Results showed that drought conditions caused significant changes in K+ uptake and translocation in a time- and genotype-specific manner, which consequently resulted in a significant difference in tissue K+ contents and drought tolerance levels between the contrasting barley genotypes. The role of K+ transporters and channels and plasma membrane (PM) H+-ATPase in barley’s adaptive response to drought stress was further investigated at the transcript level. The expression of genes conferring K+ uptake  and xylem loading (HvSKOR) in roots were all affected by drought stress in a time- and genotype-specific manner, indicating that the regulation of these K+ transporters and channels is critical for root K+ uptake and root to shoot K+ translocation in barley under drought stress. Furthermore, the barley genotypes showed a strong correlation between H+ efflux and K+ influx under drought stress, which was further confirmed by the significant up-regulation of HvHA1 and HvHA2. These results suggested an important role of plasma membrane H+-ATPase activity and/or expression in regulating the activity of K+ transporters and channels under drought stress. Taken together, it may be concluded that the genotypic difference in drought stress tolerance in barley is conferred by the difference in the ability to regulate K+ transporters and channels in root epidermis and stele.In this work, the effect of drought on K As a re climate , which t climate . Global  climate . TherefoHordeum vulgare L.) is the fourth largest cereal crop worldwide with multipurpose use as animal feed, human food and brewing material. In addition, barley is ideal for genetic studies because of its simple genetic background [Drought primarily disturbs water uptake and aggravates dehydration of the crop by disrupting the cellular osmotic equilibrium. This gradually develops into various physiological and metabolic disorders such as damaged photosynthetic activity and induced oxidative stress, all of which consequently result in yield reduction . Consideckground  and knowckground . Furtherckground . Recentlckground ,15. HowePhaseolus vulgaris [Glycine max [Zea mays [Plants have developed a series of sophisticated and effective strategies to adapt to drought conditions. Among these, osmotic adjustment coupled with induction of abscisic acid and dehydrins have conferred drought tolerance of plants by maintaining the high water potential in tissues . Osmoticvulgaris , Glycinecine max  and Zea Zea mays . Garg etZea mays  have proZea mays ,22. +) is the most abundant cation in plants and comprises 2% to 10% of a plant’s dry weight [+ uptake conferred a higher level of drought tolerance in Arabidopsis [+ transmembrane transport conferring the genotypic difference in drought tolerance in barley? If yes, what kind of K+ transporters or channels are involved in the drought tolerance?Compared with energy- and assimilate-consuming synthesis and accumulation of organic osmotic adjustment substances, taking up inorganic ions from the environment to increase cytosolic ion concentrations through transporters and channels is considered as a much more cost-effective way for intracellular osmotic adjustment . Potassiy weight . It is ny weight ,26,27. Py weight , wheat [y weight , faba bey weight  and cotty weight . Recently weight  found thbidopsis  and barlbidopsis . However+ uptake in root and translocation from root to shoot. Also, the role of K+ transporters and channels and plasma membrane (PM) proton pumps in the adaptive response of barley to drought stress was investigated by gene expression quantification. Our results showed that drought conditions caused a significant uptake of K+ in a time- and genotype-specific manner, affecting K+ regulation. This led to the conclusion that the genotypic difference in drought stress tolerance in barley is conferred by the difference in the ability to regulate K transporters and channels in root epidermis and stele.The aim of this study is to fill the above gaps in our knowledge. To achieve this, we used contrasting cultivated and wild barley genotypes to investigate the genotype-specific effect of drought on KA whole-plant experiment was undertaken to assess the genotypic variation in a range of barley genotypes under drought stress. Ten days of drought stress affected the growth of all barley genotypes, but to different extents. The most severe effect of drought on the plant growth was observed in genotypes Gairdner and XZ147, followed by Morex and XZ5, whereas WMLL and XZ141 suffered the least severe impact A. These v/m ratio) and stomatal conductance (gs). After 10 days of drought stress (stopping of watering), SPAD values of both the first and the last fully-expanded leaves were significantly reduced by a range of 20%–100%  from the last fully-expanded leaf was also significantly affected by drought stress in the sensitive or moderate tolerant barley genotypes  to 30% (in XZ147). However, no significant impact was observed on the chlorophyll fluorescence of the tolerant WMLL and XZ141 after drought stress (gs) was found in all six barley genotypes, ranging from 18% to 82% , followed by the moderate tolerant Morex and XZ5 . The sensitive Gairdner and XZ147 performed the worst, with only 28% and 18% of their gs values maintained after drought stress, in comparison to the control  in the leaves and stems were significantly decreased in response to drought stress . On aver+ uptake and root to shoot K+ translocation were then compared between the contrasting barley genotypes in terms of the tissue K+ contents and root epidermis net K+ fluxes  was measured from the mature root epidermis of all barley genotypes in the control . With the exposure time of drought stress increasing to 24 h, net K+ influxes of both moderate (Morex and XZ5) and tolerant (WMLL and XZ147) barley genotypes were also recovered to some extent (+ influxes maintained much more than the sensitive genotypes (p < 0.01). All these results indicated that the tolerance to drought is significantly correlated with the ability of K+ uptake and accumulation in barley (p < 0.01).The capability of K control A. The apss onset A. In the m−2 s−1 A. The moe extent B. Howeve+ effluxes were observed from the mature root epidermis of all six barley genotypes in the background of BSM solution  and xylem loading (HvSKOR) in roots were further compared across three contrasting barley genotypes  to find out which of these genes are contributing to the genotypic difference in K+ uptake and accumulation under drought stress. The relative transcript levels of all these genes were significantly affected by drought stress (generated by 20% PEG8000). HAK1, HAK5, KUP1 and KUP2, as members of KUP/HAK/KT transporter family, are crucial for the high-affinity K+ transport in plants [HvHAK1 was reduced approximately by half after drought treatment for 1 h, but dramatically increased afterwards  in Gairdner and Morex, whereas it was deeply suppressed in drought tolerant genotype WMLL during the entire exposure time of drought stress  in response to drought stress were further examined  than those in Gairdner and Morex at 6 d of stress onset.The results of net ion fluxes in response to drought stress showed that the great drought-induced Kt stress . Therefoexamined . The expenotypes A. In thel at 6 d A. In theure time A. This r by MIFE . On the t stress B. It is + contributes between 35% and 50% on average of the cell osmotic potential in crops [+ in response to drought stress [+ homeostasis is one of the key traits conferring the broad range of plant tolerance to abiotic stresses, such as salinity and waterlogging [+ uptake may be involved in the drought tolerance of barley [+ contents  rectifying channels such as AKT1 under normal K+ condition, and the high-affinity transporters from HAK/KUP/KT family under K+ deficiency [+ uptake, it can be hypothesized that more upregulation of AKT1 and HAK/KUP/KT family members may confer higher drought tolerance in barley. However, the present study demonstrated otherwise. Our results showed that the expression of HvAKT1 was deeply suppressed by drought stress and was much lower in the tolerant barley , known to be expressed at the xylem parenchyma interface [+ translocation from root to the shoot [SKOR severely hampers xylem K+ loading and consequently results in a dramatic decrease in shoot K+ content. It is also involved in plant adaptive response to drought stress. However, the reported experimental results remain controversial. In Arabidopsis, SKOR expression is suppressed by drought stress conditions [Zygophyllum xanthoxylum, however, ZxSKOR was induced under osmotic stress and improved drought tolerance of the plants [HvSKOR differed greatly between the contrasting barley genotypes. In the sensitive genotype Gairdner, it was significantly increased after onset of drought stress . AHA1 and AHA2 are found to express in almost all tissues and organs, functioning as housekeeping genes required for ion homeostasis [AHA1 or AHA2 has no apparent phenotypes while double knockout is lethal [+-ATPase activity was reported during early pathogen recognition events [+-ATPase was also observed in the present study. The induction of HvHA1 was observed after drought stress treatment for 1 h in Morex and WMLL, and for 6 h in Gairdner  Hitioning ,63. In tc manner , suggesteostasis . Single s lethal ,66, indiondition . In addit stress , while iGairdner B, indica complex . Taken tHordeum vulgare L.) genotypes, namely Gairdner , Morex , Wumangliuleng , XZ5, XZ141, and XZ147  were used in this study. Among these barley genotypes, Gairdner and XZ147 are sensitive, Morex and XZ5 are moderately tolerant, and WMLL and XZ141 are tolerant to drought, respectively (unpublished data). Seeds were multiplied in the field of Zhejiang University, Hangzhou. Seeds were surface-sterilized for 15 min with 10% commercial NaClO  and then rinsed for 30 min with tap water. Sterilized seeds were germinated in the dark at 26 ℃ for 48 h in the controlled growth chamber. Uniform well-germinated seeds were then planted in the vermiculite and grown with one-fifth Hoagland solution (pH6.0) [−2 