Neuronal exosomes purified from peripheral blood samples have been proposed as diagnostic tool in the setting of acute brain injury but never tested clinically. We hypothesized that exosome protein biomarkers would change over time following acute hypoxic brain injury and would predict response to therapy.SYNPO), an actin‐associated protein present in postsynaptic spines, was evaluated as a potential biomarker as well as: synaptophysin, neuron‐specific enolase, and mitochondrial cytochrome c oxidase. A secondary analysis was performed on neonatal samples collected at 8, 10, and 14 h after the initiation of therapeutic‐controlled hypothermia for acute hypoxic–ischemic encephalopathy (n = 14). Neuronal exosomes were purified from serum and protein levels were quantified using standard ELISA methods. The primary study outcomes were length of stay (LOS), discharge on seizure medication (DCMED), and composite neuroimaging score (NIS).Synaptopodin . SYNPO increased in 6/8 without DCMED and was worse or neutral in 5/5 with DCMED (P = 0.02). All four neonates with an abnormal NIS had neutral or decreasing SYNPO (P = 0.055). Other candidate biomarkers were not associated with outcomes.The slope of change in neuronal exosome This report provides the first clinical evidence that neural exosomes turn over rapidly enough in the peripheral circulation to be used as a “troponin‐like” test following acute brain injury. Optimal sampling and biomarkers likely vary with type of brain injury. Strategies to decrease adverse neurologic outcomes following acute brain injury require accurate initial diagnosis as well as efficient identification of patients with a suboptimal response to primary treatment to guide early introduction of adjuvant therapies. A common thread linking these two needs is the need for early, rapid, and accurate predictors of degree of acute brain injury and response to therapy. The search for a troponin‐like blood‐based biomarker for the diagnosis of strokeWe have developed a novel technique for purifying fetal neural exosomes from the maternal bloodstream to identify in utero neurologic injury.A secondary analysis was performed on neonatal samples collected as part of a prospective, open‐label pharmacokinetic study conducted at St. Christopher's Hospital for Children  to study the pharmacokinetics of ampicillin in a prospective cohort of term neonates undergoing therapeutic‐controlled hypothermia for HIEμL of serum was incubated with thromboplastin‐D and a cocktail of protease and phosphatase inhibitors. After centrifugation, supernates were incubated with exosome precipitation solution . To isolate the subset of exosomes from neural sources, total exosome suspensions were incubated with monoclonal IgG1 anti‐human Contactin‐2/TAG1 antibody  that had been biotinylated , and antibody‐bound exosomes were precipitated with Streptavidin‐Plus UltraLink Resin . Contactin‐2/TAG1 is a glycosylphosphatidylinositol‐anchored neuronal membrane adhesion protein of the immunoglobulin superfamily that is transiently expressed in human brain development to guide axonal connections and, in association with other proteins, promote molecular organization of myelinated nerves.Neonatal neuronal exosomes were purified from residual serum aliquots using previously described techniques.Potential protein biomarkers including synaptopodin (SYNPO), synaptophysin (SYN), neuron‐specific enolase (NSE) and mitochondrial cytochrome c oxidase (COX IV) were quantified using standard ELISA methods. The slope of biomarker levels between 8 h and 14 h was calculated to assess whether or not marker levels were rising or falling over the course of therapy. Slopes (ng/mL/hr) were utilized as we were concerned that individual values at a single time point might vary significantly with the wide ranges of gestational age and birthweight. Slopes calibrated values for each infant against their starting value and eliminated the need for repeated measures analysis. If an 8‐h sample was not available, the slope between 10 h and 14 h was substituted . One subject had only one sample, and slope could not be calculated.A neuroradiologist (EP) scored available T1/T2 and DWI magnetic resonance images from between day of life 5 and 7 in a blinded fashion according to the scoring system described by Barkovich that correlates with neuromotor outcome at ages 3 and 12 months.P < 0.05 was considered statistically significant.The primary study outcomes, selected prior to data analysis, were length of stay , discharge on seizure medication , and NIS . Long‐term neurologic outcomes were not uniformly available for this cohort. Data analyses were performed using IBM SPSS Statistics for Windows . The small sample size did not allow for adequate assessment of the normality of the data, and therefore, nonparametric statistical methods were chosen. Medians were compared using the independent samples median test. Ordinal categories are compared using Somers' d test. Bivariate correlations were assessed by Spearman's rho. A The clinical characteristics of the 14 neonates enrolled are shown . No other biomarker slope was associated with LOS. As expected, of the traditional clinical indicators , only pH was weakly correlated with LOS (Table P = 0.02). Perhaps more telling, no infant discharged on anticonvulsants had an increasing NNE SYNPO slope; all slopes in this category were neutral or decreasing. Improving levels of neuronal exosome synaptopodin were exclusively associated with medication‐free discharge. Median NSE slope was not different between discharge outcome categories. Similarly, NNE SYN and COX IV showed no specific pattern . Again as expected, there was sufficient overlap between positive and negative outcomes with traditional predictors such as pH, base excess, and APGAR scores to render them difficult to use for counseling or to make additional treatment decisions after the initial decision for the induction of controlled hypothermia (data not shown).The correlation between the slopes of the four biomarkers and LOS was assessed Table . The sloOS Table . Median OS Table A and B. P = 0.15). However, we saw a statistically significant relationship between the summary score for the diffusion‐weighted images and the slope of synaptopodin . The worst NNE SYNPO slope (−3.66) was seen in a subject with imaging results consistent with an intraventricular hemorrhage and early‐onset hydrocephalus. One subject with a deteriorating NNE SYNPO slope was not included as that neonate did not undergo DWI.No strong correlations were identified between synaptopodin slope and any individual scores or with the T1/T2‐weighted summary measure . Taken together, this evidence suggests that neural exosome biomarkers are a potentially powerful clinical tool in the classification of the severity of neonatal HIE and the response to therapy. However, the implications of this report are broader than neonatal HIE as they suggest that neuronal exosomes may have diagnostic utility in other forms of acute brain injury such as stroke or trauma.We present novel data that confirm our two initial hypotheses. First, neural exosomes can be isolated from peripheral blood samples and contain biomarkers that are associated with clinically significant short‐term neurologic outcomes. Second, the turnover of neural exosomes in the peripheral blood compartment is rapid enough that changes in exosome cargo can be assessed at clinically useful intervals to estimate therapeutic response. This allows for early discrimination between responders and nonresponders, facilitating timely adoption of additional measures where available. Finally, we were able to demonstrate that exosome‐based tests can be performed on very small clinical samples that are reasonable to collect serially from critically ill subjects (100 Exosomes are cell‐derived vesicles that consist of cytoplasm packaged in a membrane layer.Synaptopodin emerged as the most promising biomarker in this investigation. Synaptopodin is a cytoskeletal protein that is essential for the formation of dendritic spines in telencephalic neurons, and is a key mediator of synaptic plasticity, especially at excitatory synapses.Our study has several limitations. The first and most critical was the small amount of residual serum available for exploratory studies. This forced us to be parsimonious with our pilot markers, and we were only able to assess a limited candidate group. It was difficult to choose from among available candidate biomarkers as none have proven particularly clinically useful. In a recent meta‐analysis, serum and CSF concentrations of interleukin‐1b, interleukin‐6, and serum NSE were predictive of abnormal outcomes in HIE.Overall, we are confident that the data we present support further prospective studies of neural exosomes as early, noninvasive biomarkers in the setting of acute brain injury. Serial sampling may or may not prove superior to single time point testing; in prospective trials, we suggest that the first sampling point be set to coincide with primary treatment initiation. Additional markers should be investigated in addition to confirmatory studies of synaptopodin that take into account the two phases of neuronal injuryOnly one author has a potential conflict of interest. Dr. Goetzl has applied for a patent for some of the methodology described but has no current licensing or industrial applications.


