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991.
Kimberley A. Beaumont Andrea Anfosso Farzana Ahmed Wolfgang Weninger Nikolas K. Haass 《Journal of visualized experiments : JoVE》2015,(106)
Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. 相似文献
992.
Francesca Luciani Sara Galluzzo Andrea Gaggioli Nanna Aaby Kruse Pascal Venneugues Christian K Schneider Carlo Pini Daniela Melchiorri 《MABS-AUSTIN》2015,7(3):451-455
Quality by design (QbD) is an innovative approach to drug development that has started to be implemented into the regulatory framework, but currently mainly for chemical drugs. The recent marketing authorization of the first monoclonal antibody developed using extensive QbD concepts in the European Union paves the way for future further regulatory approvals of complex products employing this cutting-edge technological concept. In this paper, we report and comment on insights and lessons learnt from the non-public discussions in the European Medicines Agency''s Biologicals Working Party and Committee for Medicinal Products for Human Use on the key issues during evaluation related to the implementation of an extensive QbD approach for biotechnology-derived medicinal products. Sharing these insights could prove useful for future developments in QbD for biotech products in general and monoclonal antibodies in particular. 相似文献
993.
A comparative analysis reveals weak relationships between ecological factors and beta diversity of stream insect metacommunities at two spatial levels
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Florian Altermatt Salman A. Al‐Shami David G. Angeler Núria Bonada Cecilia Brand Marcos Callisto Karl Cottenie Olivier Dangles David Dudgeon Andrea Encalada Emma Göthe Mira Grönroos Neusa Hamada Dean Jacobsen Victor L. Landeiro Raphael Ligeiro Renato T. Martins María Laura Miserendino Che Salmah Md Rawi Marciel E. Rodrigues Fabio de Oliveira Roque Leonard Sandin Denes Schmera Luciano F. Sgarbi John P. Simaika Tadeu Siqueira Ross M. Thompson Colin R. Townsend 《Ecology and evolution》2015,5(6):1235-1248
The hypotheses that beta diversity should increase with decreasing latitude and increase with spatial extent of a region have rarely been tested based on a comparative analysis of multiple datasets, and no such study has focused on stream insects. We first assessed how well variability in beta diversity of stream insect metacommunities is predicted by insect group, latitude, spatial extent, altitudinal range, and dataset properties across multiple drainage basins throughout the world. Second, we assessed the relative roles of environmental and spatial factors in driving variation in assemblage composition within each drainage basin. Our analyses were based on a dataset of 95 stream insect metacommunities from 31 drainage basins distributed around the world. We used dissimilarity‐based indices to quantify beta diversity for each metacommunity and, subsequently, regressed beta diversity on insect group, latitude, spatial extent, altitudinal range, and dataset properties (e.g., number of sites and percentage of presences). Within each metacommunity, we used a combination of spatial eigenfunction analyses and partial redundancy analysis to partition variation in assemblage structure into environmental, shared, spatial, and unexplained fractions. We found that dataset properties were more important predictors of beta diversity than ecological and geographical factors across multiple drainage basins. In the within‐basin analyses, environmental and spatial variables were generally poor predictors of variation in assemblage composition. Our results revealed deviation from general biodiversity patterns because beta diversity did not show the expected decreasing trend with latitude. Our results also call for reconsideration of just how predictable stream assemblages are along ecological gradients, with implications for environmental assessment and conservation decisions. Our findings may also be applicable to other dynamic systems where predictability is low. 相似文献
994.
Mansoureh Malekian Steven J. B. Cooper Kathleen M. Saint Melanie L. Lancaster Andrea C. Taylor Susan M. Carthew 《Ecology and evolution》2015,5(18):3939-3953
Ongoing habitat loss and fragmentation is considered a threat to biodiversity as it can create small, isolated populations that are at increased risk of extinction. Tree‐dependent species are predicted to be highly sensitive to forest and woodland loss and fragmentation, but few studies have tested the influence of different types of landscape matrix on gene flow and population structure of arboreal species. Here, we examine the effects of landscape matrix on population structure of the sugar glider (Petaurus breviceps) in a fragmented landscape in southeastern South Australia. We collected 250 individuals across 12 native Eucalyptus forest remnants surrounded by cleared agricultural land or exotic Pinus radiata plantations and a large continuous eucalypt forest. Fifteen microsatellite loci were genotyped and analyzed to infer levels of population differentiation and dispersal. Genetic differentiation among most forest patches was evident. We found evidence for female philopatry and restricted dispersal distances for females relative to males, suggesting there is male‐biased dispersal. Among the environmental variables, spatial variables including geographic location, minimum distance to neighboring patch, and degree of isolation were the most important in explaining genetic variation. The permeability of a cleared agricultural matrix to dispersing gliders was significantly higher than that of a pine matrix, with the gliders dispersing shorter distances across the latter. Our results added to previous findings for other species of restricted dispersal and connectivity due to habitat fragmentation in the same region, providing valuable information for the development of strategies to improve the connectivity of populations in the future. 相似文献
995.
