Microfabrication Process Development for a Polymer-Based Lab-on-Chip Concept Applied in Attenuated Total Reflection Fourier Transform Infrared Spectroelectrochemistry.

Micro electro-mechanical systems (MEMS) combining sensing and microfluidics functionalities, as are common in Lab-on-Chip (LoC) devices, are increasingly based on polymers. Benefits of polymers include tunable material properties, the possibility of surface functionalization, compatibility with many micro and nano patterning techniques, and optical transparency. Often, additional materials, such as metals, ceramics, or silicon, are needed for functional or auxiliary purposes, e.g., as electrodes. Hybrid patterning and integration of material composites require an increasing range of fabrication approaches, which must often be newly developed or at least adapted and optimized. Here, a microfabrication process concept is developed that allows one to implement attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and electrochemistry on an LoC device. It is designed to spatially resolve chemical sensitivity and selectivity, which are instrumental for the detection of chemical distributions, e.g., during on-flow chemical and biological reaction chemistry. The processing sequence involves (i) direct-write and soft-contact UV lithography in SUEX dry resist and replication in polydimethylsiloxane (PDMS) elastomers as the fluidic structure; (ii) surface functionalization of PDMS with oxygen plasma, 3-aminopropyl-triethoxysilane (APTES), and a UV-curable glue (NOA 73) for bonding the fluidic structure to the substrate; (iii) double-sided patterning of silicon nitride-coated silicon wafers serving as the ATR-FTIR-active internal reflection element (IRE) on one side and the electrode-covered substrate for microfluidics on the back side with lift-off and sputter-based patterning of gold electrodes; and (iv) a custom-designed active vacuum positioning and alignment setup. Fluidic channels of 100 µm height and 600 µm width in 5 mm thick PDMS were fabricated on 2" and 4" demonstrators. Electrochemistry on-chip functionality was demonstrated by cyclic voltammetry (CV) of redox reactions involving iron cyanides in different oxidation states. Further, ATR-FTIR measurements of laminar co-flows of H2O and D2O demonstrated the chemical mapping capabilities of the modular fabrication concept of the LoC devices.

Toxicity and mechanism of nanoplastics to phytoplankton in high-latitude aquatic ecosystems of Canadian prairie: Effects of multiple environmental factors.

The potential ecological risks of nanoplastics (NPs) may be inaccurately assessed in some studies as they fail to consider the impact of environmental factors and their interactive effects. Here, the effects of six representative environmental factors (N, P, salinity, DOM (dissolved organic matter), pH and hardness) on NPs' toxicity and mechanism to microalgae are investigated based on the surface water quality data in Saskatchewan watershed, Canada. Our 10 sets of 26-1 factorial analysis reveal the significant factors and their interactive complexity towards 10 toxic endpoints from cellular and molecular levels. This is the first time to study the toxicity of NPs to microalgae under interacting environmental factors in high-latitude aquatic ecosystems of Canadian prairie. We find that microalgae become more resistant to NPs in N-rich or higher pH environments. Surprisingly, with the increase of N concentration or pH, the inhibitory effect of NPs on microalgae growth even became a promotion effect with the decreased inhibition rate from 10.5 % to -7.1 % or from 4.3 % to -0.9 %, respectively. Synchrotron-based Fourier transform infrared spectromicroscopy analysis reveals that NPs can induce alterations in the content and structure of lipids and proteins. DOM, N*P, pH, N*pH and pH*hardness have a statistically significant effect on NPs' toxicity to biomolecular. The toxicity levels of NPs across Saskatchewan watersheds are evaluated and we find that NPs could have the greatest inhibition on microalgae growth in Souris River. Our findings indicate that multiple environmental factors should be considered during the ecological risk assessment of emerging pollutants.

Rethinking the effects of micro/nanoplastics from the global environmental change and systematic perspective: An aquatic environmental system-based comprehensive assessment approach of micro/nanoplastic impacts.

