However, the procedures required to establish and maintain these systems can be challenging to implement for end users, particularly those without significant in-house engineering expertise. Here we present a module-based approach that provides an easy-to-use workflow to establish, maintain, and analyze microscale tissue constructs. Our approach begins with a removable culture insert that is magnetically coupled, decoupled, and transferred between standalone, prefabricated microfluidic modules for simplified cell seeding, culture, and downstream analysis. The modular approach allows several options for perfusion including standard syringe pumps or integration with a self-contained gravity-fed module for simple cell maintenance. As proof of concept, we establish a culture of primary human microvascular endothelial cells (HMVEC) and report combined surface protein imaging and gene expression after controlled apical stimulation with the bacterial endotoxin lipopolysaccharide (LPS). We also demonstrate the feasibility of incorporating hydrated biomaterial interfaces into the microfluidic architecture by integrating an ultra-thin (< 1 μm), self-assembled hyaluronic acid/peptide amphiphile culture membrane with brain-specific Young’s modulus (~ 1kPa). To highlight the importance of including biomimetic interfaces into microscale models we report multi-tiered readouts from primary rat cortical cells cultured on the self-assembled membrane and compare a panel of mRNA targets with primary brain tissue signatures. We anticipate that the modular approach and simplified operational workflows presented here will enable a wide range of research groups to incorporate microfluidic barrier tissue models into their work.Microfluidic barrier tissue models have emerged as advanced  H. H17]. H2 laser, Universal Laser Systems) with embedded rare earth magnets (K&J Magnetics), ii) molded PDMS microchannels, and iii) a removable cell culture insert.To achieve a “user friendly” approach to establish and analyze tissue interfaces, we developed the SEAM architecture composed of specialized modules using a laboratory prototyping method combining soft-lithography, laser machining, and lamination. As shown in Rigid plastic housing layers were constructed by laminating two laser cut pieces of PMMA  using pressure sensitive adhesive sheets . Each housing layer contained rare earth magnets (2.54 mm diameter) oriented such that the top and bottom housings had opposite magnetic poles facing one another. The cavities were designed to accept the PDMS containing channels as described below.Polydimethysiloxane  microchannels  were fabricated using standard soft-lithography replica molding techniques [μm pore diameter, Sterlitech) was laser cut, sandwiched, and laminated between two laser cut polyethylene (PE) sheets containing an open central region and pressure sensitive silicone adhesive on one face . The non-porous PE isolated the fluidic compartments and ensured that communication between the channels only occurred through the nanoporous culture membrane. The PE sheets also allowed the culture insert to be easily transferred between specialized modules.As shown in μm, Steriletch) were laser cut to create open central regions. A nanoporous track etched membrane was arranged so that a small edge extended into the open region. We adapted a liquid-liquid supramolecular self-assembly chemistry pioneered by Stupp [16V3A3K3). HA is a large molecular weight negatively charged linear polymer that is abundant in mammalian extracellular matrix. Structures are shown in For the culture insert that included a hydrated material, PE sheets and porous membranes . Membranes were then mounted onto a cleaned glass coverslip in a fluid cell filled with PBS. The Young’s modulus for the membrane was extracted from a plot of cantilever deflection versus displacement by fitting data to a spherical Hertz contact equation using a pre-calibrated AFM cantilever modified with 0.005 mm spherical probe as described by Soofi . ThickneThe attraction forces between the magnets in each housing layer sealed the PDMS channels against the culture insert and established a liquid-tight seal capable of withstanding ~ 35 kPa of applied pressure. An assembled SEAM culture platform using a track etched culture membrane insert is shown in 2).As shown in The fabrication process utilized the laser cutting and lamination processes described for the culture module. The direct seeding module consisted of a top PMMA housing with embedded magnets, access ports, and a central reservoir. A laser cut PDMS sheet  was attached to the bottom with pressure sensitive adhesive  to promote leak-free seal formation around the culture insert during magnetic compression. The bottom PMMA housing contained magnets, a fluidic channel, and a laser cut PDMS sheet defining fluidic access ports. The culture membrane was aligned onto the bottom layer over the central access port and the housings were magnetically connected. The bottom channel and top reservoir were filled with culture medium to support cell attachment, and the desired number of cells was added directly over the culture region of the carrier insert. The seeding module was incubated for 4–6 hours until cells adhered to the culture membrane. For co-cultures, the magnetically latched PMMA housings were separated and the membrane was flipped and resealed between the housings. The bottom channel was filled with appropriate culture medium to support the attached cell population, and the exposed side of the membrane was then seeded with the second cell population. After cell adhesion, the insert (with attached cells) could then be transferred to the culture module.ΔP = ρgΔz, that is reduced as the difference, Δz, between the liquid levels approaches zero. Horizontal reservoirs have been shown [Although alternative methods are emerging , conventen shown  to maintlayers 2 and 5 respectively with PMMA thickness = 2.5 mm), a fluidic isolation layer that separated the reservoirs , a microfluidic resistance network , ports to access inlet reservoirs (layer 1), and fluidic interconnection (layer 6) between the perfusion module and the magnetically connected culture module.The micro-perfusion module was fabricated using the combination of PMMA laser machining, pressure sensitive adhesive, and lamination described previously; the layer-by-layer fabrication approach allowed flexibility in prototype design. As shown in ΔPaa-bb = ρgΔz) between the inlet and outlet reservoirs induced flow from aa to bb. The meniscus moved horizontally as the liquid flowed through the circuit; since there was no change in the relative heights of the liquid within the reservoirs (Δz), and gravitational acceleration and liquid density were constant, ΔP was also constant. Flow contributions related to capillary pressure were assumed to balance because the inlet and out reservoirs were constructed from the same materials and had the same internal dimensions.As shown in ΔPaa-bb) and fluidic resistance, R, in low aspect ratio channels defined as [With the constant pressure drop  was calculated to be 2.42 μL hr-1, corresponding to 58 μL used per day (one full medium exchange in the culture channel per hour). New perfusion modules were used at the end of each culture period.The average flow rate in the network, Q-2 using the expression [The corresponding shear stress in the culture channel was calculated to be 0.00045 dyne cmpression  in Eq 2,-2 to be achieved.Shear controlled environments can be beneficial in culture maintenance applications using shear-sensitive cells such as neurons and stem cell populations . CalculaIn the embodiment shown here, the purpose of the perfusion module was to demonstrate an optional stand-alone option capable of maintaining primary human cells in culture without the need for instrument-driven pumping techniques. However, the modular nature of the SEAM platform allows conventional pumping methods, including syringe pumps, to be implemented if desired (μg mL-1) and incubated (37°C at 5% CO2) overnight to coat the membrane. The reservoir and channel were emptied via pipette, washed with PBS  and filled with appropriate culture medium (ScienCell). Primary HMVEC (ScienCell) were thawed according to manufacturer’s instructions and plated onto fibronectin-coated tissue culture plates. After one subculture, cells were trypsinized and resuspended at a concentration of 500,000 cells mL-1. 2 μL of the suspension was added to each open well (~1000 cells input) of the seeding platform directly over the culture region of the sandwiched culture insert. The cells were visualized with an inverted microscope (Olympus IX-70) to ensure uniform coverage. The seeding module was then incubated until cells adhered to the membrane (4–6 hours). Rat cortical neurons (ScienCell) were handled according to manufacturer’s instructions. Because of the fragile nature of the neurons, cells were seeded directly into a HA coated polystyrene tissue culture plate (Nunc) or onto a self-assembled membrane at a density of 30,000 cells cm-2.The culture insert was sealed in the seeding module and the bottom channel was filled with appropriate culture medium. The upper reservoir was then loaded with fibronectin  culture medium (ScienCell). HPAEpiC were then added to the reservoir at a concentration of 20,000 cells cmμg mL-1. 12-bit images were collected through ImagePro software on an Olympus IX-70 microscope with a Photometrics CoolSnap HQ2 camera. Exposure settings were chosen to minimize background fluorescence. Image processing was performed in ImageJ and settings were applied identically to all samples. Membranes were imaged directly in the device or removed and mounted onto microscope slides, covered with PBS, and sealed with a coverslip prior to imaging.Cells were fixed with 4% paraformaldehyde (Sigma) in PBS for 20 minutes followed by permeabilization with 0.1% Triton X-100 in PBS for 15 minutes. Cells were then blocked with 2% BSA in PBS for 2 hours. Primary antibodies against MAP2  or ICAM-1 , Occludin , or vascular endothelial cadherin  were introduced and incubated for 6–8 hours at 4°C. The primary antibody solution was removed and cells were gently rinsed with PBS and then incubated for two hours with secondary antibodies conjugated to Alexa 647 , or Qdot 625 . Nuclei were stained with DAPI (Life Technologies) at 15 Rat cortical neurons: Culture inserts were decoupled from the culture module with sterile forceps and placed in 150 μL of RNAzol RT (Molecular Research Center). From this point on, the RNA extraction proceeded according to manufacturer’s instructions. Cells were lysed via pipetting and the solution was certrifuged at 12,000g for 2 minutes. The supernatant was placed in an RNase-free tube and 150 μL of absolute ethanol was added to the solution. The mixture was vortexed, then placed on a Zymo-Spin IIC column, and centrifuged at 12,000xg for one minute. The column was then washed and treated with DNase to remove any contaminant DNA. RNA was eluted in 50 μL of ultrapure water. RNA quality was determined using the RNA 6000 kit on the Bioanalyzer (Agilent Technologies). For cDNA synthesis, RNA was then reverse transcribed to cDNA using the iScript cDNA Synthesis kit (Bio-Rad) according to manufacturer’s specifications. Each reaction was carried out in duplicate wells with housekeeping genes  detected in multiplex using TaqMan probes  in one well and the gene of interest detected in the other well using SYBR Green PCR master mix (Life Technologies) on a CFX96 (Bio-Rad). Gene targets included: Neurodap1, NCAM1, c-fos, trkA,  were treated as above to generate data for comparison. Data represent average with standard deviation.HMVEC: After the LPS stimulation, RNA extraction of the cells in the device was performed using the Quick RNA Microprep kit from Zymo (Cat. R1050). The culture inserts (and cell layer) were decoupled from the culture modules and immediately submerged in 600 μL of ZR buffer from the Microprep kit and centrifuged to lyse all cells. From this point on, the RNA extraction proceeded according to manufacturer’s specifications. The RNA was eluted with 10 μL of nuclease free water, and RNA quality was determined using the Agilent RNA 6000 Pico Kit . For RNA extraction from intact devices, an equivalent volume (600 μL) of lysis buffer from the RNA Microprep kit was manually pipetted into the culture module and collected. The collected solution was then loaded onto the purification columns, and extraction proceeded as directed by the manufacturer.