-free L-15 medium, expression of key genes (lhb, fshb, and gnrhr2a) was better maintained in medium containing SSR, whereas NCS tended to decrease the expression level. Although serum-free medium is adequate for many applications, serum supplement may be preferable for experiments dependent on membrane integrity. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of primary cultures of adult human epididymis epithelial cells.

PubMed

Leir, Shih-Hsing; Browne, James A; Eggener, Scott E; Harris, Ann

2015-03-01

To establish cultures of epithelial cells from all regions of the human epididymis to provide reagents for molecular approaches to functional studies of this epithelium. Experimental laboratory study. University research institute. Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement. Human epididymis epithelial cells harvested from adult epididymis tissue. Establishment of a robust culture protocol for adult human epididymal epithelial cells. Cultures of caput, corpus, and cauda epithelial cells were established from epididymis tissue of seven donors. Cells were passaged up to eight times and maintained differentiation markers. They were also cryopreserved and recovered successfully. Androgen receptor, clusterin, and cysteine-rich secretory protein 1 were expressed in cultured cells, as shown by means of immunofluorescence, Western blot, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The distribution of other epididymis markers was also shown by means of qRT-PCR. Cultures developed transepithelial resistance (TER), which was androgen responsive in the caput but androgen insensitive in the corpus and cauda, where unstimulated TER values were much higher. The results demonstrate a robust in vitro culture system for differentiated epithelial cell types in the caput, corpus, and cauda of the human epididymis. These cells will be a valuable resource for molecular analysis of epididymis epithelial function, which has a pivotal role in male fertility. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

C5a Increases the Injury to Primary Neurons Elicited by Fibrillar Amyloid Beta.

PubMed

Hernandez, Michael X; Namiranian, Pouya; Nguyen, Eric; Fonseca, Maria I; Tenner, Andrea J

2017-02-01

C5aR1, the proinflammatory receptor for C5a, is expressed in the central nervous system on microglia, endothelial cells, and neurons. Previous work demonstrated that the C5aR1 antagonist, PMX205, decreased amyloid pathology and suppressed cognitive deficits in two Alzheimer's Disease (AD) mouse models. However, the cellular mechanisms of this protection have not been definitively demonstrated. Here, primary cultured mouse neurons treated with exogenous C5a show reproducible loss of MAP-2 staining in a dose-dependent manner within 24 hr of treatment, indicative of injury to neurons. This injury is prevented by the C5aR1 antagonist PMX53, a close analog of PMX205. Furthermore, primary neurons derived from C5aR1 null mice exhibited no MAP-2 loss after exposure to the highest concentration of C5a tested. Primary mouse neurons treated with both 100 nM C5a and 5 µM fibrillar amyloid beta (fAβ), to model what occurs in the AD brain, showed increased MAP-2 loss relative to either C5a or fAβ alone. Blocking C5aR1 with PMX53 (100 nM) blocked the loss of MAP2 in these primary neurons to the level seen with fAβ alone. Similar experiments with primary neurons derived from C5aR1 null mice showed a loss of MAP-2 due to fAβ treatment. However, the addition of C5a to the cultures did not enhance the loss of MAP-2 and the addition of PMX53 to the cultures did not change the MAP-2 loss in response to fAβ. Thus, at least part of the beneficial effects of C5aR1 antagonist in AD mouse models may be due to protection of neurons from the toxic effects of C5a.

Language, culture and emotions: exploring ethnic minority patients' emotional expressions in primary healthcare consultations.

PubMed

De Maesschalck, Stéphanie; Deveugele, Myriam; Willems, Sara

2011-09-01

This study explores ethnic minority patients' expression of emotional cues and concerns in primary healthcare, and examines relationships with patient, provider and consultation attributes. 191 video-recorded consultations were analyzed using the VR-CoDES. Patients were interviewed before the consultation. Generalized Estimating Equations models (GEE) were used to test for associations. Psychosocial versus bio-medically oriented encounters contained significantly more cues (p≤0.05). Patients with poor versus good language proficiency expressed significantly less cues (p≤0.001). No significant correlations were found with patients' cultural values, patients' or physicians' gender or the presence of an interpreter. Female patients express more concerns (p≤0.05), female physicians have a higher number of concerns expressed by patients (p≤0.02). This study shows that independent of physician and diagnosis, patients' language proficiency has a more important impact on the number of cues expressed by the patient than cultural difference. Medical schools and Continuing Medical Education should focus on training programs for recognizing and handling linguistic barriers between physicians and patients. Patient education programs should encourage patients who experience language barriers to open up to physicians. In situations where language is a barrier, physicians and patients should be encouraged to use interpreters to enhance the expression of emotions. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Huperzine A, but not tacrine, stimulates S100B secretion in astrocyte cultures.

