Other methods to isolate OC cells from ascites have utilized Percoll (Amersham Biosciences) density gradients and anti- CD45 immunomagnetic beads to separate red blood cells and leukocytes from individual OC cells and cell clusters (5). Our initial trials to isolate OC cells from ascites utilized a Percoll gradient (50-10%), however, we determined that despite our initial yield of total cells being higher, these cells had a lower plating efficiency and reduced capacity to adapt to growth in culture than if we simply plated the cells as described in this paper. We suggest that this may be due to a 'culture shock' when the cells are switched from the ascitic fluid environment to a defined medium. This problem could probably be circumvented if clarified ascitic fluid were used to supplement the growth medium. Furthermore, we did not find the presence of red blood cells to adversely affect the establishment of primary OC cell cultures. Indeed, we have often received samples of ascites fluid that was extremely bloody making it difficult to easily visualize cells or cell clusters upon initial seeding of the flasks. After 3-4 days, however, the medium was changed to reveal a sub-confluent monolayer of OC cells. Leukocytes are unlikely to adapt to growth conditions required for OC cells. Thus, special procedures to reduce the number of red blood cells or leukocytes can be conducted, however in our experience they are not necessary.
of isolated first-passage RA-SFB did not significantly differ from those of the corresponding cells in conventional fourth passage. Upon stimulation with platelet-derived growth factor (PDGF) (2.5 and 5 U/ml) and IL-1 β (150 U/ml), however, the mean proliferation rates of conventional fourth-passage RA-SFB were significantly higher than those observed in first- passage cells (3.9-fold and 4.2-fold, respectively, in the case of 2.5 and 5 U/ml PDGF; 2.1-fold in the case of 150 U/ml IL-1 β ). Discussion: The advantages of negative isolation are as follows: first, there was minimal contamination with other cells, especially macrophages (< 2%); second, negative isolation avoided direct contact of SFB with mAbs and/or magnetobeads, thereby limiting possible functional alterations of the cells; and third, the FB marker mAb AS02 (anti-Thy-1; used in the parallel attempt to positively isolate SFB) identified 91% of primary-culture/first-passage normal skin-FB but only 74% of isolated primary RA-SFB, probably due the variable
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Overall, based on our data on cytogenetic analysis, PDT and mRNA expressions of target genes, we have established the sub-culturing limit beyond which replica- tive senescence was observed in primary-culture human vocal fold fibroblasts. This limit of 5 passages was critical for establishing representative in vitro models for tissue engineering approaches involving the use of primary- culture fibroblasts. For example, primary vocal fold fibro- blasts seeded in decellularized ECM scaffolds have been shown to be promising for vocal fold regeneration . The therapeutic potential of injection of autologous fibro- blasts as well as the incorporation of growth factors or biomaterial implants with fibroblasts for treating vocal fold scarring has also been targeted [9,10,40]. In those studies, autologous fibroblasts were harvested by biopsy and sub-cultured for up to the sixth passage, for up to 30 days. The current findings suggested that some of the fi- broblasts in those studies may have become senescent, compromising their findings and conclusions.
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Bradford method, were resolved by SDS-PAGE. Elec- trophoresed proteins were transferred to nitrocellulose membrane (Protran ® , Whatman ® Schleicher & Schuell, Sigma Chemical Co., St. Louis-MO, USA). The mem- branes were incubated with specific primary antibod- ies and then with HRP conjugated anti species-specific secondary antibodies. The protein signals were normal- ized using the relevant calnexin or anti GAPDH bands. Immunoreactive bands were visualized by an enhanced chemiluminescence method (Amersham Pharmacia Bio- tech, Piscataway, NJ, USA). Bands on X-ray films were then quantified using Scion Image software (Scion Corp., Frederick, MD, USA). The data were then converted to fold change (FC) of the control.
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To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as monolayers in a defined medium. Morphologically, cultured cells were epithelial in type, and similar to collecting duct principal cells. Cell pH measured fluorometrically in monolayers grown on glass slides showed recovery from acid loads in Na+-free media. Recovery was inhibited by cyanide, oligomycin A, and N-ethylmaleimide. Cyanide and oligomycin inhibited recovery less in the presence than in the absence of glucose. When cells were first acid loaded in a Na+-free medium and then exposed to external Na+, pH recovery also took place. This recovery exhibited first-order dependence on Na+ concentration and was inhibited by 5-(N- ethyl-N-isopropyl)amiloride. These studies demonstrate that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion: a proton ATP-ase and an Na+-H+ exchanger. The former may be responsible for some component of the urinary acidification observed in the papillary collecting duct in vivo; the role of the latter in acid- base transport remains uncertain.