ΔΔCt method, with GAPDH (Hs99999905_m1) serving as positive control for normalization, and the expression level without LPS stimulation served as basal level. For each cDNA generated, three technical replicate were performed with two controls without template. Determination of threshold Ct at 99% confidence was determined by multiplying the standard deviation of the NTC for each target gene by 6.965.For cDNA synthesis, 5 ng of RNA from each sample was used to generate cDNA using the Superscript VILO cDNA synthesis kit , using manufacturer’s recommended conditions. For qPCR, 100ng of cDNA was used in individual Taqman gene expression assays (Applied Biosystems). The relative expression of von Willebrand factor (Hs00169795_m1), ICAM-1 (Hs00164932_m1), Cadherin 5 (Hs00901463_m1), CXCL1 (Hs00236937_m1), CXCL2 (Hs00601975_m1), and CCL2 (Hs00234140_m1) were analyzed using the The modular approach and removable culture insert in the SEAM platform enabled workflows that combined the direct cell access provided by open cultures with the characteristic culture capabilities found in microfluidic systems. As described schematically in the workflow , culture2) using the direct seeding module. The culture surface area of the membrane was selected to accommodate a monolayer of contact inhibited cells  using an input population of 1000 cells. 1000 cells could be easily and reproducibly pipetted and helped to reduce per/assay cost. The size of the carrier membrane (7 mm x 11 mm) was designed to fit directly into the cell isolation vial of a standard RNA isolation protocol with having to be cut or otherwise manipulated.HMVEC line the blood vessels and play a critical role in mediating vascular permeability, regulation of blood flow, wound healing, and inflammation . The baca priori target specification.After cell attachment, the culture insert was transferred to the culture module and connected to the micro-perfusion module for 24 hours of culture . The perp < 0.001). ICAM-1, a surface adhesion molecule involved in the arrest of rolling leukocytes, was upregulated 2.5 fold while CXCL1 and CXCL2, chemokines involved in the recruitment of neutrophils and lymphocytes, increased 5 fold and 11 fold respectively. CCL2 has been shown to promote dendritic cell and monocyte infiltration and mRNA expression increased 6 fold compared to unstimulated control. mRNA levels of the endothelial phenotypic marker Von Willebrand Factor remained unchanged as expected for healthy control. Staining of ICAM-1 between the stimulated and unstimulated cells demonstrated that mRNA changes were translated to protein level changes in the system. Using the self-contained SEAM platform, were able to demonstrate mRNA and protein level changes resulting from controlled LPS stimulation.in vitro barrier models is to establish physiologically representative culture environments  in which to study tissue level responses. The impact of biochemical cues and mechanical stimulation within microfluidic architectures has been well documented in the literature [The overall goal of microscale terature . It is aterature –37. Eventerature ,39, techFor example, the cerebral cortex or “grey matter” plays an important role in several high level processes including movement and perception. The brain ECM has a unique composition with low percentages of fibrous proteins (e.g. collagen and fibronectin), and high percentages proteoglycans including HA ,41. To din vitro microfluidic barrier models by enabling experimental control over outside-in signaling.As shown in To support user-friendly methods to establish, maintain, and analyze microscale tissue interfaces, we developed a self-contained platform that consisted of a removable cell culture insert and distinct prefabricated modules for microfluidic operations. The reversibly sealed SEAM architecture allows the culture insert to be rapidly coupled, decoupled and transferred between modules to achieve an efficient experimental workflow. The modular also approach provides unique advantages related to i) efficient cell seeding, ii) the simple inclusion of tailored biological interfaces, iiii) robust nucleic acid isolation, iv) generation of multi-tiered readouts, and v) simplified user-friendly operation.The concept of the removable cell culture insert is a key aspect of the SEAM platform. The insert consisted of a micro-scale porous cell culture region surrounded by a non-porous carrier that facilitated transfer and coupling with different fluidic modules. The lamination of laser cut PE sheets (with pressure sensitive adhesive on one face) is compatible with commercially available micro and nanoporous membranes that have been optimized to provide low auto-fluorescence, high optical clarity, and defined pore size distributions. Materials such as ultrathin nano-membranes ,48 collaConventional microfluidic techniques to permanently seal membranes between elastomeric channels primarily use oxygen plasma bonding and have difficulties forming robust, liquid-tight seals when hydrated materials are present. With the magnetic latching and removable culture insert concept, the culture materials do not have to be directly compatible with the overall fabrication method of the architecture. We demonstrated a tethering approach to incorporate a thin, self-assembled membrane with Young’s modulus tailored to mimic the brain environment (~ 1 kPa). This interface-based liquid-liquid membrane self-assembly fabrication technique  was selein vitro assays.Our adaptation of the self-assembled membrane into the culture insert enables handling and manipulation of the suspended 900 nanometer thin membrane throughout the workflow . Thin culture membranes can help accurately represent the dynamics of cell-cell communication across a tissue interface. For example, paracrine signaling factors traveling across a 10 μm thick culture membrane requires 100x longer than with a 900 nm thick membrane. Several barrier tissues  was decoupled from the architecture using reversible magnetic latching and transferred directly to the workflow of the off-chip isolation protocol. The ability to move an entire cell population without manipulation helped limit sample losses that inevitably occur when transporting cell lysates within high surface-area-to-volume ratio microfluidic channels. Using the transfer approach, we were able to achieve RIN values approaching the maximum value of 10 from a ~1000 cell input population. RIN values were also high using in-channel lysis and collection (Average = 9.5) but resulted in ~ 2x less RNA and collection compared with culture insert transfer method. The removable culture insert technique allowed the entire cell population to be simply and rapidly transferred for RNA isolation without requiring fluid manipulation.Although we demonstrated differential gene expression readouts in this work, transfer of the insert enables a wide variety of optimized commercial techniques including analysis to explore DNA damage, genotoxicity, and whole genome transcriptomics (e.g. miRNA-Seq or RNA-Seq) that explore global pathway activation patterns that result from interaction with a drug candidate or pathogen. Even though it is well recognized that the activation of a signaling pathway at the mRNA level does not necessarily lead to protein expression and functional response, gene level readouts provide important insights to identify potentially interconnected pathways that can be used to assess potential toxicity and guide therapeutic development. qPCR-based protein assays (e.g. TaqMan Protein Assays) can be included to the workflow to directly correlate mRNA expression with protein levels and explore post-translational protein modification. In addition, the removable culture insert can be mounted onto a glass slide for high-resolution imaging of intra-cellular and surface-level protein localization. The transferable insert concept opens the door for advanced multi-tiered readouts that provide unique insights into how a tissue interacts with an external agent.In permanently sealed membrane-based systems, tissue interfaces are established by introducing cells to the interior of the device by flowing a cell suspension through the microfluidic channels that are in contact with the culture membrane; flow is stopped, and cells settle onto the membrane surface. The sedimentation process along the entire channel length makes it difficult to control initial cell density and reproducibly introduce the same number of cells to the system. Special care must also be taken to prevent the introduction of bubbles during the flow based cell introduction process. Cell seeding is significantly easier in conventional open format culture platforms, such as Petri dishes and multi-well plates, where cells are directly added to the culture surface with a pipette tip. The simplicity of the open seeding approach is replicated here by reversibly coupling a culture insert within a seeding module; a defined cell number is directly added to the culture region through the open reservoir. Direct seeding can significantly reduce the number of cells that are needed per experiment and enable the use of rare or expensive cell populations including stem cells, primary human cells, and clinical relevant samples.For example, a typical microfluidic system is seeded with ~ 20,000 primary human cells on one side of the membrane . For a cConventional culture platforms require syringe pumps to drive fluids through the platform. In an effort to make microfluidic assays accessible to a broad range of end users, we developed an option for a gravity fed module that provides micro-perfusion capabilities within our self-contained system. The micro-perfusion module can be used to maintain cell cultures or expose the apical or basolateral populations to experimental compounds. Each reservoir is individually addressable and can be used to establish physiological culture environments