Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask)

Single Cell Deposition and Patterning with a Robotic System

Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques

Negotiating the modern cross-class ‘model home’:domestic experiences in Basil Spence’s Claremont Court

This article investigates the spatial articulation of architecture and home through the exploration of current domestic experiences in Basil Spence’s Claremont Court housing scheme (1959-1962), Edinburgh. How architecture and home are both idealized and lived is the backdrop for a discussion that draws on the concept of “model home,” or physical representation of a domestic ideal. The article reads Claremont Court as an architectural prototype of the modern domestic ideal, before exploring its reception by five of its households through the use of visual methods and semistructured interviews. Receiving the model home involves negotiating between ideal and lived homes. Building on this idea, the article contributes with a focus on the spatiality of such reception, showing how it is modulated according to the architectural affordances that the “model home” represents. The article expands on scholarship on architecture and home with empirical evidence that argues the reciprocal spatiality of home

Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Enhancing apoptosis in TRAIL-resistant cancer cells using fundamental response rules

The tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in malignant cells, while leaving other cells mostly unharmed. However, several carcinomas remain resistant to TRAIL. To investigate the resistance mechanisms in TRAIL-stimulated human fibrosarcoma (HT1080) cells, we developed a computational model to analyze the temporal activation profiles of cell survival (IκB, JNK, p38) and apoptotic (caspase-8 and -3) molecules in wildtype and several (FADD, RIP1, TRAF2 and caspase-8) knock-down conditions. Based on perturbation-response approach utilizing the law of information (signaling flux) conservation, we derived response rules for population-level average cell response. From this approach, i) a FADD-independent pathway to activate p38 and JNK, ii) a crosstalk between RIP1 and p38, and iii) a crosstalk between p62 and JNK are predicted. Notably, subsequent simulations suggest that targeting a novel molecule at p62/sequestosome-1 junction will optimize apoptosis through signaling flux redistribution. This study offers a valuable prospective to sensitive TRAIL-based therapy

The impact of sexual harassment on job satisfaction, turnover intentions, and absenteeism: findings from Pakistan compared to the United States

The purpose of this study was to compare and contrast how differences in perceptions of sexual harassment impact productive work environments for employees in Pakistan as compared to the US; in particular, how it affects job satisfaction, turnover, and/or absenteeism. This study analyzed employee responses in Pakistan (n = 146) and the United States (n = 102, 76) using questionnaire data. Significant results indicated that employees who were sexually harassed reported (a) a decrease in job satisfaction (b) greater turnover intentions and (c) a higher rate of absenteeism. Cross-cultural comparisons indicated that (a) Pakistani employees who were sexually harassed had greater job dissatisfaction and higher overall absenteeism than did their US counterparts and (b) Pakistani women were more likely to use indirect strategies to manage sexual harassment than were US targets