Interpreters for the deaf in Russia     

L Godin 《American annals of the deaf》1967,112(4):595-597

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Quantifying the volume of single cells continuously using a microfluidic pressure-driven trap with media exchange     

Jason Riordon  Michael Nash  Wenyang Jing  Michel Godin 《Biomicrofluidics》2014,8(1)

We demonstrate a microfluidic device capable of tracking the volume of individual cells by integrating an on-chip volume sensor with pressure-activated cell trapping capabilities. The device creates a dynamic trap by operating in feedback; a cell is periodically redirected back and forth through a microfluidic volume sensor (Coulter principle). Sieve valves are positioned on both ends of the sensing channel, creating a physical barrier which enables media to be quickly exchanged while keeping a cell firmly in place. The volume of individual Saccharomyces cerevisiae cells was tracked over entire growth cycles, and the ability to quickly exchange media was demonstrated.Measuring cell growth is of primary interest to researchers who seek to study the effects of drugs, nutrients, disease, and environmental stress. This has traditionally been accomplished by monitoring the optical transmittance of large ensembles of cells and applying the Beer-Lambert Law.^1,2 Such population-scale measurements provide important culture statistics, but averaging obscures the behaviour of individual cells. In addition, these techniques often require cell synchronicity in order to correlate growth with specific points in the cell cycle, but synchronicity typically decays rapidly in many cell lines including Saccharomyces cerevisiae (yeast) cultures.³ Researchers have thus adopted methods that study the growth of individual cells. Quantifying cellular growth is especially challenging since proliferating cells such as yeast or Escherichia coli are irregularly shaped, and will only increase in size by a factor of two.⁴ Growth will affect the mass, volume, and density of the cell; having access to each of these characteristics is important in obtaining a complete picture of this process. Time-lapse fluorescence microscopy can provide valuable information as to the cell cycle progression of individual cells,⁵ but 2D optics requires geometric assumptions, and, thus, can provide an incomplete picture of growth.^6,7Microfluidic lab-on-chip devices with integrated sensors can provide high-resolution growth tracking of individual cells, either through mass, volume, or density monitoring.^4,7,8 Recently, a microfluidic mass sensor was used to track the buoyant mass of individual cells using a suspended microchannel resonator (SMR).^4,9 Monitoring growth can also be accomplished by tracking volume using microfluidic volume sensors⁷ operating on the Coulter principle.¹⁰ Trapping can be achieved by either (1) cycling the target back and forth through the sensor (pressure-driven⁴ and electrokinetic⁷) or (2) holding a cell in place (posts,¹¹ chevron structure,¹² and E-Field¹³). The former, dynamic approach, allows a single cell to be sampled periodically by reversing flow directions after a cell is detected. Simple in its implementation, this technique also has the ability to compensate for a drifting baseline current resulting from parasitic ionic changes within the sensing channel or other sources of noise. On the other hand, static traps allow cells to be held in place while the buffer is rapidly exchanged.¹² The ability to dynamically change cellular growth conditions during an experiment can lead to significant insight into the behaviour of cells in environments of varying salinity,¹⁴ oxidative,^15,16 or osmotic conditions,¹⁷ as well as the effect of nutrients¹⁸ and drugs.¹⁹In this work, we propose a device capable of tracking growth using high-resolution volume measurements, combining the best attributes of both types of measurement systems; continuous baseline correction and the ability to rapidly exchange cell media. This is accomplished by using a pressure-driven, feedback-based dynamic trap, whereby a cell is cycled back and forth through the sensor within a microfluidic channel. On-chip sieve valves positioned at both ends of the sensing channel are able to selectively capture a cell while the solution is being replaced. As proof of principle, the volume of several individual yeast cells was monitored over the course of their respective growth cycles, and the ability to quantify growth response to media exchange was demonstrated.Devices were fabricated using multilayered soft lithography with polydimethylsiloxane (PDMS) molding.²⁰ The completed device is pictured in Figure 1(a); full fabrication protocols are presented as supplementary material.²¹ To maximize measurement sensitivity, it is optimal to choose a channel width and height slightly larger than the dimensions of the target cell.