PubMed

Lunardi, Paula; Nardin, Patrícia; Guerra, Maria Cristina; Abib, Renata; Leite, Marina Concli; Gonçalves, Carlos-Alberto

2013-04-09

The loss of cholinergic function in the central nervous system contributes significantly to the cognitive decline associated with advanced age and dementias. Huperzine A (HupA) is a selective inhibitor of acetylcholinesterase (AChE) and has been shown to significantly reduce cognitive impairment in animal models of dementia. Based on the importance of astrocytes in physiological and pathological brain activities, we investigated the effect of HupA and tacrine on S100B secretion in primary astrocyte cultures. S100B is an astrocyte-derived protein that has been proposed to be a marker of brain injury. Primary astrocyte cultures were exposed to HupA, tacrine, cholinergic agonists, and S100B secretion was measured by enzyme-linked immunosorbent assay (ELISA) at 1 and 24h. HupA, but not tacrine, at 100μM significantly increased S100B secretion in astrocyte cultures. Nicotine (at 100 and 1000μM) was able to stimulate S100B secretion in astrocyte cultures. Our data reinforce the idea that AChE inhibitors, particularly HupA, do not act exclusively on the acetylcholine balance. This effect of HupA could contribute to improve the cognitive deficit observed in patients, which are attributed to cholinergic dysfunction. In addition, for the first time, to our knowledge, these data indicate that S100B secretion can be modulated by nicotinic receptors, in addition to glutamate, dopamine and serotonin receptors. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

PubMed

Dyszkiewicz-Konwińska, M; Bryja, A; Jopek, K; Budna, J; Khozmi, R; Jeseta, M; Bukowska, D; Antosik, P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

2017-01-01

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.

A hanging drop culture method to study terminal erythroid differentiation.

PubMed

Gutiérrez, Laura; Lindeboom, Fokke; Ferreira, Rita; Drissen, Roy; Grosveld, Frank; Whyatt, David; Philipsen, Sjaak

2005-10-01

To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.

African American Identity and a Theory for Primary Cultural Instructional Design

ERIC Educational Resources Information Center

Thomas, Michael K.; Columbus, Marco A.

2010-01-01

This article is on the strange confluence of culture, identity, learning, and systemic design. We argue that the work of instructional design is, essentially, work on culture and identity. A person's culture and identity fully and inextricably situate their thought, action, and interaction. For this reason, this inherent situatedness of culture…

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro.

PubMed

Kroehne, Volker; Tsata, Vasiliki; Marrone, Lara; Froeb, Claudia; Reinhardt, Susanne; Gompf, Anne; Dahl, Andreas; Sterneckert, Jared; Reimer, Michell M

2017-01-01

Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC)-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable analysis up to 10

Organizational Culture in a Successful Primary School: An Ethnographic Case Study

ERIC Educational Resources Information Center

Negis-Isik, Ayse; Gursel, Musa

2013-01-01

Even though they are perceived similar from outside, all schools have distinct characteristics and a culture that differ them from other schools. School culture, is one of the important factors that play role in school efficiency and success. The purpose of this study was to examine the culture of a successful school profoundly. This study was a…

In vitro cultured primary roots derived from stem segments of cassava (Manihot esculenta) can behave like storage organs.

PubMed

Medina, Ricardo D; Faloci, Mirta M; Gonzalez, Ana M; Mroginski, Luis A

2007-03-01

Cassava (Manihot esculenta) has three adventitious root types: primary and secondary fibrous roots, and storage roots. Different adventitious root types can also regenerate from in vitro cultured segments. The aim of this study was to investigate aspects of in vitro production of storage roots. Morphological and anatomical analyses were performed to identify and differentiate each root type. Twenty-nine clones were assayed to determine the effect of genotype on the capacity to form storage roots in vitro. The effects of cytokinins and auxins on the formation of storage roots in vitro were also examined. Primary roots formed in vitro and in vivo had similar tissue kinds; however, storage roots formed in vitro exhibited physiological specialization for storing starch. The only consistent diagnostic feature between secondary fibrous and storage roots was their functional differentiation. Anatomical analysis of the storage roots formed in vitro showed that radial expansion as a consequence of massive proliferation and enlargement of parenchymatous cells occurred in the middle cortex, but not from cambial activity as in roots formed in vivo. Cortical expansion could be related to dilatation growth favoured by hormone treatments. Starch deposition of storage roots formed in vitro was confined to cortical tissue and occurred earlier than in storage roots formed in vivo. Auxin and cytokinin supplementation were absolutely required for in vitro storage root regeneration; these roots were not able to develop secondary growth, but formed a tissue competent for starch storing. MS medium with 5 % sucrose plus 0.54 microM 1-naphthaleneacetic acid and 0.44 microM 6-benzylaminopurine was one of the most effective in stimulating the storage root formation. Genotypes differed significantly in their capacity to produce storage roots in vitro. Storage root formation was considerably affected by the segment's primary position and strongly influenced by hormone treatments. The storage