Silke Andrich Burkhard Haastert Elke Neuhaus Kathrin Neidert Werner Arend Christian Ohmann Jürgen Grebe Andreas Vogt Pascal Jungbluth Grit R?sler Joachim Windolf Andrea Icks 《PloS one》2015,10(9)
Epidemiological data about pelvic fractures are limited. Until today, most studies only analyzed inpatient data. The purpose of this study was to estimate incidence rates of pelvic fractures in the German population aged 60 years or older, based on outpatient and inpatient data. We conducted a retrospective population-based observational study based on routine data from a large health insurance company in Germany. Age and sex-specific incidence rates of first fractures between 2008 and 2011 were calculated. We also standardized incidence rates with respect to age and sex in the German population. Multiple Poisson regression models were used to evaluate the association between the risk of first pelvic fracture as outcome and sex, age, calendar year and region as independent variables. The total number of patients with a first pelvic fracture corresponded to 8,041 and during the study period 5,978 insured persons needed inpatient treatment. Overall, the standardized incidence rate of all first pelvic fractures was 22.4 [95% CI 22.0–22.9] per 10,000 person-years, and the standardized incidence rate of inpatient treated fractures 16.5 [16.1–16.9]. Our adjusted regression analysis confirmed a significant sex (RR 2.38 [2.23–2.55], p < 0.001, men as reference) and age effect (higher risk with increasing age, p < 0.001) on first fracture risk. We found a slight association between calendar year (higher risk in later years compared to 2008, p = 0.0162) and first fracture risk and a further significant association with region (RR 0.92 [0.87–0.98], p = 0.006, Westfalen-Lippe as reference). The observed incidences are considerably higher than incidences described in the international literature, even if only inpatient treated pelvic fractures are regarded. Besides which, non-inclusion of outpatient data means that a relevant proportion of pelvic fractures are not taken into account. Prevention of low energy trauma among older people remains an important issue. 相似文献
996.
Emanuela Zannin Riccardo Pellegrino Alessandro Di Toro Andrea Antonelli Raffaele L. Dellacà Luciano Bernardi 《PloS one》2015,10(6)
Background
It is not known whether parasympathetic outflow simultaneously acts on bronchial tone and cardiovascular system waxing and waning both systems in parallel, or, alternatively, whether the regulation is more dependent on local factors and therefore independent on each system. The aim of this study was to evaluate the simultaneous effect of different kinds of stimulations, all associated with parasympathetic activation, on bronchomotor tone and cardiovascular autonomic regulation.Methods
Respiratory system resistance (Rrs, forced oscillation technique) and cardio-vascular activity (heart rate, oxygen saturation, tissue oxygenation index, blood pressure) were assessed in 13 volunteers at baseline and during a series of parasympathetic stimuli: O2 inhalation, stimulation of the carotid sinus baroreceptors by neck suction, slow breathing, and inhalation of methacholine.Results
Pure cholinergic stimuli, like O2 inhalation and baroreceptors stimulation, caused an increase in Rrs and a reduction in heart rate and blood pressure. Slow breathing led to bradycardia and hypotension, without significant changes in Rrs. However slow breathing was associated with deep inhalations, and Rrs evaluated at the baseline lung volumes was significantly increased, suggesting that the large tidal volumes reversed the airways narrowing effect of parasympathetic activation. Finally inhaled methacholine caused marked airway narrowing, while the cardiovascular variables were unaffected, presumably because of the sympathetic activity triggered in response to hypoxemia.Conclusions
All parasympathetic stimuli affected bronchial tone and moderately affected also the cardiovascular system. However the response differed depending on the nature of the stimulus. Slow breathing was associated with large tidal volumes that reversed the airways narrowing effect of parasympathetic activation. 相似文献997.
Steve Kelling Alison Johnston Wesley M. Hochachka Marshall Iliff Daniel Fink Jeff Gerbracht Carl Lagoze Frank A. La Sorte Travis Moore Andrea Wiggins Weng-Keen Wong Chris Wood Jun Yu 《PloS one》2015,10(10)
Volunteers are increasingly being recruited into citizen science projects to collect observations for scientific studies. An additional goal of these projects is to engage and educate these volunteers. Thus, there are few barriers to participation resulting in volunteer observers with varying ability to complete the project’s tasks. To improve the quality of a citizen science project’s outcomes it would be useful to account for inter-observer variation, and to assess the rarely tested presumption that participating in a citizen science projects results in volunteers becoming better observers. Here we present a method for indexing observer variability based on the data routinely submitted by observers participating in the citizen science project eBird, a broad-scale monitoring project in which observers collect and submit lists of the bird species observed while birding. Our method for indexing observer variability uses species accumulation curves, lines that describe how the total number of species reported increase with increasing time spent in collecting observations. We find that differences in species accumulation curves among observers equates to higher rates of species accumulation, particularly for harder-to-identify species, and reveals increased species accumulation rates with continued participation. We suggest that these properties of our analysis provide a measure of observer skill, and that the potential to derive post-hoc data-derived measurements of participant ability should be more widely explored by analysts of data from citizen science projects. We see the potential for inferential results from analyses of citizen science data to be improved by accounting for observer skill. 相似文献
998.
Yiying Cai Hui Leck Tze Peng Lim Jocelyn Teo Winnie Lee Li Yang Hsu Tse Hsien Koh Thuan Tong Tan Thean-Yen Tan Andrea Lay-Hoon Kwa 《PloS one》2015,10(10)
Background
Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.Methods
100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (105 CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (TRLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of TRLU values was determined. External validation was further done using 50 additional CR-GNB.Results
Predictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (TRLU = 0.81, UA = 91%), and lowest for AB (TRLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.Conclusion
We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations. 相似文献999.
The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin. 相似文献
1000.
G Correia de Faria Santarém R de Cleva Marco Aurélio Santo Aline Biaseto Bernhard Alexandre Vieira Gadducci Julia Maria D’Andrea Greve Paulo Roberto Santos Silva 《PloS one》2015,10(6)