s−1, temperature of 22/18 ℃ (day/night), and relative humidity of 60%. Barley seedlings were grown to have two fully-expanded leaves (for 12 days) prior to the drought treatment, in order to make the difference in the phenotypic performance as significant as possible between treatments and barley genotypes. Thereafter, irrigation was stopped for half the barley seedlings to generate a drought condition, whilst the others were well irrigated to the water content of 40% . Ten days later, shoots and roots of barley plants under both control (water content of ∼40%) and drought (water content of ∼4%) conditions were sampled to investigate the morphological performance as well as the physiological parameters.Six barley ( (pH6.0)  in the cPrior to the harvest of the biomass, the number (no.) of wilting leaves and the total number leaves from each of the drought-stressed plants were counted. The leaves that were ≤ 50% wilted were counted as 0.5 and those that were > 50% wilted as 1. The ratio of wilting leaves was then calculated according to the equation: ratio of wilting leaves = no. of wilting leaves/no. of total leaves. The shoot of the counted plant was then excised, dried at 75 ℃ in the oven for 2 days, and weighted for the dry weight. Five replicates were randomly taken for each barley genotype.v/m = (Fm–Fo)/Fm) was recorded at a saturating actinic light (660 nm) with an intensity of 1100 µmol m−2 s−1. Stomatal conductance was measured from the latest fully-expanded leaf with a porometer . All three measurements were conducted on the middle part of the fully-expanded leaves following the manufacturer’s instructions. Ten replicates were randomly taken for each barley genotype under either control or drought condition.Leaf chlorophyll content was measured from both the first and the last fully-expanded leaves with a SPAD meter . Chlorophyll fluorescence was measured from the latest fully-expanded leaf with a portable fluorimeter . Plants were dark-adapted for 30 min prior to measurement. The maximum quantum efficiency of photosystem II (FFresh leaves from plants under control or drought condition were collected and weighed immediately for their fresh weight (FW). The weighed leaves were then soaked in deionized water for 2 h at room temperature, and their turgid weights (TW) was weighed subsequently. Dry weights (DW) were determined after drying for 2 days at 75 ℃. RWC was calculated using the following formula: RWC% = (FW − DW) / (TW − DW) × 100%. Five replicates were determined for each barley genotype under either control or drought conditions.After harvesting, the first and last fully-expanded leaves, stems, and roots were collected separately and immediately stored in the 1.5 mL centrifuge tubes at −20 ℃. Tissue sap was extracted through the freeze-thawing method according to Tomos et al. . After c3 at 120 ℃ for 2 h. The digested solution was then diluted with deionized water and analyzed for K+ concentration using atomic absorption spectrophotometry . Tissue K+ contents were then calculated with the K+ concentrations in the diluted solution and tissue dry weights.Dried barley leaves or roots were weighed and digested with HNO+ and H+ fluxes were measured using the MIFE technique , essentially as described in our previous publications [2; pH was adjusted to 6.0 with HCl or KOH). Three-day seedlings (with only ∼ 1 cm long coleoptile) with 7 ~ 8 cm long roots, which can be easily handled and immobilized in the measuring chamber, were used for ion flux measurements. Ion-selective microelectrodes containing specific liquid ion exchanger  in the tips were positioned 50 µm above the root surface. Ion fluxes were measured by a slow  square-wave movement of electrodes between two positions, close to (50 µm) and away from (100 µm) the root surface. Net ion fluxes were measured from the mature root (∼20 mm from the root tip) epidermis. The voltage difference between two positions was recorded by the MIFE CHART software and converted to the electrochemical potential difference using the calibrated Nernst slope of the electrodes. Net ion fluxes were then calculated from the electrochemical potential difference using cylindrical diffusion geometry with the MIFEFLUX program [Net Kications ,74. Barl program ,76. For the transient ion flux measurements, roots of intact seedlings were horizontally immobilized on a glass slide in a petri dish containing BSM 1 h prior to the measurement. Transient ion fluxes were measured for 5 min under the control condition  as the steady initial net fluxes. Subsequently, BSM solution was replaced by 20% PEG8000 in the background of BSM solution, and transient ion flux responses were recorded for another 50 min. The period of time for changing the solution (∼2 min) was discarded from the data analysis. Eight individual roots were measured for each barley genotype.For the steady-state ion flux measurements, 3-day-old seedlings were pretreated with 20% PEG8000 in the background of BSM for 1 h, 6 h and 24 h prior to the ion fluxes measurements. Seedlings without PEG treatment were used as the control. Ion fluxes were measured for 10 min, and the steady-state fluxes were calculated by averaging the values of the last 5 minutes. Eight individual roots were measured for each barley genotype.+ transmembrane transport and plasma membrane ATPase were determined in the barley genotypes of Gairdner, Morex and WMLL. Barley seedlings were grown in aerated one-fifth Hoagland solution (pH6.0) until the first leaf was fully expanded (for 7 days) to ensure enough root segments for gene expression analysis. Then, drought stress was induced using polyethylene glycol 8000  in the background of one-fifth Hoagland solution (pH6.0). Two drought levels were used as control (CK) and 20% PEG8000. The solution was renewed every day. After onset of drought treatment for 1 h, 6 h, 1 d and 6 d, root segments (∼50 mm from the root tip) of barley plants under both control and drought conditions were collected for RNA extraction and qRT-PCR. Three biological replicates were determined for each genotype.The transcriptional levels of genes for K−△△Ct method using Beta-tubulin as the internal standard [Total RNA was extracted by using MiniBEST Plant RNA Extraction Kit  following the manufacturer’s instructions. RNA concentrations were determined by NanoDrop2000 spectrophotometer  and qualities were detected by agarose gel electrophoresis. The cDNA was synthesized from total RNA (1 ug) using PrimeScript RT Reagent Kit  and was used as templates for qPCR amplification. qPCR amplification was performed with LightCycler 480 II  using SYBR Green Supermix . The primer sequences are listed in the standard ,78, and p < 0.05, 0.01 or 0.001level, respectively.Statistical analyses were performed using SPSS Statistics 20 . All data in the figures were given as mean ± SE. Significance of differences was determined by Student’s t-test. One to three asterisks indicate the significance at + influx and H+ efflux, compared to the sensitive genotype Gairdner. The current results demonstrated that root K+ uptake and root to shoot K+ translocation confer the osmotic adjustment and contribute to the genotypic difference in drought tolerance levels in barley. Gene expression measurements showed that the expression of genes conferring K+ uptake  and xylem loading (HvSKOR) in roots were all affected by drought stress in a time- and genotype-specific manner, which consequently led to a significant genotypic difference in root and shoot K+ contents. These results indicated that the regulation of the K+ transporters and channels is critical for root K+ uptake and root to shoot K+ translocation in barley under drought stress. Furthermore, the barley genotypes showed a strong correlation between H+ efflux and K+ influx under drought stress, which was further confirmed by the significant up-regulation of HvHA1 and HvHA2, suggesting an important role of PM H+-ATPase activity and/or expression in regulating the activity of K transporters and channels under drought stress. Taken together, the ability to regulate potassium transport across plasma membrane in roots is critical for drought tolerance in barley.Our MIFE measurements revealed that drought tolerant barley genotype WMLL exhibited a significantly larger K


Radix balthica and the marine amphipod Orchestia gammarellus. The challenge of phenomics is significant but so too are the rewards, and it is particularly relevant to the urgent task of assessing complex organismal responses to current rates of environmental change. EmbryoPhenomics can acquire and process data capturing functional, temporal, and spatial responses in the earliest, most dynamic life stages and is potentially game changing for those interested in studying development and phenomics more widely.Phenomics has the potential to facilitate significant advances in biology but requires the development of high-throughput technologies capable of generating and analysing high-dimensional data. There are significant challenges associated with building such technologies, not least those required for investigating dynamic processes such as embryonic development, during which high rates of temporal, spatial, and functional change are inherently difficult to capture. Here, we present EmbryoPhenomics, an accessible high-throughput platform for phenomics in aquatic embryos comprising an Open-source Video Microscope (OpenVIM) that produces high-resolution videos of multiple embryos under tightly controlled environmental conditions. These videos are then analysed by the Python package Embryo Computer Vision (EmbryoCV), which extracts phenomic data for morphological, physiological, behavioural, and proxy traits during the process of embryonic development. We demonstrate the broad-scale applicability of EmbryoPhenomics in a series of experiments assessing chronic, acute, and multistressor responses to environmental change  in >30 million images of >600 embryos of two species with markedly different patterns of development—the pond snail  EmbryoPhenomics is an open-source technology platform for high-throughput phenome screening of aquatic embryos. This paper