We regret any confusion or inconvenience these errors may have caused.In the article “The High Prevalence of Diabetes in a Large Cohort of Patients Drawn From Safety Net Clinics,” some figure notations and table footnotes were incorrect. Corrections were made to our website on June 21, 2016, and appear online at


Using this fluorescence HSI (fHSI) system, we were able to accurately resolve the presence and concentration of at least 7 fluorescent dyes in solution. We also demonstrate high spectral unmixing precision, signal linearity with dye concentration and at depth in tissue mimicking phantoms, and delineate 4 fluorescent dyes in vivo. Our approach, including statistical background removal, could be directly generalised to broader spectral ranges, for example, to resolve tissue reflectance or autofluorescence and in future be tailored to video rate applications requiring snapshot HSI data acquisition.The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter‐based imaging systems. Hyperspectral imaging (HSI) facilitates the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for HSI is typically complex, bulky and expensive. We sought to overcome these limitations using a novel robust and low cost HSI camera based on a spectrally resolved detector array (SRDA). We integrated this HSI camera into a wide‐field reflectance‐based imaging system operating in the near‐infrared range to assess the suitability for  Originally conceived for remote sensing >5 has required the use of narrowband emitters such as quantum dots The ability to detect multiple fluorescent labels targeted to different processes is highly advantageous across a range of length scales in biomedical imaging: from studying complex molecular interactions in cells at super‐resolution To date, translating the power of fHSI into applications in humans, such as endoscopic and intra‐operative imaging, has been challenging due to the need for complex, bulky and expensive instrumentation with limited frame rate. Nonetheless, the ability to characterize both endogenous tissue contrast based on diffuse reflectance and to identify the presence and concentration of exogenous small molecule fluorescent dyes Spectrally resolved detector arrays (SRDAs) – where the spectral filters are deposited on a glass substrate either in front of Here, we evaluated the performance of an SRDA‐based HSI camera for fluorescence imaging, with a view to future clinical application. While the SRDA filters can be deposited pixel‐wise for real‐time ‘snapshot’ imaging 22.1The HSI camera SRDA (Imec) monolithically integrates a set of 100 Fabry‐Pérot etalon filters between 600 and 1000 nm  on a CMOS sensor  packaged within a camera system . The Fabry‐Pérot etalon filters are row‐wise deposited on the sensor to form spectral bands; each spectral band occupies 8 pixel rows. Transmission efficiencies of up to 73 % have been reported for the filters in this HSI camera SRDA Ke is the conversion gain of the sensor, peakI the peak photon intensity measured with the calibrated photodetector, t the integration time and pixelA the pixel area where DN(λ) is the digital number of the averaged, dark subtracted pixels within the spectral bands, −1nm−1, DN is the digital number output of the camera, λ the peak wavelength output from the monochromator and peakI is the peak intensity recorded by the inspection spectrometer in μWcm−2.where SR is the baseline corrected relative spectral response of the camera in DNμW2.2The HSI camera was mounted onto a strong aluminium frame  above the sample using a custom adapter, designed to minimize mechanical vibrations. The system was enclosed in a light tight box. Sample translation for line scan imaging was achieved using a motorized translation stage , which was synchronized to the process of image acquisition. The sample was consecutively translated a distance equivalent to that of one spectral band before each image acquisition. Our alignment procedure has been detailed elsewhere <2 %). Six LEDs of each type were connected in series equidistant around the internal rim of the ring and photographic diffuser paper  was placed underneath. We compared data acquired with and without crossed linear polarisers  to compare hardware specular reflection removal with our background removal methods implemented in software.Sample illumination was provided by a custom LED ring of 50 mm internal radius. Three colors of high power LEDs with peak wavelengths 590, 660, and 732 nm  were mounted on a copper ring, each LED controlled via a separate current controller  with negligible intensity drift  substantially affected the uniformity of the illumination increasing to 29 % at 660 nm due to a combination of the removal of the diffusion paper and the low quality of the polarizing filters.The power density at the sample was calibrated using an optical power meter  centrally placed in the sample area. Spatial illumination uniformity was mapped separately for each LED color at 100 mW/cm2.3For every experiment, a dark and ‘flat’ HSI cube were acquired respectively by turning off all LEDs or by illuminating the sample prior to addition of the fluorescent dyes. Raw data is available by contacting the corresponding author of the manuscript. Initial pre‐processing steps (see Supporting Methods for more details) include: dark subtraction, to account for any sensor non‐uniformities; edge correction, to account for a slight mismatch with the filter edges and pixel positions ; and spatial vignetting correction, using an HSI cube acquired under broadband illumination in the sample plane to provide a vignetting correction mask for each spectral band. Finally, background removal is performed to address the presence of specular reflections in our data, which comprise 1–5 % of the image pixels in well plate and tissue phantom data. Strong specular reflections in the well plates arise due to meniscus effects in the droplets containing fluorescent dyes, and moist or wet areas in the tissue phantoms. For mouse skin, which is more diffusely reflective, less than 0.003 % of image pixels are affected. A ratiometric thresholding method was developed to remove areas of strong specular reflection from our quantitative analysis (see Supporting Methods).a) were then subtracted to produce background removed endmembers  according to r=a −aVTV . This approach has the advantage of removing not only specular reflections but also any other sources of stray background light.We tested two software background removal methods, which exploit the spectral dimension of the information collected in the HSI cube, and compared the result to the inclusion of crossed linear polarisers in the hardware, which remove only surface specular reflections. The advantage of performing background removal in software, assuming we are able to achieve a comparable performance for reflectance removal as in hardware, is the potential to extract information from the diffuse reflectance light in a clinical scenario. The ‘brute force’ software approach mimics the action of applying optical band‐pass filters to block the reflectance light. The maximum signal intensity of the reflected LED illumination for the power levels chosen in this study was first determined by imaging the uniform reflectance target under our standard illumination conditions. For spectral bands 9–17 and 21–35, which would be adversely affected by reflectance of the LED illumination, bands where the signal intensity exceeded 10 % of the measured maximum reflectance intensity were then rejected from the analysis. The spectral bands rejected were kept fixed throughout the study. Orthogonal subspace projection (OSP) ‘Endmembers’ are the spectra to be fitted using spectral unmixing. To account for system‐dependent variations in the recorded spectral profiles, we used the spectra of the fluorescent dyes recorded by the wide field fHSI system in a single reference measurement 2.4A 40 μM dilution of each of AF610, AF647, AF700 and AF750  was prepared in phosphate buffered saline (PBS) . A well plate  containing 30 μL of each dye in a separate well, along with a PBS control was used to acquire the ‘reference’ HSI cube (with and without crossed polarizers), from which endmembers could be extracted. A ‘flat’ HSI cube of a well plate containing only PBS was recorded for OSP background removal. Plates containing both dilution series of each dye (1:2 steps from 10 μM to 625 nM) and mixtures of different dyes were then imaged for performance evaluation. Two duplicates of each concentration and two PBS blanks were pipetted in opposite directions along two rows of the well plate, to ensure repeatability within the FOV. Absorption and fluorescence spectra, as well as the accuracy of the dilutions, were verified using microplate readers . A further well plate was imaged containing 40 μM dilutions of three additional fluorescent dyes to the Alexa Fluors: Cyanine 7.0; Sulfo‐Cyanine 7.0; and Cyanine 7.5 . 