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Primary culture—cytokine production. To determine the effect of RBC on PBMC cytokine production, primary cultures were stimulated with OKT3 for 3 days. Cytokine production from PBMC increased with the addition of increasing amounts of RBC (Fig. 1A, B, and C). The levels of cytokine production from unstimulated cells were 17.7 ⫾ 9.4 pg/ml for IL-␤, 37.9 ⫾ 6.5 pg/ml for TNF-␣, and 74.8 ⫾ 19.2 pg/ml for IFN-␥. IL-1␤ (Fig. 1A) production increased from 328 ⫾ 109 pg/ml in the PBMC-alone group to 462 ⫾ 154 pg/ml in the 10:1 mixtures of RBC and PBMC (P ⫽ 0.02) and to 624 ⫾ 171 pg/ml in the 50:1 mixtures (P ⫽ 0.003). The IL-1␤ production in the 2:1 group was increased compared to that in the PBMC-alone group, but the difference was not significant statistically (P ⫽ 0.07). TNF-␣ (Fig. 1B) production increased from 1,174 ⫾ 391 pg/ml in the PBMC-alone group to 1,377 ⫾ 459 pg/ml in the 2:1 mixture (P ⫽ 0.02), to 1,624 ⫾ 541 pg/ml in the 10:1 mixture (P ⫽ 0.02), and to 1,946 ⫾ 266 pg/ml in the 50:1 mixture (P ⫽ 0.02). IFN-␥ (Fig. 1C) production increased from 2,925 ⫾ 975 pg/ml in the PBMC-alone group to 4,181 ⫾ 1,394 pg/ml in the 2:1 mixture (P ⫽ 0.03), to 6,850 ⫾ 2,283 pg/ml in the 10:1 mixture of RBC and PBMC (P ⫽ 0.01), and to 8,733 ⫾ 1,309 pg/ml in the 50:1 mixtures of RBC and PBMC (P ⫽ 0.0003). The increases in cytokine production at higher RBC-to-PBMC ratios varied for the different cytokines. IFN-␥ production showed the largest increase, threefold in the 50:1 mixture of RBC and PBMC relative to the PBMC-alone group. TNF-␣ production had a 1.7-fold increase, followed by IL-1␤, which had a 1.9-fold increase.
The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin’s lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recom- binant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the prolif- eration of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4 R24C oncogene in transforming MEF cells.
This study explores the cultural barriers and incentives early career primary teachers experience regarding Masters-level primary mathematics CPD. Its focus is the teachers’ analyses of their experiences within both their Initial Teacher Education (ITE) Higher Education Institution (HEI) and primary school setting. Data was collected through questionnaires and follow-up, in-depth individual interviews. Findings indicate that the role of significant others, such as the Senior Leadership Team, can be influential and that key aspects of the early career phase of a teacher’s career can also be important. Implications for practice in both primary schools and HEI ITE providers are discussed.
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To investigate drug effects on post-injury sprouting, mature cultures were injured using an established scratch model of injury. This in vitro model of complete transection injury is capable of eliciting substantial axonal sprouting at 24 hours post- injury. Whilst this injury is unable to replicate the complexity of the injured brain, it is a powerful tool for investigating drug effects on neuronal regeneration, screening for treatment targets and identifying new injury biomarkers (Chung et al., 2002, Chuckowree and Vickers, 2003, Loov et al., 2013). In this study Epo D was added to the culture media immediately after transection injury. The administration of a high concentration of Epo D (100 nM) resulted in a substantial reduction of the axonal sprouting response by approximately 50%, in respect to vehicle. This suggests that the stabilization of microtubules, evidenced by the initial increase in acetylated tubulin and loss of Tau reduces the number of axons that are able to respond to injury with the formation of axonal sprouts. Conversely, the administration of lower concentrations of the drug (0.1nM, 1nM) significantly increased the number of Tau immunolabeled sprouts directed into the injury site. These data indicate that Epo D promotes polymerization but not stabilization of microtubules, and increases the ability of neurons to regenerate.