demonstrates its application in experiments assessing the sensitivity of aquatic embryos to environmental stress, consisting of more than 600 embryos and more than 30 million images. www.embryophenomics.org), a new open-source technological platform comprising high-throughput bioimaging hardware that produces high-resolution video of multiple, developing embryos maintained under controlled environmental conditions and software for automatically quantifying embryo responses from these videos. We demonstrate the broad applicability of EmbryoPhenomics using four experiments assessing responses to global change  in which we generate data for more than 600 embryos produced from video comprising more than 30 million images. EmbryoPhenomics was used to capture functional, temporal, and spatial change in morphological, physiological, and behavioural responses in the earliest, most dynamic life stages and addresses a serious bottleneck in biology. Such capabilities are urgently required, particularly within the context of assessing the response of embryos to the current unprecedented rates of global environmental change.Phenomics is the collection of high-dimensional phenotypic data on an organism-wide scale, and it requires high-throughput technologies. However, a lack of technologies for efficiently visualising and measuring whole-organism responses to different environments represents a serious challenge for biologists. This challenge is most apparent when studying complex responses, such as those occurring during the dynamic period of embryonic development, when the phenotype changes markedly through time. Here, we present EmbryoPhenomics ( Phenomics is the acquisition of high-dimensional phenotypic data on an organism-wide scale . It is bTechnologies for acquiring high-resolution phenomic data enable scales of data collection  and combThe rewards of phenomics will arguably be greatest when used to quantify aspects of the phenome for which current methods are most limiting. Embryonic development is a dynamic process with high levels of intraindividual temporal, spatial, and functional change, meaning that traditional approaches to its quantification at the whole-organism level can never be thorough. Furthermore, in addition to high levels of intraindividual change, embryonic development also exhibits high levels of interindividual variation , and theThere are good examples of when automated longitudinal observation of developing embryos has been used effectively for commercial applications. Automated noninvasive imaging and analysis of cytokinetic patterns in early-stage cell cleavages in human embryos can predict survival to the blastocyst stage with >93% sensitivity . ConsequRadix balthica, a freshwater gastropod mollusc, and Orchestia gammarellus, a marine amphipod crustacean.Here, we present EmbryoPhenomics, an open-source high-throughput platform for phenomics in aquatic embryos that consists of an Open-source Video Microscope (OpenVIM), with experimental control over the embryonic environment, and a Python package Embryo Computer Vision (EmbryoCV) for high-dimensional measurement of phenomic traits from video data sets. The EmbryoPhenomics platform enables the quantification and integration of interindividual and intraindividual temporal change in morphological, physiological, and behavioural traits with high resolution and to an unprecedented scale in a largely automated workflow. We demonstrate the application of EmbryoPhenomics for quantifying integrated organismal responses to environmental change, using experiments assessing i) chronic, ii) acute, and iii) interactive effects of environmental stressors. Furthermore, we show the efficacy and transferability of this platform using two ecologically important species with radically different patterns of development: www.embryophenomics.org) consists of open-source hardware  and software . The EmbryoCV software performs analysis and extraction of data from every image sequence acquired  chronic, ii) acute, and iii) interactive effects. Furthermore, we demonstrate the transferability of the platform using two ecologically important species with different patterns of development: workflow .R. balthica embryos were placed in three temperatures  and recorded using OpenVIM for 30 s at 20 frames per second every hour for the duration of their development. This generated a total of 17.73 million images for all 144 embryos and these were subsequently analysed by EmbryoCV. The embryo was successfully identified and measured by the Image Analysis stage of EmbryoCV in 16.32 million of the acquired images (92%) and in 143 of the 144 embryos recorded using the OpenVIM , rates of movement , and heart rates  but also revealed a marked difference in the direction and magnitude of responses between temperature increments  fluctuations at all temporal frequencies in the embryo. Consequently, a reduction in the Total Energy proxy trait  of R. balthica embryos to an elevated temperature of 36°C. OpenVIM recorded individual embryos for 30 s at 20 frames per second every 20 minutes for 24 h. This generated a total of 7.7 million images for all 183 embryos, and these were subsequently analysed by EmbryoCV. We assessed the ability of EmbryoCV to quantify the occurrence of lethal end points (time to death) within developmental stages that exhibit different forms of biological response. In addition to the core EmbryoCV processes of Image Analysis and Data Integration, an additional Data Analysis process—identifyLethalEndPoint—was used to identify time to death for each embryo from the data that had been collected in the preceding stages, including size, movement, and energy within different frequency bands.To assess the capability of the EmbryoPhenomics platform for measuring extreme  biological responses, we used an acute 24-h exposure of three developmental stages . The earliest developmental stage (E3) had a lower LT50 (8.6 h ± 1.82) and LT75 (4.11 h ± 0.98) than the two later stages , indicating a greater sensitivity to thermal stress. These early stage embryos exhibited a ciliary-driven spinning behaviour, and their lethal end point coincided with a loss of osmotic control, made visual by a rapid increase and subsequent decrease in embryo area. Automated peak identification was used in the EmbryoCV function identifyLethalEndPoint to identify this lethal end point. E7-stage embryos possess a weak heartbeat and a transitionary form of locomotion consisting of part ciliary-driven gliding and part muscular crawling on the egg capsule wall, whereas E9-stage embryos possess a strong heartbeat, radula movements, and muscular contractions. Lethal end points in E7- and E9-stage embryos were indicated by a loss of cardiac function and a cessation of gross embryo movements. The EmbryoCV identifyLethalEndPoints function identified this response in these later developmental stages and detected lethal end points via a decrease in the relative energies within specific summed frequency bins within the energy proxy traits.Of the 183 embryos studied, 93% were correctly identified as exhibiting a lethal end point. Concordance with manually determined lethal end points was high for all three developmental stages  to contrasting temperatures  and salinities . OpenVIM recorded embryos for 30 s at 20 frames per second for 24 h. This generated a total of 4.3 million images for all 288 embryos, and these were subsequently analysed by EmbryoCV  and significant effects of, and a significant interaction between, temperature and salinity on growth rate .The ability of the EmbryoPhenomics platform to detect complex multifaceted responses within multistressor experiments was assessed using a 24-h exposure of E3-stage EmbryoCV . There wIn a salinity of 0, EmbryoCV identified increases in both cumulative movement and growth rate that were associated with an increase in temperature from 20°C to 25°C; however, from 25°C to 30°C, the increase in growth rate was markedly reduced despite a similar increase in movement. At a salinity of 7, both growth rate and movement were reduced at each temperature, relative to the 0-salinity treatment. Furthermore, the proportionate magnitude of response comparing 20°C to 25°C and 25°C to 30°C was more pronounced in salinity 7, with a significant interaction in the response of embryo growth rates to temperature and salinity. This suggests that the ability to tolerate elevated temperatures was compromised by the addition of a salinity stress.The EmbryoCV software and OpenVIM hardware have been developed to be versatile and extendible to a wide range of species and scientific applications. A core process within EmbryoCV is the use of a multiresolution blockwise signal quantification that is capable of quantifying embryonic traits with different functional forms and of capturing embryo responses as holistic proxy measures.R. balthica, the embryo exhibits both ciliary-driven rotation and a muscular crawling behaviour within the egg capsule. Furthermore, R. balthica possesses a two-chambered globular heart and undergoes the process of torsion in which its mantle cavity (where the heart is located) rotates by 180°. The automated quantification of cardiac function in R. balthica is demonstrated in Experiment 1, and here, we apply the same process to quantifying the response of cardiac function in the intertidal amphipod O. gammarellus during a 24 h period in contrasting temperatures  and approximately four times faster in 20°C than 15°C. Such high rates of acceleration in the physiological ontogeny of cardiac function in response to a 5°C increase in temperature are interesting and suggest a high thermal sensitivity of O. gammarellus.OpenVIM recorded R. balthica embryos to combined thermal and saline stress and revealed an inhibitory effect of salinity on growth and movement that contrasted with the effects of elevated temperature. The generation of high-dimensional data is a prerequisite to phenomics, and owing particularly to the dynamic nature of embryonic development, this capability will facilitate advances in our understanding of this pivotal life stage. EmbryoCV and OpenVIM are