20 μL of 40 μM dye dilutions prepared in ethanol –to avoid aggregation of the cyanine dyes – were imaged.−1 at 633 nm. A 0.75 % volume of 0.5 mgml−1 nigrosin  was then added to provide an absorption coefficient of 0.05 cm−1 at 633 nm. While liquid, the phantom base material was poured into multiple petri dishes  to depths of either 0.2 or 1 cm. Fluorescent target inclusions were made using transparent plastic straws with an internal diameter of 0.3 cm . The transparent straws were cut into 1 cm long pieces and one end of each piece sealed with a glue gun . Eight 1 cm long straw pieces were inserted into a 1 cm thick phantom slab, with the glue sealed end directed towards the bottom of the petri dish; half of the straw inclusions were filled (50 μL) with a 40 μM solution of each of the fluorescent dyes  in equal part phantom base material and PBS and half were filled with PBS and phantom base material. A second equivalent phantom was prepared with all the straw inclusions filled with the PBS and phantom base material mixture for the ‘flat’ HSI cube acquisition.For phantom experiments, 1.5 % agar solution  was mixed with intralipid emulsion  to provide a scattering coefficient of 5 cm2.5All animal procedures were conducted under a project license issued under the United Kingdom Animals (Scientific Procedures) Act, 1986 (70‐8214). The project license was reviewed by the local Animal Welfare and Ethical Review Board and the specific experimental protocol was approved locally by the Named Animal Care and Welfare Officer at the Cancer Research UK Cambridge Institute under compliance form number CFSB0964 and conducted by individuals holding personal licenses. 40 μM dilutions of each of the AlexaFluor dyes and an equal parts mixture of all four (from 30 μM dilutions) were prepared in a 1:1 solution of PBS and phenol‐red‐free matrigel . Experiments were performed in nude mice (n=2) maintained under inhaled anaesthesia using isoflurane mixed with 100 % oxygen for a maximum of 2 hours for the entire study. During imaging on the fHSI system, the mouse was placed on a heat pad  within a bespoke 3‐D printed animal holder. 30 μL of each dye preparation was injected subcutaneously using a Hamilton syringe into the flank of the mouse. The contralateral side of the mouse was used as an imaging control for acquisition of the ‘flat’ HSI cube. Images were also acquired using an IVIS200 (Perkin Elmer) per standard operating procedures to verify successful injection and placement of the fluorescent dyes using a filter‐based fluorescence imaging system.33.1in vivo imaging of exogenous fluorescent dyes. The monolithically integrated spectral filters are formed from Fabry‐Pérot etalon cavities deposited over 8 pixel rows on the sensor . Line scanning requires an acquisition time of up 4 minutes per HSI cube, but allows us to maximize the spectral and spatial information available in our SRDA. While acceptable in the present system, we would need to trade off spectral and spatial resolution to transition into a pixelwise filter deposition for future clinical implementations.To provide an initial proof‐of‐concept for fHSI relevant to clinical applications, we decided to create a simple reflectance‐based demonstrator system, which mimics typical sample and illumination geometry. This allowed us to specifically capture and explore the hardware and software challenges of performing fHSI without adding all of the complexities that would be associated with a fully functional endoscopic or intraoperative system. We mounted the HSI camera above a motorized translation stage Figure A. The sa−2 in the sample plane for all experiments. Light from all LEDs simultaneously illuminated the full FOV throughout the data acquisition.HSI traditionally uses either broadband white light or narrowband filters for illumination 3.3Having implemented and calibrated the system hardware, we then sought to evaluate the imaging performance in a well‐controlled sample. Four fluorescent dyes initially used to assess fHSI performance were AlexaFluor (AF) 610, 647, 700 and 750. The dyes were chosen to have a relatively high spectral peak separation (∼50 nm) and a range of quantum yields, with variable overlap between their fluorescence emissions and the reflectance of the LED excitation light . The conventional filter‐based reflectance removal was mimicked mathematically in software by ‘brute force’ exclusion of the spectral bands encompassing the peaks of the LED excitation. Orthogonal subspace projection (OSP) removes the principal components (PCs) of the ‘flat’ HSI cube (acquired before fluorescent dye administration) from the fHSI data cube to produce new endmembers To extract the fluorescent dye composition at a given spatial location within the fHSI data cube, the data was processed as illustrated Figure The pre‐processed HSI cube was analysed using non‐negativity constrained least squares (NNLS) spectral unmixing based on data extracted from the ‘endmember’ HSI cubes Figure B. The fi3.3.22 values for all linear fits to the LS scores of the whole dilution series across the range of dyes .We evaluated the impact of different background removal methods used in data pre‐processing on data quantification. ROIs of 65 pixels radius were manually placed over the wells in the well plate image to extract an average LS score over each well. Figure In addition to the LS score, we defined the spectral unmixing precision (SUP): the ratio of the LS score for the correct abundance map to the sum of scores recorded from the same spatial location across all other fluorescent dye abundance maps. For each background removal method, the spectral unmixing precision is compared to the ground truth Figure B. Again,To assess the response of the system in the case of more overlapping spectra, we introduced a well plate containing 3 additional fluorescent dyes  with significant spectral overlap with both the reflectance light and the existing AlexaFluor dyes. The abundance maps for each fluorescent dye is shown in Figure To quantify the repeatability of the fHSI data, a well plate containing 4 wells of 30 μL of 40 μM dilutions of AF647 and 750 was imaged 8 consecutive times over a 35 minute time period. The coefficient of variation of the unmixed fluorescence signal  with time was found not to exceed 6 % for any of the wells. Based on the low coefficient of variation, we can conclude that photobleaching of the dyes does not occur at the illumination levels and acquisition times used in this study. Well plates containing 30 μL of 40 μM dilutions of AF610, AF647, AF700 and AF750 were also imaged on four separate occasions over a 6 month period; the average coefficient of variation of the unmixed fluorescence signal from the dyes over the different imaging sessions was 16 %. The larger variations in the fluorescence signals is likely due to the different positioning of the well plate within the camera FOV between imaging sessions.3.42 values of the linear fits. Spectral unmixing precision was also evaluated  in the flank of a nude mouse maintained under anesthesia. Prior to imaging on the fHSI system, the successful injection and placement of the fluorescent dyes was verified on a filter based optical imaging instrument  records both spatial and spectral information in a single data set. We hypothesized that the use of SRDAs could provide a compact and cost effective approach to acquisition of fHSI data, which would aid future clinical application of the approach. We first performed a technical characterization of the novel SRDA‐based HSI camera. Our HSI camera calibration explored the impact of illumination angle of incidence on the spectral response of the monolithically integrated filters in vivo in mice. We compared our in vivo to images from a traditional filterbased fluorescence system, which reinforces the advantage of