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Claims of renewed enthusiasm and increased job satisfaction after coteaching are well documented (Bacharach et al., 2012; Gallo-Fox & Scantlebury, 2016; Nilsson & Van Driel, 2010). As discussed in Chapter Two (Subsection 2.3.2) a host of scholars including Murphy and Beggs (2010) and Gallo- Fox and Scantlebury (2016) acknowledged the impact of coteaching on enthusiasm for teaching science and on having gained greater subject knowledge and on-site professional development. Findings from the coteaching music study resonate with those of Murphy and Beggs (2005a), Carlisle (2010), Kerr (2010) and Gallo-Fox and Scantlebury (2016) which, considering initial opposition to music and to coteaching music in particular, indicated quite a transformation in attitude amongst teacher participants (Chapter 4, Section 4.3; Chapter 5, Section 4). This coteaching music study adds to current research by confirming the impact of coteaching on sustained enthusiasm for music over three years. Findings revealed in Chapter Four (for example Chapter 4, Section 3) document the prioritization and preservation of a vibrant music programme such that music, a subject, once perceived as possessing marginal value was elevated to a status alongside mathematics and English. Music, three years after the coteaching music study was completed, still retained its core curricular status such that all children could access an incremental, developmental, sequential primary music programme and a high-quality music lesson at least once each week. The long-term effect of teacher enthusiasm for music was also evident in the expansion of the classroom music programme. At the beginning of the third residency the school principal reported that the school appointed a teacher with a specialist interest in music and a special needs assistant with keyboard skills, demonstrating its commitment to the preservation of music education (Personal communication with school principal, September 2016). Sustained teacher enthusiasm for music is also evident in the support of two further music coteaching partnerships one of which resulted in the school winning a prestigious Feis Cheoil silver cup for choral singing at the time of writing this study. Evidence of such collective and extended support for music in the form of a positive Whole School Evaluation Report (2016) by the Inspectorate of the Department of Education and Skills has already been mentioned in Chapter Four (Subsection 4.2.6).
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The present study investigates the associations between “un- der primary control cultural discrepancy”, “over primary con- trol cultural discrepancy” and the sense of well-being in Israeli subjects using three indicators of well-being: low level of de- pression, low level of anxiety, and high level of self-esteem. First, it was hypothesized that 1) discrepancy between the per- ceived ought primary control and the perceived actual primary control will be negatively related to well being. This discrep- ancy was termed as “general primary control cultural discrep- ancy”. This prediction is in line with cognitive dissonance the- ory (Festinger, 1957); “self-ideal” discrepancy (Rogers, 1951; Rogers & Dymond, 1954); and the self discrepancy theory (Higgins, 1987). Dividing the discrepancy to its components: “under primary control cultural discrepancy” versus “over pri- mary control cultural discrepancy” it was hypothesized that 2) “under primary control cultural discrepancy” would be nega- tively correlated with well being, but “over primary control cultural discrepancy” would not be related to well being.
Sodium fluoride (NaF), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitor cocktails, dithiothreitol (DTT), 0.01% poly-L-lysine solution, Per- coll ® , sterile filtered dimethylsulfoxide Hybri-Max ® (DMSO), Triton X-100, paraformaldehyde (PFA), annex- inV-fluorescein isothiocyanate (FITC) apoptosis detec- tion kit and all reagent-grade chemicals for buffers were purchased from Sigma (St Quentin Fallavier, France); DMEM (1 g/L), MEM and Neurobasal media, B-27 Sup- plement, 200 mM L-glutamine, 5,000 units of penicillin (base) and 5,000 μ g of streptomycin (base)/mL (PS) mix- ture, 0.5 g/L Trypsin/0.2 g/L EDTA 4Na, Fetal Bovine Serum, Certified (FBS), Horse Serum, NuPAGE ® Novex ® Bis-Tris Mini Gels, NuPAGE ® LDS 4X LDS Sample Buffer, NuPAGE ® Sample Reducing Agent (10X), NuPAGE ® MES SDS Running Buffer and NuPAGE ® Antioxidant, iBlot ® Gel Transfer Device (EU), the Prolong Gold antifade reagent with 4’,6-diami- dino-2-phenylindole (DAPI) and the Zenon mouse IgG labelling kit from Gibco-Invitrogen (Fisher Bioblock Scientific distributor, Illkirch, France); the imidazolo- oxindole compound C16 from Merck Chemicals Calbio- chem ® (Nottingham, UK). For western blot, primary antibodies and secondary anti-rabbit IgG antibody con- jugated with horseradish peroxydase were purchased from Cell Signalling (Ozyme, St Quentin Yvelines, France) excepted anti-P T451 -PKR from Eurogentec (Sera-
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PKCs were not inhibited by Jak inhibitor (Figures 1C, D). Pretreatment with anti-CD40 antibody or CD40 siRNA significantly attenuated cytokine production and activation of signal molecules in the co-culture system, but did not completely inhibit. This implies that inflam- matory cytokines secreted by cell-to-cell interaction of both cell surfaces may re-activate each other or that other signal pathways maybe exist. There are also reports that Jak/STAT 701 signaling pathway is involved in early events of cytokine stimulation in astrocytes , and that various cytokines and their receptors are expressed via the Jak/STAT1 701 pathway in brain section of patients with MS . Therefore, we focused on the Jak/STAT 701 cytokine signaling pathway. Jak/STAT1 701 was not involved in Rac/Ca 2+ /PKCs pathways (Figures 1C, D and 2A). Activities of Jak/STAT 701 showed diphasic responses (Figure 3A, B). It can be inferred that Jak/ STAT1 701 , which is weakly activated early (peak activity at 3 and 10 min, respectively) after co-culturing, is induced by interaction of CD40-CD40L. And, our data also infer that Jak/STAT 701 , which is strongly activated late (peak activity at 6 h) after co-culturing, is evoked by cytokines secreted via the Rho-family pathway. Therefore, our data suggest that cytokines produced in co-cultured-astrocytes are mainly induced by signaling via Ca 2+ /PKCs/MAP kinases/STAT1 727 downstream of Rho-family GTPases, and cytokine-induced astrocyte re-activation leads to further cytokine production via the Jak/STAT1 701 path- way. Evidence of this event is supported by our data that anti-TNFR1 antibody as well as anti-CD40 antibody sup- pressed activation of Jak/STAT1 701 and induction of cyto- kine mRNAs in co-cultured-astrocytes. This indicates that TNF-a bound to TNFR1 re-activates astrocytes via the
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Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA’s performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most con- taminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis.