²² However, yeast cells are asymmetrically shaped and tend to tumble as they traverse the sensor. Preliminary testing suggested this effect could be mitigated by having cells flow along trajectories far from the electrodes (through buoyancy), where electric field is more uniform. Thus, a channel height of 20 μm was chosen as a compromise. Channel height increases to 28 μm in the wider part of the central and bypass channels, a result of using a mold made out of reflowed photoresist.²³ Channel width was set at 25 μm through the sensor, and widens to 80 μm at the sieve valves to facilitate valve actuation, which requires a high width to height ratio.²⁰ The fluidic layer is integrated in a 35 μm thick PDMS spin-coated layer, above which sits a 50 μm tall valve channel in a 4 mm PDMS layer. Tubing connects I1 and I2 to a common inlet vial, V1 and V2 to vials filled with deionised water and O1 and O2 connect to empty vials (not pictured). Inlet pressures I1 and I2, and valve pressures V1 and V2 are controlled with manual regulators (SMC IR2000-N02-R and SMC IR2010-N02-R); outlet pressures are computer-controlled (SMC ITV-1011). This pressure scheme is detailed elsewhere.²⁴ Current pulses caused by transiting particles/cells (Figure 1(d)) were acquired by applying a 50 kHz, 220 mV AC voltage between a pair of electrodes and measuring the drawn current. This frequency is sufficiently elevated to avoid the electrical double layer capacitance at the electrode-electrolyte interface,²⁵ but low enough to avoid sensitivity to cell impedance or substrate.²⁶ The electrical setup used for these experiments has been described previously.^24,27 A temperature controller maintains the device at 30 °C.Open in a separate windowFIG. 1.(a) Micrograph of the microfluidic device. Two parallel bypass channels are connected by a sensing channel with sensing electrodes. Pressure is applied at inlets (I1, I2) and outlets (O1, O2) to control flow conditions. Valves (V1, V2) are positioned over each end of the sensing channel. Food coloring is used to highlight the valve (red) and fluidic layers (blue). (b) Flow mode: valves are unpressurized, and cells flow freely through the device. (c) Trapping mode: valves are pressurized to capture a cell within the central channel. Pressure-driven flow cycles the cell back and forth across the sensor. (d)Typical current pulses measured for a yeast cell.The cell capture, media exchange, and detection process occurs as follows. A cell suspension is loaded into the bypass channel and made to flow through the central sensing channel by imposing a pressure gradient (Figure 1(b)). Cells flowing through the sensor are observed optically; once a cell of interest is observed (a cell without a bud), valves are sealed (V1 = V2 = 35 psi). This stops all flow through the sensor, and enables bypass channels to be flushed and replaced with fresh media. After 2 min, valve channels are pressurized to 24 psi where they compress the channel to a sufficient height to physically restrict the passage of yeast cells, while allowing the media to flow through the central channel (Figure 1(c)). The pressure gradient between bypasses causes the media in the central channel to be flushed out, while the target cell is physically trapped. Replacing the media in the central channel takes 2 min. At this stage, a pressure-driven feedback-based dynamic trap can be initiated. In this dynamic trap mode, the pressure settings at O1 and O2 are adjusted to redirect the cell back and forth through the sensor, based on current pulses measured from cells transiting through the sensor. Through custom LabView® software, these outlet pressure settings are feedback-adjusted to maintain a speed of 250 μm/s in both directions at a detection frequency of 30 cells/min (Figure 1(d)). To minimize the effects of channel stretching/shrinking, the sum of pressures at O1 and O2 is held constant. This precaution was taken since the sensing channel structured within the flexible PDMS polymer will alter its geometry based on internal pressure.²⁸ The short central channel ensures steady nutrient replenishment from the bypasses. For example, a glucose molecule takes ∼4 min to diffuse from the bypass to the electrodes. In practice, Taylor-Aris dispersion will reduce this replenishment time considerably. Based on video analysis, 25% of the central channel''s media is replenished every pressure reversal (video presented as supplementary material²¹). Polystyrene microspheres of 3.9 ± 0.3 μm, 5.6 ± 0.2 μm, and 8.3 ± 0.7 μm (NIST size standards) were used to calibrate the sensor, and obtain the current pulse-to-volume calibration for every solution (supplementary material²¹). The validity of this calibration method is discussed elsewhere.