The study on micro/nanoplastic pollution should embrace complexity. Here, we aim to develop an aquatic environmental system-based comprehensive assessment approach of micro/nanoplastic impacts (ACAM) to evaluate the effects of micro/nanoplastics on aquatic ecosystems from the global environmental change (GEC) and systematic perspective. A case study for freshwater systems in Saskatchewan, Canada was conducted to evaluate the comprehensive effects of multiple GEC factors (polystyrene-nanoplastics (PS-NPs), N, P, salinity, dissolved organic matter (DOM), pH, hardness) on Asterococcus superbus based on ten ecologically relevant endpoints. It is found that at the cellular level, PS-NPs and N had an antagonistic interaction on microalgal growth in the Saskatchewan freshwater ecosystem; at the molecular level, the PS-NP-induced changes in lipid composition in microalgae were regulated by P, DOM, and pH. The significance ranking of factor effects suggested that instead of PS-NPs pollution, the fluctuations in pH level, DOM and N concentrations should be paid attention to first in Saskatchewan. Under the combined impact of PS-NPs and other GEC factors, microalgae at station 14 (Qu'Appelle River near highway 56) might have the minimum growth rate with [-0.048, 0.094] d-1 in Saskatchewan. These findings demonstrate the efficacy of the developed ACAM in a more comprehensive and context-specific assessment of MNP risks, providing new insight for the management of MNP pollution.

Chemical Imaging of Mass Transport Near the No-Slip Interface of a Microfluidic Device using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy.

Mass transport in geometrically confined environments is fundamental to microfluidic applications. Measuring the distribution of chemical species on flow requires the use of spatially resolved analytical tools compatible with microfluidic materials and designs. Here, the implementation of an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) imaging (macro-ATR) approach for chemical mapping of species in microfluidic devices is described. The imaging method is configurable between a large field of view, single-frame imaging, and the use of image stitching to build composite chemical maps. Macro-ATR is used to quantify transverse diffusion in the laminar streams of coflowing fluids in dedicated microfluidic test devices. It is demonstrated that the ATR evanescent wave, which primarily probes the fluid within ∼500 nm of the channel surface, provides accurate quantification of the spatial distribution of species in the entire microfluidic device cross section. This is the case when flow and channel conditions promote vertical concentration contours in the channel as verified by three-dimensional numeric simulations of mass transport. Furthermore, the validity of treating the mass transport problem in a simplified and faster approach using reduced dimensionality numeric simulations is described. Simplified one-dimensional simulations, for the specific parameters used herein, overestimate diffusion coefficients by a factor of approximately 2, whereas full three-dimensional simulations accurately agree with experimental results.

Biochemical Alterations in White Matter Tracts of the Aging Mouse Brain Revealed by FTIR Spectroscopy Imaging.

White matter degeneration in the central nervous system (CNS) has been correlated with a decline in cognitive function during aging. Ultrastructural examination of the aging human brain shows a loss of myelin, yet little is known about molecular and biochemical changes that lead to myelin degeneration. In this study, we investigate myelination across the lifespan in C57BL/6 mice using electron microscopy and Fourier transform infrared (FTIR) spectroscopic imaging to better understand the relationship between structural and biochemical changes in CNS white matter tracts. A decrease in the number of myelinated axons was associated with altered lipid profiles in the corpus callosum of aged mice. FTIR spectroscopic imaging revealed alterations in functional groups associated with phospholipids, including the lipid acyl, lipid ester and phosphate vibrations. Biochemical changes in white matter were observed prior to structural changes and most predominant in the anterior regions of the corpus callosum. This was supported by biochemical analysis of fatty acid composition that demonstrated an overall trend towards increased monounsaturated fatty acids and decreased polyunsaturated fatty acids with age. To further explore the molecular mechanisms underlying these biochemical alterations, gene expression profiles of lipid metabolism and oxidative stress pathways were investigated. A decrease in the expression of several genes involved in glutathione metabolism suggests that oxidative damage to lipids may contribute to age-related white matter degeneration.

Quasar: Easy Machine Learning for Biospectroscopy.