versatile, extendible, and open-source technologies applicable to a range of species and study designs. Our aim is that these resources will develop a community of users, in a similar model to that seen with the Open Selective Plane Illumination Microscopy (OpenSPIM) project [Here, we demonstrate the ability of the open-source EmbryoPhenomics platform to quantify complex phenome-level responses of large numbers of developing aquatic embryos. EmbryoPhenomics is versatile and we demonstrate its application to a broad range of experimental designs, ranging from 2-week to 24-h experiments, and demonstrate its application to two species. These experiments include assessment of responses to acute and chronic thermal stress, arguably one of the greatest current threats to biodiversity and one to which early life stages can have heightened sensitivity . EmbryoP project . In cont project , 28, botR. balthica but also the elongated, tubular heart of O. gammarellus. The typical maximum cardiac rate of R. balthica at 20°C is 1.5 Hz, whereas O. gammarellus has a higher cardiac rate of 3.5 Hz with sustained periods of diapause, presenting challenges in the effective manual quantification of rate. Here, for O. gammarellus, to account for the skew introduced by diapause, beat-to-beat timings were recorded manually, and the rates calculated from the median timing were closely correlated with the frequency measurements produced by EmbryoCV. For R. balthica, direct comparisons of manually determined rate, i.e., counts of heartbeats, were closely correlated with EmbryoCV determined rates. Movements of the embryo meant that the heart and other features of interest were not always visible in the image. Consequently, EmbryoCV applies spectral frequency analysis across different areas of the embryo and attempts to identify biologically relevant frequencies for modelling heart rate. These data then inform the fitting of models to heart rate, and the resultant cardiac rates were closely correlated to manual measures for both species in Experiments 1 and 4. A similar workflow also underpins the automated identification of lethal end points across the three developmental stages of R. balthica studied in Experiment 2. Here, developmental stage–specific algorithms identifying rapid increases in size indicative of osmotic control, rapid reductions in energy within particular frequency bands, or a combination of the two and were highly effective at producing classifications of lethal end points closely aligned with those ascertained manually. Further optimisation and mining of the data produced by EmbryoPhenomics will allow tailored end points to be identified, including responses that, while present, are not immediately apparent to human observers in the multitemporal video produced by OpenVIM.Concordance between manually and EmbryoCV-determined measures of embryo spatial and temporal characteristics was high . The bloN > 600) in experimental treatments ranging from long- (>240 h) to short-term (24 h) exposures. Of the >30 million images acquired across the four experiments described here, EmbryoCV successfully extracted phenome-level data from 95%, producing high dimensional data describing morphological, physiological, and behavioural embryo responses.Phenomics offers the potential to facilitate rapid advances in our understanding of biology via the generation of a quantity and quality of phenotype-level data that is more appropriate to addressing questions focused on understanding the complexities of organismal biology. The EmbryoPhenomics platform provides the technological capability for the study of organismal development in a way that captures temporal, spatial, and functional diversity at both interindividual and intraindividual levels—a task identified as a major challenge in biology , 11. HerImage acquisition in the EmbryoPhenomics platform is achieved using OpenVIM, a modular, custom assembled video microscope that enables long-term automated image acquisition of large numbers of aquatic embryos. OpenVIM is modular and can incorporate a range of different components, enabling a wide range of operating parameters Figs . The motImage acquisition is performed using the Multidimensional Acquisition function in MicroManager. A sequence of images is acquired of each individual embryo in succession, and this process is repeated for the duration of the experiment using a Beanshell script . Images www.embryocv.org), a Python [The software component of the EmbryoPhenomics platform is EmbryoCV .Command: Validating egg identification: an optional stage during which a user interface is used for modifying the egg ROI size, shape, and position prior to beginning the more computationally demanding aspects of the analysis.experiment.validateEggsCommand: Quantify embryo traits: measurement of size, shape, position, and multiresolution blockwise signals are made from every image within each image sequence and stored to the embryo results files. Multiresolution blockwise signals of mean pixel intensity are produced at different spatial resolutions 1 x 1 (whole embryo ROI), 2 x 2, 4 x 4, 8 x 8, and 16 x 16. Power spectral densities within different temporal frequencies are then calculated using Welch’s method from the signal module of Scipy , 37 to pexperiment.quantifyAllEmbryosCommand: Integrate embryo traits: raw measurements from the preceding step are integrated to form biologically relevant measures, including time-specific measurements such as minimum, maximum, and mean size and movement at each time point during the experiment but also global measurements such as growth rate. Frequency analysis is also applied to the blockwise signals generated in the preceding step to quantify energy within different frequency bands. At this stage, data are transformed into a dynamically accessible XArray Dataset.experiment.savePhenomeMeasuresForAllEmbryos(‘pathToSave’)Command: Generate summary reports: produces PDF reports for individual embryos, including developmental time series of time, movement patterns, and energy within different frequency bands.experiment.generateSummaryReports(‘pathToSave’)Command: R. balthica) or linear (O. gammarellus) model. Time series of cardiac rates, including parameters from the model are output.Quantify and model cardiac rates: identify cardiac rates from within the frequency data output and fit either a segmented (experiment.measureHeartRateFoAllEmbryos(‘pathToSave’)Command: R. balthica embryos, using either reductions in energy within particular frequency bands or sudden increases in size indicative of a failure of osmotic control or a combination of the two.Quantify lethal end points: use data from previous processes to identify lethal end points in different stage embryos, optimised to work with E3-, E7-, and E11-stage analysis.identifyLethalEndPointsCommand: Focussed data analysis optional steps:quantifyAllEmbryos (-1) and data are stored to disk as a Pandas Dataframe via pickle. A high proportion (>95%) of the 30.03 million images acquired by OpenVIM across the four experiments analysed were successfully analysed by EmbryoCV.The experiments described in this paper were analysed on Apple Mac computers . The most computationally intensive stage of EmbryoCV is lEmbryos , during N = 617) were randomly selected from Experiment 3 and were presented to users (N = 7) for manual analysis in Fiji [Assessment of the accuracy of the embryo size measurements produced by EmbryoCV was performed by comparison of manual and automated measurements of embryo area . Images  in Fiji  with a s in Fiji  user intR. balthica is significantly slower than in O. gammarellus and more regular in its rhythm. Consequently, manual counts of the number of heartbeats visible within the video at a particular time point were considered a reliable benchmark against which to compare the heart frequency identified for R. balthica embryos by EmbryoCV.Heart rate measurements performed by EmbryoCV were validated by comparison with heart rate measurements made by performing manual video analysis. The heart rate of O. gammarellus has an irregular heart rhythm, including extended periods of asystole; therefore, it was necessary to record the timing of individual heartbeats to enable a representative heart frequency to be calculated via analysis of beat-to-beat timings. A Fiji macro  and maintained in the laboratory for a minimum of 7 days prior to experiments. Snails were maintained at 15°C in a 20 L aquaria containing Artificial Pond Water  with 90 mg Ca2+/litre [Experiments 1–3 used 2+/litre . Water wO. gammarellus were collected by hand from Mount Batten beach (50.35° N 4.13° W) and were maintained in a 1-L aquaria on damp filter paper at 15°C and were fed carrot ad libitium. Embryos were harvested from mothers within 48 h of collection via extraction using a paint brush from the brood pouch. They were then staged and inserted into a prewarmed multiwell plate at the relevant treatment temperature . Water was changed every 2 days. Automated image acquisition using OpenVIM and subsequent image analysis using EmbryoCV was performed for each experiment as outlined in S1 Code(ZIP)Click here for additional data file.S2 Code(TXT)Click here for additional data file.S3 CodeInterface provides users ability to measure and record embryo (polygon tool) and egg outlines (ellipse), with automated saving of results, for comparison with EmbryoCV-determined measures. EmbryoCV, Embryo Computer Vision.(DOCX)Click here for additional data file.S4 Code(DOCX)Click here for additional data file.S1 VideoOpenVIM, Open-source Video Microscope.(MP4)Click here for additional data file.S1 TableOpenVIM, Open-source Video Microscope.(PDF)Click here for additional data file.S1 TextOpenVIM, Open-source Video Microscope.(PDF)Click here for additional data file.S1 DataEmbryoCV, Embryo Computer Vision.(CSV)Click here for additional data file.S2 DataEmbryoCV, Embryo Computer Vision.(CSV)Click here for additional data file.S3 DataEmbryoCV, Embryo Computer Vision.(CSV)Click here for additional data file.S4 Data(CSV)Click here for additional data file.S5 Data(CSV)Click here for additional data file.