²⁹ Care was taken to limit trajectory-based variations in signal: the device is positioned with electrodes at the top of the sensing channel, and with the negatively buoyant cells/particles flowing along the bottom. Based on previous experimental and theory work, we found that signal amplitude can vary as much as 3.5 fold for different heights.²⁷ The effect of trajectory on current pulse amplitude has also been reported elsewhere.^30,31 In this work, buoyancy is used to ensure that the cell flows along a trajectory at the same distance from the electrodes for every measurement.Saccharomyces cerevisiae (BY4743 Mat a/alpha, genotype: his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0 ade2::LEU2/ade2::URA3) was cultured to exponential phase at 30 °C in an incubator/shaker in yeast bacto-peptone (YPD) with 2% w/v glucose, supplemented with 0.2 M NaCl, 0.05% bovine serum albumin (BSA) and 42 mg/l adenine. Sodium chloride was added to enable the current pulse measurement, at a concentration where cells are viable;³² BSA was used to prevent cell agglomeration; adenine was supplemented since this particular yeast mutant does not produce its own supply. A cell suspension was introduced into the device, from which a cell at the early stages of its cell cycle was captured, and dynamically trapped for 100 min. Three typical cell growth results are shown in Figure 2(a). Since the culture was not synchronized, this leads to variability between “initial” cell volumes: there is a 27% difference in initial volume between the cells identified by red squares and green triangles. This is caused by (1) optical limits, whereby cells chosen for study are not all at the exact same cell cycle stage and (2) differences in the age of the mother cell: the more buds a mother cell has produced, the larger it becomes.³³ On average, captured yeast cell demonstrated a doubling time consistent with growth rates under ideal incubator/shaker conditions; nutrient depletion, electric field, and shear stresses are not affecting growth. Optical inspection of budding cells confirms that most growth is occurring at the daughter cell, as expected.³³ An elevated signal-to-noise ratio allows for high resolution volumetric measurements (4 μm³); cell asymmetry⁷ and trajectory variability^27,30,31 lead to a relative standard deviation of 6% for cells and 4% for microspheres of similar size. While mass or protein synthesis methods have indicated linear³⁴ or exponential^4,6,35,36 growth curves, volume-based methods have suggested sigmoidal patterns.^7,37 Prior to daughter cell emergence, and later in the cycle as the daughter cell emerges, volumetric growth rate declines.³⁸ In this work, it is difficult to ascertain with mathematical rigor the shape of the growth profile; however, for each cell, volume increases steadily throughout the growth cycle before declining near the end of the cycle.Open in a separate windowFIG. 2.(a) Growth curves for 3 cells trapped in succession. Simultaneous optical and electrical measurements allow cell cycle stage to be correlated with volume. Pictures of cell corresponding to the red squares are presented in 15 min increments. A cell is cycled through the sensor every 2 s. For clarity, each data point for yeast volume represents the average of data points over a period of 5 min, with standard deviation. (b) Demonstration of an interrupted growth cycle, where YPD + 0.2 M NaCl was replaced with 0.2 M NaCl at 40 min, and then again returned to YPD + 0.2 M NaCl at 80 min. The media exchange process takes 4 min.To demonstrate our ability to easily exchange media while maintaining a trap, the solution was exchanged 40 min into a yeast growth cycle; culture media was replaced with a pure saline solution 0.2 M NaCl + 0.05% BSA, and then replaced again with culture media at 80 min (Figure 2(b)). Cell growth is halted temporarily while in saline solution, before resuming normal growth thereafter. The cell cycle time is extended by this period. The cell volume drifts downward after the initial solution change at 40 min. Though this drift lies within our uncertainty bounds, cellular responses to osmotic shock on similar timescales have been documented elsewhere.³⁹ This result demonstrates an ability to quickly exchange cell media, and observe cellular response.In conclusion, we have demonstrated a microfluidic device capable of maintaining a dynamic, pressure-driven cell trap, which can monitor cellular volume over the cell cycle. Concurrent optical microscopy allows for real-time visual inspection of the cells. In addition, sieve valve integration provides for the exchange of media or the addition of drugs. Such a platform could also be key in cancer cell cytotoxicity assays,⁴⁰ where growth response to anticancer drugs could be monitored.  相似文献   