Data volumes collected in many scientific fields have long exceeded the capacity of human comprehension. This is especially true in biomedical research where multiple replicates and techniques are required to conduct reliable studies. Ever-increasing data rates from new instruments compound our dependence on statistics to make sense of the numbers. The currently available data analysis tools lack user-friendliness, various capabilities or ease of access. Problem-specific software or scripts freely available in supplementary materials or research lab websites are often highly specialized, no longer functional, or simply too hard to use. Commercial software limits access and reproducibility, and is often unable to follow quickly changing, cutting-edge research demands. Finally, as machine learning techniques penetrate data analysis pipelines of the natural sciences, we see the growing demand for user-friendly and flexible tools to fuse machine learning with spectroscopy datasets. In our opinion, open-source software with strong community engagement is the way forward. To counter these problems, we develop Quasar, an open-source and user-friendly software, as a solution to these challenges. Here, we present case studies to highlight some Quasar features analyzing infrared spectroscopy data using various machine learning techniques.

Probing Heterogeneity in Attenuated Total Reflection Surface-Enhanced Infrared Absorption Spectroscopy (ATR-SEIRAS) Response with Synchrotron Infrared Microspectroscopy.

The heterogeneity of metal island films electrodeposited on conductive metal oxide modified internal reflection elements is shown to provide a variable attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) response. A self-assembled monolayer of a ferrocene-terminated thiol monolayer (FcC11SH) was formed on the gold islands covering a single substrate, which was measured using both a conventional spectrometer and a custom-built horizontal microscope. Cyclic voltammetry and ATR-SEIRAS results reveal that the FcC11SH-modified substrate undergoes a reversible electron transfer and an associated re-orientation of both the ferrocene/ferrocenium headgroup and the hydrocarbon backbone. The magnitude of the absorption signal arising from the redox changes in the monolayer, as well as the IR signature arising from the ingress/egress of the perchlorate counterions, is shown to depend significantly on the size of the infrared beam spot when using a conventional Fourier transform infrared spectrometer. By performing equivalent measurements on a horizontal microscope, the primary cause of the differences in the signal level is found to be the heterogeneity in the density of gold islands on the conductive metal oxide.

Microsecond Resolved Infrared Spectroelectrochemistry Using Dual Frequency Comb IR Lasers.

A dual infrared frequency comb spectrometer with heterodyne detection has been used to perform time-resolved electrochemical attenuated total reflectance surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS). The measurement of the potential dependent desorption of a monolayer of a pyridine derivative (4-dimethylaminopyridine, DMAP) with time resolution as high as 4 µs was achieved without the use of step-scan interferometry. An analysis of the detection limit of the method as a function of both time resolution and measurement coadditions is provided and compared to step-scan experiments of an equivalent system. Dual frequency comb spectroscopy is shown to be highly amenable to time-resolved ATR-SEIRAS. Microsecond resolved spectra can be obtained with high spectral resolution and fractional monolayer detection limits in a total experimental duration that is 2 orders of magnitude less than the equivalent step-scan experiment.

Implementing reasonable adjustments for -disabled people in healthcare services.

Disabled people are one of the groups in society with the greatest health needs, yet they experience some of the most significant barriers to accessing healthcare services. This article describes examples of how three healthcare services have met the Equality Act 2010 duty to make reasonable adjustments for disabled people, so that they are not disadvantaged in accessing these services. Each of these services identified disabled patients, and considered and recorded the specific reasonable adjustments that were required. In doing so, they took time to fully understand the needs of the individual from their perspective. The services collaborated and coordinated the provision of reasonably adjusted care by communicating effectively with other health and social care providers, working together as a team, and treating disabled people as individuals.

Disabled people's experiences of accessing reasonable adjustments in hospitals: a qualitative study.