such as air-liquid interface culture, which is relevant in models for the cornea, skin, and lung. Our equipment-free perfusion option also makes the platform amenable for use in BSL3/4 facilities where syringe pumps are difficult to implement because of the risks of aerosol generation and issues arising from problematic decontamination . Eqs 1 aThe laser cutting and lamination based fabrication process used here is ideal for design iterations and prototype development; with experience, a batch or 5–10 modules can be cut, aligned, and assembled in less than five minutes. Once a design has been finalized, modules can be scaled up for small batch production using hot embossing techniques or translated to injection molding for larger scale manufacturing. An inventory of mix-and-match modules to support experimental studies can established at either the lab prototype and advanced prototype stage. End users can thus easily introduce capabilities as needed.The magnetic coupling technique used throughout also provides a simple approach to connect different tissue modules together (e.g. heart to lung) through a magnetic manifold to explore multi-module drug or toxin interactions . ExperimThe key objectives of the SEAM platform were to create i) a simple but functional architecture to explore cell interactions to external stimuli, ii) extend experimental capabilities by incorporating physiologically relevant culture interfaces, iii) streamline the cell introduction process and iv) simplify experimental workflows including imaging and nucleic acid analysis. Taken as a whole, the easy to use magnetically sealed modules and SEAM workflow could help to make compartmentalized microfluidic approaches more accessible to the scientific community and contribute to advances in barrier tissue research.S1 Fig3V3K3 peptide sequence as designed by Stupp et al. b) Chemical structure of hyaluronic acid used in the supramolecular self-assembly process to make suspended membranes.a) Chemical structure of peptide amphiphile with 16 carbon alkyl tail and A(TIF)Click here for additional data file.S2 Figa) Representative AFM scan used to determine thickness of the self-assembled membrane. b) Representative AFM characterization curve to determine the Young’s modulus of the self-assembled membrane. C) Molecular weight cutoff (90% retention in donor channel) of membrane determined by quantitating transport of fluorescent dextran across the membrane from a donor to acceptor channel over 24 hours. Data represented by mean and standard deviation.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Figa) Bioanalyzer RNA integrity number for off-chip RNA extraction using in channel lysis  and culture insert transfer method . A RIN value 10 represents intact RNA. b) RNA quantity from on-chip lysis (17.3 ± 1.17 ng) and collection compared with culture insert transfer method (34.1 ± 1.38 ng). Data represented as mean and standard error. * p<0.05.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Video(MOV)Click here for additional data file.


Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.Clinical laboratories are increasingly using molecular tests for methicillin-resistant  Staphylococcus aureus is an important human pathogen and is responsible for healthcare-, community- and livestock-associated colonisation and infection S. aureus (MRSA), which also exhibit phenotypic resistance to related β-lactam antibiotics including flucloxacillin, cefoxitin and oxacillin. As such there is a great demand for rapid tests to detect MRSA.S. aureus (MSSA) becomes MRSA when it acquires the SCCmec  genetic element. This element contains the mec gene that encodes the protein PBP2A (penicillin binding protein 2A). Co-colonising coagulase-negative staphylococci (CNS) are thought to act as the reservoir for the mec gene with S. fleurettii suggested to be the origin mec elements mec elements and MSSA means that nucleic acid tests (NATs) for mecA and an MSSA-specific gene can give false positive results. NATs for MRSA therefore need to specifically detect the insertion of the SCCmec element into the orfX gene of S. aureus. This testing is performed by amplifying with a primer in the SCCmec element and a primer in the orfX gene of the S. aureus chromosome S. aureus isolates are phenotypically resistant but produce false negative results using currently marketed real-time PCR assays orfX-SCCmec junctions, as to date at least twenty types have been identified worldwide orfX-SCCmec sequence if it is to be detected. The prevalence of different junction types is not well known, but it is suggested that types i, ii, iii, iv, v and vii, account for over 98% of worldwide strains tested Methicillin-sensitive www.twistdx.co.uk).RPA is a novel, isothermal nucleic acid amplification chemistry As the price of whole-genome sequencing fell significantly after our initial study, it became possible to expand the scope of our investigation and use whole genome sequencing to further identify the precise reasons why our RPA multiplex failed to detect some of the isolates as MRSA.orfX-SCCmec junctions and the sequences of SCCmec elements circulating in hospitals it remains to be seen if one assay will be effective as a screening tool. Rapid whole-genome sequencing has revolutionised the investigation of MRSA outbreaks and transmission Failing to identify carriage of MRSA in hospital inpatients will have significant consequences for the individual patient and for general infection control. With the high variability in both Central Manchester University Hospitals NHS Foundation Trust (CMFT) is a large academic trust comprising of specialist hospitals for children, dentistry and ophthalmology, together with a large teaching hospital and community services. MRSA isolates were obtained from clinical samples collected via standard hospital screening procedures from the Trust. A total of 726 isolates were collected from 726 patients at CMFT between December 2008 and June 2009 (n = 580) and between July 2009 and November 2009 (n = 146).Isolates were first screened using a multiplex RPA test. The CMFT standard screening procedure was to collect nasal and groin swabs and combine to inoculate 15 ml nutrient broth  containing 7.5% NaCl. Broths were incubated for a minimum of 18 hrs at 35°C in air and then subcultured on MRSA Select agar plates (BD Diagnostics). In addition to usual diagnostics, a single, pink, colony was picked from each positive plate after 24 hours incubation, streaked onto an anonymised blood agar plate and incubated for a further 24 hours. A lack of suitable biocontainment facilities at TwistDx meant that plates were scraped and bacteria resuspended and boiled for 20 minutes. Lysed bacteria were then diluted 1∶1000 in sterile distilled water for use in RPA reactions.orfX-SCCmec junction type to determine the best combinations for multiplexing. Potential confounding SNPs were identified by BLAST searches and oligonucleotides targeted to the most conserved regions of each junction. OrfX was compared to CNS using BLAST to identify the most divergent sequences from S. aureus to minimise the risk of false positives caused by amplification of methicillin resistant CNS. To determine the range of SCCmec element types that TwistAmp MRSA was able to detect, 15 prototypic strains (SCCmec types I-XI were tested with the assay SCCmec types I-IV and VI-VIII. The SCCmec type V prototype strain (WIS) was not detected by TwistAmp MRSA and was later identified as containing a type of junction, xii, not covered by the assay. Performing a BLAST alignment of all SCCmec type V entries in GenBank other than WIS  revealed that they all contained type iii junctions, suggesting that they would be successfully detected by the multiplex.RPA primers differ from PCR only in length, with 30–38 bases being optimal for efficient recombinase filament formation. TwistAmp exo probes are typically 46–52 bases long, with a THF ≥30 bases from the 5′ end and ≥15 bases from the 3′ end. A fluorophore and a quencher are positioned either side of the THF such that cleavage by exonuclease III separates the two and fluorescence increases. TwistAmp exo probes have a C3-spacer or similar block at their 3′ end to prevent them amplifying DNA unless cleaved. Numerous overlapping primers were tested empirically with 25 copies of PCR product for each  strains  for SCCm6 copies of genomic DNA from S. saprophyticus (ATCC 43867), S. epidermidis (ATCC 35983) and S. hominis (ATCC 51624) and gave no signal. We developed a multiplex RPA reaction that detected, but did not differentiate, junctions i, ii, iii, iv, v and vii. Isolates that were positive by this test were typed by uniplex assays for junction ii, i, iii, iv, v and vii. 50 µl RPA reactions pellets that included the primers and probes were freeze-dried by TwistDx Ltd, Babraham, Cambridge. For the multiplex reaction an internal control sequence was also included to confirm that the reactions had worked. This internal control DNA was designed with the junction i and iii primers at opposite ends with the sequence detected by the control probe in between . The sequence type (ST) of each isolate was determined using the online MLST database (http://saureus.mlst.net/). eBURST (http://eburst.mlst.net/) was used to determine founding genotypes. The spa type of each isolate was determined using methods described previously We used the MLST scheme previously developed mec regions were found by searching the GenBank Nucleotide database (February 2013). Features were extracted and manually curated into a protein fasta file and analysed using OrthoMCL v2.0. Maximal discrimination between similar proteins was achieved by using an inflation parameter (I) of 13. OrthoMCL could not distinguish between different Ccr and Mec allotypes due to their high amino acid similarity.57 high quality annotated sequences of MRSA SCCorfX-SCCmec junction were run on an E-Gel 2% gel System (Invitrogen) and examined for quality of DNA and degree of degradation. DNA from 24 (80%) of the 30 isolates was judged to be of sufficient quantity and quality for library making and sequencing. Unfortunately the other six isolate cultures were unrecoverable and so further DNA could not be sourced. The isolates were sequenced on an Illumina Genome Analyzer II, with 101 base pair reads and a paired-end insert size of 400 bp to an average coverage of 100-fold. It is regrettable that we were not able to sequence all 30 undetected isolates and that culturable isolates are not available for them. When this study was started the cost of whole-genome sequencing was prohibitive and it was only the subsequent precipitous drop in cost that allowed us to re-evaluate what it was possible to do with the samples that we had collected. The sequence reads were trimmed and corrected by Phred+33 quality values using Quake De novo sequence assembly was carried out using Velvet mec types in GenBank determined using BLASTN on a custom database. Manual reordering was performed by visualisation of BLAST results in ACT orfX-SCCmec junction was not fully assembled 20–30 iterations of IMAGE mec types were annotated using RATT mec sequences have been uploaded to GenBank [accession numbers HF569093–HF569116]. Unfortunately