Book reviews     

Paul Smeyers  Gaston Mialaret  José A. Ibáñez-Martín  Glen A. Eyford  Akio Miyadera 《International Review of Education/Internationale Zeitschrift für Erziehungswissenschaft/Revue internationale l'éducation》1990,36(2):261-270

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How prevalent are plagiarized submissions? Global survey of editors     

Pippa Smart  Thomas Gaston 《Learned Publishing》2019,32(1):47-56

Most analyses of plagiarism focus on published content and do not report on the prevalence of plagiarism in submitted articles. Fears over large‐scale plagiarism, particularly in articles submitted by authors for whom English is a second language, have only been investigated in small publishing communities or using duplication‐checking analysis, which does not separate legitimate from unacceptable duplication. This research surveyed journal editors from around the world to ascertain recent (past year) experiences of plagiarized and/or duplicated submissions. We then compared their experiences to their assumptions about global levels of plagiarism. The survey received 372 responses, including 119 from Asian editors, 112 from European editors, and 57 from editors in North America. The respondents estimated that c.15% of all submissions contained plagiarized or duplicated content, although their own experiences were in the range of 2–5% of submissions. Of the respondents, 42% reported no incidence of plagiarized or duplicated submissions in the past year. Asian editors experienced the highest levels of plagiarized/duplicated content, although most of the problem articles were resolved, indicating that most of the identified duplication constituted relatively minor problems, rather than fraudulent plagiarism.  相似文献   

Engaging Undergraduate Students in Research Activities: Are Research Universities Doing a Better Job?   总被引：1，自引：0，他引：1  

Shouping Hu  George D. Kuh  Joy Gaston Gayles 《Innovative Higher Education》2007,32(3):167-177

Engaging undergraduate students in research activities has been advocated as an innovative strategy to improve American higher education (Boyer Commission, Reinventing undergraduate education: A blueprint for America’s research universities. The Carnegie Foundation for the Advancement of Teaching, Stony Brook, NY, 1998). This study compared the frequency of undergraduate student research experiences at different types of colleges and universities from the early 1990s through 2004. The results indicate that the frequency of student research experiences increased since 1998 at all types of institutions and that students at research universities were not more likely than their counterparts elsewhere to have such experiences. The findings were consistent across major fields. To live up to their claims, research universities must find additional ways to involve undergraduates in research with faculty members. Shouping Hu is Associate Professor of Higher Education at Florida State University. He received his M.S. degree in Economics and Ph.D. in Higher Education from Indiana University. His research and scholarship focuses on postsecondary access and persistence, college student experience, and higher education finance. George D. Kuh is Chancellor’s Professor of Higher Education and Director of the Center for Postsecondary Research at Indiana University Bloomington. He received his Ph.D. degree from the University of Iowa. His research focuses on the quality of undergraduate education. Joy Gaston Gayles is Associate Professor in Adult and Higher Education at North Carolina State University. She received her Bachelor’s degree from Shaw University, Master’s degree from Auburn University, and Ph.D. in Higher Education from The Ohio State University. Her research focuses on college student learning and development.  相似文献   

Do more distant collaborations have more citation impact?   总被引：1，自引：0，他引：1  

Önder Nomaler  Koen Frenken  Gaston Heimeriks 《Journal of Informetrics》2013,7(4):966-971