BACKGROUND: The UK Equality Act 2010 requires providers of health services to make changes or 'reasonable adjustments' to their practices in order to protect disabled people from discrimination or disadvantage when accessing care. Existing evidence suggests that despite this legislation, health services are not always providing reasonably adjusted care for disabled people. This paper presents the perspectives of disabled people themselves in relation to their experiences of accessing reasonable adjustments in hospitals in England. METHODS: Twenty-one semi-structured interviews were held with disabled people who had a recent experience of hospital care in England. Participants were asked about the extent to which the hospital provided reasonably adjusted care, and if necessary, how they thought the provision of reasonable adjustments could be improved. Each interview was anonymised and transcribed, and the data analysed using thematic analysis. RESULTS: Participants reported mixed experiences about whether and how reasonable adjustments were provided: some shared positive examples of good practice; others spoke about difficult encounters and limited provision. Recommendations made include a need for culture change in how reasonable adjustments are perceived and enacted; improvements in identifying the needs of disabled people; improvements to the hospital environment and the provision of information; and the need to involve disabled people themselves in the process of change. CONCLUSIONS: Gaps remain in how reasonable adjustments are provided for disabled people accessing hospital care. It is important for hospital staff to listen to the perspectives of disabled people about the provision of reasonable adjustments, and make improvements as necessary. Hospital staff could also do more to share good practice in relation to the provision of reasonable adjustments to effectively inspire and embed positive change.

Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectromicroscopy Using Synchrotron Radiation and Micromachined Silicon Wafers for Microfluidic Applications.

A custom-designed optical configuration compatible with the use of micromachined multigroove internal reflection elements (µ-groove IREs) for attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and imaging applications in microfluidic devices is described. The µ-groove IREs consist of several face-angled grooves etched into a single, monolithic silicon chip. The optical configuration permits individual grooves to be addressed by focusing synchrotron sourced IR light through a 150 µm pinhole aperture, restricting the beam spot size to a dimension smaller than that of the groove walls. The effective beam spot diameter at the ATR sampling plane is determined through deconvolution of the measured detector response and found to be 70 µm. The µ-groove IREs are highly compatible with standard photolithographic techniques as demonstrated by printing a 400 µm wide channel in an SU-8 film spin-coated on the IRE surface. Attenuated total reflection FT-IR mapping as a function of sample position across the channel illustrates the potential application of this approach for rapid prototyping of microfluidic devices.

Negotiating identity: a qualitative analysis of stigma and support seeking for individuals with cerebral palsy.

PURPOSE: The current research investigates how adults with cerebral palsy construct their personal and social identities in the face of stigma when support seeking, and considers the dilemmas they might face when doing so. METHOD: Participants were 28 adults with cerebral palsy who completed an online survey reporting on their identity as a person with cerebral palsy and their experiences of stigma when seeking and accessing support. RESULTS: Qualitative analyses indicated that the majority of participants sought support to help manage their cerebral palsy. Of these, half reported experiencing stigma in these environments, although they largely continued seeking support despite this. The majority viewed both their personal identity (i.e. as a unique individual) and their social identity (i.e. as a person with cerebral palsy) as important to their sense of self. However, how participants constructed their identity also appeared to vary according to context. While they appeared to value being seen as an individual to receive support that was unique to their needs (their personal identity), they also reported valuing the group to facilitate coping with stigma (their social identity). Yet, despite their utilities, enacting their identity in each of these ways was associated with costs. In order to access desired support, they had to incorporate their social identity as similar to other disabled people, which led to stigmatization through feelings of difference to the non-disabled. Conversely emphasizing individuality and difference from the disabled stereotype was associated with concerns about the degree to which their suitability for support might be questioned by their care provider. CONCLUSIONS: As has been observed in many fields, stigma can complicate identity. In this domain, people with cerebral palsy face a number of threats in how they construe their identity, both in navigating stigma and maintaining access to needed support. Implications for Rehabilitation Stigma in help and support settings remains a significant problem for adults with cerebral palsy (CP). This creates dilemmas regarding how they view and construct their identity in the contexts of stigma and support-seeking. It is important for specialists to recognize that the needs of adults with CP are unique, and so provided services should be tailored to the individual. Healthcare providers should also encourage their patients to actively engage with online disability support groups in order to build a meaningful social identity with other people with CP.

Centralized mouse repositories.

Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.

Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die.

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3-dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells.

Role of prodomain in importin-mediated nuclear localization and activation of caspase-2.

Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.

A novel Apaf-1-independent putative caspase-2 activation complex.

Caspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome.

The role of cytochrome c in caspase activation in Drosophila melanogaster cells.

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.