Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using  Early detection of blood stream infections is essential for providing effective treatments. Here the authors present a system integrating DNAzyme sensors, droplet microfluidics and a high-throughput 3D particle counter that can detect specific, single bacterial cells in blood within a few hours. Blood stream infections (BSIs) are a major cause of morbidity and mortality. Sepsis resulting from a BSI annually affects over 18 million people worldwide and 700,000 in the United States, with a mortality rate of 30–40% refs  Sepsis a1124126891011121314151617We present herein a platform technology called Integrated Comprehensive Droplet Digital Detection (IC 3D) that is able to selectively detect bacteria from millilitres of unprocessed  blood at single-cell sensitivity in a one-step, and culture- and amplification-free reaction within 1.5–4 h.in vitro evolution to specifically react with the lysates of target bacteria, leading to a rapid fluorescence signal20Our IC 3D system integrates real-time DNAzyme sensor technology, droplet microfluidics and a high-throughput 3D particle counter system . DNAzymein vitro from a vast random library using a combinatorial approach called in vitro evolution2422in vitro evolution of a vast DNA library against crude extracellular matrix (CEM) components of target bacteria as complex targetsDNAzymes  are synthetic single-stranded DNA oligonucleotides with catalytic activities22E. coli used herein as a model system, DNAzymes will bind to target molecules produced by bacteria and cleave the substrate. The cleavage event  and was not statistically different than other clinical control isolates . Nonetheless, this points out the need to rigorously validate the sensor specificity using larger number of patient samples in the future. This also suggests that by including appropriate positive and negative selection targets in the in vitro evolution process, it is feasible to generate DNAzyme sensors that can distinguish different strains of the same bacterium species, which will need to be validated in the future work. As our goal is to develop a ‘mix-and-read’ assay that uses whole blood with no or minimal sample processing , we further tested the sensor performance in blood and found that our Fluorescein/Dabcyl-modified DNAzyme sensors produced a sufficiently high fluorescence signal in response to E. coli spiked in blood that was diluted by sensor solution to various volume ratios  and shorten the detection time. The ‘droplet microfluidics’ system enables the generation and manipulation of monodisperse, picolitre-sized  liquid droplets in an immiscible carrier oil fluid 2829303132103131In our study, droplet microfluidics were fabricated using standard soft lithography and operated following previously established procedures (see ‘Fabrication of droplet-based microfluidic device’ in Methods)−1), when encapsulated in picolitre droplets, each droplet will contain one or no bacterium. Therefore, it is critical to test if our system is able to detect a single bacterium in a droplet. Specifically, bacteria were statistically diluted to achieve a range of concentrations from 10 to 107 ml−1 spiked in HEPES buffer or blood and compartmentalized in droplets. In some experiments, bacteria were stained with Syto17 (red), which allows us to co-localize the bacterium with the DNAzyme sensor signal (green) in the same droplet to determine false-positive and -negative rates. In this section, fluorescent droplets were imaged by conventional fluorescent  microscopy or counted on chip in a 1D microfluidic channel at a throughput of ~200 droplets per s using a custom-built confocal microscope equipped with APD detectors (see ‘1D on-chip detection system’ in E. coli K12 that is lysed in a droplet (5 μm in diameter) within 8 min  from ~70,000 droplet counts in triplicate experiments, which we performed using E. coli K12 as positive target and sensor alone or control bacteria as negative controls  of the cuvette. The excitation light generated by a diode laser (469 nm) is focused at the volume of observation that is typically positioned relatively close to the inside wall of the cuvette. The emission from the sample is collected by the same objective, transmitted through the set of dichroic filters, focused by a lens into a pinhole and then collimated by a second lens to the photomultiplier tube (PMT). The optics of the microscope is designed to measure a relatively large volume (100 pl) in about 0.01 ms. The rotation of the tube in a spiral motion for about 100 s allows us to effectively explore about 1 ml of the tube. We emphasize that using this optical setup, we are penetrating only 150 μm into the sample. Therefore, strongly scattering samples such as whole blood (even before dilution) that have a transmittance at 500 nm of about 10% for a 250-μm path length can be easily handled.We have demonstrated above that the encapsulated DNAzyme sensor system can detect target bacteria in a rapid manner with single-cell sensitivity in the droplet. However, the 1D on-chip droplet counting system  and other particle counting systems including flow cytometry suffer from low throughput: they typically operate at 1,000 s particles per s and are only able to analyse a total of 100,000 s to 1 million droplets 31E. coli K12 and DNAzyme sensors can be detected at single-droplet sensitivity from a typical 2 ml sample volume within 3 min measurement time , large volume biological samples. The IC 3D system simultaneously satisfies numerous important bioanalytical parameters including sensitivity, selectivity, assay time, throughput and robustness, which is a long-standing unmet challenge in biodetection.in vitro selection, which can be used to quickly generate specific DNAzymes for, in principle, any complex targets, including slowly growing organisms . Given that BSIs and sepsis can be caused by several different types of bacteria and fungi, our ongoing effort aims to expand the sensor set through in vitro DNAzyme sensor selection to detect the pathogen species that are most commonly recovered from BSIs. In particular, the nonbiased screening using bacteria as a complex target without prior knowledge of any specific target molecules bypasses the tedious process of purifying and identifying target molecules from extremely complex mixtures and permits the rapid development of sensors for new bacterial strains in an unanticipated outbreak. This addresses a major challenge faced by existing techniques including PCR that rely on the detection of pre-identified target genes or other biomarkers given the rapid and complex evolving mechanisms associated with bacteria. Although the identification of specific bacterial biomarkers that bind to DNAzymes to trigger substrate cleavage is neither necessary for our assay to operate nor the focus of this paper, they can be identified by using affinity purification coupled with mass spectrometry, which is part of our ongoing work.In our proof-of-concept system, the real-time and ‘mix-and-read’ DNAzyme sensors were obtained by Moreover, the compartmentalization of a single bacterium in a droplet significantly increases the concentration of target molecules, permitting rapid detection and single-cell sensitivity without signal amplification processes such as PCR. Compartmentalization of target-specific reactions is a critical step to ‘light up’ the droplet ‘reactors’ that contain target bacteria so that they can be detected by the 3D particle counting system.Furthermore, our 3D particle counting system for single-droplet detection in millilitre volumes within minutes bypass many challenges faced by 1D on-chip counting systems and flow cytometry that suffer from limited throughput and high false-positive/negative rates. The exceptional reliability, accuracy and throughput uniquely distinguish the IC 3D from the conventional droplet microfluidic systems . The IC 3D can provide absolute quantification of target bacteria in blood at a broad range of concentrations from 1 to 10,000 bacteria per ml within ~1.5–4 h (droplet generation (≪40 min)+DNAzyme sensor reaction (~45 min for ‘yes or no’ and ~3.5 h for absolute quantification)+3D particle counting (3–10 min)+data processing (5 min)) with single-cell sensitivity and an exceptional LOD in the single digit regime. We summarized the major performance specifications in We believe that the IC 3D system would be relatively inexpensive compared with existing analytical equipment including PCR and flow cytometry. For instance, according to the potential manufacture of this instrument, the predicted cost of all parts of the 3D particle counter is about $1,000 including optics, detection PMT and computer interface see .Furthermore, to enable multiplex and parallel detection of multiple pathogens, we plan to develop a device comprised of multiple laser sources and detectors capable of reading at different wavelengths. The multiplex system would permit simultaneous reading of multiple sensors (labelled in different colors) coded for different pathogens. A carousel could also be added to our apparatus to accommodate multiple sample vials for carrying out parallel tests.Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae that are among the most prevalent antimicrobial-resistant pathogens.Finally, future integration of other sensing methods  with droplet microfluidics and a 3D particle counter may potentially serve as a platform for rapid detection and analysis of almost any type of low abundant markers in biological samples including cells , extracellular vesicles , viruses  and molecular markers . In particular, the IC 3D system can potentially integrate fluorogenic substrates2O. Next, 20 U of T4 DNA ligase was added and the mixture was incubated at room temperature for 1 h. The ligated DNA products in the mixture were concentrated by ethanol precipitation and purified by 10% denaturing polyacrylamide gel electrophoresis. A mutant DNAzyme, used as a control, was also synthesized (mRFD-EC1: 5′-CACGCTGTACGGATGGAGTCGCGAGCCTGCGACCGGAAATGAAAGATCTTTCGCGTTTTGCTCATGCGATGGATTTTTTACAGTGGGCAGCTCCGTCCG-3′) and ligated to the substrate as described above.The DNAzyme construct used in this study consists of a fluorogenic substrate ) and a catalytic sequence (RFD-EC1: 5′-CACGGATCCTGACAAGGATGTGTGCGTTGTCGAGACCTGCGACCGGAACACTACACTGTGTGGGATGGATTTCTTTACAGTTGTGTGCAGCTCCGTCCG-3′)A schematic of the fluidic chip design and operating mechanism is shown in For the high-throughput droplet generation , two PDME. coli K12 or clinical isolates were obtained through serial dilution or, at extremely low numbers (1–50), through a microinjection system, and were spiked in 20% fresh donor blood. Bacteria-spiked blood sample was then microencapsulated with DNAzyme sensor and bacterial lysis agent (lysozyme) using a droplet microfluidic device. Unprocessed whole blood can also be directly encapsulated if diluted by the sensor solution to various blood concentrations to reduce the background signal. On-chip dilution is well established39−1 lysozyme in 10% blood.Different cell numbers of −1. Briefly, the 20% blood samples were prepared by diluting with phosphate-buffered saline  then spiking with E. coli K12 cells. The DNAzyme solution was prepared at a concentration of 500 nM in 2 × RxN buffer containing 2 mg ml−1 lysozyme. The bacteria-spiked 20% blood samples and DNAzyme solution were injected via respective inlets at flow rates ranging from 0.5 to 3 μl min−1, whereas the oil phase was injected at flow rates ranging from 1 to 5 μl min−1. Uniform picolitre-sized droplets were generated at a rate of approximately 2,000 Hz by flow focusing the resulting continuous (oil) phase. To optimize the DNAzyme reaction, different-sized droplets (from 5 to 100 μm in diameter) were generated by tuning the microfluidic channel size and flow rate. A 2-mm magnetic stir bar was placed inside a syringe and was gently rotated by a portable magnetic stirrer (Utah Biodiesel Supply) to prevent precipitation of the blood cells. High-throughput blood encapsulation was achieved by the two-layer droplet microfluidic device with an eight-channel droplet generation structure (flow focusing) on the bottom layer and three inlets on the top layer (two for blood sample and one for DNAzyme solution). The 20% blood samples were injected through the two inlets at a flow rate of 10 μl ml−1 then split into eight flow streams. Each blood stream was mixed with 500 nM DNAzyme in 2 × RxN buffer containing 2 mg ml−1 lysozyme that was injected through the other inlet. This aqueous mixture was then passed down to the bottom layer through the interlayer connecting hole to the eight-channel flow focusing structure into the oil phase (injected from the bottom layer).Specifically, for microencapsulation of blood samples via the droplet microfluidic device, HFE-7500 fluorocarbon oil (Dow Corning) containing 1.8% (w/w) perfluorinated polyethers with polyethyleneglycol surfactant was used as a continuous phase. Aqueous and oil phases were injected into the microfluidic device via pressure equalization tubes . In all microfluidic experiments, PHD 2000 syringe pumps (Harvard Apparatus) were used to inject liquids at flow rates ranging from 0.5 to 5 μl min−1 range. The vertical and rotational motions are produced, respectively, by the Linear Actuator series  and a VEXTA stepping motor model PK233PB . These motors are connected to a stage holding the transparent cuvette containing the sample. The excitation light generated by lasers is focused at the volume of observation. The excitation focus is positioned inside the cuvette and relatively close to the wall of the cuvette, at a distance of about 1 mm from the wall. This distance can be adjusted so that detection of particles and analysis could be done even in highly scattering media.A 3D particle-counting prototype instrument was built to our specifications (ISS Inc), comprised of a two-channel setup to allow simultaneous red and green fluorescence detection for the rapid quantification of the total number of fluorescent particles in a large volume sample . The appThe excitation sources are two diode lasers emitting at 469 nm (ISS Inc) or at 532 nm (Aquaplan). Thus, a particle fluoresces when in the volume of observation. The use of a confocal microscope in combination with simple mechanical motions of the sample container in front of the objective provides the means to move and analyse a sample containing particles through an observation region without requiring a complex optical system comprised of moveable optical components, such as translating optical sources, mirrors or photodetectors. The excitation light from the two lasers are combined in one path through a set of dichroic filters ZT532nbdc and Z470rdc (Chroma Technology Corporation) and directed through a 20 × 0.4 numerical aperture air objective (Newport) to the same volume of excitation.Fluorescence emitted from the sample is collected by the same objective, transmitted through the set of dichroic filters, focused by a lens into a large pinhole (diameter =2 mm), and then collimated by a second lens to the detectors. A dichroic beam splitter T550lpxr-25mmNR (Chroma Technology Corporation) separates the emission beam into two light paths before its detection by two PMTs . Two emission filters  are located in front of each PMT. The signal from the PMT is sent to the analog to digital converter and to the acquisition card (IOTech). The sampling frequency is set to 100,000 Hz, corresponding to a time resolution of 10 μs. We systematically optimized the performance of 3D particle counter including various ranges of PMT (200–800), RPM , droplet size  and scanning time (1–10 min).E. coli K12 or clinical isolates were obtained through serial dilution or, at extremely low numbers (1–50 CFU), through a microinjection system  were spiked in 100 μl of 20% fresh donor blood. For microinjection experiments, specifically, cultured E. coli K12 were diluted with fresh and cold LB (to reduce their mobility) from 107 to 109 times to be easily isolated as single cells in the Luria Broth (LB). To collect a bacterium, a glass microcapillary was loaded in a manual microinjector  and the tip of the capillary was adjusted to the position of the target cell under a microscope  using a TransferMan NK2. Single E. coli K12 cells were sucked out one by one manually by moving the xyz stage and subsequently were added into blood. Bacteria-spiked blood sample was then microencapsulated with 250 nM DNAzyme using the microfluidic device. In a typical experiment, 100 μl of generated droplets were then mixed with 1.9 ml of pre-made 10% blood droplets (with DNAzyme sensors but not bacteria) in a cuvette and incubated for various time at room temperature. Following the incubation, 2 ml of droplets were analysed for 3–10 min using 3D particle counter at a rotating speed of 400 r.p.m. while translating vertically at a speed of 10 mm s−1. Note that that the time of the measurement can be expanded to decrease the errors if necessary20Droplets containing bacteria in the blood sample, DNAzyme sensor and lysozyme were collected in a cuvette following droplet generation. A 2-ml collected droplet solution contains bacteria at various numbers ranging from 2 to 200,000 cells . Specifically, different cell numbers of In our 3D particle counting system, the detection of a ‘hit’ is based on a pattern-recognition algorithm .Supplementary Figures 1-7, Supplementary Tables 1-3 and Supplementary MethodsBlood droplet generation


Visual short-term memory (VSTM) enables the representation and manipulation of information no longer present in the sensorium. VSTM storage has long been associated with sustained increases in univariate activity  across a broad network of frontal and parietal cortical areas . Indeed, one alternative possibility is that V1–V4 encode stimulus-specific representations during distractor blocks, but at a level of anatomical or physiological granularity that is inaccessible to the multivoxel decoding approaches used in this study , r = −0.70) and sIPS (r = −0.59). This result suggests that both V1–V4 and sIPS contribute to VSTM storage. Instead, If V1–V4 and/or sIPS contribute to VSTM performance, then delay period activation patterns in these regions should correlate with participants’ memory performance . In ExpeIn our view, the data reported by


Alzheimer’s disease (AD) is a neurodegenerative disorder. The pathohistological features in AD are intracellular accumulation of neurofibrillary tangles and extracellular senile plaques. Plaque deposition leads to recruitment and activation of microglial cells, which induces neuroinflammation and drives neurodegeneration. Recent evidence show that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are highly neurotoxic and correlate with disease severity.Hence, preventing formation of soluble Aβ and its interaction with neurons is a major goal in AD. Despite massive efforts, how extracellular factors regulate assembly of Aβ peptide and neurotoxic activity of Aβ species is still largely undefined. Recent studies indicate that Extracellular Vesicles (EVs), including exosomes and PM-derived microvesicles (MVs), may influence Aβ neurotoxicity. Our findings reveal that production of microglial MVs (m-MVs) is strikingly high in patients with mild cognitive impairment and AD as compared to healthy controls and positively correlates with markers of neurodegeneration and hippocampal atrophy. Furthermore we found that MVs isolated from the CSF of AD patients are toxic to cultured hippocampal neurons. Through in vitro studies we demonstrate that the m-MVs promote generation of neurotoxic soluble species from almost inert Aβ aggregates, which is mediated by lipid components of MVs. Our findings suggest that m-MVs favor formation of neurotoxic Aβ species throughout the brain, possibly representing the mechanism behind transynaptic spread of Aβ in AD. On the other hand, studies conducted by Yuyama et al 2012, 2014 & An et al 2013 suggest that exosomes produced by neurons may exert opposite action by neutralizing neurotoxicity of soluble Aβ. To verify if the overall effect of exosomes and MVs on Aβ neurotoxicity may vary depending on parental cell type we are currently studying the influence of EVs (exosomes & MVs) derived from distinct brain cells on Aβ toxicity and assembly.