frozen stocks of these bacteria were no longer available at CMFT by this time. With the sequence information that we have published however, researchers will be able to order synthetic versions of novel junction regions with which to test any new assays that they may wish to develop.Extracted DNA from isolates undetectable by RPA to the orfX-SCCmec junction multiplex RPA. orfX-SCCmec junction uniplex-RPA testing showed that most (653) of these 696 isolates were orfX-SCCmec junction-ii, with the other orfX-SCCmec junction types tested only representing a minority of cases .Of the 726 MRSA isolates tested, 696 (96%) could be detected by mec sequences available in GenBank were compared using OrthoMCL (mec elements with the most frequent occurring proteins being mec (100% strains), an uncharacterised protein , IS431 transposase (89.5%), ugpq (87.7%), ccrA (84.2%), ccrB (82.5%) and maoC (82.5%). Strains clustered based on the SCCmec types proposed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) To understand better the conservation of proteins in MRSA, the proteins from the 57 MRSA SCCOrthoMCL . A core orfX-SCCmec isolates for which the junction was undetectable by RPA and these underwent further analysis using MLST and spa typing. Only 24 of the isolates were recoverable for further DNA extraction for second-generation sequencing. Unfortunately, because isolates had been anonymised and the cultures taken from them boiled, it was not possible to return to CMFT and re-grow any strains that had poor quality DNA. spa and SCCmec types (where available) within these 28 isolates. Eight STs in total were found, none of which were closely related except for ST30, which is a single locus variant (SLV) of ST36. 18 spa types were found among the 28 isolates tested. Sequencing of the SCCmec elements demonstrates conserved homology for many of the isolates but with one cassette displaying no homology and variable levels among the others  and 36 (10 isolates). ST36 belongs to clonal complex 30 (CC30). All, but one of the isolates were  working . Seven o working . This cand Tn554  and S3a. element  and S3b.ansposon .orfX-SCCmec junction-undetectable isolates. It is now responsible for 85% of MRSA bacteraemia cases in UK hospitals. Nine isolates belong to CC22, a common and widespread group that carries SCCmec type IV. Eight ST22-MRSA-IV strains were sequenced and four of them had elements that have a different structure to previously published type IV cassettes. CMFT201 shows the characteristic type IV features of a class 2 ccr gene complex and class B mec gene complex. However, similar to the variant type II elements discussed above, there is a further class 1 (A1B1) ccr complex situated at the 5′ end of the cassette (mec elements available in GenBank demonstrates remarkable homology to an unpublished strain 45394F (GU122149)   and S4a.U122149)  with themec element shares partial homology with two existing strains; ZH47 ccrC along with the normal class 2 ccr gene complex. The larger SCCmec element in CMFT3119 is due to the addition of an arc gene cluster. Therefore overall CMFT3119 also bears similarity to isolate M1, which also has a class B mec complex, class 2 ccr complex and part of the arginine catabolic mobile element (ACME) often found in S. epidermidis and S. saprophyticus. This may be a subtype of IVh as the J1 region bears homology to ST22-MRSA-IVh, and some isolates belonging to this genotype can contain a truncated ACME element An additional novel variant is only demonstrated by CMFT3119. The sequenced SCCThe three further isolates sequenced from this clonal complex had smaller changes but of a great enough magnitude to still prevent molecular detection with RPA .mec element. ST149 has previously been described in Malta where it appears to be common mec elements hsd complex.The three isolates belonging to ST149, part of CC5, also have a type IVk SCCorfX-SCCmec junction types iv and v, leading to no clear amplified fragment generation.The two ST59 isolates belong to the major community-associated CC59 lineage, a clonal group that has become widespread in the Asia-Pacific region. CC59 strains have been described in several countries including the UK spa type t843, previously reported in bovine and more recently in humans in the UK, Denmark, Ireland and Germany CMFT540 belongs to the clonal lineage CC130 with pepF, more commonly found in type II cassettes.The single ST772-t657 isolate is known as the Bengal Bay clone or WA MRSA-60, a multiply-resistant Panton-Valentine Leukocidin-positive CA-MRSA that is becoming increasingly prevalent in India, where it has spread into hospitals mec element types is required. MRSA SCCmec elements display a high level of protein diversity resulting from significant nucleotide diversity within shared proteins. Despite this we have shown that an RPA assay can be used to assess the prevalence of the orfX-SCCmec junction types described by Huletsky et al, in a large UK teaching hospital Molecular methods for the detection of MRSA that are currently on the market make an attractive alternative to the slower methods of using chromogenic selective MRSA agar or by enrichment in a 7.5% NaCl nutrient broth mec element orfX-SCCmec junction type ii. Although orfX-SCCmec junction types and SCCmec types do not easily correlate, orfX-SCCmec junction type ii usually corresponds to SCCmec element types I, II or IVd A previous study at a UK hospital found the majority of molecularly characterised MRSA isolates carry a type IV SCCmec elements that may occur. PCR and RPA primers are therefore being developed for a genetically highly variable location. It has also been difficult to identify composite SCC-SCCmec elements as they would either produce a large number of PCR fragments that were difficult to interpret or have a very unusual hybridisation pattern mec elements exists which leads to very low mapping coverage  isolates but not often seen outside of this clonal group for MRSA. However, they are seen more commonly in coagulase negative staphylococci. arc and opp genes are homologs of genes that are recognised bacterial virulence factors and encode an arginine deaminase pathway and ABC transporter systems respectively. The speG gene of ACME has also been shown to be a potential virulence factor by allowing the bacterium to circumvent polyamine hypersensitivity arc cluster can be found on the chromosome of S. aureus but the ACME-arc cluster inserts as an SCC-like element adjacent to the SCCmec in some strains. Animal models have suggested that the presence of ACME clusters in ST8-MRSA-IVa strains leads to an improved fitness and ability to colonise the skin and mucous membranes The composite SCC-SCCde novo assembly approach is a reliable method to identify novel SCCmec element sequences.The remaining six isolates that were sequenced all contained relatively minor changes but each change was sufficient to interrupt RPA primer binding or lead to excessively large fragments . The numS. aureus and the speedier time to results that the molecular assays provide has to be balanced against their reduced sensitivity. However, clinical laboratories need to be cautious in adopting fully molecular assays at present – it is likely that 4–5% of MRSA will be missed. We suggest that further uptake occurs only in the knowledge that a phenotypic assay is used to confirm negative isolates. We await larger sequencing studies to provide more information on the ultra-conserved areas of the cassette that can be used for primer design.To effectively reduce post-operative infection rates all surgical patients are screened for MRSA colonisation. In addition all medical admissions are also screened in most hospitals to reduce overall levels of colonisation and the risk of bacteraemia. Effective isolation and treatment of patients with MRSA colonisation requires adequate identification of resistant Figure S1orfX-SCCmec junction multiplex reactions. Fluorescence is generated by the cleavage of TwistAmp exo probes that have hybridised to amplicon produced by opposing primers. Primers are designed to amplify junction types i, ii, iii, iv, v and vii. a) Signal from the orfX probe (FAM) indicating the presence of the junction sequence. b) Signal from the internal control probe (TAMRA) showing that the reactions have worked. Two negative, no template, controls (NTC) were run. Reactions were run at 39°C for 20minutes in Twista portable real-time fluorometers (www.twistdx.co.uk). The strips of 8×0.2 ml tubes were removed from Twista, agitated and replaced after 4 and 6minutes – these are visible as spikes in fluorescence. Because RPA reactions are viscous and run at relatively low temperatures, agitation is necessary to disperse amplicons if there are not many starting template molecules.Typical fluorescence (arbitrary units) curves for RPA (TIF)Click here for additional data file.Figure S2mec elements. Presence or absence of proteins in SCCmec published sequences on GenBank using OrthoMCL to determine homology. Conventional gene names are shown for each orthologue group with uncharacterised  proteins listed with a relevant reference strain and locus tag in table S2.Binary heatmap showing presence or absence of proteins within MRSA SCC(TIF)Click here for additional data file.Figure S3a) Comparison between strain with novel SCCmec from CMFT and SCCmec type II reference strain shown on ACT (26) demonstrating very similar homology for the majority of the 3′ end of the cassette. b) Close-up view of 5′ end of SCCmec demonstrating main divergence with additional class 1 ccr complex inserted in CMFT2.Type II variant: homology between strains MRSA252 (NC_002952) and CMFT strain 2. (TIF)Click here for additional data file.Figure S4mec from CMFT and SCCmec type IVk strain on GenBank focusing on the 3′ end, which demonstrates considerable homology. b) Close-up view of 5′ end of SCCmec demonstrating the only area of significantly reduced homology; with absence of hsdR and hsdM in CMFT201.Type IVk: homology between strains CMFT201 and 45394F (GU122149). a) Comparison between strain with novel SCC(TIF)Click here for additional data file.Figure S5mec for CMFT3119 compared to strains ZH47 (AM292304) and M1 (HM030720). The cassette of CMFT3119 shows significant homology to ZH47 with the addition of an arc gene cluster similar to that found in M1.Type IVh variant SCC(TIF)Click here for additional data file.Figure S6mec types in MRSA. a) VCF view on Artemis (27) for all 24 MRSA isolates sequenced (rows) with variants compared to MRSA252 shown as coloured vertical lines. Area shaded in pink is for SCCmec region. There appears to be little variation but b) is the same region in pink shown with a variation of varB (50) that displays areas of zero coverage as grey. The low variation seen in a) is due to a significant reduction in mapping depth over this region. c) BAM view of same reads of CMFT540 piled onto completed assembly of CMFT540 (only SCCmec is annotated).Importance of assembly for examining SCC(TIF)Click here for additional data file.Table S1mec element prototype strains used.SCC(DOCX)Click here for additional data file.Table S2Locus tags for uncharacterised protein clusters in (DOCX)Click here for additional data file.


Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation. GSH is converted back to GSSG during this activity. A cellular redox cycle is completed when GSSG is recycled back to its reduced form by GSSG-reductase at expense of NAPDH.The tri-peptide gamma-glutamyl-cysteinyl-glycine (glutathione) is the major cellular antioxidant to combat reactive oxygen species (ROS). There are three major reservoirs of glutathione inside eukaryotic cells: ~90% in cytosol, ~10% in the mitochondria, and <1% in the endoplasmic reticulum. In cytoplasm, under normal physiological conditions, the ratio of reduced glutathione (GSH) to the oxidized disulphide form (GSSG) varies from 10:1 to 100:1  and the hydroxyl radical (•OH).ROS are a natural side product of oxidative phosphorylation, being produced at many positions along the electron transport chain in the mitochondria  and endogenous GSH. This allowed direct assessment of identifiable cells in a native, accessible environment in which the normal cell-cell interactions are maintained. In order to explore the role of GSH during aminoglycoside toxicity, we exposed organotypic cochlear explant cultures to the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) to deplete cellular GSH  mice. We used live imaging in Meister, , then chThe animals were sacrificed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Auditory bullae were isolated from C57BL/6 mice at postnatal days 4–6 (P4–P6), at hearing onset (P14–P16) and in both young (P33–P47) and aged  adults. Auditory bullae were removed and transferred into Leibovitz's L15  and then a window opened in the apical turn  supplemented with 1% fetal bovine serum  and maintained at 37°C in a 5% CO2/95% air atmosphere.For the aminoglycoside and BSO experiments that required longer incubations, cochleae from P4 to P6 mice were dissected out and placed in explant culture. Auditory bullae were removed and transferred into medium 199  supplemented with penicillin (10 U/ml) and fungizone (25 ng/ml). The cochleae were isolated from the surrounding cartilagenous capsule, Reissner's membrane was cut and removed but stria vascularis was maintained. The cochlea was cut into three and the apical, middle and basal turns then placed onto Cell-Tak® cell and tissue adhesive -coated Matek®  culture dishes. Cell-Tak® was diluted in 0.1M NaHCODuring aminoglycoside exposure experiments, cochlear explant cultures were maintained in HEPES-buffered Hanks Balanced salt solution (10 mM HEPES) adjusted to pH 7.3 with NaOH.Monochlorobimane (MCB) is a non-fluorescent bimane which is freely permeable across the cell membrane and forms a fluorescent adduct when combined with reduced glutathione (GSH) in a reaction catalyzed by glutathione s-transferase. MCB  was prepared as a 50 mM stock solution in DMSO and used at a final concentration of 50 μM. Our initial experiments confirmed that this reaction reached steady-state within 40 min  for 16–18 h. BSO is an irreversible inhibitor of gamma-glutamylcysteine synthetase  over the different cell types in the organ of Corti in single image planes XY. Care was taken to ensure that ROIs were placed within the boundaries of individual cells and for each image file  the following cells were sampled: 3–5 inner hair cells, 12–15 outer hair cells, 4–6 pillar cells, and 4–6 inner sulcus cells. The mean background intensity was measured from regions devoid of cells from within the same image stacks using 4 different ROIs. All mean intensity values from the ROIs were exported to Excel. The background values were subtracted from the measured MCB-GSH values within each data set. The images were not manipulated in any way and measurements were made from single planes. Ratios were calculated from the absolute measurements from individual cells after background subtraction. Unless indicated data are presented as the mean ± SEM and the statistical tests used are either Student's paired t-tests, with significance set at p < 0.05 or ANOVA with post-hoc Tukey-Kramer's, with significance set at p < 0.05.Images were analyzed offline using IQ software . The mean MCB-GSH fluorescence intensity was quantified by selecting regions of interest  for 18 h. After the BSO treatment, GSH content was evaluated in each cell type in cochlear explant cultures. GSH was reduced by between 65 and 85% across the different cell types with inner hair cells showing the least reduction and outer hair cells the greatest (Supplementary Figure Hair cells do not survive in the ex vivo whole auditory bulla preparation. A window was opened in the apical region to allow optical access to the cells and for the MCB loading that was used to assess GSH content. Using live multiphoton imaging we determined cellular GSH concentrations in individual cells, overcoming some limitations of biochemical and histological approaches to measuring GSH in intact cells and tissue (Sun et al., In this study, we evaluated cellular GSH distribution in organ of Corti cells at different ages, from P5 to ~1 year old animals, using an ex vivo whole auditory bulla preparation has been used previously both in guinea pigs (Mammano and Ashmore, ex vivo context. Here we first characterized the hair cell status in this preparation using the expression of the hair cell marker myosin VIIA and assessed the stereocilial bundles using phalloidin. These same assays were applied after MCB assessment of cellular GSH content to confirm the normal cellular status of all the preparations used in this study.The GSH is one of the most important cellular antioxidants. GSH is distributed in mitochondrial, cytosolic and ER pools where it maintains exogenous antioxidants such as vitamins C and E, peroxide and glutaredoxins in their reduced and active forms. Alterations in cellular GSH have been linked to variety of neurodegenerative diseases such as Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis and others (Schulz et al., GSH is the major free radical scavenger in the brain, and it has found at higher levels in glial cells compared to the neurons (Keelan et al., GSH content in hair cells isolated from the guinea pig cochlea was measured using MCB (Sha et al., in vivo (Garetz et al., GSH levels have been strongly implicated in providing a protective role in the cochlea and our data showing raised cellular GSH in inner hair cells correlates with this assertion. Food deprivation results in a reduction in endogenous GSH and has been correlated with enhanced auditory dysfunction (Hoffman et al., 2+ may well be responsible for catastrophic mitochondrial depolarization (Esterberg et al., Here we evaluated the role and potential importance of cellular GSH during aminoglycoside ototoxicity. We performed these longer term experiments in cochlear explant cultures from postnatal day 4 to 6 mice. Explants were pre-treated with BSO for 18 h in order to deplete GSH and depletion was confirmed by MCB labeling. After 18 h of BSO-treatment explants were then challenged with 1 mM neomycin for 3 or 6 h. Compared to controls not treated with BSO, there was no significance difference in hair cell survival or death with either 3 or 6 h of neomycin exposure. If anything, we identified a trend for hair cell survival in the BSO-treated explants at the earlier 3 h time point, which was not present after 6 h treatment. The simplest explanation for these data is that ROS production is not as major a determinant of aminoglycoside-induced hair cell death as has been suggested. A number of reports have begun to indicate that mechanisms other than ROS generation may be critical in promoting cell death during ototoxicity. Recent work has indicated that aminoglycosides may block cytoplasmic ribosomal activity (Francis et al., An alternative explanation for the lack of effect of GSH depletion on neomycin-induced hair cell death describe here, is that when cellular GSH is reduced, other free radical scavenging systems take over so that that GSH depletion does not cause any additional hair cell death. Although this is possible, we predict that we should still have observed some effect of the GSH depletion in these experiments. A further investigation into potential regulation of other cellular antioxidants present in sensory hair cells is warranted.In summary, using live imaging to measure cellular GSH levels we observe increased cellular GSH in inner hair cells compared to outer hair cells, but also higher levels in supporting cells compared to the outer hair cells. These data are consistent with the enhanced sensitivity of outer hair cells compared to inner hair cells with various types of cellular stress, from noise, ototoxic drugs to aging. However, when we tested the role that cellular GSH plays during neomycin-induced hair cell death we were unable to provide positive data in support of GSH-dependent protection. Further work is required to determine whether other ROS scavenging systems are able to provide sufficient buffering to cover for the lack of GSH in our experimental condition or whether we need to consider that ROS play a lesser role in aminoglycoside-induced hair cell death than has been previously suggested.