We did not identify any statistically significant associations between water consumption or consumer product use practices and urinary phthalate metabolite concentrations in our adolescent group, and directions of correlation coefficients differed by individual phthalate compound. While phthalate exposure was widespread, these results highlight the challenges in examining phthalate exposure determinants and emphasize the need for further investigation into understanding exposure sources and potential health risks from chronic low-level exposures.Phthalates are plastic softeners that have been linked to several adverse health outcomes. The relative contributions of different sources to phthalate exposure in populations in different regions and at different life stages is unclear. We examined the relationships between water consumption, consumer product use, and phthalate exposure among 40 adolescents  in Cluj-Napoca, Romania. Interviewers administered a questionnaire about drinking water consumption and use of phthalate-containing consumer products. Four common phthalates were measured in representative samples of participants’ municipal drinking water and consumed bottled water using gas chromatography-mass spectrometry. Urine samples were collected from participants and analyzed for the corresponding phthalate metabolites. Relationships between different exposure measures were assessed using nonparametric tests . Diisobutyl phthalate, dibutyl phthalate, and bis(2-ethylhexyl) phthalate were commonly detected in bottled water, but generally not the municipal drinking water samples. Mono- Phthalates are plastic softeners found in a range of industrial and commercial products including flooring tiles, varnishes, food packaging materials, and personal-care products . In addiPhthalate exposure has been linked to several health outcomes across adult and child populations, the most well-studied being obesity. Associations with obesity have been reported in male children, male and female adolescents, and female adults . Addition-octyl phthalate (DOP) in China [Exposure to phthalates in drinking water is an emerging area of public health concern. Studies suggest ingestion of water, as well as food, are important routes of exposure for phthalates ,11. Phthin China . Bottledin China .Our study was conducted in the city of Cluj-Napoca, Romania where, like much of the European Union (EU), the municipal drinking water system is primarily composed of HDPE or PVC pipes. A prior study suggested phthalate concentrations in municipal drinking water may be of public health concern . RomaniaPrevious phthalate biomonitoring studies focused on pregnant or lactating women ,14 and cThe overarching goal of the study was to better understand the relationships between water consumption and consumer product use and phthalate exposure among adolescents in Romania. The specific objectives were (1) characterize drinking water consumption patterns of the adolescent population; (2) analyze tap and bottled water samples for the presences of phthalates; (3) evaluate the association between water consumption behaviors and the presence and concentrations of urinary phthalates metabolites; and (4) assess whether consumer product use was associated with phthalate biomarker concentrations.Study protocols were approved by the Human Investigation Committee at Yale University and the Environmental Health Center (Centrul de Mediu si Sanatate) in Cluj-Napoca, Romania. Potential participants were identified from the Environmental Health Center’s existing database of adult participants of previous drinking water studies who had consented to be recontacted regarding continuing or related drinking water studies. The adult participants were contacted by phone and asked whether they had a child between the ages of 12 and 17 currently resided in the cities of Cluj-Napoca or Floreşti. Adults with children of eligible ages were given further information on the study purpose and activities and asked permission to recruit their child. If permitted, we contacted their adolescent child by phone call to ascertain his or her interest and eligibility. Those who agreed were interviewed in their native language—Romanian or Hungarian—either at the subject’s home or the Environmental Health Center depending upon the subject’s preference. The study was approved by the Ethics Committee of Centrul de Mediu si Sanatate (registration number 290) on 6 April 2017 and Yale University (HIC #2000020986) on 19 May 2017.We used a standardized, structured questionnaire to obtain information on demographics , health status  drinking water behavior at home and at school , and other known phthalate sources from the subject’s consumer product use . Questions were designed to understand the magnitude and frequency of contact with phthalate-containing consumer products, particularly personal care products. We queried about several personal care products  and created a score with three categories: low , medium (5–7 used weekly), and high (8–9 used weekly) for analysis.For those reporting bottled water use, we queried brand names and consumption frequency of the bottled water both in general and specifically within the 24 h prior to the urine collection. We also asked whether the water was uncarbonated (“still”) or carbonated (“gas”) and queried the storage conditions  for the bottled water for both general and previous 24-h consumption. Respondents identified their main reason for using bottled or tap water, and their opinions of the drinking water.n = 33), we collected a 1-L sample either from their kitchen tap or, if a study participant served by the same public water system had already provided a sample, the previously collected sample was used, and no new sample was collected. In total, we collected 10 tap water samples, which were used to represent tap water for all participants primarily consuming tap water. Samples were collected in precleaned glass containers, transported back to the lab on ice, and extracted upon arrival.For participants reporting tap water as their primary drinking water source within the 24 h before the home visit (n = 27) reported 16 unique combinations of brand, type (still or gas), and storage conditions (room temperature or refrigerated). Subsequently, we analyzed one sample for each of these 16 conditions (n = 16). We purchased the corresponding brands and recreated the reported storage conditions in the Environmental Health Center lab prior to analysis, to best represent the water consumed by participants. In the laboratory refrigerator, unopened in their original containers, we stored both gas (n = 3) and still (n = 5) samples at 1 to 4 degrees Celsius. We also stored the both gas (n = 5) and still (n = 3) samples at room temperature in a cupboard not in direct sunlight. No one reported storage conditions in direct sunlight in the previous 24 h of consumption, so we did not create these storage conditions.Participants with any bottled water use within the previous 24 h . The extraction cartridges were preconditioned by first washing with one column volume of ethyl acetate, vacuuming for 10 s, then washing with two column volumes of methanol. We then passed 250 mL samples through the column and vacuum dried the cartridge. The compounds were eluted with ethyl acetate. We transferred the samples into 1.5 mL vials, covered the vials with aluminum foil, closed the vial cap, and analyzed with GC-MS.N,O-bis (trimethylsilyl) trifluoracetamide with 1% trimethylchlorosilane (BSTFA with 1% TMCS) and 0.9 mL of ethyl acetate were added to each sample. The vials were covered with aluminum foil and placed in thermoreactor at 65 °C for 1 h. The samples were transferred into 1.5 mL vials and coated with aluminum foil before placing the vial cap and ultimately analyzed with GC-MS.We analyzed all urine samples for the urine metabolites corresponding to the parent phthalate compounds measured in water, namely MnBP, MEHP, MBzP, MEOHP, and MEHHP, according to Kim et al., 2014 , with mig. A total of 1-mL of urine was transferred to a 100 mL volumetric flask and filled it to its nominal value with distillated water. A 2-mL aliquot of the diluted sample was transferred into a new vial, with 6 mL of picric acid and 0.4 mL sodium hydroxide (10%) added. After 20 min at room temperature, the sample was analyzed for creatinine by molecular spectrophotometry.To correct for urine dilution, all samples were analyzed for creatinine. Samples were centrifuged for 15 min at 1600× A blank and a laboratory control sample was analyzed for every 10 water or urine samples analyzed. Phthalates were not detected in any of the blank samples, indicating that no phthalate contamination occurred during sample analysis. The values for the control were all confirmed to be between the limits of a Shewhart Control Chart.We conducted descriptive statistics to characterize the drinking water and consumer product use in the study population. We also calculated summary statistics to describe the distribution of phthalate concentrations in water and urine samples. Samples with phthalate concentrations below the detection limit were assigned a value corresponding to half the limit of detection.We estimated exposure to each phthalate compound occurring in the past 24 h from drinking water