Artificial proteins, named αRep, binding tightly and specifically to EGFP are described. The structures of αRep–EGFP complexes explain how αRep recognize their cognate partner. Specific αRep can be used for biochemical or live cells experiments. A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes. Protein–protein interactions are essential to most biological functions in the cell. Information concerning localization, trafficking, activation states and interaction partners of proteins in living cells is crucial to understand the complexity of cellular networks. Technologies that allow modulating or preventing protein interactions offer powerful tools to investigate pathways but could also be applied to target deregulated signalling cascades in diseases .in vivo and are compatible with intracellular applications. For example, the selection of binders from naive libraries of ScFv allowed the generation of intrabodies able to detect specific conformations of the small GTPase ras-related in brain 6 (Rab6)  and a favourable negative −T∆S  contribution. The favourable entropic energy (−T∆S) suggests that binding is mainly driven by hydrophobic interactions. This result is corroborated by the structural analysis showing that complex formation involves 10 hydrophobic residues located between the N-cap and the fourth repeat (−1) and entropic contributions . The favourable enthalpic contribution reflects the important involvement of hydrogen bonds in this interaction, as corroborated by the structure: the bGFP-C–EGFP complex involves 165, 154 and 156 contacts .Although the binding affinities of bGFP-A and bGFP-C for EGFP are very similar, the crystal structures of the complexes showed they bind very differently to the EGFP protein. Despite their different binding modes, the two binders mainly interact with EGFP via the randomized surface residues situated on their concave surfaces. A more detailed analysis of the interfaces between the αRep proteins and EGFP revealed that different types of interactions stabilize the complexes. The thermodynamic parameters of binding obtained by ITC are presented in h repeat A. The thcontacts D with 9,In order to determine whether artificial αRep proteins could be used in living cells, we characterized the αRep–EGFP interactions in a cellular context.in vitro for the interaction, αRep proteins should be able to selectively bind their target within a complex mixture such as crude cell extract. To confirm this hypothesis, we set up a pull-down experiment. αReps can easily be expressed and biotinylated in E. coli by the addition of an Avi-tag sequence to the N-terminus. The individual previously purified biotinylated bGFPs and a non-relevant αRep used a control, were incubated with a cell lysate of HEK 293 cells expressing EGFP. αReps were captured on streptavidin-agarose beads and aliquots of the remaining unbound supernatants were kept for analysis (depleted lysate). Following washing steps, the bound αRep complexes were denatured with SDS and analysed by western blot using an anti-GFP antibody , a protein involved in Sfic αRep . The facThe comparison of the structures of GFP in complex with bGFP-A and bGFP-C is very instructive. Using the same protein fold containing randomized residues at the same positions, αRep proteins were able to form very different complexes with the same target protein. The GFP surfaces bound by the two αRep proteins only partially overlap. The extended concave surface of bGFP-A is sufficiently large to accommodate the cylindrical shape of EGFP with an unexpected orientation, interacting with one end of the cylinder as well as with side chains located along the side of the barrel.The structures of both αRep–GFP complexes clearly indicate that the target recognition is specific of the conformation of the native protein. This was also generally observed with binders selected out from other repeat protein libraries ,19,26,31The available structures of GFP/nanobodies ,48 can bThe superposition of all known αRep structures ,31 showsThese αRep-based GFP binders can be easily produced in large quantities and are very stable proteins. We show here that the affinity and specificity of three different αReps for their GFP target are sufficient to use them as new tools to purify GFP-fusion proteins from a cell extract in pull-down experiments.Natural HEAT repeat proteins are found in various organisms from prokaryotes to humans. The restricted sub-family used for the consensus design of αRep is however more common in prokaryotic species. It was thus unclear how those artificial proteins would behave in a eukaryotic expression system. The intracellular eukaryotic expression of αRep variants confirms that they can be stably expressed in mammalian cells. No specific localization could be observed for αRep proteins. They are soluble in the cell cytoplasm and can diffuse into the nucleus.All three αRep variants selected against the EGFP were able to co-localize with EGFP inside different cell compartments. Thus, αReps accurately report the localization of a target protein in living cells without forming any aggregates or causing toxicity, which are often observed when using intrabodies. Therefore, αRep proteins seem to be fully appropriate to explore intracellular processes in living cells, by interacting directly with endogenous proteins. As overexpression of recombinant protein binders inside the cell may induce a high background signal due to the presence of an excess of unbound binders. Strategies have been devised to match the expression level of the binder relatively to its endogenous target . Here, cDetailed structural information provided by the crystal structures will provide the basis for future, more elaborate design. For example, the EGFP-binding αRep described in the present study could be easily fused to other targeted components to create hetero-bifunctional intracellular reagents. In this respect, the high foldability (low propensity to aggregation) of these αRep binders is critical both for efficient production and for their future use as generic EGFP-binding domains in engineered multi-domain proteins.


The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end-of-century open ocean pH reductions. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different p CO2 levels for four weeks: 400 μatm (pH 8.0), 800 μatm (pH 7.7), 1000 μatm (pH 7.6), or 2800 μatm (pH 7.3).Ocean acidification as a result of increased anthropogenic COp CO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated p CO2. Elevated p CO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with p CO2, with numerous processes significantly affected by mechanical stimulation at high versus low p CO2 .At the end of the four week exposure period, oysters in all four p CO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of p CO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.Oyster physiology is significantly altered by exposure to elevated The online version of this article (doi:10.1186/1471-2164-15-951) contains supplementary material, which is available to authorized users. Calcite was undersaturated (Ωc <1.0) only at the highest p CO2 level and aragonite was undersaturated at the two highest p CO2 levels (1000 μatm and 2800 μatm).The nt Table . Averagep CO2, as measured by buoyant weight, was not significantly different among treatments (p >0.05)  shell length growth was negatively impacted by elevated p CO2 of 2400 and 4000 μatm  synthase), implying a relatively decreased need for readily available carbohydrate resources. At both p CO2, fewer lipid metabolism proteins were differentially abundant (four proteins at 400 μatm and three at 2800 μatm) compared to carbohydrate metabolism. Mechanical stimulation is a transient stress and carbohydrates represent a more readily available resource compared to lipids for a short-term response. The different changes observed in response to mechanical stimulation illustrate that an additional stress at high p CO2 necessitated greater resource use and a need to access energy reserves. Given the resources needed to sustain a response to an environmental stress, this increase in energetic demand could be detrimental.At the metabolic pathway level, there were different proteomic responses to mechanical stimulation between the two e stress  and livee stress . In foode stress . The samp CO2 treatment had decreased levels of many proteins associated with oxidative metabolism and had a much larger antioxidant response. Four mitochondrial NADH dehydrogenases were detected at lower levels after mechanical stimulation in the high p CO2 oysters, while at low p CO2 three were detected at higher levels and one was decreased. Expression levels of cytochrome b-c1 complex, cytochrome c, and cytochrome c oxidase subunit 5B were elevated in the 2800 μatm after mechanical stress (and were not affected at low p CO2). These abundance changes suggest that at high p CO2 mechanical stress caused a decrease in electron supply from NADH to the electron transport chain (ETC) but an increase in the transfer of electrons to the terminal oxidase, while at low p CO2 more electrons entered the ETC from NADH. In marine invertebrates ROS production occurs during forward electron transport  A, GST 3, GST μ 3, thioredoxin domain-containing protein 17, and dual oxidase 2) compared to just one protein at low p CO2 (GST Ω-1). These changes are further evidence that ocean acidification alters the cellular balance between energy resource supply and oxidative stress, especially upon exposure to an additional exogenous stressor.In contrast to the proteomic response to mechanical stimulation at 400 μatm, oysters from the elevated iewed in ) and a dp CO2 perhaps reflecting different resource availability in responding to the cellular damage inflicted by the additional stress. Changes to the normal apoptotic and stress responses could be detrimental to oysters, highlighting the importance of considering other environmental conditions when examining biological impacts of ocean acidification.Ocean acidification appears to significantly impact apoptotic and cellular stress responses of oysters exposed to mechanical stress  in combination with ocean acidification could have significant implications for survival and potential for adaptation.Stress from mechanical stimulation impacted abundance of proteins involved in many of the processes that were influenced by elevated p CO2 to better understand the physiological trade-offs that occur during this particular stress response. Shell growth was not affected by ocean acidification after one month, but elevated p CO2 did affect the integrity of the deposited shell material. Relative amounts of fatty acids and glycogen content, which are necessary for continued survival and execution of other energy-consuming processes, were also unaltered at elevated p CO2. Mortality in response to acute heat shock remained unaffected as well. The proteomic profile of C. gigas gill tissue was significantly altered by ocean acidification, elucidating the molecular physiological costs of elevated environmental p CO2. These changes in proteomic profile suggest that oysters experience shifts in their energy budget as they allocate resources to combat extended exposure to ocean acidification. From a proteomics perspective, ocean acidification also affected C. gigas’s response to mechanical stress. Shell integrity and response to a second stress become important in a dynamic environment, and in this way ocean acidification may decrease C. gigas fitness under chronic exposure. This research demonstrates the utility in applying proteomics technology to a study of environmental stress response in a wild population, which could lead to developing more targeted tools for studies of environmental toxicology and building models for response to p CO2 stress.Ocean acidification is frequently portrayed as being detrimental to marine calcifiers, but its effects on invertebrates range well beyond changes to the calcification process. In this study, a wide variety of processes and responses were assessed in the Pacific oyster’s response to elevated p CO2 values of 400 μatm, 800 μatm, 1000 μatm, or 2800 μatm. The system and control of water chemistry has been previously described in detail .All raw data accompanying this manuscript is also available through the data compilation on the biological response to ocean acidification through Ocean Acidification International Coordination Centre Additional file 1: Figure S1: Representative indents made during micromechanical testing for the (A) 400 μatm and (B) 2800 μatm p CO2 and 7 oysters from 800 μatm. (XLSX 66 KB)Additional file 2: Table S1: Raw and normalized (proportion) fatty acid data for 8 oysters each from 400 and 2800 μatm Additional file 3: Table S2: ProteinProphet output for each technical replicate. Information for each protein includes percent coverage by sequenced peptides, total number unique peptides, total independent spectra , and peptide sequences. (XLSX 28 MB)p CO2-exposed oysters); fold change for treatment/control oysters (i.e. Fold Diff OA is [2800 avg NSAF]/[400 avg NSAF]); columns for each of the three treatment comparisons with an asterisk indicating if the protein is >5-fold higher or lower levels; SwissProt annotation, e-value, and gene description; proteins responsible for enrichment and the treatment comparisons in which they are enriched; a column indicating in which stress treatment proteins are differentially abundant . In the fold difference columns “up” signifies that the protein was only detected in oysters from the 2800 μatm treatment , mechanical stress at 400 μatm treatment , or in the mechanical stress at 2800 μatm ; “down” represents proteins that were only detected in the other treatment for each comparison. (TXT 384 KB)Additional file 4: Table S3.: Protein abundance values (NSAF) for each oyster for the 1 616 proteins identified. Also included are average abundance values across treatments , and GO Slim annotations. (TXT 4 MB)Additional file 5: Table S4: p CO2 (B), and mechanical stress at high p CO2 (C). Red lines represent pathways that are more prevalent in the stress treatments and blue lines represent those that are less prevalent. In the key, the different colored lines represent different metabolic pathways that are affected by oyster exposure to ocean acidification and/or mechanical stimulation. Figures are also available on FigShare with input files for iPath2 to allow for interactive exploration of the data [Additional file 6: Figure S2: Representation of key metabolic pathways that are significantly affected by ocean acidification (A), mechanical stress at low the data . p CO2 of 2800 μatm (“OA”), mechanical stress after a one month exposure to 400 μatm (“Mech Stress 400 μatm”), and mechanical stress after a one month exposure to 2800 μtam (“Mech Stress 2800 μatm”). Table includes enriched GO term, number of proteins contributing to that GO term, p-value indicating degree of enrichment, the SwissProt accession numbers for those proteins, the fold enrichment for each GO term, and the false discovery rate (FDR). (TXT 9 KB)Additional file 8: Table S5: Enriched biological processes for proteins >2-fold differentially abundant in the stress responses to elevated