In vitro Cultured Primary Roots Derived from Stem Segments of Cassava (Manihot esculenta) Can Behave Like Storage Organs

PubMed Central

Medina, Ricardo D.; Faloci, Mirta M.; Gonzalez, Ana M.; Mroginski, Luis A.

2007-01-01

Background and Aims Cassava (Manihot esculenta) has three adventitious root types: primary and secondary fibrous roots, and storage roots. Different adventitious root types can also regenerate from in vitro cultured segments. The aim of this study was to investigate aspects of in vitro production of storage roots. Methods Morphological and anatomical analyses were performed to identify and differentiate each root type. Twenty-nine clones were assayed to determine the effect of genotype on the capacity to form storage roots in vitro. The effects of cytokinins and auxins on the formation of storage roots in vitro were also examined. Key Results Primary roots formed in vitro and in vivo had similar tissue kinds; however, storage roots formed in vitro exhibited physiological specialization for storing starch. The only consistent diagnostic feature between secondary fibrous and storage roots was their functional differentiation. Anatomical analysis of the storage roots formed in vitro showed that radial expansion as a consequence of massive proliferation and enlargement of parenchymatous cells occurred in the middle cortex, but not from cambial activity as in roots formed in vivo. Cortical expansion could be related to dilatation growth favoured by hormone treatments. Starch deposition of storage roots formed in vitro was confined to cortical tissue and occurred earlier than in storage roots formed in vivo. Auxin and cytokinin supplementation were absolutely required for in vitro storage root regeneration; these roots were not able to develop secondary growth, but formed a tissue competent for starch storing. MS medium with 5 % sucrose plus 0·54 μm 1-naphthaleneacetic acid and 0·44 μm 6-benzylaminopurine was one of the most effective in stimulating the storage root formation. Genotypes differed significantly in their capacity to produce storage roots in vitro. Storage root formation was considerably affected by the segment's primary position and strongly

Identification of cellular compartments involved in processing of cathepsin E in primary cultures of rat microglia.

PubMed

Sastradipura, D F; Nakanishi, H; Tsukuba, T; Nishishita, K; Sakai, H; Kato, Y; Gotow, T; Uchiyama, Y; Yamamoto, K

1998-05-01

Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic

Increased production of 4 kDa amyloid beta peptide in serum deprived human primary neuron cultures: possible involvement of apoptosis.

PubMed

LeBlanc, A

1995-12-01

The etiology of the amyloid beta peptide in sporadic Alzheimer's disease (AD) is not known. Amyloid beta peptide (A beta), a proteolytic product of the amyloid precursor protein (APP), is deposited in the senile plaques and cerebrovascular tissues of individuals with either sporadic or familial AD (FAD). Increased A beta production from mutant APPs in FAD fosters the hypothesis that overexpression of A beta plays a primary role in the pathogenesis of AD. The absence of APP mutations in sporadic AD which displays identical pathological features than FAD such as synapse and neuronal loss, senile plaques and neurofibrillary tangles, suggests other causes for overexpression and/or deposition of A beta. To investigate the effect of neuronal death on APP metabolism and A beta secretion, human primary neuron cultures were induced to undergo apoptosis by serum deprivation. Serum deprived neurons display shrunken and rounded morphology, contain condensed chromatine and fragmented DNA, which are characteristic of apoptosis. In serum deprived neurons, metabolism of APP through the nonamyloidogenic secretory pathway is decreased to 20% from 40% in control cultures whereas 4kDa A beta is increased three- to fourfold. The results suggest that human neurons undergoing apoptosis generate excess A beta and indicates a possible mechanism for increased A beta in the absence of APP mutations.

Deaf Culture. NETAC Teacher Tipsheet

ERIC Educational Resources Information Center

Siple, Linda; Greer, Leslie; Holcomb, Barbara Ray

2004-01-01

It often comes as a surprise to people that many deaf people refer to themselves as being members of Deaf culture. The American Deaf culture is a unique linguistic minority that uses American Sign Language (ASL) as its primary mode of communication. This tipsheet provides a description of Deaf culture and suggestions for effective communication.