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Greater sensory stimulation in advertising has been postulated to facilitate attention and persuasion. For this reason, video ads promoting health behaviors are often designed to be high in “message sensation value” (MSV), a standardized measure of sensory intensity of the audiovisual and content features of an ad. However, our previous functional Magnetic Resonance Imaging (fMRI) study showed that low MSV ads were better remembered and produced more prefrontal and temporal and less occipital cortex activation, suggesting that high MSV may divert cognitive resources from processing ad content. The present study aimed to determine whether these findings from anti-smoking ads generalize to other public health topics, such as safe sex. Thirty-nine healthy adults viewed high- and low MSV ads promoting safer sex through condom use, during an fMRI session. Recognition memory of the ads was tested immediately and 3 weeks after the session. We found that low MSV condom ads were better remembered than the high MSV ads at both time points and replicated the fMRI patterns previously reported for the anti-smoking ads. Occipital and superior temporal activation was negatively related to the attitudes favoring condom use (see Condom Attitudes Scale,  Consistent with this hypothesis, medial temporal and prefrontal response to low MSV anti-tobacco video ads was positively correlated with subsequent frame recognition accuracy Despite the evidence that high MSV may disrupt the processing of ad arguments, campaign designers often include high MSV inputs to capture attention and without considering possible negative effects of these distractors A second goal of the research was to determine whether the superiority of low MSV ads previously demonstrated for anti-tobacco video ads Thirty-nine healthy subjects were recruited through advertising and screened for psychiatric, neurological or medical illnesses. Exclusion criteria included presence of DSM-4-TR Axis 1 psychiatric disorder rd similarly trained individual.We identified 71 ads that encouraged condom use by populations at-risk for unplanned pregnancy or sexually transmitted Diseases (STDs). Most of the ads were commercials produced by condom manufacturers , who share a common goal with government-sponsored programs to encourage their target audiences to engage in safe-sex practices. Any references to particular manufacturer brand names were cut or digitally blurred. All 71 videos were scored for MSV by two raters who were trained on using the validated rating procedure We selected 32 complete ads (ranging between 26 to 66 [x¯ = 42.2] seconds in length) from the set of 71 drawn equally from above and below the collection’s median MSV score of 6 . The length of the ads was not different between high and low MSV categories . We created two sets of 16 ads, each consisting of 8 “high MSV” and 8 “low MSV” ads, with no difference in MSV between these two sets . The mean MSV of the low subgroup was 3.6 , and the mean MSV of the high subgroup was 8.8 . MSV values were significantly different between low and high MSV ads . Four of the selected videos were produced by firms that sell condoms , while the rest were developed by non-profit or government public health agencies  for network and cable broadcast (e.g. MTV).Wang et al. Subjects were given the task see  of viewiFive minutes after the video task, subjects were given the Frame Recognition Test (FRT), a forced-choice recognition memory task designed to probe episodic memory of the videos they had seen in the scanner. This task was adapted from an electroencephalography study of the retention of TV commercials Three weeks following the fMRI session, participants returned for a second Frame Recognition Test to examine decay and to assess whether initial differences between high MSV and low MSV ads remained. The FRT presented during this session (“delayed FRT”) used the same design as the FRT that immediately followed the ad-viewing task. However, each participant was presented with a different set of 96 still-frames .Subjects were questioned about their attitudes towards condoms immediately before and after viewing the video task using an abridged version of the Condom Attitude Scale (CAS) All visual tasks were programmed in the Presentation  stimulus presentation package and rear-projected to the center of the visual field using a PowerLite 7300 Video projector system  that was viewed through a mirror mounted on the scanner head coil. The video soundtrack was delivered through Silent Scan 2100 MRI-compatible headphones . Before the imaging session, participants were instructed to watch all the videos carefully, that their memory of the videos would be tested after a delay and that their performance on this test was an integral part of the experiment. Participants performed the FRT approximately five minutes after the video task. The FRT was repeated 3 weeks later at a second session.Correct target recognition–ZFalse alarms, which reflects how well subjects correctly distinguish targets from foils Correct target recognition+ZFalse alarms. Br = 0 indicates no bias, Br<0 indicates liberal bias (i.e. tendency of saying “Yes” when uncertain), Br>0 indicates conservative bias (i.e. tendency of saying “No” when uncertain). Paired t-tests were performed comparing subject performance on Session 1 vs. Session 2 FRT (immediate vs. delayed sessions), and high MSV vs. low MSV. Independent t-tests were performed comparing gender performance.Statistical analyses were performed using the IBM Statistical Package for the Social Sciences (IBM SPSS version 21). Subjects’ performance on the FRT was evaluated using the Discrimination Index, Pr = ZMRI data were collected on the University of Pennsylvania’s Siemens Trio  3 Tesla whole body system using a 32-channel head coil. BOLD fMRI gradient-echo echo-planar sequence was acquired with the following parameters: TR = 3000 ms, TE = 32 ms, flip angle = 90, matrix = 64×64, FOV = 220 mm × 220 mm, slice thickness/gap = 3.4/0 mm, 46 slices. After BOLD fMRI, a 5-min magnetization prepared, rapid acquisition gradient echo T1-weighted image  was acquired covering the whole brain for spatial normalization and anatomical overlay of functional data www.fmrib.ox.ac.uk/fsl). Images were slice time-corrected, motion-corrected to the median image using tri-linear interpolation with six degrees of freedom fMRI data were preprocessed and analyzed using FEAT, part of FSL  with local autocorrelation correction high MSV>low MSV contrast) would have a weaker or negative functional relationship with the brain regions required for successful memory encoding of the videos, specified by the low MSV>high MSV contrast in the High MSV condition than the low MSV condition. In the first step of this analysis, based on methods detailed by Gitelman et al. The PPI analysis was designed to test the hypothesis that greater visual processing would interfere with memory encoding of the ads. Thus, brain regions most associated with visual stimulation , we utilized a second functional bilateral mask as a target region of interest (ROI). This seed mask was created by using the voxels surviving cluster correction (z>2.3 p<0.05) in the low MSV>high MSV contrast. As seen by the red-tinted voxels in To compare the strength and direction of averaged interactions involving the OCC (seed) and the MTG/IFG (ROI target) between low MSV vs. high MSV conditions, we performed a second-level t-test analysis using a procedure modified from Hartley et al To further characterize differences between the two PPI distributions, we divided both conditions’ voxel distributions into two sets of 5 quintiles and used paired t-tests to compare the means within each pair of corresponding quintiles. This analysis involved the following procedure: Using the OCC mask as the seed region, a PPI z-score was determined for each voxel within the MTG/IFG  for each subject. Within the target ROI, the voxel/seed PPI distributions were broken into two sets of quintiles, one set for each condition.The goal of this analysis was to determine whether differences in interaction between low MSV versus high MSV were driven by (1) a relative increase in positively interacting voxels during low MSV, (2) a relative increase in negatively interacting voxels during high MSV, or (3) a combination of these mechanisms. We also sought to determine if low MSV’s more positive PPI mean could be associated with a weaker overall associative relationship between the seed and target ROI relative to high MSV, a result which would disprove our hypothesis that low MSV videos improve the cooperation between regions involved in input of sensory information and downstream encoding of this information.A two-way repeated ANOVA revealed that memory performance was significantly better during the immediate post-task FRT session than the 3-week delayed session , and low MSV ads were bettered remembered than high MSV ads , but no interaction between Session and MSV . Subjects responded significantly less conservatively during low MSV than high MSV ads . There were no significant bias differences between sessions . An independent t-test on gender revealed no significant differences between males vs. females in discrimination or response bias . Examining the pre- and post-task CAS measure, there was no main effect of PSA exposure  or interaction of task and gender . There was a strong main effect of gender, with females exhibiting more positive condom attitudes than men .Data were thresholded at Z = 2.3 and cluster-corrected for multiple comparisons at p<0.05. Compared to the high MSV videos, low MSV videos were associated with greater response in the prefrontal cortex, including the inferior and middle frontal gyri, and the temporal cortex, including the bilateral middle temporal gyrus . There wAveraging the z-scores (interaction with