Participants with complete accelerometer data often represent a low proportion of the total sample and, in some cases, may be distinguishable from participants with incomplete data. Because traditional reliability methods characterize the consistency of complete data, little is known about reliability properties for an entire sample. This study employed Generalizability theory to report an index of reliability characterizing complete (7 days) and observable (1 to 7 days) accelerometer data.Cross-sectional.Accelerometer data from the Study of Early Child Care and Youth Development were analyzed in this study. Missing value analyses were conducted to describe the pattern and mechanism of missing data. Generalizability coefficients were derived from variance components to report reliability parameters for complete data and also for the entire observable sample. Analyses were conducted separately by age  and daily wear time criteria .Participants with complete data were limited (<34%) and, most often, data were not considered to be missing completely at random. Across conditions, reliability coefficients for complete data were between 0.74 and 0.87. Relatively lower reliability properties were found across all observable data, ranging from 0.52 to 0.67. Sample variability increased with longer wear time criteria, but decreased with advanced age.A reliability coefficient that includes all participants, not just those with complete data, provides a global perspective of reliability that could be used to further understand group level associations between activity and health outcomes. Wearable monitors overcome many of the limitations of self-report methods and are now regularly used to objectively assess free-living physical activity (PA) in children and adolescents. Accelerometers, such as the Actigraph, are a common type of wearable monitor that, in contrast to other objective tools , are capable of characterizing the frequency, intensity, duration, and time of daily PA. Despite the accelerometer's appeal as a field-based assessment tool, several data management and processing challenges exist One specific challenge accelerometer end-users must contend with involves missing data. Missing data are inherent to nearly all free-living accelerometer studies and can exist as repeated episodes within a day or across entire day(s). Based on Rubin's taxonomy Several techniques have been used to recover missing data from individual monitoring days and some of these methods have yielded unbiased estimates of PA dn) ≤ 7)) exist but have yet to be explored in PA research. Reporting a reliability coefficient that includes all participants, not just those with complete data, provides a global perspective of reliability that may be useful in understanding group-level associations between MVPA and health outcomes.Cronbach and colleagues dn  =  7) and observable (1 ≤ dn ≤ 7) accelerometer data. All analyses were conducted using daily levels of MVPA, which were available from a large prospective study of youth at 9, 11, 12, and 15 yrs of age.To advance reliability research, the current study details the application of G theory to report an index of reliability using complete (http://www.nichd.nih.gov/research/supported/seccyd/pages/overview.aspx). Accelerometer data were collected across a 7-day monitoring period using 1-minute epochs at mean ages of 9, 11, 12, and 15 yrs. ActiLife software (version 6.4.3) was used to detect and remove daily non-wear intervals between the hours of 7:00 and 22:00. Similar to other studies Accelerometer data from the Study of Early Child Care and Youth Development were analyzed to address the study objective. The original monitoring protocol was approved by each participating university's ethics committee  and written consent was obtained from each participant. Details regarding the enrollment procedure and research protocol are available from the study's website  and observable (1 ≤ dn ≤ 7) accelerometer data were compared using G theory methods. Although G theory has been described in the literature 2pô)), the facet (day (2dô)), and the interaction term which is confounded with unsystematic or unmeasured error (2 pdô). Derived variance components were then used to calculate two types of error (absolute (2Δô) and relative (2δô)). Absolute, or criterion-referenced, error is the error involved in using a participant's mean score as an estimate of their universe score  + (2 pdô/d)), whereby d is the harmonic mean of To address the study's primary objective, reliability coefficients using complete (2 pS) minus participant variance (2δô  =  2 pS – 2 pô) (Equation 2). In contrast, relative error is associated with norm-referenced interpretations of measurement and equals the variance of the observed mean score for participants  (2CompleteEp  =  2 pô/[2 pô + (2 pdô/dn)]) and, specific to this study, a separate coefficient that characterized all observable data (1≤ dn ≤ 7) (2ObservedEp  =  2 pô/2 pS). Coefficients range from 0 to 1. In PA research, reliability coefficients ≥ 0.80 are desirable. Standard error of the mean (SEM), which provides an indication of the uncertainty associated with each measure, was calculated for each condition by taking the square root of the absolute error term. The SEM is expressed in the same metric unit of measurement and represents a 68% CI for the participant's universe score. Variance components, error estimates, and reliability coefficients were derived using EXCEL macros created by the corresponding author , the mean absolute difference in MVPA between 6- and 12-hr criteria approximated 3 mins, and the absolute percent error (APE) for MVPA increased with advancing mean age . APE estimates were derived using MVPA levels from the 6- and 12-hr wear time criteria (((|MVPA6 hrs – MVPA12 hrs|)/MVPA12 hrs) × 100).Missing data characteristics are reported in 2δô and 2 pS) are reported in Total variance in MVPA increased with longer wear time criteria and decreased with increasing age. In general, total variance was distributed in a similar manner across conditions (p × d interaction term > participant term > day term). The relatively large contribution of variance from the p × d interaction term (ranging from 47 to 68%) reflects the wide range of individual variability in daily free-living MVPA levels across days, but also includes unexamined error. The participant term reflects inter-individual variation and explained a majority of the remaining error (ranging from 27 to 47%). Ideally, the participant term would account for the largest proportion of error because it represents true score variance. In comparison, a relatively low amount of variation was attributable to the day term (1 to 9%), suggesting little variability in MVPA across monitoring days. Variance components and error estimates of MVPA (a priori given that each coefficient (2CompleteEp and 2ObservedEp) characterized unique data. Across age and wear time conditions, reliability coefficients derived from complete data  were relatively higher compared to coefficients derived from observed data  ; pn = 834 (11 yrs), pn = 713 (12 yrs), and pn = 611 (15 yrs)) and 7 monitoring days even when variance components were derived with missing data in the G study 2ObservedEp) ranged from 0.52 to 0.67. A similar index of reliability would be expected among separate samples given similarities in both sample size and pattern of missing data.Reliability methods were expected to produce different coefficients  nd ≤ 7) . Ep2Comp2ObservedEp) that included all observable data satisfying daily wear time criteria. To date, several perspectives of reliability have been reported in the literature A 7-day monitoring period is recommended for PA research because week and weekend data are included Although data imputation could have been used in this study to increase the proportion of youth with complete accelerometer records, the primary aim was to report a reliability coefficient that characterized all observable (non-imputed) data satisfying daily wear time criteria. Under most age and wear time conditions, the missing data in this study were indistinguishably MAR or NMAR suggesting data imputation may produce biased summary statistics and reliability estimates. Ideally, missing data would be MCAR and represent a simple random proportion of the entire sample. Given the inherent challenges of specifying the pattern of missing data, few studies have directly examined the implications of missing accelerometer data on reliability parameters. In an accelerometer simulation study involving girls, Catellier et al 2 pS and 2 pô increased as daily wear time criteria became more stringent, indicating greater variability in MVPA within the sample using the 12-hr threshold compared to the more conservative 6-hr threshold. As a general tenet of reliability, increased variability among individuals produces higher reliability estimates 2CompleteEp) at each mean age. In this study, estimates of 2 pS at a given wear time duration  progressively declined with increasing mean age, suggesting youth levels of MVPA may become more homogeneous with age. Certain caveats do exist however when reporting trends in reliability and error estimates. For example, the accelerometer data included in this analysis originated from a prospective study design; however, the sample size and composition at each mean age (and across wear time conditions) varied. Therefore, these trends should be interpreted judiciously given that each age and wear time condition was comprised of unique data.Accelerometer data included in this study were processed using ActiLife software to identify complete monitoring days using four daily wear time criteria  between 7:00 and 22:00. This approach was selected not with the intent to identify a specific wear time threshold, but rather to report the effect of wear time inclusion criteria on factors that influence reliability estimates and levels of MVPA . At each mean age, Relatively small absolute differences in MVPA were found between 6- and 12-hr wear time criteria at each mean age, ranging from 2.8 to 3.7 mins. The relative stability in MVPA estimates found in this study can likely be attributed to the similarities in mean wear time duration. Across conditions, the accelerometer was worn, on average, for nearly 80% of the 15-hr monitoring period. Although daily patterns of MVPA were not specifically examined in the current study, it is quite possible that the additional 1.5 hrs of daily wear time reported using the 12-hr criteria (compared to the 6-hr criteria) occurred during the latter portions of the day, a period when activity levels decline among girls and boys dn  =  7) and an unbalanced design using all observable data (1≤ dn ≤ 7). Reporting a global index of reliability is novel to PA research and may prove useful for investigators interested in reporting parameter characteristics of an entire sample, rather than a sub-sample with complete data. Future research may consider applying a reliability coefficient, like the one described here (2ObservedEp), to Spearman's disattenuation formula G theory was applied in this study to report an index of reliability using a balanced design with complete data Click here for additional data file.Table S2Variance component equations and estimates.(DOCX)Click here for additional data file.