increased spectral sampling for multiplexed fluorescence imaging.Integrating the camera into a reflectance‐based widefield imaging system provided the first proof‐of‐concept for fHSI using an SRDA. We exploited the spectral component of the HSI data cube for background removal using OSP. Our results demonstrated simultaneous imaging and unmixing of up to seven commonly used fluorescent dyes with highly overlapping emissions in pure solutions. When prepared in mixtures, fluorescent dyes naturally ‘bleed over’ i. e. the emission of one excites another, which affects any attempt to quantify concentration of highly overlapping dyes (independent of the imaging system used). We therefore focused on extracting quantitative data of four relatively well spectrally separated dyes. The presence of individual dyes at a given location could be identified based on majority decisions in our data: in superficial well plates; at depths of several millimetres in tissue mimicking phantoms; and in vitro, achieving this in vivo is more challenging as sample topology and the optical properties of tissue can modulate the recorded fluorescence spectra and this must be carefully accounted for, especially when imaging at depth in vivo imaging applications, the abundance maps would need to be reconstructed in real time; transferring our data processing algorithms onto graphical processing units could achieve the required processing speeds. Finally, while pushbroom imaging is acceptable for static  samples , for video‐rate applications such as endoscopic and intra‐operative imaging, a pixel level deposition of filters is preferable, requiring a trade‐off between spectral and spatial resolution.Although our fHSI system shows promise, there remain some limitations to this study. Firstly, the system performance is limited by the low QE and dynamic range of the commercial CMOS image sensor used in the HSI camera. Future integration of the optical filters onto a scientific grade sensor could overcome the QE limitations. Dynamic range could be extended using a sensor with spatially programmable exposure times or through application specific filter design to exclude reflected excitation light. Secondly, while we were able to demonstrate quantitative imaging in solutions in vitro and in vivo in mice. These data are highly encouraging, indicating that in addition to removing reflected light, fHSI combined with OSP could in the future enable diffuse reflectance and autofluorescence to be separately resolved from fluorescent dyes in clinical applications. Exploiting SRDAs, fHSI using targeted molecular imaging fluorescent contrast agents could find application in endoscopic cancer diagnosis and intra‐operative tumour resections, improving disease classification and margin identification respectively.In summary, we have designed, characterized and applied a novel SRDA‐based HSI camera in a wide‐field reflectance‐based fluorescence imaging system. We demonstrated that our fHSI system shows promise for multiplexed fluorescence imaging in solutions Additional supporting information may be found in the online version of this article at the publisher's website.We thank Dale Waterhouse for scientific advice and comments on the manuscript, lab manager Fiona Morgan and the staff at BRU Cambridge Cancer Institute. We also thank Andy Lambrechts and the hyperspectral team at Imec for helpful discussions. This work was funded by CRUK , the EPSRC‐CRUK Cancer Imaging Centre in Cambridge and Manchester (grant no. C197/A16465) and the EU FP7 agreement FP7‐PEOPLE‐2013‐CIG‐630729. Additional funds were provided by the University of Cambridge MRC Confidence in Concept Award. ASL thanks the EPSRC, the George and Lillian Schiff Foundation and the Foundation Blanceflor for studentship funding.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Ecological theory predicts that if animals with very similar dietary requirements inhabit the same landscape, then they should avoid niche overlap by either exploiting food resources at different times or foraging at different spatial scales. Similarly, it is often assumed that animals that fall in different body mass modes and share the same body plan will use landscapes at different spatial scales. We developed a new methodological framework for understanding the scaling of foraging  by applying a combination of three well‐established methods to satellite telemetry data to quantify foraging patch size distributions: (1) first‐passage time analysis; (2) a movement‐based kernel density estimator; and (3) statistical comparison of resulting histograms and tests for multimodality. We demonstrate our approach using two sympatric, ecologically similar species of African ducks with quite different body masses: Egyptian Geese , and Red‐billed Teal. Contrary to theoretical predictions, the two species, which are sympatric throughout the year, foraged at almost identical spatial scales. Our results show how ecologists can use GPS tracking data to explicitly quantify and compare the scales of foraging by different organisms within an animal community. Our analysis demonstrates both a novel approach to foraging data analysis and the need for caution when making assumptions about the relationships among niche separation, diet, and foraging scale. Available energy, resources, habitat structure, and interspecific interactions have long been known to be important influences on community composition  with landscape and community ecology. While we have the beginnings of a “first‐principles theory” with roots in body mass, body plan , and metabolic rate Brown, , 1995, tBody mass and plan can usefully explain and predict many key elements of an animal's ecology, including such things as its metabolic rate, resource demands, locomotory performance, life expectancy, and reproductive rate  treat body mass as both a driver and a response variable, making it difficult to distinguish between the causes (in evolutionary time) and the consequences  of body mass. Most empirical analyses of such hypotheses have not yet gone beyond the use of body mass distributions as the primary data source  foraging effort within a community of sympatric animals occurs across multiple scales, rather than being centered around a single scale; and (2) there are scale breaks  in the spatial extents of foraging movements.While there is already considerable support for the idea that most vertebrates focus their foraging efforts on resource‐rich patches, with longer “commuting” movements between foraging patches  and Egyptian Geese (Alopochen aegyptiaca).One of the reasons why telemetry data have not been used to test community‐level hypotheses about foraging and body size is presumably that appropriate frameworks for doing so have not been established. However, telemetry data have the potential to let the animals themselves tell us where food is in their environment and how they use it, rather than attempting to measure food distributions ourselves. In this paper, we address this methodological gap, presenting a novel approach to the quantitative, empirical analysis of scale and foraging behavior. We demonstrate our approach using a combination of dietary information and GPS satellite telemetry data from two African ducks: Red‐billed Teal . We used 22‐g PTTs for Red‐billed Teal (RBT) and 32‐g PTTs for Egyptian Geese (EG). Note that Egyptian Geese are ducks in the family Tadorninae (shelducks), rather than true geese. PTTs were set to capture GPS location data every 2 hr (EG) and 4 hr (RBT)  may be important for certain life history stages  to the foraging area data (creating a mean‐shifted data set) and tested whether it differed from the original data. Second, we pooled the original data set with each mean‐shifted data set in turn and tested whether Silverman's test could detect bimodality.3D = 0.5, p = .37; n = 457 and 1,269 for Red‐billed Teal and Egyptian Goose, respectively).The Red‐billed Teal data yielded 457 different foraging extent polygons and the Egyptian Goose data 1,269 polygons. The density distributions of log‐transformed foraging area extents for the two species showed almost complete overlap  and for Red‐billed Teal as 0.7 (six modes). These values are well below the recommended p ≥ .95 cut‐off value for accepting a hypothesis of multimodality.Silverman's test for multimodality, run from 1 to 30 modes, gave the highest probability of