OCC seed) across all MTG/IFG voxels revealed that both conditions shared a negative overall PPI, with low MSV less negative than high MSV (−0.46 vs. −0.95). This finding was marginally significant .The analysis by quintiles revealed an asymmetrical voxel distribution, as reflected by the increasing asymmetry between low MSV and high MSV from Quintile 1 to Quintile 5. That is, while the MTG/IFG voxels with the most negative z-score interactions with OCC during the low MSV condition did not differ from that during high MSV , the most positive quintile of voxels was significantly greater during low MSV than high MSV . As seen in This study extends findings from anti-smoking PSAs The symmetrically bilateral occipitoparietal activation associated with high MSV ads, extends findings first reported in Langleben et al. Finally, we used PPI to explore the hypothesis that relative to high MSV videos, low MSV videos allow for more efficient transfer of visual information inputs processed in the OCC and subsequently encoded in the MTG . By contrast, we hypothesized that high MSV videos should be associated with reduced interaction between these regions by reallocating brain processing resources away from the regions involved in processing and remembering ad messages For the most part, the distribution of PPI z-scores was negative between the regions of interest during viewing of either high MSV or low MSV videos. Studies disagree to what extent the negative interactions between the occipital and rostral cortices are part of normal brain activity. There is some evidence that negative interactions may imply a dysfunctional inhibitory process. For example, relative to healthy control subjects, hyperactivation of the ventrolateral prefrontal cortex in patients with schizophrenia has been found to diminish top-down cognitive control normally mediated by the anterior cingulate cortex Despite the controversy about whether to interpret negative interactions as evidence of increased or decreased functional connectivity, our PPI analysis shows the negative interaction between the OCC and MTG/IFG regions to be weaker during the low MSV than high MSV at a marginally significant (p<0.06) level. This was especially prominent at the upper end of the PPI distribution , suggestAlthough our results suggest that low MSV ads are more efficacious than the high MSV ads, they come with a number of caveats and limitations. Similarly to prior neuroimaging studies In conclusion, we confirmed prior reports


Micropterus salmoides) to quantify the magnitude of intraspecific variability in functional traits and niche  between age classes. Stable isotope analyses (δ13C and δ15N) were also used to determine the association between individual trophic ecology and intraspecific functional trait variability. We observed that functional traits were highly variable within the population (mean coefficient variation: 15.62% ± 1.78% SE) and predominantly different between age classes. In addition, functional and trophic niche overlap between age classes was extremely low. Differences in functional niche between age classes were associated with strong changes in trophic niche occurring during ontogeny while, within age classes, differences among individuals were likely driven by trophic specialization. Each age class filled only a small portion of the total functional niche of the population and age classes occupied distinct portions in the functional space, indicating the existence of ontogenetic specialists with different functional roles within the population. The high amplitude of intraspecific variability in functional traits and differences in functional niche position among individuals reported here supports the recent claims for an individual-based approach in functional ecology.Investigations on the functional niche of organisms have primarily focused on differences among species and tended to neglect the potential effects of intraspecific variability despite the fact that its potential ecological and evolutionary importance is now widely recognized. In this study, we measured the distribution of functional traits in an entire population of largemouth bass ( Biodiversity studies have primarily focused on the role of species richness , a top predator freshwater fish species that has been introduced in more than 50 countries and reported to display ecological impacts (Cucherousset and Olden 2 private pond where angling was prohibited. This pond has been stocked approximately 15 years before sampling with largemouth bass and roach (Rutilus rutilus), a native cyprinid prey fish. Seine netting was used in July 2010 to collect largemouth bass and the pond was fully drained in October 2010 for maintenance purposes, ensuring that all individuals of the population were captured . Collected specimens were euthanized using an overdose of anesthetic and preserved at −18°C.Largemouth bass (M), standard body length (Bl), body depth (Bd), caudal peduncle minimal depth (CPd), maximal caudal fin depth (CFd), caudal fin surface (CFs), eye diameter (Ed), distance between the center of the eye to the bottom of the head (Eh), total gut length (Gl), maximal gill raker length (GRl), head depth along the vertical axis of the eye (Hd), distance from the top of the mouth to the bottom of the head along the head depth axis (Mo), distance between the insertion of the pectoral fin to the bottom of the body (PFi), body depth at the level of the pectoral fin insertion (PFb), pectoral fin length (PFl), pectoral fin surface (PFs), body width (Bw), mouth depth (Md), and mouth width  at the Cornell Isotope Laboratory .In the laboratory, a set of 19 measurements describing the morphological characteristics of individuals was performed on each specimen directly using a scale and a digital caliper or through picture analyses (ImageJ). This set included mass  based on the minimum number of individuals within the three age classes. In addition, we tested whether intraspecific variability affects the estimates of niche size obtained with a restricted number of individuals. We thus computed functional niche size on 10,000 random subsets of 15 individuals from the entire population. This sample size is similar to the number of individuals considered per species in study on functional diversity in fish communities  on the functional traits measured on all individuals  and trophic niche position differ between age classes using PERMANOVA and Kruskal–Wallis tests. Stable isotope niche size and niche overlap were quantified using the convex hull area (TA) in δ13C-δ15N bi-plot space  were tested using Pearson correlations with Bonferroni corrections for multiple tests. All statistical analyses were conducted in R , age-1 (n = 64) and ≥age-2 (n = 8), respectively, and total length did not overlap between age classes. High intraspecific variations were observed for each of the 16 functional traits  than age-0 (n = 33) individuals in the population, they had a smaller observed functional niche size than age-0 individuals. The smallest observed functional niche size was displayed by ≥age-2 individuals , 7.90 ‰ (±0.06 SE) and 9.41 ‰ (±0.17 SE) for age-0, age-1 and ≥age-2, respectively) significantly differed between age classes , −26.74 ‰ (±0.14 SE) and −26.67 ‰ (±0.21 SE) for age-0, age-1, and ≥age-2, respectively) differed significantly between age classes  filled slightly less trophic space than age-1 individuals (TA = 6.39 and SEAc = 1.68) and these two age classes filled more trophic space than ≥age-2 , each age class could also be considered to occupy distinct trophic niche as there was no or only little trophic niche overlap between age classes .Trophic niche position significantly differed between the three age classes  and to δ15N values .The present study demonstrated that the variation in the functional attributes of individuals within a top predator population was high, principally driven by ontogenetic shifts (differences among age classes) coupled to interindividual variability (differences within age classes). Overall, the level of overlap in functional niche among the three age classes was null or extremely low. Specifically, the patterns of ontogenetic niche shift demonstrated that age-0 individuals overlapped only slightly with age-1 individuals that displayed a totally distinct niche than ≥age-2 individuals. Additionally, we found that age-0 and age-1 individuals significantly differed in terms of functional niche size, that is, the amount of space occupied in the functional space. All of these observations indicated that these three age classes should be considered as distinct functional entities when investigating the functional properties of populations or the functional diversity of communities as this source of variation could disproportionally affect the estimates of functional diversity. Furthermore, variations of 16 functional traits within the population were associated with significant changes in stable isotope values. A vast majority of animal species may modify their trophic resource use during ontogeny  preying upon invertebrates or insects in the littoral zone (Winemiller δ13C values.Particularly, there was a strong association between ontogenetic trophic niche shift and several functional traits. The significant positive relationship between ish Post . During ish Post  to mainlish Post , leadingSeveral studies have demonstrated that intraspecific variation in functional traits could be negligible compared to interspecific variation (McGill et al.