Deletion of the hilD 3′UTR increases the hilD mRNA level, suggesting that the hilD 3′UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3′UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3′UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3′UTR mRNAs, suggesting that the hilD 3′UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3′UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3′UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3′UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.Long 3′ untranslated regions (3′UTRs) are common in eukaryotic mRNAs. In contrast, long 3′UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3′UTR of 310 nucleotides in  Posttranscriptional regulation of gene expression plays essential roles in cellular physiology in all domains of life. During the last few decades, special attention has been paid to mRNA regulation, and a variety of regulatory mechanisms have been described in both prokaryotes and eukaryotes. A difference in posttranscriptional regulation between eukaryotes and prokaryotes is the region of the mRNA molecule involved: while in eukaryotes the interaction of proteins or RNA molecules occurs with sequences located either in the 5′ untranslated region (5′UTR) or in the 3′ untranslated region (3′UTR) , posttraSalmonella enterica is a Gram-negative bacterium that has played a historic role in bacterial genetics and in the study of host–pathogen interactions. More recently, Salmonella has also become a model organism for the study of RNA-mediated regulation   ,7. The tinvasion .Salmonella chromosome known as Salmonella pathogenicity island 1 (SPI-1)  . Express (SPI-1) . A numbeitiation , and theitiation , physicaitiation , likely itiation . Regulatpression ,15, and entified .hilD mRNA possesses a long 3′UTR of 310 nucleotides, and provide evidence that the hilD 3′UTR is a target for the RNA degradosome and for the RNA chaperone Hfq. Lack of the hilD 3′UTR permits SPI-1 expression under conditions that are not favorable for invasion and retards Salmonella growth, suggesting that the hilD 3′UTR may have physiological significance.Here we show that S. enterica strains listed in Supplementary Table S1 belong to serovar Typhimurium, and derive from the mouse-virulent strain ATCC 14028. For simplicity, S. enterica serovar Typhimurium is usually abbreviated as S. enterica. Two Escherichia coli strains were used: DH5α  and CC118 λ pir [phoA20 thi-1 rspE rpoB argE(Am) recA1 (λ pir)]. Targeted gene disruption was achieved using pKD4 or pKD13  sites generated by excision of Kmr cassettes  as template  LB without salt and incubation at 37°C with shaking (200 rpm) until O.D.600 = 2–2.5. When required, kanamycin sulfate (50 μg/ml) or chloramphenicol (20 μg/ml) were added to the culture medium. Green plates were prepared according to Chan et al. (Luria-Bertani (LB) broth was used as standard liquid medium. For standard conditions (SPI-1 induction) , culturen et al. , except hilD Δ3′UTR allele, the Kmr gene of pKD13 was amplified with primers JVO5462 and JVO5463 (Supplementary Table S2) and inserted in the Salmonella chromosome by λ Red recombination, deleting a 230 nt fragment of the hilD 3′UTR starting at the first nucleotide after the hilD stop codon. The Kmr gene was removed by recombination of flanking FRT sequences mediated by pCP20-encoded FLP recombinase, leaving a scar of 82 nt. As a result, a shorter 3′UTR of 162 nt was produced without eliminating the native Rho-independent transcriptional terminator. The same strategy was used to construct hilD 3′UTR deletion alleles, using different forward primers (Supplementary Table S2) and keeping JVO5463 as the reverse oligo for amplification. The hilD 3′UTR50end allele was constructed using primer pairs JVO5462 and hilD50endDO.For construction of the hilD Δ3′UTRsl (scarless) allele was constructed by overlapping polymerase chain reactions using oligonucleotides EPF097, EPF100, EPF103 and EPF104 (Supplementary Table S2). The resulting amplicon contained the hilD coding sequence followed by the 3′UTR transcriptional terminator (without the 3′UTR) and approximately 500 bp of nearby DNA, flanked by SacI and XbaI restriction sites. The polymerase chain reaction (PCR) product was cloned into the SacI–XbaI sites of the suicide plasmid pDMS197, and propagated in E. coli CC118 lambda pir. The pDMS197 derivative was transformed into E. coli S17-1 lambda pir, and several transformants were used as donors in matings with S. enterica 14028 harboring a KmR cassette in place of the hilD 3′UTR (strain SV6189). TcR transconjugants were selected on E plates supplemented with tetracycline. Several TcR transconjugants were grown in nutrient broth (without NaCl) containing 5% sucrose. Individual tetracycline-sensitive segregants were then screened for kanamycin sensitivity and examined for the incorporation of the mutant hilD allele (hilD Δ3′UTRsl) by PCR amplification with oligonucleotides EPF097 and EPF100 and sequencing of the PCR product.The hilD from a heterologous promoter was achieved by replacing its native promoter with the PLtetO promoter (cat gene and PtetOL was amplified by PCR using pXG1 as template (LtetO-hilD UP and PLtetO-hilD DO (Supplementary Table S2). The PCR product was treated with DpnI to remove template traces. The construction was then inserted in the S. enterica chromosome by λ Red recombination .Expression of promoter . A fragmtemplate . The pribination  and Cmr gfp alleles was achieved by electroporation of pXG1-derived plasmids into S. enterica ATCC 14028. To generate the gfp allele carrying a hilD 3′UTR, a PCR product containing the hilD 3′UTR coding sequence downstream of gfp was generated by overlapping PCRs. Two PCR products were obtained using primer pairs EPF043–EPF044 (Supplementary Table S2) and pXG1 as template, and primers EPF045 and EPF046 (Supplementary Table S2) and genomic DNA from S. enterica ATCC 14028 as template. Purified PCR products were subsequently used as templates for amplification with primers EPF043 and EPF046. The resulting product was cloned into Nhe-I digested pXG1 vector, rendering plasmid pIZ1988. To generate the gfp allele with a 3′UTR deletion (Δ3′UTR) the same procedure was used. The primer pairs used were EPF043 and EPF047 (Supplementary Table S2), with pXG1 as template, and primers EPF048 and EPF046 (Supplementary Table S2) with SV6190 genomic DNA as template. The final PCR product was cloned into pXG1 to obtain pIZ1989. Plasmids pIZ1988 and pIZ1999 were constructed using E. coli DH5α as host. Transfer to S. enterica was achieved by electrotransformation of ATCC 14028, selecting Cmr colonies. Introduction of pIZ1988 and pIZ1999 generated strains SV7440 and SV7441, respectively.Expression of 600 ∼ 2) was centrifuged at 16 000 g, 4°C, for 5 min. The pellet was resuspended in 100 μl of a solution of lysozyme . Cell lysis was facilitated by three consecutive freeze-thaw cycles. After lysis, RNA was extracted using 1 ml of Trizol reagent , according to manufacturer's instructions. Finally, total RNA was resuspended in 30 μl of RNase-free water for subsequent uses. The quality of the preparation and the RNA concentration were determined using a ND-1000 spectrophotometer .A 2 ml aliquot from a culture of the appropriate strain grown to stationary phase , and four independent clones of each product were sequenced using external primers.Rapid amplification of cDNA 3' ends (3′RACE) was performed as described by Argaman et al. . The hil+ membranes  were performed by vacuum using 0.05 M NaOH. Filters were then hybridized using an internally labeled [(α-32P)UTP] riboprobe specific for the first (5′) 300 nucleotides of the hilD coding sequence. Hybridization was carried out at 65°C. As a control of RNA loading and transfer efficiency, the filters were hybridized with a riboprobe for the RNase P mRNA gene (rnpB). Images of radioactive filters were obtained with a FLA-5100 imaging system , and quantification was performed using the MultiGauge software (Fujifilm).Ten micrograms of RNA was loaded per well and electrophoresed in denaturing 1% agarose formaldehyde–3-(N-morpholino)propanesulfonic acid (MOPS) gels. Transfer and fixation to Hybond-N3–sodium dodecyl sulfate (SDS) permeabilization procedure . Approximately 3.5 × 108 bacterial cells were collected by centrifugation  and suspended in 100 μl of Laemmli sample buffer . Proteins were resolved by Tris–Tricine–polyacrylamide gel electrophoresis (PAGE) using 12% gels. Conditions for protein transfer have been described elsewhere . For routine cultivation, 60 μg/ml penicillin and 100 μg/ml streptomycin were added to the culture medium. The day before infection, ∼1.5 × 105 HeLa cells were seeded, using 24-well plates . Each well contained 1 ml of tissue culture medium without antibiotics. Cells were grown at 37°C, 5% CO2 to obtain 80% confluency. One hour before infection, the culture medium was removed and replaced by 0.5 ml fresh tissue culture medium without antibiotics. Bacteria were grown in LB to an O.D. ≈ 0.4 and added to reach a multiplicity of infection (MOI) of 50:1 bacteria/HeLa cell. HeLa cells were infected for 30 min, washed three times with PBS, incubated in fresh tissue culture medium containing 100 μg/ml gentamicin for 1.5 h, and washed three times with PBS. Numbers of viable intracellular bacteria were obtained by lysing infected cells with 1% Triton X-100 (prepared in PBS) and subsequent plating. Invasion rates were determined as the ratio between viable intracellular bacteria and viable bacteria added to infect the HeLa cells. Each assay was run in triplicate, and data shown are the relative invasion rates and standard deviations of five independent experiments. The Student's t test was used to determine