One explanation for these differences in findings is the cultural context of these studies. Yariv’s study, based in Israel, acknowledges the increasing pressure to get results but also notes the level of autonomy amongst the teachers and the lack of formality in the assessment system. However, as already highlighted in this study’s literature, the performative culture of the UK seems to hold every employee to account, which are often externally audited by such agencies as Ofsted, local authorities or the DfE. Using the literature review on panopticism, it could be argued that senior managers in the UK are aware their own judgments are often judged to check on their capability of running a school and therefore there is a pressure to deliver the reality of the observations, whether that is emotionally difficult or not. One aspect of Yariv’s research that is evident in this research is the notion of a vicious cycle (Yariv, p.457, 2009). For this research, rather than there being situations where there are untold truths, which leads to a continuation of poor practice, Vanessa, Tina and Nicola describe situations where their already low confidence is heightened by negative feedback unaccompanied by emotional support. This will be explored further later.
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We have studied the role of gap junction-mediated intercellular communication on the steroidogenic response of bovine (BAC) and human (HAC) adrenal fasciculo-reticularis cells in culture to corticotropin (ACTH). Indirect immunofluorescence analyses showed that intact human and bovine adreno-cortical tissue as well as HAC and BAC in culture
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Analyzing different approaches to the typology of culture, we can see that culture consists of many subsystems, and components of those subsystems closely interact with one another. Each component acts as the necessary con- dition for the functioning of culture. Application of methods do not violate fundamental mutual relations and enable outlining of simpler blocks and also enable description of important features inside those blocks. Thus, outlin- ing of the types of culture is the methodological way to analyse and study them. Outlining of such types of culture as traditional, modern, and dynami- cally developing culture enables clari Þ cation of special features of culture in general. Division of culture as the system into simpler subsystems, which are characterized by certain features, enables analysis of the very phenomenon of culture in detail. Besides that, in this study the importance of values and value orientations as an integral characteristic of culture is stressed.
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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 the PHC setting. In the forthcoming health system reform, healthcare professionals will face challenges both at organizational level and professional status. Results of the study can serve as background knowledge for leaders and policy makers who seek interventions to improve performance before the implementation of a new national healthcare scheme.
variables, only on the supervariable) and also due to findings that Chinese individuals are more sensitive to contextual relationships and tend to process information holistically. Yoon et al. (2000) reasoned that, as there is a cultural bias for holistic processing that remains well-preserved across the lifespan among the Chinese, it is reasonable to expect smaller decrements in performance, compared with the Americans on memory tasks associated with this type of processing. Yoon et al. (2000) also used a composite variable for memory and results were consistent with Levy and Langer (1994); namely, significant effects for age and culture, and main effects were qualified by a significant interaction effect. Yoon et al. (2000) then replicated the analysis for each memory test and found that: a) old Chinese Canadian did perform better than old Anglo speakers on only two (immediate and delayed recall) out of four tests (there were no differences in memory for complex figures and abstract design); and b) the young Chinese Canadians outperformed the old Chinese Canadians (in Levy’s results both old and young Chinese performed equally well). Yoon et al. (2000), however, utilized different memory tests, but the measures on aging stereotypes were taken from Levy and Langer (1994). Consistent with Levy and Langer (1994), Chinese individuals hold more positive views of aging than their Canadian counterparts. However, two measures did not achieve
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The aims of this study were to develop a reliable cul- ture method for use in human bladder biopsy specimens. Specifically, we characterised the immunocytochemical features of the urothelial and myofibroblast cell layers during cell culture and subsequent passage. To separate different cell types, we used a cell enrichment technique known as magnetic activated cell separation of human cells (MACS). This technique enables isolation of func- tionally active cells, the isolation of rare cells and the recovery of intact cells with minimal stress during sorting . To strengthen the characterisation of these cells, we undertook immunocytochemical analysis of full thickness bladder samples, taken at open surgical procedures.