Internationally co-authored papers are known to have more citation impact than nationally co-authored paper, on average. However, the question of whether there are systematic differences between pairs of collaborating countries in terms of the citation impact of their joint output, has remained unanswered. On the basis of all scientific papers published in 2000 and co-authored by two or more European countries, we show that citation impact increases with the geographical distance between the collaborating counties.  相似文献   

Short-term tracking of performance and health-related physical fitness in girls: The Healthy Growth in Cariri Study     

Simonete Pereira Da Silva  Gaston Beunen  António Prista  José Maia 《Journal of sports sciences》2013,31(1):104-113

Abstract

The main purpose of this study was to track the performance and health-related physical fitness of girls from Brazil's Cariri region. In the “Healthy Growth in Cariri Study”, 294 girls from public and private schools were divided into four age cohorts – 8, 10, 12, and 14 years – and followed for three consecutive years, with an assessment every 6 months. Shuttle run, hand grip, standing long jump, trunk lift, curl-up, 12-min run, and fatness were used to rate physical fitness performance and health-related components on each of six occasions. Tracking was done in a stepwise manner, using auto-correlation, by modelling the individual history of change in performance of each girl, and using Foulkes and Davies’ γ-coefficient. SPSS 18.0 and TIMEPATH were used for data analysis. Auto-correlations evidenced low-to-moderate values in almost all components of performance and health-related physical fitness. Intra-individual tracking analysis showed large variation in all fitness components as a result of a wide spread in individual history of change in fitness performance. Population estimates of γ were low in all tests. Our results show low-to-moderate tracking of physical fitness components of girls. A wide range of intra-individual and inter-variability in fitness development was observed.  相似文献   

Methodological issues associated with longitudinal research: Findings from the Leuven Longitudinal Study on Lifestyle,Fitness and Health (1969 – 2004)     

Lynn Matton  Gaston Beunen  Nathalie Duvigneaud  Katrien Wijndaele  Renaat Philippaerts  Albrecht Claessens 《Journal of sports sciences》2013,31(9):1011-1024

Abstract

Longitudinal studies provide unique opportunities but are also faced with several limitations. The purpose of this study was to document three of these issues (“imperfect” design, evolution of data collection methods, representativeness) by means of the Leuven Longitudinal Study on Lifestyle, Fitness and Health (LLSLFH). The LLSLFH (1969 – 2004) comprises observations on males between 12 and 18 years and at 30, 35, 40, and 47 years, and on females at 16 and 40 years. In the most recent phase of the study, spouses and offspring were also included. The different phases and evolving research questions throughout the LLSLFH required an appropriate adaptation of the research design. The associated evolution of data collection methods largely reflects the changing ideas about physical fitness, body composition, and physical activity, the continuing search for new and better measurement techniques, and the need for adaptations with age. Ongoing study participants are representative in terms of body composition and, except for adolescence in males, also physical activity. No straightforward answer can be given concerning physical fitness. In both sexes, socio-economic status is above average. When informed about the possible “pitfalls” of longitudinal research in advance, several measures could be taken to prevent or limit them as much as possible.  相似文献   

Review Article: Recent advancements in optofluidic flow cytometer     

Cho SH  Godin JM  Chen CH  Qiao W  Lee H  Lo YH 《Biomicrofluidics》2010,4(4):43001

There is an increasing need to develop optofluidic flow cytometers. Optofluidics, where optics and microfluidics work together to create novel functionalities on a small chip, holds great promise for lab-on-a-chip flow cytometry. The development of a low-cost, compact, handheld flow cytometer and microfluorescence-activated cell sorter system could have a significant impact on the field of point-of-care diagnostics, improving health care in, for example, underserved areas of Africa and Asia, that struggle with epidemics such as HIV∕AIDS. In this paper, we review recent advancements in microfluidics, on-chip optics, novel detection architectures, and integrated sorting mechanisms.  相似文献   

Assessment of conceptual complexity     

Mary H. Beaven  Gaston A. Mendoza 《Assessment Update》1991,3(6):12-13

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