the statistical significance of the differences observed.HeLa cells (ATCC CCL2) were cultured in tissue culture medium .GFP expression was monitored by flow cytometry of live cells. Bacteria were grown overnight at 37°C in LB with shaking, diluted into fresh medium (1:100), and incubated at 37°C to reach and O.D.hilD transcription start point was identified previously (in silico annotation of the S. enterica genome identified a putative long intergenic region between hilD and the neighbor gene hilA (http://www.xbase.ac.uk/colibase/). We determined the hilD transcription stop point by 3′RACE and mapped the hilD transcriptional stop around 300 nucleotides downstream the hilD stop codon, immediately after a U-rich region . In silico analysis of the 3′UTR , which might indicate a functional role for this non-coding RNA region.The eviously , defininn Figure . Furthern Figure  predictst Figure . AltogethilD 3′UTR might play a role in hilD expression. To test this hypothesis, we constructed a strain carrying a modified hilD 3′UTR (hilD Δ3′UTR). In this strain, a 230 nt fragment of the hilD 3′UTR, starting at the first nucleotide after the hilD stop codon, was deleted leaving the Rho-independent transcriptional terminator intact. The deleted fragment was replaced with the pKD13 scar (82 nucleotides)  300 nucleotides of the hilD coding sequence. As shown in Figure hilD Δ3′UTR mRNA is shorter than the hilD native mRNA. More importantly, the level of hilD mRNA was found to be ∼11-fold higher in the absence of the 3′UTR than in the wild-type strain, suggesting that the presence of the 3′UTR somehow reduces the hilD mRNA level. To exclude the possibility that the hilD mRNA increase detected in the hilD Δ3′UTR strain could be caused by a stabilizing effect of the scar sequence, we constructed a strain in which the 3′UTR was cleanly deleted. Higher levels of hilD mRNA were detected in the scarless strain . The specificity of the hilD probe was confirmed using a strain carrying a SPI-1 deletion, and no signal was detected by northern blot .Long 3′UTRs have been poorly studied in prokaryotes, while eukaryotic 3′UTRs often play physiological roles . We thuseotides) , resultieotides) . To testhilD 3′UTR is involved in hilD expression, we compared the levels of hilD mRNA in isogenic hilD 3′UTR+ and hilD Δ3′UTR strains in which hilD was transcribed from a heterologous promoter, PtetOL  gene on plasmid pXG1 (tetOL promoter (hilD Δ3′UTR downstream of gfp (pIZ1989) was also constructed. Both plasmids were introduced in S. enterica and E. coli, and GFP levels were monitored in single bacterial cells by flow cytometry. A representative experiment is shown in Figure Salmonella enterica cells presented a mean fluorescence of 58.4 for cells expressing GFP-3′UTR and 85.3 for cells expressing GFP-Δ3′UTR. In turn, E. coli cells showed a mean fluorescence of 96.9 for cells containing GFP-3′UTR and 132.0 for cells lacking the 3′UTR (GFP-Δ3′UTR). Thus, the presence of the hilD 3′UTR downstream of gfp resulted in lower fluorescence intensity (indicating lower GFP production) in both S. enterica and E. coli. This ability of the hilD 3′UTR to reduce expression of a heterologous gene supports the view that the 3′UTR can act as an independent module.To further confirm that the mid pXG1 , yieldinpromoter . As a cohilD mRNA is more abundant in the absence of its native 3′UTR and HilD is a positive regulator of SPI-1, we wondered whether increased level of hilD mRNA resulted in increased SPI-1 expression. For this purpose, we analyzed the expression of four SPI-1 genes  in isogenic hilD 3′UTR+ and hilD Δ3′UTR strains. Western blotting was used to monitor InvF, InvH, SipB and SipC levels using protein variants tagged with the 3xFLAG epitope. The levels of all four proteins were higher in hilD Δ3′UTR strains compared with the hilD 3′UTR+ background . Hence, overproduction of hilD mRNA in the absence of its native 3′UTR correlates with SPI-1 overexpression, suggesting that the hilD 3′UTR may promote hilD mRNA turnover.Because d Figure , suggesthilD 3′UTR necessary for mRNA turnover, we constructed a set of mutants containing partial deletions of the 3′UTR. The mutants conserved 200, 150, 100, 50 and 25 nucleotides of the region immediately downstream of the hilD stop codon, followed by the 82 nt pKD13 scar and the Rho-independent transcriptional terminator . We also constructed a mutant containing the 82-nt pKD13 scar immediately after the hilD stop codon, followed by the last (3′) 50 nucleotides of the 3′UTR and the transcriptional terminator . SPI-1 expression was monitored by measuring the β-galactosidase activity of a sipC::lac fusion in the 3′UTR deletion mutants. Results shown in Figure sipC expression levels similar or identical to the wild type, indicating that deletion of downstream regions does not impair hilD mRNA turnover. Hence, signals for 3′UTR-mediated mRNA decay must be located within nucleotides 1–100.The mutants containing 200, 150 and 100 nucleotides from the 5′ region of the 3′UTR showed sipC::lac expression , suggesting that some signals for hilD mRNA turnover had been lost upon deletion of nucleotides 25–50 and 50–100.The mutants containing only 50 and 25 nucleotides of the 5′ region of the 3′UTR showed moderate increases in sipC::lac expression was higher than in the wild type but lower than in the mutant lacking the whole 3′UTR (hilD Δ3′UTR)  25 nucleotides of the 3′UTR.In the mutant containing 25 nucleotides of the 3′UTR, ) Figure , suggestsipC::lac expression level detected in the mutant containing the last 50 nucleotides of the 3′UTR (3′UTR50end) was similar to that of the whole-deletion mutant (hilD Δ3′UTR), supporting the view that downstream regions of the hilD 3′UTR do not play a relevant role in hilD mRNA turnover.The hilD mRNA turnover: mutants containing 50 nucleotides of the 3′UTR, either from the upstream region (3′UTR50) or the downstream region (3′UTR50end), have deletions of the same length but undergo disparate levels of sipC expression , and clear-cut correlations between mRNA abundance and SPI-1 expression levels were found.The levels of To define the region(s) of the sipC::lac expression at low levels, similar to those of the wild type, suggesting that signals for mRNA decay are contained within nucleotides 1–100 after the hilD stop codon.In summary, conservation of the upstream (5′) 100 nucleotides of the 3′UTR is sufficient to keep hilD mRNA might be a target for mRNA degradation by the RNA degradosome. If such was the case, the increased hilD mRNA level detected in the absence of the 3′UTR might be the consequence of reduced mRNA turnover. Hence, we reasoned, inactivation of factors involved in RNA degradation might suppress the differences observed in the level of hilD mRNA with and without its native 3′UTR. Salmonella enterica mutants carrying deletions in genes encoding ribonuclease E (RNase E), ribonuclease G (RNase G), polynucleotide phosphorylase (Pnp), or poly(A) polymerase I (PapI) were used. Because RNase E is essential in S. enterica, we used a mutant carrying a deletion that eliminates only the C-terminal region of the protein N↓AU, where N identifies any nucleotide, and ↓ indicates the cleavage site . Absence of the hilD 3′UTR caused a strong increase in invF expression under non-invasive conditions , as expected under non-invasive conditions. However, the hilD Δ3′UTR mutant showed a higher invasion rate  60 nucleotides. Signals for degradosome-mediated turnover appear to be located within the upstream 100 nucleotides of the hilD 3′UTR, and the upstream (5′) 25 nucleotides seem to be especially relevant .The ability of the residues . The hilt Figure . These thilD 3′UTR might be a direct target for the RNA degradosome, we have not been able to detect differences in the half-lives of hilD mRNA with and without 3′UTR (data not shown), opening the possibility for alternative, more indirect models. One such model is the involvement of an unstable small RNA (sRNA) in the control of hilD mRNA turnover. Stopping transcription with rifampicin would cause fast disappearance of the sRNA, and the sRNA effect on mRNA stability would be masked. It seems also likely that the hilD mRNA increase detected in the absence of the 3′UTR . Growth retardation is a well known consequence of SPI-1 overexpression (hilD gene itself is subjected to transcriptional control, most regulatory systems that control SPI-1 expression through hilD appear to act at the posttranscriptional or the postranslational levels (hilD 3′UTR by the degradosome may contribute to maintain steady-state levels of hilD mRNA, thus contributing to cellular homeostasis. Efficient turnover of hilD mRNA may be especially relevant under non-invasive conditions, when the growth retardation caused by SPI-1 expression is not compensated by the possibility of colonizing an animal niche.The relevance of the pression , probablpression . HilD isl levels , and thehilD RNA turnover may fuel the speculation that downstream regions of the 3′UTR might contain additional regulatory signals. For instance, Chao et al. have described that the 3′UTR region of hilD mRNA appears enriched upon Hfq co-inmunoprecipitation, defining a candidate region for a new 3′UTR-derived sRNA (hilD 3′UTR might not only provide a target for hilD mRNA turnover but be also the source of a sRNA. In support of this speculation, certain 3′UTRs are known to be a source of small regulatory RNAs in S. enterica (hilD 3′UTR as a prokaryotic example of a 3′UTR with functional capacity.The abundance of RNase E targets in the upstream 100 nucleotides of the 3′UTR and the evidence that this region is sufficient for ved sRNA . Hence, enterica . This ouSupplementary Data are available at NAR online.