multimodality for Egyptian Geese as Our comparisons of real data for each data set to an identical data set with a small constant added indicated that our methods can detect differences as small as 0.06 between the mean log areas of the actual versus mean‐shifted home‐range polygons for Egyptian Geese and 0.07 for the smaller data set, Red‐Billed Teal. Combining the actual Egyptian Goose data set and a mean‐shifted version of the same data similarly indicated that Silverman's test would successfully identify bimodality beyond a difference in the mean log areas of 0.07.4Although FPT, kernel density home‐range estimation, statistical comparison of frequency distributions, and tests for multimodality are all well‐established and widely used techniques, they have not previously been applied together in this context. What is particularly novel about our approach is the idea of defining foraging patches from telemetry data, rather than trying to quantify food abundance itself, and using these data to compare how different animals use and respond to the same spatial patterns of resource distributions in their landscape.Our sensitivity analyses suggest that the methods are capable of distinguishing quite small differences between and within species. Silverman's test is known to be a conservative test for modality, but could easily replaced by a less conservative test when applying the framework; the current difficulty with alternatives, such as BCART , is that no alternative test has been proven to provide a fair balance between type I and type II errors. Similarly, the number of individuals from which telemetry data would be needed to provide a fair representation of population‐level trends in foraging areas  is unclear and needs further testing.The unexpected outcome of our analysis demonstrates the potential value of our proposed methods. Contrary to what textural discontinuity theory would predict, we found that our two study species foraged at identical scales, or more precisely, that the frequency distributions  of extents of foraging bouts were indistinguishable. Detailed analysis of their movement trajectories suggests strong similarities  our methods permit clear, direct comparison of patterns in foraging behavior that span multiple different scales; and (2) the methods are capable of identifying similarities and differences that may challenge existing theories and assumptions. Although we have presented an ensemble of existing methods rather than a new method per se, our overall approach has the potential to make novel contributions to the further development of theory.The assumption that organisms that fall into different body mass modes use landscapes at different scales is seldom questioned in landscape and community ecology. While we have not yet analyzed a data set for an entire ecological community, our methods suggest that comparisons of the scales of foraging using the methods that we have presented can provide valuable tests of assumptions about niche separation and its relationship to the landscape ecology of the study group. Multiscale analysis of the foraging patterns of different individuals and species carries the intriguing potential to provide a “missing link” between individual movement data, community‐level data, and species range data Cumming, . We lookNone declared.


It is documented that well‐modeled Monte Carlo dose calculation algorithms are more accurate than traditional correction‐based algorithms or convolution algorithms at predicting dose distributions delivered to heterogeneous volumes. This increased accuracy has clinical implications for CyberKnife, particularly when comparing dose distributions between the ray‐tracing and Monte Carlo algorithms. Differences between ray‐tracing and Monte Carlo calculations are exacerbated for highly heterogeneous volumes and small field sizes. In this study, the anthropomorphic thorax phantom from the Radiological Physics Center was used to validate the accuracy of the CyberKnife Monte Carlo dose calculation algorithm. Retrospective comparisons of dose distributions calculated by ray‐tracing and Monte Carlo were made for a selection of CyberKnife treatment plans; comparisons were based on target coverage and conformality. For highly heterogeneous cases, such as those involving the lungs, the ray‐tracing algorithm consistently overestimated the target dose and coverage. In our sample of lung treatment plans, the average target coverage for ray‐tracing calculations was 97.7%, while for Monte Carlo, the average coverage dropped to 69.2%. In each plan comparison, the same beam orientations and monitor units were used for both calculations. Significant changes in conformality were also observed. Isodose prescription lines and subsequent target coverage selected for treatment plans calculated with the ray‐tracing algorithm may be different from comparable treatment plans calculated with Monte Carlo, and as such, may have clinical implications for dose prescriptions.PACS number: 87.53.Wz The CyberKnife robotic radiosurgery system  is routinely used in the treatment of early stage and/or inoperable lung cancer, in addition to intracranial tumors, which can be treated by many radiosurgical modalities. Radiosurgical accuracy is achieved through the use of frameless real‐time image guidance and dynamic tumor tracking throughout the entire respiratory cycle.(deff); there are no corrections for changes in electron transport or lateral scatter disequilibrium that may develop in the presence of low‐density heterogeneities. The following equation represents the calculation of dose output (cGy/MU) for the ray‐tracing algorithm currently used in CyberKnife treatment planning:Despite the highly accurate targeting capabilities of CyberKnife, until recently the only dose calculation algorithm available for CyberKnife treatment planning was a simple ray‐tracing pathlength correction algorithm. In the ray‐tracing algorithm, off‐center ratios (OCR), tissue‐phantom ratios (TPR), and collimator output factors (OF), measured under reference conditions, are corrected for patient geometry. Tissue heterogeneities are accounted for through a central axis effective depth calculation  To address these issues, a Monte Carlo dose calculation algorithm is now commercially available for CyberKnife treatment planning. This algorithm utilizes a dual source model with parameters derived from clinically measured central axis percent depth dose curves, beam profiles, and collimator output factors.The ray‐tracing algorithm is quite sufficient for dose calculations involving homogeneous structures such as the brain; however, more robust heterogeneity corrections are required for accurate dose calculations in highly heterogeneous structures such as the lungs.,,6 The increased accuracy of Monte Carlo dose calculation may have clinical implications for CyberKnife dose prescriptions. Protocols for CyberKnife treatment planning, which use dose distributions calculated with the ray‐tracing algorithm, may need to be reevaluated for Monte Carlo calculations. In this study, we aim to quantify the differences expected between ray‐tracing and Monte Carlo calculations for CyberKnife treatment planning.The Radiological Physics Center (RPC) anthropomorphic thorax phantom was used to validate the accuracy of the CyberKnife Monte Carlo dose calculation algorithm (Multiplan 2.1.0). The RPC thorax phantom is designed to simulate the treatment of a solitary left lung mass.To evaluate the clinical implications of adopting Monte Carlo treatment planning for CyberKnife, a retrospective comparison was made between dose distributions calculated by the ray‐tracing and Monte Carlo algorithms. A sample of 18 lung treatment plans, treated with CyberKnife at Parkview Comprehensive Cancer Center, was selected for this comparison. The lung plans were designated as either peripheral or central according to the guidelines from the Lung Cancer Stereotactic Radiotherapy vs. Surgery (STARS) Trial. Targets are considered peripheral if they reside greater than two centimeters from mediastinal, pulmonary, and vertebral structures; all other targets are considered central. In each case, a clinically acceptable treatment plan was developed using the ray‐tracing algorithm. The beam sets for each ray‐tracing plan were then recalculated using the Monte Carlo algorithm . In this fashion, the beam orientations