BAG3 promotes the phenotypic transformation of primary rat vascular smooth muscle cells via TRAIL.

PubMed

Fu, Yao; Chang, Ye; Chen, Shuang; Li, Yuan; Chen, Yintao; Sun, Guozhe; Yu, Shasha; Ye, Ning; Li, Chao; Sun, Yingxian

2018-05-01

Under normal physiological condition, the mature vascular smooth muscle cells (VSMCs) show differentiated phenotype. In response to various environmental stimuluses, VSMCs convert from the differentiated phenotype to dedifferentiated phenotype characterized by the increased ability of proliferation/migration and the reduction of contractile ability. The phenotypic transformation of VSMCs played an important role in atherosclerosis. Both Bcl-2-associated athanogene 3 (BAG3) and tumor necrosis factor-related apopt-osis inducing ligand (TRAIL) involved in apoptosis. The relationship between BAG3 and TRAIL and their effects the proliferation and migration in VSMCs are rarely reported. This study investigated the effects of BAG3 on the phenotypic modulation and the potential underlying mechanisms in primary rat VSMCs. Primary rat VSMCs were extracted and cultured in vitro. Cell proliferation was detected by cell counting, real-time cell analyzer (RTCA) and EdU incorporation. Cell migration was detected by wound healing, Transwell and RTCA. BAG3 and TRAIL were detected using real-time PCR and western blotting and the secreted proteins in the cultured media by dot blot. The expression of BAG3 increased with continued passages in cultured primary VSMCs. BAG3 promoted the proliferation and migration of primary rat VSMC in a time-dependent manner. BAG3 significantly increased the expression of TRAIL while had no effects on its receptors. TRAIL knockdown or blocking by neutralizing antibody inhibited the proliferation of VSMCs induced by BAG3. TRAIL knockdown exerted no obvious influence on the migration of VSMCs. Based on this study, we report for the first time that BAG3 was expressed in cultured primary rat VSMCs and the expression of BAG3 increased with continued passages. Furthermore, BAG3 promoted the proliferation of VSMCs via increasing the expression of TRAIL. In addition, we also demonstrated that BAG3 promoted the migration of VSMCs independent of TRAIL

Development of a new culture system for human lymphokine-activated killer cells: comparison with a conventional static culture method.

PubMed

Murata, M; Yano, T; Yoshino, I; Togami, M; Sogabe, M; Yasumoto, K; Sugimachi, K; Kimura, G; Nomoto, K

1991-10-01

We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7 x 10(7) cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

PubMed

Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

2009-09-01

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air-liquid interface method for nasal drug transport study.

PubMed

Cho, Hyun-Jong; Choi, Min-Koo; Lin, Hongxia; Kim, Jung Sun; Chung, Suk-Jae; Shim, Chang-Koo; Kim, Dae-Duk

2011-03-01

P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method. MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner. These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

PubMed

Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

2016-11-30

The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N -acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

PubMed Central

Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

2016-01-01

The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes. PMID:27916916

Perceptions of culturally competent diabetes management in a primary care practice.

PubMed

Kirk, Julienne K; Hildebrandt, Carol; Davis, Stephen; Crandall, Sonia J; Siciliano, Alissa B; Marion, Gail S

2014-01-01

To evaluate whether clinicians consider the impact of culture on diabetes management, a survey was mailed to 300 randomly selected patients > or = 50 years with type 2 diabetes and 153 surveys were returned. Data were correlated with A1C values. African Americans (AA) and non-Hispanic whites (NHW), (91.9%, 97.0%) respectively, reported clinicians discussed benefits of controlling blood sugar but did not discuss effects of cultural issues on glucose control (< or = 50%). AAs perceived clinicians were more accommodating of their cultural preferences than did NHWs (49.2% versus 30.6%) (P < .05). Females (51.9%) (P < .01) reported that clinicians acknowledged the importance of their cultural beliefs with a slightly higher percentage for African American females (54.8%) versus non-Hispanic White females (48.6%). Understanding the patient's and clinician's views of cultural beliefs as they relate to diabetes self-management can provide perspectives to guide care.

Role of Organizational Culture on the Performance Primary School Teachers

ERIC Educational Resources Information Center

Suharningsih; Murtedjo

2017-01-01

This study aims to identify and examine the role of organizational culture on teacher performance. In the present study examined the role of organizational culture with teacher performance. In accordance with the study design, namely the survey, the data collected in this research is quantitative data. The data is extracted and obtained through…