APOE) gene has been implicated as one of the genes susceptible to temporal lobe epilepsy (TLE), but the association is inconsistent. We carried out a study to investigate the association of APOEε4 allele with a subtype of TLE-nonlesional mesial temporal lobe epilepsy (NLMTLE) in Han Chinese people.Apolipoprotein E (APOE polymorphisms were genotyped using polymerase chain reaction (PCR) DNA sequencing. We compared the frequency of APOEε4 allele and carrying status between NLMTLE patients and control subjects to test for the association of APOEε4 allele with NLMTLE clinical status.The study consisted of total 308 NLMTLE patients and 302 controls in Han Chinese. The APOEε4 allele was significantly associated with the risk of NLMTLE. No effect of APOEε4 allele was found on the age of onset, duration of epilepsy, or frequency of seizure. Moreover, there was no association between APOEε4 allele and hippocampal sclerosis (HS) or febrile convulsion (FC) history.Carrying status of APOEε4 allele was a possible risk factor for NLMTLE, and further study with a larger sample is needed to warrant this finding.Our study provided an evidence that  Therefore, susceptibility genes may play some important roles in the pathogenesis, especially may be involved in specific aspects of clinical phenotypes and pathological characteristics, or through gene–gene interactions.Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy with a variety of clinical manifestations and underlying etiology.7The APOE gene expression is upregulated after brain damage from various causes.8 There are 3 allelotypes ε2, ε3, and ε4 in APOE. In the presence of APOEε4 allele, brain's ability to repair and to form new synapses is impaired.9 The link between APOE polymorphisms, especially APOEε4 allele, and TLE have recently drawn great attention.10Apolipoprotein E (APOE) is a critical protein for the maintenance and repair of injured cell membrane, as well as neurite growth, dendritic remodeling, and synaptogenesis.APOEε4 allele was related to the phenotypic severity of TLE such as early onset of habitual seizures, increased risk of verbal learning deficit, longstanding seizures which affect memory performance and increased risk of postictal confusion.14 However, some other studies did not find the association between APOE polymorphisms and TLE.18 TLE can be classified into mesial TLE (MTLE) and lateral TLE (LTLE) by the origin of seizure or into lesional and nonlesional by etiology. Nonlesional mesial temporal lobe epilepsy (NLMTLE) is a unique form of TLE because of its resistance to drug therapy.19 A better understanding of the molecular role of APOE in the development of NLMTLE may help to understand the pathogenic mechanism and to develop antiepileptogenic therapies. Here, we reported a study to examine the association of APOEε4 allele with the specific NLMTLE in Han Chinese population.Recent studies indicated that This study was reviewed and approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University in China in accordance with the Declaration of Helsinki. Informed consent was obtained from each subject enrolled.20 Unrelated healthy subjects were recruited from individuals who came for routine physical examination at the clinics of Beijing Tiantan Hospital, Capital Medical University from June 2005 to January 2010, and subjects without family history of neurological diseases were randomly selected as controls. Both NLMTLE patients and controls were of Han Chinese ancestry and did not have a history of hypertension, hyperlipidemia, and diabetes.This study consisted of 308 patients with NLMTLE and 302 healthy controls. All patients were those who visited and diagnosed at the clinics at Epilepsy Center in Beijing Tiantan Hospital, Capital Medical University from June 2005 to January 2010. Diagnosis of NLMTLE was made based on the clinical history, electroencephalogram, and magnetic resonance imaging (MRI). The inclusion criteria included: seizure semiology consistent with MTLE; anterior and mesial temporal ictal or interictal discharges, without generalized or extra temporal discharges; no lesion such as cerebral tumor, cortical dysgenesis, vascular lesion or malformation, or posttraumatic scar other than atrophy and increased signal in hippocampal formation identified by MRI; and without epilepsy family history. In this study, MRI was performed by using sequences and slices designed to optimize visual detection of hippocampal sclerosis (HS), following the protocol described by Kanemoto et al.APOE polymorphisms were genotyped using polymerase chain reaction (PCR)-DNA sequencing. Venous blood was drawn from each individual and stored at −80 °C. Genomic DNA was extracted from whole blood using standard method. A 292-bp PCR fragment spanning APOE polymorphisms was amplified by using the following primers: 5′-AAC AAC TGA CCC CGG TGG CG-3′; 5′-ATG GCG CTG AGG CCG CGC TC-3′. PCR conditions used for amplification were 94 °C for 8 minutes, followed by 35 cycles of 94 °C for 45 seconds, 65 °C for 45 seconds, and 72 °C for 45 seconds. The final extension step at 72 °C was lengthened to 8 minutes. PCR products were tested by 1.2% Agarose Gel Electrophoresis and the products were purified by DNA rapid purification kit . DNA sequencing PCR reaction mixture (5 μL) consisted of templates DNA 2 μL, sequencase 2 μL, and primer 1 μL (5′AAC AAC TGA CCC CGG TGG CG 3′). PCR conditions used for sequence were 95 °C for 2 minutes, followed by 35 cycles of 95 °C for 3 seconds, 50 °C for 3 seconds, and 60 °C for 90 seconds. The final extension step at 60 °C was lengthened to 5 minutes. The products were then sequenced using an ABI 3730 × L DNA Sequencer .APOE polymorphisms was assessed using χ2 test. Tests for differences in frequency of allele, carrier of APOEε4, and genotype by disease status and by gender were performed using the χ2 test or Fisher exact test when appropriate. Mann–Whitney U-test was used for quantitative clinical variables as variance of some variables were slightly different by APOEε4 carrying status, and normality checking was not appropriate due to smaller sample size in APOEε4 carriers; Chi-square tests were used in comparison of qualitative clinical variables. Multiple logistic regression was used to test for the association of APOE allele with the risk of NLMTLE.The Stata statistical software Windows Release 12.0 was used for statistical data analysis. Hardy-Weinberg equilibrium of P = 0.099). The patient group consisted of 184 males and 124 females, whereas the control group included 165 males and 137 females (P = 0.203). There was no significant difference in mean age and sex ratio between NLMTLE patient group and control group. In the patient group, the age of seizure onset was 19.9 ± 11.8 years, duration of epilepsy was 9.5 ± 8.0 years, and frequency of seizure was 5.7 ± 4.2 times/month , likely due to the sparse sample of limited sample size in some genotypes. When classifying the sample subjects by APOEε4 allele carrying status . The difference in the APOEε4 carrying status was statistically significant . When adjusted for sex and age in multiple logistic regression analysis, carrying of APOEε4 allele was significantly associated with NLMTLE disease (Table P = 0.044). In the NLMTLE patient group, we did not observe any significant difference in clinical variable between APOEε4 allele carriers and noncarriers (Table P = 0.181), duration of epilepsy (P = 0.858), and frequency of seizure (P = 0.100) did not differ in NLMTLE patients by APOEε4 allele carrying status, and neither was different for the presence of HS (P = 0.139) and FC history (P = 0.228).The frequency of genotype in both patients and controls were in Hardy–Weinberg equilibrium. The frequency of APOE polymorphisms with NLMTLE disease status in Han Chinese population. Although there was not significant association of NLMTLE with APOE genotype likely due to limited sample size, we found that carrying of APOEε4 significantly increased the risk of NLMTLE in Han Chinese population. The proportion of APOEε4 carriers in controls was 12.58%, which was consistent with that in other studies, suggesting that our controls were representative in Han Chinese population. To our knowledge, this is the first report on the association between ApoEε4 and NLMTLE in Han Chinese population.In this study, we examined the association of APOE as a potential susceptibility gene to TLE. Animal study has shown that brain's ability to repair damage and form new synapses was impaired in presence of APOEε4 allele.21 The APOEε4 allele promotes the intracerebral accumulations of β-amyloid, which plays a central role in increasing neuronal susceptibility to damage.22 Studies on expression and presence of β-amyloid precursor protein and β-amyloid have been performed and demonstrated a link between APOEε4 allele and TLE using temporal lobe specimens.25 Further studies found the APOEε4 allele might play an active role in modulating some clinical features of TLE. APOEε4 allele was associated with earlier onset of TLE in a meta-analysis of 7 published studies, carriers of APOEε4 allele tended to have 5 years earlier than noncarriers in onset of the disease,26 risk of posttraumatic seizures,11 refractory complex partial seizures,27 verbal learning deficit,12 memory loss,13 and postictal confusion.14 However, there are other studies that failed to find a significant association between TLE and APOE polymorphisms in several populations including Han Chinese people.28Our finding was consistent with previous studies that showed APOE genotype or allele in both the controls and patients is very similar to that previously found in the general population in Han Chinese people.31 TLE is one of complex human disorders that are clinically and genetically heterogeneous. The pathogenic effect of each putative “susceptibility gene” is small or modest, and environmental factors may play a part role in the development of TLE. The related studies on APOE and TLE also showed inconsistent results, probably due to the clinical heterogeneity of the patients sample. According to the focus of seizure origins, TLE can be classified into MTLE and LTLE subtype. The clinical and pathological feature may be different between MTLE and LTLE. As for the etiology MTLE is further classified into leisonal and nonleisonal subtype. HS is the most common histopathologic form of nonleisonal MTLE. Although the pathomechanism of HS is unclear, some studies show that early precipitant events such as febrile convulsion are correlated with HS. This indicates that susceptibility gene may play a role in the development of HS and MTLE. Genetic study of pure type of nonlesional MTLE alone may be helpful for identifying risk variants. We have seen that previous study18 in Han Chinese population has mixed all of patient samples with different subtypes such as nonlesional TLE and developmental lesions, therefore not comparable with our observations.It is important to emphasize that in our study the proportion of each 32 It should be noted that our study fulfill the fundamental requisites and has minimal of those limitations. First, as the frequency of APOE allele varies among different ethnic populations, control subjects were carefully matched for ethnic background in our study. Second, it was reported that the frequency of ApoEε4 allele in general Han Chinese was 3.5% to 12.9%, which was consistent with that in our study sample. In the meantime, the proportion of APOEε4 allele carrying was 12.58% in controls, which was close to population-based sample in multiple ethnic group including Han Chinese (14.25%).31 Our sample size provided enough statistical power (80%) to detect an allelic association with an modest effect size (OR = 2.0) for a 10% probability of exposure in controls at a level of 0.05. In addition, the association may have arisen by chance, or may be artifactual because of the weaknesses of study design and selection bias.34 Focusing on subtype of TLE such as NLMTLE may reduce the disturbance of possible underlying etiology. Although we provided an evidence that the APOEε4 allele was a risk factor for NLMTLE, we did not find any significant association of APOEε4 allele carrying status with the age at onset of seizures, duration of epilepsy, frequency of seizure, HS, and FC history in NLMTLE patients. It is worth noting that there were some trends of association with age at onset of seizure, frequency of seizure. A further study with a larger cohort of patients is needed. Our study illustrated APOE might be a possible susceptibility gene for NLMTLE. Further studies in independent population samples should be investigated to confirm the association; and molecular study is also needed to understand the mechanism of this genetic association in the future.Association studies seem to be a common approach to identify genes associated with complex human diseases such as TLE. However, we are aware of those limitations such as sample size, selection bias, etc.