and monitor units for each respective pair of calculations were identical, only the resulting dose distributions changed.Comparisons between the Monte Carlo and ray‐tracing dose distributions were based on the dose statistics parameters available in Multiplan: prescription dose coverage, conformality index (CI), and new conformality index (nCI). These are some of the same parameters typically used to judge whether or not a plan is clinically acceptable for treatment. Prescription dose coverage is simply defined as the fraction of the tumor volume that receives at least the prescription dose. The CI represents the ratio of the tissue volume receiving at least the prescription dose to the tumor volume receiving at least the prescription dose, while the nCI is the CI multiplied by the ratio of the total tumor volume to the tumor volume covered by the prescription isodose line.A clinically acceptable treatment plan, calculated with the Monte Carlo algorithm, was developed for the thorax phantom following the dose prescription and planning guidelines provided by the RPC. A dose of 6 Gy, prescribed to the 79% isodose line, was delivered to the planning target volume (PTV) Fig. . The RPC97.7%±1.7%, while for the Monte Carlo calculations, the average coverage dropped to 69.2%±15.2% for the same isodose prescription line. A significant change in conformality was also observed, which again indicates the inaccuracy of the ray‐tracing algorithm in heterogeneous volumes.In every lung treatment plan that was analyzed, the ray‐tracing algorithm overestimated the dose and coverage of the target volume. Table 75%±4%, a drop of 9% when compared to the ray‐tracing algorithm (Table To achieve target coverage comparable to that attained for the ray‐tracing plans, the Monte Carlo calculations required a prescription line of There is also a shift in the standard deviation of each of the parameters listed in Tables Several factors could potentially influence the discrepancy observed between ray‐tracing and Monte Carlo calculations including tumor location and PTV composition. Figure 28%±15%, as compared to the initial ray‐tracing calculations. Considerable spread was observed in the coverage difference between ray‐tracing and Monte Carlo for our sample of lung treatment plans (Fig. There can be significant differences between ray‐tracing and Monte Carlo calculations for CyberKnife treatment planning, particularly for treatments involving heterogeneities such as the lungs. This is due to the tendency of the ray‐tracing algorithm to overestimate the dose to the target for heterogeneous volumes. The magnitude of overestimation is influenced by the location of the tumor within the lung and amount of lung tissue contained within the target volume. On average, in our sample of lung treatment plans, the Monte Carlo calculations resulted in a decrease in target coverage of approximately ans Fig. . In geneStereotactic lung radiosurgery protocols that currently specify ray‐tracing as the appropriate dose calculation algorithm may need to evaluate a reduced dose prescription when transitioning to the Monte Carlo algorithm. For example, a 6000 cGy prescription, calculated with the ray‐tracing algorithm, may actually be delivering significantly less dose to large portions of the target. The discrepancy between ray‐tracing and Monte Carlo dose distributions may need to be investigated on a case‐by‐case basis. There does not seem to be a uniform scaling factor that can be used to accurately transform a ray‐tracing dose prescription into an equivalent Monte Carlo dose prescription for all cases.The authors thank Mu Young Lee, Ph.D. and Miroslav Nikolic, Ph.D. of Accuray, Inc.


Europe). In most settings, however, the majority of intervention responses to sexual‐ and drug‐related risks tend to give rise to two parallel approaches to intervention. With some notable exceptions in a few major urban centres , the vast majority of sexual healthcare services have not tended to address the harms associated with substance use among gbMSM. Similarly, conventional substance use prevention, treatment and care services tend to do so with little regard for an individual's sexuality and/or sexual behaviour. The Five key implementation “gaps” that are currently hindering our efforts to adapt interventions to address the harms that are associated with the sexualized use of substances among gbMSM are described below.Gap 1: The spectrum of substance use is diverse among gbMSM who experience drug‐ and sexual‐related harmsA growing body of epidemiological and behavioural research indicates that the use of both stimulants  and depressants , used alone or in combination, are among the primary contemporary drivers of HIV and other sexually transmitted and blood‐borne infections (STBBI) experienced by some populations of gay, bisexual and other men who have sex with men (gbMSM) sexualized substance use enhances or detracts from gbMSM's ability to prevent or reduce harm. For instance, little is known about the treatment and care needs of substance‐dependent gbMSM versus those who display more episodic substance use patterns configurations of substance use  may enhance or reduce sexual‐ and drug‐related risk Gap 2: Life course perspectives are critically neededCommunity drug use patterns vary significantly over time, underscoring the importance of implementing services that maintain “nimble” and evidence‐informed adaptations that are responsive to the needs of today's gbMSM. Furthermore, while previous research has been helpful in identifying how some sub‐groups of gbMSM are more likely to use substances, little is known about how the social and cultural contexts of vs. episodic users) is needed to optimize implementation strategies for the right group of gbMSM at the right time.Gap 3: Healthcare providers’ perspectives remain absentThere are limited understandings about how substance use patterns occur across the life course of gbMSM's lives, including how key transitional periods  coincide with or shape substance use trajectories. Monitoring the patterns of substance use that produce sexual‐ and drug‐related HIV risks among various population sub‐groups of gbMSM . Likewise, substance use care providers will also benefit from opportunities to better understand how contexts of sexualized substance use are associated with a combination of sexual‐ and drug‐related harms among some groups of gbMSM. Better engaging healthcare providers will also provide opportunities to identify the actionable levers available in a given setting to improve care and health outcomes among gbMSM who use drugs.syndemics among gbMSM, that is, interrelated health inequities  that are produced and reinforced by structural inequities such as stigma and barriers to care. Nevertheless, public health policy and community‐based interventions that address the patterns of sexual‐ and drug‐related HIV risks experienced by gbMSM within the context of supporting comorbid health issues  may provide gbMSM with the support they need to engage with other intervention modalities, including clinic‐based services  and/or uptake of pharmacological regimens .Gap 5: A variety of ethical questions remain unansweredThere are real shortcomings to our current approach to addressing There are a variety of ethical considerations that require careful attention as interventions are adapted to address drug‐ and sexual‐related harms experienced by gbMSM. For example, previous discourse in this area has challenged the public health impetus to intervene “on” gbMSM's sexual lives, a population who has experienced decades of public health intervention and surveillance. Integrating community‐based principles  with key team science principles  may allow those working in this area to address the ethical challenges “head on.”1Filling the key implementation “gaps” identified above will provide new opportunities to effectively and ethically address the harms that are associated with the sexualized use of substances among gbMSM. As funding commitments are dedicated towards the growing field of implementation science , the onus will be on integrated and multi‐disciplinary teams  to collect, monitor and respond to “feedback” (i.e. adaptations) in ways that optimize (and effectively integrate) the substance use and sexual healthcare intervention “landscapes” for gbMSM.None to declare.RK drafted and finalized the manuscript.


