Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Resolving how the early signaling events initiated by cell–cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell–cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting single-cell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell.Singapore-MIT AllianceAmerican Association for Cancer Research. Pancreatic Cancer Action NetworkMassachusetts Institute of Technology. Department of Electrical Engineering and Computer Science (Frank Quick Faculty Research Innovation Fellowship

Monoclonal Invariant NKT (iNKT) Cell Mice Reveal a Role for Both Tissue of Origin and the TCR in Development of iNKT Functional Subsets

Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-γ, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα paired with unique Vβ7 or Vβ8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity

Multiple Stressors in the Environment: The Effects of Exposure to an Antidepressant (Venlafaxine) and Increased Temperature on Zebrafish Metabolism

Aquatic organisms are continuously exposed to multiple environmental stressors working cumulatively to alter ecosystems. Wastewater-dominated environments are often riddled by a myriad of stressors, such as chemical and thermal stressors. The objective of this study was to examine the effects of an environmentally relevant concentration of a commonly prescribed antidepressant, venlafaxine (VFX) [1.0 μg/L], in addition to a 5°C increase in water temperature on zebrafish metabolism. Fish were chronically exposed (21 days) to one of four conditions: (i) 0 μg/L VFX at 27°C; (ii) 1.0 μg/L VFX at 27°C; (iii) 0 μg/L VFX at 32°C; (iv) 1.0 μg/L VFX at 32°C. Following exposure, whole-body metabolism was assessed by routine metabolic rate (RMR) measurements, whereas tissue-specific metabolism was assessed by measuring the activities of major metabolic enzymes in addition to glucose levels in muscle. RMR was significantly higher in the multi-stressed group relative to Control. The combination of both stressors resulted in elevated pyruvate kinase activity and glucose levels, while lipid metabolism was depressed, as measured by 3-hydroxyacyl CoA dehydrogenase activity. Citrate synthase activity increased with the onset of temperature, but only in the group treatment without VFX. Catalase activity was also elevated with the onset of the temperature stressor, however, that was not the case for the multi-stressed group, potentially indicating a deleterious effect of VFX on the anti-oxidant defense mechanism. The results of this study highlight the importance of multiple-stressor research, as it able to further bridge the gap between field and laboratory studies, as well as have the potential of yielding surprising results that may have not been predicted using a conventional single-stressor approach.This study was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC: RGPIN-2015-05643) as well as the Canada Foundation for Innovation (CFI 34317)

Automatic correction of hand pointing in stereoscopic depth

In order to examine whether stereoscopic depth information could drive fast automatic correction of hand pointing, an experiment was designed in a 3D visual environment in which participants were asked to point to a target at different stereoscopic depths as quickly and accurately as possible within a limited time window (≤300 ms). The experiment consisted of two tasks: "depthGO" in which participants were asked to point to the new target position if the target jumped, and "depthSTOP" in which participants were instructed to abort their ongoing movements after the target jumped. The depth jump was designed to occur in 20% of the trials in both tasks. Results showed that fast automatic correction of hand movements could be driven by stereoscopic depth to occur in as early as 190 ms.This work was supported by the Grants from the National Natural Science Foundation of China (60970062 and 61173116) and the Doctoral Fund of Ministry of Education of China (20110072110014)

Concurrent adaptation to opposing visual displacements during an alternating movement.

It has been suggested that, during tasks in which subjects are exposed to a visual rotation of cursor feedback, alternating bimanual adaptation to opposing rotations is as rapid as unimanual adaptation to a single rotation (Bock et al. in Exp Brain Res 162:513–519, 2005). However, that experiment did not test strict alternation of the limbs but short alternate blocks of trials. We have therefore tested adaptation under alternate left/right hand movement with opposing rotations. It was clear that the left and right hand, within the alternating conditions, learnt to adapt to the opposing displacements at a similar rate suggesting that two adaptive states were formed concurrently. We suggest that the separate limbs are used as contextual cues to switch between the relevant adaptive states. However, we found that during online correction the alternating conditions had a significantly slower rate of adaptation in comparison to the unimanual conditions. Control conditions indicate that the results are not directly due the alternation between limbs or to the constant switching of vision between the two eyes. The negative interference may originate from the requirement to dissociate the visual information of these two alternating displacements to allow online control of the two arms

Impacts of wastewater treatment plant effluent on energetics and stress response of rainbow darter (Etheostoma caeruleum) in the Grand River watershed

The final publication is available at Elsevier via https://doi.org/10.1016/j.cbpb.2017.11.011. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/The objective of this study was to assess the effects of municipal wastewater treatment plant effluent on the energetics and stress response of rainbow darter (Etheostoma caeruleum). Male and female rainbow darter were collected upstream and downstream of the Waterloo WWTP in the Grand River watershed, ON, Canada. To assess the effects of wastewater treatment plant effluent on whole-body and tissue specific metabolic capacity, closed-chamber respirometry and muscle-enzyme activity analyses were performed. Plasma cortisol was also collected from fish before and after an acute air-exposure stressor to evaluate the cortisol stress response in fish exposed to additional stressors. Male and female rainbow darter collected downstream of the effluent had higher oxygen consumption rates, while differences in enzyme activities were primarily associated with sex rather than collection site. No impairment in the cortisol stress response between downstream and upstream fish was observed, however baseline cortisol levels in female fish from the downstream site were significantly higher compared to other baseline groups. Stress-induced cortisol levels were also higher in female fish from both sites when compared to their male counterparts. Overall, this study demonstrates that chronic exposure to WWTP effluent impacts whole-body metabolic performance. This study was also able to demonstrate that sex-differences are a key determinant of various metabolic changes in response to physiological stress, thereby, providing a novel avenue to be considered and further explored.Natural Sciences and Engineering Research Council (Grant RGPIN-2015-05643)Canada Foundation for Innovation (Grant CFI 34317