using the following equation.This exposure variable was calculated separately for each of the phthalates. We evaluated associations between the calculated exposure to the parent phthalate in water and the corresponding urinary metabolite concentrations with Spearman rank correlation coefficients. We stratified the urinary metabolites by water consumption and consumer product usage of the subjects, and compared these ranges using the Kruskal–Wallis test.We recruited 20 female and 20 male adolescents for this study. The mean age of the participants was 15 years (SD = 1.8) and the average body mass index was 20.89 (SD = 3.8). Mirroring their age, participants were most likely to have 8 to 9 years of education (48%), followed by 5 to 7 years (27%), and 10 to 11 years (25%). The participant’s parents were most likely to have 15 to 19 years of education , followed by 8 to 14 years , and 20 to 25 years .Phthalates were generally not detected in the tap water samples , with onPhthalate exposure was prevalent: all but two participants had detectable levels of at least one phthalate metabolite . PercentCalculated exposure to parent phthalates from drinking water based on Equation (1) yielded the following median and interquartile ranges. DBP exposure = 5.12 µg ; DEHP = 2.37 µg ; and DiBP = 1.67 µg . Water exposure estimates were not correlated with urinary concentrations of corresponding metabolites . DBP, the parent compound of MnBP, was most highly detected in the water samples, but we did not observe a correlation between them.p < 0.1).n = 15), conditioner (n = 13), and make-up (n = 11), though of these individuals, some reported use of make-up (55%) and body lotion (53%) daily. Neither consumer product use nor demographics  were associated with phthalate metabolite concentrations. Those in the highest category of personal care product use had the highest concentrations of phthalate metabolites, but this relationship was not statistically significant.Our study used questionnaires, water samples, and urine phthalate metabolite concentrations to examine if water consumption influences phthalate exposure in adolescents. Most municipal water samples had no detectable levels of phthalates, while phthalates were commonly detected in bottled water samples. Phthalate metabolites were detected in 38 of the 40 participants urine samples. We did not find a significant association between drinking water phthalate exposure, consumer product usage, and urinary metabolite concentrations.While we observed concentrations of DiBP of 0.084 and 0.104 µg/L in two of the municipal water samples collected in Cluj-Napoca and no detectable levels of DBP or DEHP, a previous study found 0.05 µg/L DBP in one sample in Dâmbu Rotund and values up to 0.3 µg/L DiBP in the water throughout Florești, Gruia, and Dâmbu Rotund areas of Cluj-Napoca . There mBottled water storage conditions may play an important role in the presence and concentrations of phthalates in drinking water. Previous studies have demonstrated that bottle water samples stored at higher temperatures have higher concentrations of phthalates ,26. Consp < 0.10). The elevated levels in those who use plastic wrap for food storage is consistent with the evidence suggesting phthalates can migrate from plastic food storage material into the food, and potentially ingested [p < 0.10) even though MBzP’s parent compound BzBP can migrate from food storage materials and has been detected in food products [We contextualized water consumption behaviors with other consumer product use linked to phthalate exposure . We obseingested . Howeverproducts .There were several behaviors we might have expected to see an increasing correlation with phthalate metabolite concentrations and did not. Such behaviors include microwaving food in plastic containers  and incrThe phthalate concentrations observe in our adolescent participants were in a similar range of values reported in other populations, though differences could be observed depending on the compound and the population. The median concentration of MnBP (24 µg/g creatine) was higher than that observed in Austrian children ages 7 to 15 (12 µg/g creatinine)  and simiOur adolescents had a higher geometric mean concentration of MnBP (49.57 µg/g creatinine) compared to Korean adolescents (33.14 µg/g creatinine) . The adoPrevious studies conclude younger age groups are more vulnerable and have higher exposure to phthalates compared to adults ,17,18,19While it appears that our adolescents generally had comparable or lower concentrations of phthalate metabolites compared to other studies of European adults, differences may be related to a small sample size or intercountry differences in exposures, or differences in urine collection methods . We would need additional data to fully understand Romanian adolescent and adult exposure and provide further context for comparison.Limitations of this study include the low percent detection of phthalates metabolites in urine, and the sample size of 40, both of which limited our power and statistical analysis. Additionally, in our bottled water analyses, we recreated water storage conditions, instead of using a sample of the actual bottled water participants consumed. To address this limitation, we matched the brand and storage conditions to the best of our abilities. Another limitation is the use of morning first urine sample instead of 24 h urine sample.One of the strengths of this study is the use of urine metabolite analysis, consistent with methods promoted by DEMOCOPHES studies, facilitating comparisons with other populations ,32,33. IAnother strength of this study includes the examination of an understudied group—adolescents. Adolescents are especially important to understand plasticizer exposure because of their endocrine system vulnerability. The endocrine system becomes increasingly active throughout puberty until operating at full maturity . DisruptPhthalate exposure was nearly ubiquitous in our study population; phthalates were detected in bottled water samples, but generally not municipal water. We did not identify water consumption or consumer product use as major sources contributing to phthalate exposure. Further investigation into the contribution of different sources to overall exposure could help inform exposure mitigation strategies across different population groups and life stages. Further studies should examine whether this low-level but potentially continuous exposure from multiple sources could pose a potential health threat.


Persons with musculoskeletal disorders frequently seek care in family medicine clinics. However, musculoskeletal education provided in medical schools is often considered insufficient. The implementation of a collaborative model that integrates physiotherapists into teaching clinics may benefit the musculoskeletal training of medical residents. This paper describes a model developed in a family medicine teaching clinic by examining the interprofessional educational and collaborative activities implemented in this model. The model allowed to provide physiotherapy services, involve the physiotherapist in the training of family medicine residents and enhance interprofessional collaboration, particularly for the management of persons with musculoskeletal disorders. We examined interprofessional educational and collaborative activities implemented in this model using clinical and administrative data. The ethics review board of the In 2009, a physiotherapist was hired to join a primary care team in a university-affiliated Family Medicine Group (U-FMG) located in Québec City, Canada. The primary care team included approximately twenty-eight medical residents, seventeen family physicians, one physiotherapist as well as nurses, social workers, and nutritionists. This initiative aimed to 1) provide physiotherapy services, 2) involve the physiotherapist in the training of family medicine residents, and 3) enhance interprofessional collaboration.Collaboration between physiotherapists and family medicine residents/physicians first took the form of medical referral to physiotherapy. A total of 1,321 referrals were made between 2009 and 2015, for an average of 220.2 ± 44.0 (mean ± standard deviation) per year . MoreovePhysiotherapists contributed to the training of residents by providing 8.9 ± 4.5 planned teaching lessons  and 36.0 ± 17.0 observation sessions per year. These observation sessions allowed residents to spend a few hours with the physiotherapist to gain insight into their clinical practices. Physiotherapists also took part in a weekly formal interprofessional meeting, during which a resident discussed a specific patient’s case with an interprofessional group.This innovative collaborative model in which physiotherapists are integrated in a family medicine teaching clinic encompasses a variety of interprofessional activities. Based on our experience, such an innovative model has the potential to enhance the musculoskeletal training of medical residents and the management of patients with MSDs in primary care. Consequently, we believe that this model has implications for researchers, decision makers and clinicians. Identifying barriers and enablers to the implementation of this model and assessing its efficacy could serve as suggestions for future research.