Arabidopsis thaliana (arabidopsis). We describe the different somatic embryogenesis systems developed for arabidopsis and discuss the roles of transcription factors and chromatin modifications in this process. We describe how these somatic embryogenesis factors are interconnected and how their pathways converge at the level of hormones. Furthermore, the similarities between the developmental pathways in hormone‐ and transcription‐factor‐induced tissue culture systems are reviewed in the light of our recent findings on the somatic embryo‐inducing transcription factor BABY BOOM.Somatic embryogenesis is a form of induced plant cell totipotency where embryos develop from somatic or vegetative cells in the absence of fertilization. Somatic embryogenesis can be induced in vitro by exposing explants to stress or growth regulator treatments. Molecular genetics studies have also shown that ectopic expression of specific embryo‐ and meristem‐expressed transcription factors or loss of certain chromatin‐modifying proteins induces spontaneous somatic embryogenesis. We begin this review with a general description of the major developmental events that define plant somatic embryogenesis and then focus on the transcriptional regulation of this process in the model plant  EvidencAt that time, scientists working in the field had already recognized the potential of in vitro embryogenesis as a plant propagation tool. In vitro embryogenesis is now a standard biotechnology tool, with applications in both industrial and academic laboratories. Somatic embryos retain the genotype and ploidy of the donor explant and are used to clonally propagate plants for different applications, including scaling‐up of breeding material for testing, and for shortening the breeding cycle of highly heterozygous plants and plants with long life cycles. It is also used to regenerate transgenic plants after transformation. SE is often the preferred clonal propagation tool for plant breeding, as plantlets can be obtained in a single step rather than through the sequential regeneration of shoots and roots that is required during de novo organogenesis. The higher throughput of this technique and the potential for storage of cultures and embryos also contribute to its utility over de novo organogenesis and rooted cuttings. Such advantages have led to significant increases in production efficiency and uniformity, and in the quality of crop germplasm, especially in the forestry sector , histogenesis (differentiation), and conversion (germination) from embryo to plantlet.Arabidopsis thaliana (L.) Heyhn. (arabidopsis) to develop a framework for understanding plant SE. We begin with a general explanation of the different developmental concepts and events that define SE, describe the different SE systems developed for arabidopsis, and then discuss the roles of transcription factors (TFs) and chromatin‐modifying proteins in this process. Where relevant, we also include observations on other regeneration systems and model plants to augment this overview.The in vitro embryogenesis field has developed through the years to include studies aimed at obtaining and improving embryogenesis, as well as studies aimed at understanding how in vitro embryogenesis is initiated and maintained. What has become clear is that, although gametophytic and somatic embryos differ in origin, they share many commonalities, including the treatments used to induce embryogenesis and the developmental changes that lead to the production of embryos  and/or abiotic stress treatments. The mechanism underlying auxin‐ and stress‐induced SE is not known, but both treatments induce biosynthesis of endogenous auxins, which is thought to be an important early step in the switch to totipotent growth Feher, .Somatic embryos, as with all in vitro and zygotic embryos, are bipolar structures with an apical pole (the future shoot) and a basal pole (the future root), each with its own meristem, and an independent provascular system. This bipolarity distinguishes somatic embryos from ectopic or adventitious organs, such as shoots and roots, which are unipolar structures with a (lignified) vascular connection to the underlying explant. Somatic embryos also accumulate species‐specific storage products that are not found at other stages of plant development, although the extent to which they do so is often determined by the ability of the culture conditions to mimic the storage product accumulation phase of seed development. The absence of trichomes, which can be found on the first leaves of some plants, is often used as a morphological marker for somatic embryo formation, although trichome formation can be misleading as it can be delayed initially.2.1The developmental steps that take place after a cultured explant is induced to undergo SE have been well described at the histological level. Two developmental routes can be followed, termed indirect and direct embryogenesis Fig. , althougThe second pathway, direct embryogenesis, is less well defined, as it is characterized by the absence of a callus phase. In this system, the explant shows less prolific and more regular compact cell divisions. Single or multiple cells in single or multiple cell layers divide and bulge to develop into morphologically recognizable embryos without further treatment  is required to convert the explant into a somatic embryo  are embryogenic cells formed in tissue culture? Regardless of their origin, single or multiple cells in the explant can be converted into cells with embryo identity, but whether this involves direct transdifferentiation to an embryogenic state or redifferentiation to one or more intermediary identities is not known   that regulate embryo identity and maturation  proteins LEC1 and LEC2 were the first TFs shown to induce SE when ectopically expressed in seedlings /GROUNDED (GRD)  clade of AP2/ERF TFs that was initially identified as a marker for the induction of haploid embryo development from Brassica napus immature pollen grains  and six other AIL/PLETHORA (PLT) genes  or RAP2.4, also induces SE  is a homeodomain TF that is expressed in flower and shoot meristems, where it induces stem cell fate in a non‐cell‐autonomous manner  in an activation tagging screen for genes that induce somatic embryo formation from root callus . Indirect SE proceeds more slowly and is not initially associated with BBM‐mediated LAFL expression. LEC1 expression was only detected once embryos started to appear from the callus. At the seed‐to‐seedling transition, LAFL genes are repressed by chromatin‐modifying proteins (see below). The inability of BBM to directly activate LAFL genes after germination suggests that the chromatin state of these loci might be one factor that determines whether embryos are formed via direct or indirect SE. Constitutive overexpression of LEC1, L1L or LEC2 in arabidopsis appears to induce direct SE, as a callus phase has not been described .The effect of WUS on regeneration capacity has been investigated in several studies. Ectopic LEC2, Stone et al., WIND1, Ikeuchi et al., BBM, Boutilier et al., WUS, Zuo et al., The co‐occurrence of callus, adventitious organs and somatic embryos in seedlings that overexpress regeneration‐promoting TFs  TF gene. LEC2 activates expression of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the YUCCA2 and 4 (YUC) genes, which encode key enzymes in the auxin biosynthesis pathway  , which encodes a non‐canonical Aux/IAA protein  in somatic embryo tissue, although direct gene regulation was not studied  function during early embryogenesis, is important for SE induction from callus. PLT2 may be required for the establishment of a lateral root identity in the callus during indirect SE. Whether PLT2 or other AIL genes are also required for 2,4‐D‐induced direct SE is not known.The role of WUS for 2,4‐D‐induced SE was investigated using a 2,4‐D system from immature zygotic embryos where embryogenic clusters develop mostly from the shoot apex and to a lesser extent from the cotyledon petioles  or whether they are required to initiate processes downstream of 2,4‐D is not clear yet.From these studies we can conclude that 2,4‐D‐induced SE relies on the LAFL and PLT2 TFs, but whether mutations in these genes simply affect the identity of the explant  and SWINGER (SWN) or VERNALIZATION 2 (VRN2) and EMBRYONIC FLOWER 2 (EMF2) form callus on the shoot apex, which eventually gives rise to indirect somatic embryo formation and ectopic roots , which deposit K27me3 marks on histone H3, were found to be required for stem cell pluripotency , the somatic embryos formed after 2,4‐D treatment contain root apical meristems and are viable. Transient activation of CLF in a clf swn CLF‐GR line abolishes somatic embryo initiation, but does not affect somatic embryo development at a later stage, showing that PRC2 represses the formation and not the outgrowth of somatic embryos from seedling tissues , BBM, SHOOT MERISTEMLESS (STM) and WUS  double mutants, which also fail to form true leaves and develop embryo‐like proliferations in the root and shoot apical meristem regions . Double mutants in the PRC1 subunits AtBMI1a/b and AtRING1a/b show retarded germination, fail to form true leaves, and develop callus‐ and embryo‐like structures  and histone deacetylases (HDACs). The first indication that histone deacetylation plays a major role during SE was provided by Tanaka, Kikuchi, and Kamada , who shosee Fig. .pkl mutant seedlings retain embryonic traits after germination and produce somatic embryos from a number of seedling tissues, coinciding with de‐repression of the LEC genes  also mediates cross‐talk between histone methylation and acetylation. The B. napus  and histone acetylation play an important role in SE by regulating the expression of embryonic and meristematic genes. The extent to which chromatin reprogramming is required prior to SE probably depends on the explant and culture system in which SE is induced, and might also require additional chromatin modifications such as changes in DNA methylation, which are often associated with SE (De‐la‐Peña et al., 6Plant cell totipotency has intrigued scientists for decades. The first protocols used stress or growth factor treatments for SE induction and the process was studied at the histological level. SE can now be induced by embryo‐ and meristem‐expressed TFs, as well as TFs involved in wound repair. Molecular analysis identified cross‐regulation between the different SE‐inducing TFs, as well as a role for hormone pathways in SE induction. Genetic analysis has shown that mutations in SE‐inducing TFs reduce the efficiency of 2,4‐D‐induced SE, but it is not clear whether the TFs are required downstream of 2,4‐D or whether SE is impaired due to altered explant identity. An inducible gene silencing approach could be used to address this point.Somatic embryos can develop directly or indirectly via a callus phase. Whether this callus has lateral root identity, similar to organogenic callus, is a topic for future studies. Our work on BBM suggests that the transcriptional accessibility of embryo genes in different tissues might determine whether embryos are formed via direct or indirect SE. Both types of SE, as well as organogenesis, coexist in different 2,4‐D‐ and TF‐induced tissue culture systems, suggesting that these types of regeneration are mechanistically linked. A study on BBM has shed a light on this phenomenon, as BBM induces organogenesis or SE depending on its dose, but whether the same or a different set of genes is involved in these different types of regeneration is not known. Finally, multiple studies show that SE‐inducing genes are repressed through histone methylation and deacetylation during the transition from embryo to seedling development. Loss‐of‐function mutants in the chromatin modification proteins that regulate these marks undergo spontaneous SE, which is accompanied by upregulation of key embryo identity genes, including genes for SE‐inducing TFs. Whether there are major differences between the chromatin‐level reprogramming that occurs during direct and indirect SE and between SE and adventitious organogenesis remains to be investigated. The fact that only a small fraction of the cells in an explant are induced to form somatic embryos and that these embryogenic cells are difficult to recognize at an early stage complicates a detailed study of the chromatin changes, such as the one performed by She et al.  on cellsThe authors declare no conflict of interest.