Hypertension is common after acute stroke onset. Previous studies showed controversial effects of early blood pressure (BP) lowering on stroke outcomes. The aim of this study is to assess the effects of early BP lowering on early and long-term outcomes after acute stroke.A meta-analysis was conducted with prospective randomized controlled trials assessing the effects of early BP lowering on early and long-term outcomes after acute stroke compared with placebo. Literature searching was performed in the databases from inception to December 2013. New evidence from recent trials were included. Outcomes were analyzed as early (within 30 days) and long-term (from 3 to 12 months) endpoints using summary estimates of relative risks (RR) and their 95% confidence intervals (CI) with the fixed-effect model or random-effect model.p = 0.03). However the results showed that early BP lowering had no evident effect on early neurological deterioration, early death within 7 days, long-term death, early and long-term dependency, early and long-term combination of death or dependency, long-term stroke recurrence, long-term myocardial infarction and long-term CVE.Seventeen trials providing data from 13236 patients were included. Pooled results showed that early BP lowering after acute stroke onset was associated with more death within 30 days compared with placebo (RR: 1.34 and 95% CI: 1.02, 1.74, The new results lend no support to early BP lowering after acute stroke. Early BP lowering may increase death within 30 days after acute stroke. Elevated blood pressure (BP) is common in acute phase of stroke onset; about more than 75% of patients with acute stroke have elevated blood pressure at presentationPrevious studies about the effects of early BP lowering on the outcomes after acute stroke had given conflicting results. Several randomized controlled trials (RCTs) suggested that early BP lowering after acute stroke was safeFor recent years, some new studies with large sample focusing on the early BP lowering after acute stroke have still noted inconsistent results: the COSSACS study which enrolled patients with either ischemic stroke or hemorrhagic stroke, and the PRoFESS study which enrolled patients with ischemic stroke suggested safe but non-significant benefit of early BP interventionWe conducted the meta-analysis by reviewing published literature. We searched the following data sources: MELINE via Pubmed (from 1966 to December 2013), Science Citation Index , Cochrane Database of Systematic Reviews (CDSR) and the Cochrane Central Register of Controlled Trials (CENTRAL) with the search terms, key words or medical subject heading which included “blood pressure lowering”, “hypertension”, “anti-hypertension”, “antihypertensive” and “acute” and “stroke”, “cerebrovascular disease”, “cerebral infarction”, “brain infarction”, “cerebral ischemia”, “brain ischemia”, “cerebral hemorrhage”, “intracranial hemorrhage” and “randomized controlled trial”. The search was limited to trials in human beings and that published in English. In addition, we also screened the references of identified articles and previous reviews or meta-analyses on blood pressure lowering in acute stroke.We included the eligible trials which met the following criteria: (1) participants were aged 18 years or older with acute ischemic or hemorrhagic stroke; (2) study design was prospective randomized controlled trial; (3) intervention compared with placebo was initiated within 7 days of stroke onset; (4) intervention aimed to lower blood pressure or intervention achieved BP reduction; (5) one or more functional outcomes were reported, such as death or dependency. We excluded the studies with the patients of subarachnoid hemorrhage, studies without available full-text or relevant data, studies about ongoing trials and those written in languages other than English.We extracted the data from each trial by reviewing published papers and even requesting detailed information from original authors if needed. Outcomes including early neurological deterioration (END), death, dependency, death or dependency, stroke recurrence, myocardial infarction and combined vascular events (CVE) were collected. We defined END and dependency as the original authors' definition in each study , and we stratified the outcomes to two phases as early outcome (within 30 days) and long-term outcome (3 to 12 months). We also defined CVE as the original authors' definitions .Two authors (HW and XR) conducted the literature searches, data extraction and study assessment independently. Any disagreement in study selection or data collection was discussed by HW, XR and YT until agreement was reached. We also discussed the quality of trials such as randomization, blinded method, eligibility criteria and intention-to-treat analysis before they were finally included.2 statistic. Potential heterogeneity was explored in estimates of treatment effect with univariate meta-regression and by comparing summary results obtained from the studies. A two-sided p value less than 0.05 was regarded as significant for all analyses. If the value of I2 was >50% in the analysis, the heterogeneity between studies was defined as significant, and the random-effect model was used for analyses. Otherwise, fixed-effect models was used. Funnel plots were used for analyses of bias. The Cochrane Collaboration's Review Manager software package  was used for the meta-analyses.Individual study relative risks (RR) and 95% confidence intervals (CI) were calculated from event numbers extracted from each trial before data pooling. In calculation of risk ratios, the total number of patients randomly assigned in each group was used as the denominator. Summary estimates of RR ratios were obtained with fixed-effect models or random-effect models. The percentage of variability across studies attributable to heterogeneity beyond chance was estimated with the IThe literature search and the review of previous meta-analyses or reviews finally yielded 17 studies which met the inclusion criteria . The chaThe trials had a sample size ranging from 18 to 4071 patients, of which eleven were multi-centre randomized controlled trials (RCT) and the others were single centre RCT. Eight trials2I = 0%, p>0.05).Six trials mentioned early neurological deterioration (END) after early BP lowering in acute stroke . Of them2I = 72%, p = 0.03) and it was due to the discrepant data from the BEST trials, however, there was no clinical reason to exclude it from the analysis and no other potential subgroup analysis to eliminate the heterogeneity.Three studies recorded early death within 7 days after early BP lowering in acute stroke, which enrolled the patients within 36 to 72 hours after stroke onset . Overallp = 0.03, without significant heterogeneity between studies). Considering the broad span of admission time after stroke onset in the trials included in the analysis, we narrowed the inclusion criteria for the remaining 10 trials which enrolled the patients within 24 to 72 hours after stroke onset (excluding the Willmot's study), and the pooled results were not changed and the adverse effect of early BP lowering on death within 30 days was still evident (data not shown).Eleven studies followed up early death for 14 to 30 days after early BP lowering in acute stroke, which enrolled the patients within 24 to 120 hours after stroke onset . Pooled Fourteen studies followed up long-term death from 3 to 12 months after acute stroke, which enrolled the patients within 6 to 120 hours after stroke onset . No effeFive studies reported early dependency and early death or dependency, which enrolled the patients within 24 to 72 hours after stroke onset . Pooled Nine studies reported long-term dependency and long-term death or dependency, which enrolled the patients within 6 to 120 hours after stroke onset . Pooled Seven studies considered the long-term effect of early BP lowering on stroke recurrence and combined vascular events (CVE)  and six We used funnel plots to evaluated the publication bias. It showed no evident publication bias in the majority of outcomes such as END within 72 hours, END within 14 days, early death within 30 days, long-term death, early and long-term dependency, early and long-term combination of death or dependency and long-term stroke recurrence (data and plots not shown). Asymmetric funnel plots were shown in few results such as early death within 7 days, long-term MI and long-term CVE, which just only yielded neutral results in our analysis (data not shown).Whether to lower high blood pressure early after acute stroke or not still remains debated. One cochrane meta-analysis failed to analyze the effect of BP lowering on stroke because the author could not obtain enough dataThis analysis assesses early neurological deterioration after early BP lowering in acute stroke, which has not been discussed in previous reviews or meta-analyses. How ever, limited trials reported relevant data so that no certain effect can be found. So far, we could not draw any conclusion that early BP lowering after acute stroke is safe enough. More RCTs concerning BP lowering on early neurological deterioration within 72 hours are needed.Considering potential confounding effect in previous meta-analyses because they included several trials without achieving BP reduction indeed  of the patients were enrolled within 72 hours after stroke onset and the intervention or placebo was assigned in the acute phase of stroke. Further more, the PRoFESS study was a 2×2 factorial design trial, which also had sufficient statistical power to detect the independent effect of BP lowering on outcomes after stroke. Therefore, the PRoFESS subgroup analysis can reasonably represent a single trial to test the effects of BP lowering on outcomes after acute stroke. On the other hand, the period of intervention in the PRoFESS study was longer (90 days) than other studies because of its initial aim for the secondary stroke prevention. We can not exclude the confounding effects due to mixed effects of short-term and long-term BP lowering. In our analysis, we found that the data from PRoFESS study showed different trends of effects of BP lowering on early death within 30 days after acute stroke  and longThis updated meta-analysis differentiate from previous studiesHowever,there are still several limitations in this analysis. Firstly, we could not obtain enough the data from the original authors, so several pooled outcomes could not be evaluated with sufficient studies and data, and it may affect our results more or less. Secondly, we could not make further subgroup analysis between ischemic stroke and hemorrhagic stroke because of the unavailable data of individual patients in each trial. But we did a crude subgroup analysis by different types of stroke with insufficient data and found that early BP lowering had a trend to increase early death within 30 days in ischemic stroke, which was similar to the combined result, however, no trend could be found in hemorrhagic stroke (data and figure not shown). Thirdly, we also could not make further subgroup analysis of the effects between different timing of intervention for the above reason. Finally, we could not assess the effects of different types of antihypertensive agents because of limited data available for detailed subgroup analyses. Therefore, more studies concerning the above problems are required in future and they will help to clarify the specific question more clearly on BP lowering after acute stroke, such as whether or when to intervene and which agent to use.In conclusion, this updated meta-analysis with new evidence lend no support to early blood pressure lowering after acute stroke. It indicates that early BP lowering significantly increases death within 30 days after acute stroke.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


Blockade of inhibitory immune checkpoints (ICPs) improves survival in melanoma patients. Interestingly, the expression of these ICPs in most tumor tissues including melanoma is restricted to only a subset of tumor-infiltrating immune cells (TIICs). Here, we utilize single cell mass cytometry (CyTOF), functional profiling and TCR sequencing of freshly isolated cells to evaluate the phenotypic, functional and genetic heterogeneity of TIICs in patients (n=43) with advanced melanoma to define the subsets of immune cells involved in ICP-mediated regulation.RM). Expression of some ICPs  in TIICs is primarily restricted to TRM cells and to CD16+ subset of inflammatory/patrolling myeloid cells. Functional analysis of freshly isolated tumor T cells using multiplex luminex ELISA revealed that despite expression of multiple ICPs, tumor-associated T cells are functional, but have an altered cytokine profile compared to circulating T cells. In contrast to blood T cells, tumor infiltrating T cells secrete lower levels of IL-2, IFN-γ and TNF-α, but comparable levels of IL4, IL5 and IL17.Using paired blood and tumor samples from melanoma patients and CyTOF analysis with 36 different markers, we show that T and myeloid cells within tumor metastases are distinct from their circulating counterparts. T cells within metastases are highly enriched for CD45RO+, CD69+, CD103+ tissue resident CD8+ memory T cells (TRM cells without recirculation has been extensively demonstrated in the form of lack of equilibrium in parabiotic mice that share a systemic circulation. The finding that melanoma metastatic lesions are enriched for TRM cells suggested the possibility that each of the metastatic lesions may contain a distinct microcosm of T cells without equilibration, in spite of shared systemic circulation within the same host, analogous to the biology in parabiotic mice. In order to test this directly, we compared the TCR sequences of memory CD4 and CD8 T cells isolated from patients with biopsies of multiple metastatic sites. Using high throughput deep sequencing of rearranged TCRβ loci, we find that each biopsy site has a distinct TCR usage with only partial overlap with the other metastatic site from the same patient.Tissue restriction of murine TTogether these data suggest that immunity to melanoma is highly regional and enriched for tumor-resident memory T cells creating genetically unique immune-microenvironments within each metastatic lesion. Regional nature of immunity within metastases has several implications for current strategies to harness and measure tumor immunity.