How combinations of gene-environment interactions collectively give rise to genotype-environment interactions is not fully understood. To shed light on this problem, we genetically dissected an environment-specific poor growth phenotype in a cross of two budding yeast strains. This phenotype is detectable when certain segregants are grown on ethanol at 37°C (‘E37’), a condition that differs from the standard culturing environment in both its carbon source (ethanol as opposed to glucose) and temperature (37°C as opposed to 30°C). Using recurrent backcrossing with phenotypic selection, we identified 16 contributing loci. To examine how these loci interact with each other and the environment, we focused on a subset of four loci that together can lead to poor growth in E37. We measured the growth of all 16 haploid combinations of alleles at these loci in all four possible combinations of carbon source (ethanol or glucose) and temperature (30 or 37°C) in a nearly isogenic population. This revealed that the four loci act in an almost entirely additive manner in E37. However, we also found that these loci have weaker effects when only carbon source or temperature is altered, suggesting that their effect magnitudes depend on the severity of environmental perturbation. Consistent with such a possibility, cloning of three causal genes identified factors that have unrelated functions in stress response. Thus, our results indicate that polymorphisms in stress response can show effects that are intensified by environmental stress, thereby resulting in major genotype-environment interactions when multiple of these variants co-occur. Saccharomyces cerevisiae cross progeny show extremely poor growth specifically on ethanol at 37°C. This environment differs from the standard condition used for culturing budding yeast in both its carbon source (ethanol as opposed to glucose) and temperature (37°C as opposed to 30°C). We provide evidence that poor growth on ethanol at 37°C is caused by a number of predominantly additive loci that individually exhibit gene-environment interactions with both carbon source and temperature. These loci show their largest effects when carbon source and temperature are simultaneously modified, indicating their effect magnitudes may be influenced by the severity of environmental stress. Consistent with this possibility, we clone three causal genes and find they encode functionally unrelated components of stress response. Our work suggests that polymorphisms in stress response can contribute additively to genotype-environment interactions that vary in intensity across conditions in a stress level-dependent manner.Determining the genetic and molecular mechanisms that give rise to genotype-environment interaction (‘GxE’) is important for many areas of biology, including agriculture, evolution, and medicine. To help advance knowledge regarding this topic, we dissect the genetic basis of an example of GxE in which certain  Saccharomyces cerevisiae suggests GxE can arise due to not only individual loci that show gene-environment interactions, but also sets of loci that show environment-dependent epistatic interactions ) using Burrows-Wheeler Aligner (BWA) version 0.7.7-r441 [S1 Fig). Additionally, we found that two YJM NILs possessed nearly identical sets of introgressed regions, suggesting a technical error on our part during the recurrent backcrossing process. Only one of these NILs was included in our analyses .The NILs were genotyped by Illumina sequencing. Whole genome libraries were constructed using the Illumina Nextera kit, with each library tagged with a unique barcode for multiplexing. Each library was sequenced to an average per site genomic coverage of at least 21X on a NextSeq with 100 base pair (bp) x 100 bp reads. The BY and YJM parent strains were also sequenced to an average per site genomic coverage of ~100X, and these data were used to identify 57,402 high confidence SNPs. Reads for the NILs were mapped to the S288c genome  to a MATα hoΔ his3Δ version of YJM. Sporulation and selection for MATa segregants was performed as described for the initial F2 population.YJM F2B7 random segregants and 45 additional F2B7s that grew poorly on E37 were genotyped by sequencing to an average per site coverage of at least 5X using the same method described for the BY and YJM NILs. An additional 96 YJM F2B7 random segregants were genotyped at the five loci that had been introgressed into YJM NIL 3 using PCR and restriction enzyme typing. All reactions are provided in S6 Table. Fisher’s exact tests were then performed in R, using two-by-two matrices in which the first row contained the counts of BY and YJM alleles among the 45 F2B7s showing poor growth in E37, and the second row contained the counts of BY and YJM alleles among the 192 YJM F2B7 random population. Allele counts were measured at a single site for each locus that showed maximal allelic enrichment among the 45 F2B7s that grow poorly in E37.96 YJM F2B7 population, we grew each of these individuals on all possible combinations of carbon sources—glucose and ethanol—and temperatures—30 and 37°C. Individuals were pinned onto agar plates and then grown in the appropriate condition for three days. The plates were then imaged using the BioRAD Gel Doc XR+ Molecular Imager. The dimensions of all the images were set at 13.4x10 cm (WxL) and imaged under white Epi illumination with an exposure time of 0.5 seconds. The images were then exported as tiff files with a publishing resolution of 300dpi. To measure the pixel intensity of each colony, ImageJ [http://research.stowers.org/imagejplugins/index.html; S5 Fig). The Circ Background option was used to control for background noise. The average pixel intensity was determined by dividing the total pixel intensity by the area of the circle examined (7845 pixels2). Five biological replicate measurements using different randomized designs were taken for each F2B7 in each condition (S7 Table). The median pixel intensity among these five replicates was then used in downstream analyses (S8 Table).To further analyze growth in the F, ImageJ  was used2B7s in a particular condition, we implemented full factorial ANOVAs in R. Specifically, we modeled the median pixel intensity of the F2B7 segregants in each condition as a function of all possible additive and epistatic effects involving the four loci. The model was specified using the statement: lm(median_pixel_intensity_for_each_condition ~ genotype_at_locus_I * genotype_at_locus_VII * genotype_at_locus_X_1 * genotype_at_locus_X_2). ANOVA tables were then obtained using the anova function. In addition to the terms provided by R, we computed the percent of phenotypic variance explained for each locus by dividing the sum of squares associated with a particular term by the sum of squares total . Respectively, the fractions of phenotypic variance explained by all genetic effects (R2G) or only additive genetic effects (R2A) were computed by summing the fractions of phenotypic variance explained by all genetic terms or only additive genetic terms in a given model.To measure the additive and epistatic effects of the Chromosome I, VII, X_1, and X_2 loci among the F2B7s as a function of how many YJM alleles they carried. This model assumes complete additivity with loci showing equal effect sizes. These linear models were fit in R using the lm function in R with the statement lm.Within each condition, we modeled the median pixel intensities of the FkanMX, and 60 bp of the genomic region downstream of the transcribed portion of the gene, such that the entire coding and the promoter region of a given gene was replaced (S9 Table). All engineerings were performed in YJM NIL 3 and involved replacement of the BY allele of a given gene with the YJM allele. Each putative allele replacement was verified by Sanger sequencing. Controls were also generated to ensure that inserting kanMX near each gene was not responsible for our findings.All transformations were conducted using standard PCR-based techniques . Allele S. cerevisiae strains were downloaded from the Saccharomyces Genome Database , as well as from different S. cerevisiae resequencing projects [http://www.yeastgenome.org/blast-fungal). The putative RNA binding motifs of YGR250C were then identified from domain predictions available through InterPro (http://www.ebi.ac.uk/interpro/protein/P53316) [DNA sequences for other projects . DNA seq/P53316) .S1 Fig(TIF)Click here for additional data file.S2 FigOne YJM NIL, which is denoted as YJM NIL 2*, was excluded from further study as it appears to be a replicate of YJM NIL 2.(TIF)Click here for additional data file.S3 Fig2B7s are plotted. The Chromosome I, VII, X_1, and X_2 loci show statistically significant differences in their frequencies between the two populations , while the locus on Chromosome XVI did not (XVI: p ≤ 0.341). The significant loci are denoted with ‘***’.Frequencies of the BY alleles at each locus in the populations of poorly growing and control F(TIF)Click here for additional data file.S4 FigA) Amino acid differences between BY and YJM are shown with either a black line  or a red line . The three predicted RNA recognition motifs within YGR250C are labeled in purple. (B) The causal amino acid polymorphism in YJM is highlighted in red and other sites that differ from S. cerevisiae are highlighted in grey. Based on presently available genomes from recent resequencing projects [Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 TableColumn 2 shows the replicate number, and Columns 3 through 6 labeled ‘G30’,’G37’,’E30’, and ‘E37’ are the average pixel intensity of the colonies measured under the four conditions. The genotype at a locus is labeled as ‘0’ if the individual carries the BY allele or ‘1’ if it carries the YJM allele.(XLS)Click here for additional data file.S8 TableColumns 2 through 5 labeled ‘G30_med’, ‘G37_med’, ‘E30_med’, and ‘E37_med’ are the median pixel intensity measurements from the five replicates, which were used in the analyses described in the paper. The genotype at the four loci are labeled as ‘0’ if the individual carries the BY allele or ‘1’ if it carries the YJM allele.(XLSX)Click here for additional data file.S9 TablekanMX drug resistance cassette, which is used for drug selection and are same in all of the PCR primers. Base pairs shown in green varies depends on which genes are being replaced. For allele replacements on the positive strand, primers labeled GeneX MAR1 and GeneX MAR2 were used to amplify the promoter and the coding region of the gene being replaced with a 60bp tail overlapping the first 60bp of the kanMX cassette. KanMX amplification primer 3 and GeneX MAR4 were used to amplify the kanMX cassette with a 60 bp tail that is homologous to the genomic region downstream of the transcribed portion of the gene. The two PCR products were then co-transformed into the yeast cell and selected for G418 resistance. For allele replacements on the negative strand, primers labeled GeneX MAR1 and kanMX amplification primer 2 were used to amplify the kanMX cassette with a 60 bp tail that is homologous to the genomic region downstream of the transcribed portion of the gene. GeneX MAR3 and GeneX MAR4 were used to amplify the promoter and the coding region of the gene being replaced with a 60bp tail overlapping the last 60bp of the kanMX cassette. Expected PCR amplicons are provided in S2 Note.The first 11 rows show how the primes were designed to make allele replacement primers. Base pairs shown in black are sequences found in the (XLSX)Click here for additional data file.S10 TableAll the genomes were downloaded from SGD.(XLSX)Click here for additional data file.S1 Note(DOCX)Click here for additional data file.S2 NotekanMX are in black.Sequences corresponding to the target locus are in green, while sequences corresponding to (DOCX)Click here for additional data file.