Impacts on Metabolism and Gill Physiology of Darter Species (Etheostoma spp.) That Are Attributed to Wastewater Effluent in the Grand River

The effluent from municipal wastewater treatment plants is a major point source of contamination in Canadian waterways. The improvement of effluent quality to reduce contaminants, such as pharmaceuticals and personal care products, before being released into the environment is necessary to reduce the impacts on organisms that live in the river downstream. Here, we aimed to characterize the metabolic and gill physiological responses of rainbow (Etheostoma caeruleum), fantail (Etheostoma flabellare), and greenside (Etheostoma blennioides) darters to the effluent in the Grand River from the recently upgraded Waterloo municipal wastewater treatment plant. The routine metabolism of darters was not affected by effluent exposure, but some species had increased maximum metabolic rates, leading to an increased aerobic scope. The rainbow darter aerobic scope increased by 2.2 times and the fantail darter aerobic scope increased by 2.7 times compared to the reference site. Gill samples from effluent-exposed rainbow darters and greenside darters showed evidence of more pathologies and variations in morphology. These results suggest that darters can metabolically adjust to effluent-contaminated water and may also be adapting to the urban and agricultural inputs. The modification and damage to the gills provide a useful water quality indicator but does not necessarily reflect how well acclimated the species is to the environment due to a lack of evidence of poor fish health.This research was supported through the Natural Science and Engineering Research Council (#RGPIN-2015-05643) and the Canadian First Research Excellence Fund Global Water Futures initiative (PI: Mark Servos). R.H. was supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology

Tissue storage affects lipidome profiling in comparison to in vivo microsampling approach

Low-invasive in vivo solid-phase microextraction (SPME) was used to investigate the lipid profiles of muscle tissue of living fish. Briefly, mixed mode SPME fibers were inserted into the muscle for 20 min extraction, and then the fibers were desorbed in an optimal mixture of solvents. The obtained lipid profile was then compared and contrasted to that obtained with employment of ex vivo SPME and solid-liquid extraction (SLE) from fish muscle tissue belonging to the same group of fish, following a one-year storage period. Ex vivo SPME analysis of stored muscle samples revealed 10-fold decrease in the number of detected molecular features in comparison to in vivo study. Moreover, in vivo microsampling enabled the identification of different classes of bioactive lipids, including fatty acyls, not present in the lipid profile obtained through ex vivo SPME and SLE, suggesting the alterations occurring in the unbound lipid fraction of the system under study during the storage and also indicating the advantage of the in vivo extraction approach.This work was supported by Environment Canada through the Environmental Damages Fund (Grant EC-129114)

Influence of operating parameters on the biodegradation of steroid estrogens and nonylphenolic compounds during biological wastewater treatment processes

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Environmental Science & Technology, copyright © American Chemical Society after peer review. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/es901612v.This study investigated operational factors influencing the removal of steroid estrogens and nonylphenolic compounds in two sewage treatment works, one a nitrifying/denitrifying activated sludge plant and the other a nitrifying/denitrifying activated sludge plant with phosphorus removal. Removal efficiencies of >90% for steroid estrogens and for longer chain nonylphenol ethoxylates (NP4−12EO) were observed at both works, which had equal sludge ages of 13 days. However, the biological activity in terms of milligrams of estrogen removed per day per tonne of biomass was found to be 50−60% more efficient in the nitrifying/denitrifying activated sludge works compared to the works which additionally incorporated phosphorus removal. A temperature reduction of 6 °C had no impact on the removal of free estrogens, but removal of the conjugated estrone-3-sulfate was reduced by 20%. The apparent biomass sorption (LogKp) values were greater in the nitrifying/denitrifying works than those in the nitrifying/denitrifying works with phosphorus removal for both steroid estrogens and nonylphenolic compounds possibly indicating a different cell surface structure and therefore microbial population. The difference in biological activity (mg tonne−1 d−1) identified in this study, of up to seven times, suggests that there is the potential for enhancing the removal of estrogens and nonylphenols if more detailed knowledge of the factors responsible for these differences can be identified and maximized, thus potentially improving the quality of receiving waters.Public Utilities Board (Singapore), Anglian Water Ltd, Severn Trent Water Ltd, Thames Water Utilities Ltd, United Utilities 393 Plc and Yorkshire Water Services

Symmetric Sensorimotor Somatotopy

BACKGROUND: Functional imaging has recently been used to investigate detailed somatosensory organization in human cortex. Such studies frequently assume that human cortical areas are only identifiable insofar as they resemble those measured invasively in monkeys. This is true despite the electrophysiological basis of the latter recordings, which are typically extracellular recordings of action potentials from a restricted sample of cells. METHODOLOGY/PRINCIPAL FINDINGS: Using high-resolution functional magnetic resonance imaging in human subjects, we found a widely distributed cortical response in both primary somatosensory and motor cortex upon pneumatic stimulation of the hairless surface of the thumb, index and ring fingers. Though not organized in a discrete somatotopic fashion, the population activity in response to thumb and index finger stimulation indicated a disproportionate response to fingertip stimulation, and one that was modulated by stimulation direction. Furthermore, the activation was structured with a line of symmetry through the central sulcus reflecting inputs both to primary somatosensory cortex and, precentrally, to primary motor cortex. CONCLUSIONS/SIGNIFICANCE: In considering functional activation that is not somatotopically or anatomically restricted as in monkey electrophysiology studies, our methodology reveals finger-related activation that is not organized in a simple somatotopic manner but is nevertheless as structured as it is widespread. Our findings suggest a striking functional mirroring in cortical areas conventionally ascribed either an input or an output somatotopic function