Cracking reactions were improved with microwave heating, while deoxidation and dehydrogenation reactions were enhanced by activated carbon, which creates a reduction environment. Consequently, these reactions generated H2-rich syngas formation. The approach implemented in this study revealed higher H2, syngas yield and that the overall LHV of products has huge potential in the transformation of biomass into high-value synthesis gas.This study focuses on the use of a microwave reactor that combines biomass pyrolysis, at mild temperature, with catalytic reforming of the pyrolytic gas, using activated carbon, for generating hydrogen-rich synthesis gas. The traditional pyrolysis of biomass coupled with the reforming of its pyrolytic yields were also conducted using an electrically heated reactor. The bio-oil attained from conventional pyrolysis was higher in comparison to the yield from microwave pyrolysis. The reforming of the pyrolytic gas fraction led to reductions in bio-oil yield to <3.0 wt%, with a simultaneous increase in gaseous yields. An increase in the syngas and H The escalating concerns over energy supply security and deterring environmental implications related with fossil fuels consumption have made searching for sustainable and alternative energy resources very attractive in the past few decades  and increase in quality of bio-oil was detected in microwave heating  with microwave-assisted pyrolysis of biomass (MAPB) to maximize the yield of HA suite of biomass samples, i.e., bamboo, gumwood, pine and rosewood, were used in this study, which were native to Huzhou, Zhejiang Province, China. Prior to characterization and testing, about 1.0 kg of each sample was prepared using the standard procedures to ensure the representativeness of the samples and further milled to 212 μm  at 900°C for an hour to minimize its volatile and moisture content and ensure they remains <0.5 wt%, to avoid interactions between the pyrolytic yields and gaseous constituents of activated carbon.Coconut-derived activated carbon  was used as the reforming agent for upgrading the pyrolytic products. Activated carbon's capacity for absorbing microwave irradiation makes it a suitable reforming and deoxygenating agent. Before use in the reactor, the activated carbon was heated isothermally in an inert environment . The procedures are adopted from literature  with a maximum power of 3 KW. K-type thermocouple enclosed in an Niobium alloy tube was used for operating temperature detection. A customized quartz tube with similar diameter as the vertical tube furnace for conventional pyrolysis was used as the microwave reactor. Experimental set-up is illustrated in 2 (200 ml/min) was used to sustain an anaerobic atmosphere. In each test, ~5.0 g of air-dried sample was added into the feeder placed on top of the furnace, which was dropped into the reactor after the furnace temperature reached the pre-set temperature. This helps maintain an isothermal reaction at the desired temperature for 15 min. The tests were also carried out at 600, 700, and 800°C. The condensable volatiles were collected by using dichloromethane in an ice bath, whilst gas product was collected using gasbags.CPB was conducted in a vertical tube furnace . The isothermal section of the reactor cavity was 10.0 cm long with a supporter in the middle and the inner diameter of the tube was 5.0 cm. Temperature detection was done using a K-type thermocouple. NProximate and intrinsic analyses of char was conducted. The bio-oil was evaluated using a gas chromatograph mass spectrometry . The GC's oven temperature was programmed to remain isothermal at 60°C for 2 min before the temperature was raised up at a heating rate of 10°C/min to 280°C and kept isothermal for 2 min. During the analysis, split mode was adopted with a split ratio of 50:1. For the correlation of the molar concentrations to the chromatographic areas, further quantification was performed using the response factors and retention time of the detected compounds. The C1–C5 hydrocarbons and other major gases of the gas yield from pyrolysis were analyzed with a gas chromatograph  equipped with 2 TCD detectors, 1 FID detector, 1 HP-AL/s capillary column and 8 molecular sieve columns.5H7.8O3. It is also expected that the maximum amount of H2 from biomass pyrolysis should be lower than the total hydrogen content of around 6.5 wt%.Properties of the activated carbon and biomass are detailed in Ig), peak temperature (TP), and burnout temperature (TB) of the samples. Generally, combustion profile of biomass is characterized by 2 peak temperatures, which can be attributed to the burning of cellulose & hemicellulose at 300–340°C and lignin at 420–480°C, respectively , more biphenylene, naphthalene, phenanthrene, fluorene, anthracene, and their derivatives were formed. It is clear that the side chains of phenol or benzene derivatives were cracked at high pyrolysis temperatures. In addition to this, Polycyclic Aromatic Hydrocarbons (PAHs) were also detected in the bio-oil. styrene, a valuable chemical in industry, was produced at temperatures above 700°C. It is found that phenol, furanmethanol, cresol, catechol, and derivatives were the main compounds existing in bio-oil, which are similar to constituents of conventional pyrolytic bio-oil. However, some sulfur-containing compounds were generated in microwave pyrolytic bio-oil, such as cyclic octaatomic sulfur, dimethyl tetrasulfide or dimethyl trisulfide. Furthermore, the content of cyclic octaatomic sulfur in microwave bio-oil increased with temperatures, indicating sulfur migrates from biomass to bio-oil under microwave radiation. Similar observations about benefits of moderate microwave treatment for breaking the C–S bonds has been discovered by other studies  and CO2 (3–9 vol%) fractions, and higher CO (49–67 vol%) and CH4 (15–22 vol%) contents. The syngas fraction was discovered to increase with increasing temperature from 600 to 800°C and the C2–C4 hydrocarbon gases were found in minor quantities. The release of volatiles from biomass, thermal cracking of bio-oil at higher temperatures and reactions between various pyrolytic compounds consequently results in the gaseous product formation  and decrease in CO2 concentration. In addition, there was an increase in the H2:CO ratio with increasing temperature resulting from H2 increase   in comparison to the syngas yield from conventional pyrolysis under similar conditions . Low LHV could also be attributed to the higher extent of decomposition of biomass under microwave heating, leading to the chars containing less carbon. LHV of the microwave pyrolytic gas products was around 15–26 MJ/kg. This is much higher than that of gaseous fraction derived from conventional pyrolysis, as listed in LHV of pyrolytic products and its ratio to LHV of biomass were calculated and illustrated in LHVs of products derived via different pyrolysis methods are also illustrated in Normally carbon materials, for instance char and activated carbon, can be applied as catalysts for heterogeneous reactions under microwave irradiation  which promotes CO formation due to activated carbon reaction with COFurthermore, It has been established that microwave heating promotes various catalytic reactions (Motasemi et al., 2 selectivity and reduced yield of bio-oil. The highest bio-oil yield was lower than 3 wt%, and the highest gaseous fraction was around 76 wt%, which contains 85 vol% of syngas and 46.7 vol% of H2. The thermal cracking and reforming reactions were improved by microwave irradiation, resulting in the production of a H2-rich syngas. The activated carbon enabled reforming process favors dehydration and deoxidation reactions by the creation of a non-oxidizing atmosphere, which further promotes hydrogen production. The findings of this study demonstrate that the coupling of activated carbon enabled reforming with microwave-assisted pyrolysis favors the conversion of biomass into H2-rich gas and raises energy conversion efficiency.To summarize, it is found that microwave-assisted pyrolysis coupled with activated carbon enabled reforming resulted in an enhanced yield of gaseous product, higher HAll datasets generated for this study are included in the article/supplementary material.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine efficacy and safety of patient-controlled intravenous analgesia (PCIA) with tramadol and lornoxicam for postoperative analgesia, and its effects on surgical outcomes in patients following thoracotomy.The records of patients who underwent thoracotomy for lung resection between January 2014 and December 2014 at our institution were reviewed. The patients were divided into 2 groups according to postoperative pain treatment modalities. Patients of the patient-controlled epidural analgesia (PCEA) group (n = 63), received PCEA with 0.2% ropivacaine plus 0.5 μg/mL sufentanil, while patients in the PCIA group (n = 48), received PCIA with 5 mg/mL tramadol and 0.4 mg/mL lornoxicam. Data were collected for the quality of pain control, incidences of analgesia related side effects and pulmonary complications, lengths of thoracic intensive care unit stay and postoperative hospital stay, and in-hospital mortality.Pain at rest was always controlled well in both groups during the 4-day postoperative period. Patients in the PCIA group reported significantly higher pain scores on coughing and during mobilization in the first 2 postoperative days. The incidences of side effects and pulmonary complications, in-hospital mortality and other outcomes were similar between groups.PCIA with tramadol and lornoxicam can be considered as a safe and effective alternative with respect to pain control and postoperative outcomes for patients underwent thoracotomy. So far, the benefits of TEA on the length of hospital stay and the in-hospital mortality are still controversial.,5 On the other hand, epidural catheterization may require additional anesthesia preparation time and may fail, may be contraindicated as well as associated with rare, but serious complications.,7 Therefore, intravenous analgesia is also widely used for pain control after thoracotomy due to its technical simplicity and safety.Thoracotomy is well recognized as one of the most painful surgical procedures. Adequate pain relief after thoracic surgery is of particular importance, not only for keeping the patient comfortable but also for reducing the incidence of postoperative complications. Tramadol has been shown to offer a similar analgesic potential to opioid in patients receiving patient-controlled intravenous analgesia (PCIA) following thoracotomy. Its relative lack of sedative and respiratory depressive effects makes it especially suitable for analgesic use after thoracic surgery.Tramadol is a centrally acting analgesic with 2 different mechanisms of action. One enantiomer exerts a predominantly weak μ opioid effect, whereas the other inhibits norepinephrine and serotonin reuptake, activating descending monoaminergic inhibitory pathways.,11 Efficacy and safety of intravenous application of tramadol with NSAIDs have been documented in patients who have undergone the thoracic procedure.,13 However, no study has looked at post-thoracotomy pain control as well as postoperative outcomes of PCIA with tramadol and an NSAID compared to patient-controlled epidural analgesia (PCEA).Lornoxicam, a member of the oxicam group of nonsteroidal anti-inflammatory drugs (NSAIDs), was reported to improve postoperative pain control and decrease tramadol consumption as well as the incidence of side effects when administered with tramadol by PCIA after abdominal surgery.At our hospital, as an alternative technique to PCEA, tramadol has been administered in combination with lornoxicam by PCIA for pain management following thoracic surgery. Therefore, we performed this retrospective study primarily to examine the differences in postoperative pain intensity and side effects between patients who received PCIA or PCEA after thoracotomy for lung resection. A secondary aim was to determine whether postoperative outcomes differed significantly between the 2 analgesia regimens.2Following the approval from our hospital's Ethics Committee (registration number: 2013-61), we reviewed the medical records of 124 consecutive patients who underwent elective posterolateral thoracotomy for lung resection between January 2014 and December 2014 at our institution. After excluding patients under 18 years of age, patients with a chronic pain condition or with regular consumption of opioids for more than 2 weeks preoperatively, and those had their epidural catheter dislodged within the first 2 days after surgery, the remaining 111 patients were included in this study. The patients were divided into 2 groups according to their postoperative pain treatment modalities. Patients in the PCEA group (n = 63), received PCEA with ropivacaine plus sufentanil, while patients in the PCIA group (n = 48), received PCIA with tramadol–lornoxicam combination.The decision for postoperative analgesic modality was based on the outcomes of preoperative evaluation, and conversation between the attending anesthesiologist and patient about benefits and risks of both techniques. All patients gave written consent for the analgesic technique that was to be used.In the PCEA group, a thoracic epidural catheter was placed at the level of T5-T7 using a loss of resistance technique before the induction of general anesthesia. The catheter was inserted cephalically 4 to 5 cm and fixed in place. A 3 mL test dose of 2% lidocaine was administered to exclude accidental intrathecal injection. Then an additional 1% ropivacaine 5 mL was administered. The level of sensory block was aimed at T2-T10, tested by pinprick method 20 minutes later. Epidural administration of 1% ropivacaine 5 mL was repeated every 60 minutes during surgery.General anesthesia was induced with 2 to 3 mg/kg of propofol, 2 to 4 μg/kg of fentanyl, and a nondepolarizing muscle relaxant, either vecuronium (0.1 mg/kg) or atracurium (0.5 mg/kg) was used to facilitate endotracheal intubation with a double-lumen tube, and the lungs were ventilated with a mixture of 50% oxygen/air. Anesthesia was maintained with sevoflurane, fentanyl, and remifentanil as necessary. After completion of surgery, the double-lumen tube was removed and the patient was shifted to the postanesthetic care unit (PACU) for close observation for at least 2 hours, and then transferred to the thoracic intensive care unit (TICU).Postoperative pain treatments were initiated at the end of surgery. In the PCEA group, patients received a 0.2% ropivacaine solution with 0.5 μg/mL sufentanil. The epidural infusion was delivered at a rate of 3 to 5 mL/h with a 2 to 3 mL bolus and 20-minutes lockout interval. Patients in PCIA group received a 10-mL combination of 5 mg/mL tramadol and 0.4 mg/mL lornoxicam as a loading dose, followed by a continuous infusion at a rate of 1 to 2 mL/h, and a bolus of 1 to 2 mL with a lockout interval of 15 minutes.The acute pain service (APS) is responsible for postoperative pain management at our Hospital. The visual analog scale (VAS) was used for assessing the degree of pain relief during patient controlled analgesia (PCA) treatment. The VAS ranged from 0 = no pain to 10 = unbearable pain. The nursing staff in the surgical ward measured pain intensity every 4 hours while the PCEA or PCIA was in use. The APS routinely reviewed each patient 2 times per day, and whenever necessary. VAS scores at rest, on coughing, and during mobilization (defining as changing body position) were recorded. If patients reported a pain score at rest equal to or greater than 4 on 2 consecutive time points, the ward nurse paged an APS member, who then visited the patient and adjusted the PCA settings as needed until the patient had a pain score at rest of less than 4, which was considered adequate analgesia. Supplemental morphine 5 to 10 mg was administered intravenously if the adjustment of the infusion pump still resulted in insufficient pain relief. Side effects of analgesia were also assessed daily. Antiemetic or antipruritic drugs were prescribed on an as-required basis. Ondansetron 4 to 8 mg was intravenously administered every 12 hours to treat nausea and vomiting. Intravenous diphenhydramine 20 mg was used every 12  hours for pruritus. The PCEA or PCIA protocol was maintained until patients were switched to oral analgesics. All patients were questioned as to their satisfaction with the quality of analgesia using a 4-point scale  at the end of PCA treatment period.Patients’ charts were analyzed for demographic data including gender, age, height, weight, American Society of Anesthesiologists (ASA) status, preoperative forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1), type and duration of surgery. Information about the maximum VAS values at rest, during coughing and mobilization in PACU, for the day of surgery  and for postoperative days 1 (POD 1) through 4 (POD 4), and side effects of each analgesic modality including nausea and/or vomiting, pruritus , oversedation , urine retention, hypotension (systolic blood pressure <90 mm Hg or >20% decrease from baseline), and respiratory depression  were also collected. Additional parameters included requirement for supplemental intravenous morphine, duration of PCA use, patient satisfaction, occurrences of pulmonary complication as well as thoracic bleeding requiring reoperation, need for postoperative packed red blood cells (PRBCs) transfusion, lengths of TICU and postoperative hospital stay, and in-hospital mortality.2.1t test. Chi-square test was used to analyze categorical variables between the 2 groups. A P-value of less than .05 was considered statistically significant. All statistical analysis was performed with the use of SPPS 17.0 for Windows .Continuous data are expressed as mean and standard deviation. Categorical data are described with percentages. Comparisons of continuous variables were performed using Student unpaired 3The 2 groups were similar with respect to gender, age, weight, and height. There were no differences in ASA status, preoperative FVC, FEV1, type, and duration of surgery between the groups . The duration of PCA use and patients overall satisfaction with postoperative analgesia were similar in both groups (Table Supplementary morphine was required during the study period in 21 patients (43.8%) in the PCIA group and 14 patients (22.2%) in the PCEA group. There was a significant difference between the 2 groups  and inducible cyclooxygenase-2 (COX-2) and consequently inhibits the production of prostaglandins and reduces the development of sensitization process. Compared with other oxicams, lornoxicam has high-efficiency potential, good gastrointestinal tolerance, and a short plasma half-life of 3 to 5 hours. Moreover, it has also been reported not to exert a serious nephrotoxic effect.,19 Finally, as far as we know, this is the first study to compare the effect of PCIA using the combination of tramadol and an NSAID with PCEA on postoperative outcome.There are several differences between our present research and previously published literature. First, both TEA and intravenous analgesia in our study were given in the form of PCA, which has been demonstrated to be able to provide better pain control with fewer adverse effects. Meanwhile, we used a low-dose background infusion in addition to PCA bolus because studies have shown this decreased the need for rescue analgesics and enhanced postoperative analgesia and patient satisfaction.,21In the present study, patients in the PCIA group reported higher pain scores on coughing and mobilization during the first 2 days after surgery when compared with the PCEA group. The possible explanation is the epidural administration of opioid provides segmental analgesia and improves the quality and duration of sensory block produced by local anesthetic.,22Despite improved pain control with PCEA, overall patient satisfaction did not differ between the 2 groups. This underscores that patient satisfaction is influenced by complex factors. One important reason is the similar incidence of side effects between the 2 groups. It is notable that none of our patients developed respiratory depression, which probably attributes to the concurrent administration of 2 drugs with different modes of action in both groups. The synergistic effects could reduce the total amount of each drug needed, resulting in fewer unwanted side effects. In our study, there was no patient developed excessive bleeding and required reoperation in each group during the postoperative period. In addition, the percentage of patients requiring PRBCs transfusion after surgery was comparable in the 2 groups. These results were consistent with previous reports which have indicated that lornoxicam had no effect on postoperative bleeding, bleeding time, or blood transfusions requirements.,25Increased risk of bleeding is one of the major concerns with perioperative NSAIDs therapy.,26–28 In the present study, we found the types of pain treatment did not affect the incidence of clinical respiratory complications, the length of hospital stay and in-hospital mortality. More studies are needed before definite conclusions can be drawn.Researchers have compared the outcomes between TEA and intravenous analgesia after thoracic procedure. Some strongly suggest improved outcomes with the use of TEA, whereas others have found no improvement.A few limitations of our study should be mentioned. First of all, we cannot exclude the possibility of selection bias for the type of analgesia due to the retrospective nature of the study. Second, postoperative FVC and FEV1 values were not collected because pulmonary function test was not reperformed after surgery in all patients, so we were not able to examine the effects of PCEA and PCIA on lung function. Last, as perioperative coagulation profiles could not be obtained in this retrospective analysis, it is impossible for the authors to evaluate accurately the risk of postoperative bleeding associated with lornoxicam use.This retrospective study has demonstrated that PCIA with tramadol and lornoxicam can be considered as a safe and effective alternative with respect to pain control and postoperative outcomes for patients underwent thoracic surgery. Certainly, collecting data in a prospective manner is required to verify the current results.Conceptualization: Juying Jin, Su Min.Data curation: Juying Jin, Su Min.Formal analysis: Juying Jin.Investigation: Juying Jin, Qibin Chen, Dong Zhang.Methodology: Juying Jin, Su Min.Project administration: Juying Jin, Qibin Chen, Dong Zhang.Resources: Juying Jin, Su Min.Software: Su Min.Supervision: Su Min.Validation: Qibin Chen, Dong Zhang.Visualization: Qibin Chen, Dong Zhang.Writing – original draft: Juying Jin.Writing – review and editing: Juying Jin.