Blunt injuries to visceral organs have the potential to lead to delayed pseudoaneurysm formation or organ rupture, but current trauma and surgical guidelines do not recommend repetitive imaging. This study examined the incidence and timing of delayed undesirable events and established advisable timing for follow‐up imaging and appropriate observational admission.NOM) in our institution were included and retrospectively reviewed.Patients with blunt splenic (S), liver (L), or kidney (K) injury treated with non‐operative management  patients underwent transcatheter arterial embolization. Delayed undesirable events occurred in four patients: three patients with splenic pseudoaneurysm on hospital day 6–7 and one patient with splenic delayed rupture on hospital day 7. The second follow‐up computed tomography scan carried out 1–2 days after admission did not show any significant findings that could help predict undesirable results of delayed events. The patients with delayed events had longer continuous abdominal pain than that of event‐free patients (P = 0.04).From January 2013 to January 2017, 57 patients were admitted with documented blunt visceral organ injuries and 22 patients were excluded. Of 35 patients  treated with NOM patients at hospital day 6–7 and tended to be associated with high‐grade splenic injuries and continuous symptoms. Repetitive screening of these patients 6–7 days after injury might be warranted because of the potential risk of delayed events.Undesirable delayed events were recognized on follow‐up computed tomography scans in 11.4% of  Recently, as the quality of non‐operative management (NOM) has developed, blunt injuries to visceral organs such as spleen, liver, or kidney are tending to be managed non‐operatively with a high rate of success.Some reportsThe purpose of this study is to examine the incidence and timing of such delayed undesirable events and to establish advisable timing of follow‐up imaging and appropriate duration for observation or disposition of patients with blunt visceral organ injuries.This study was undertaken retrospectively in the Department of Trauma and Critical Care of Osaka City University Hospital . During the period January 2013–January 2017, all patients aged 16 years or older who were admitted to our institution because of blunt liver, kidney, or splenic injuries were included. We reviewed the patients' demographics, injury descriptions, values of laboratory data on admission, volume of blood products used in the 24 h after admission, timing of follow‐up CT scans, management techniques, and outcomes obtained from the patients' medical records.In our institution, the patients who have clinical findings or suspicion of abdominal trauma, if focused abdominal sonography for trauma (FAST) is positive and the patient's unstable hemodynamics makes their transfer to the CT scan room or operating room difficult, we immediately carry out resuscitative surgery in the emergency unit without a pan‐scan CT. If FAST is positive and hemodynamics are stable or controlled, or if FAST is negative, a contrast‐enhanced CT (CECT) scan is carried out to assess the injuries at the time of hospital admission.If the CECT findings show contrast blush in the arterial phase or pooling in the dynamic phase, we basically plan transcatheter arterial embolization (TAE) to embolize the sites of bleeding, even if the hemodynamic status is stabilized without intervention.For the patients with an AAST Trauma Organ Injury Scale of grade III–IV or in those treated with TAE, a follow‐up CT scan is undertaken on day 1 or 2 following hospital admission and then approximately 1 week after the injury if clinical symptoms or vital signs remain stable. For the patients with AAST Trauma Organ Injury Scale of grade I–II, the follow‐up CT scan is carried out approximately 1 week after the admission. If the patients have intolerable pain, sudden onset of pain, or positive FAST findings during the observation period, we carry out an extra emergent CT scan.Including the timing of ambulation, the activity level of the patients after admission is determined essentially on the basis of the clinical symptoms or the results of follow‐up CT scan. The pseudoaneurysm is defined as diameter >7 mm.Transcatheter embolization for trauma patients is always carried out by interventional radiologists. For injuries of visceral organ arteries, we usually use gelatin sponges with concomitant use of pushable coils if needed. The exact procedure depends on the hemodynamic conditions, injured sites, and concomitant injuries of the patients. If the hemodynamic status of the patient is sufficiently stable and there are no concomitant injuries, we often choose distal embolization and selectively embolize the artery as close as possible to the injured sites. If the condition of the patient or the injured sites does not permit a long procedural time or selective embolization, we choose proximal artery embolization for the abbreviated intervention.During the study period, 57 patients were diagnosed as having liver, splenic, or kidney injuries. Eighteen patients were excluded as their hemodynamics had not been stabilized and they were treated with immediate laparotomy. We also excluded four patients because of severe brain injury that had not recovered, as indicated by not attaining a Glasgow Coma Scale score >8 during hospital admission, and patients with spinal or pelvic injuries who had needed continuous bedrest. Finally, 35 patients treated with NOM were included in this study. Of the 35 patients, 14 patients underwent TAE; the other 21 patients had no TAE indications and were observed carefully.U‐test. A value of P < 0.05 was considered statistically significant. Data were analyzed using IBM spss Statistics, version 22 .All statistical data are presented as median (25–75% interquartile range) or number as the results of statistically non‐normal distribution of the collected data. Non‐parametric numerical data were compared using the Mann–Whitney During the period January 2013–January 2017, 57 patients with documented blunt splenic (S), liver (L), or kidney (K) injury were admitted in our hospital. Twenty‐two patients were excluded described as above, and 35 patients  treated with NOM were included . All delayed events were detected in splenic injury, and delayed events occurred in 23.5% of splenic injured patients. As Table As the initial TAE procedure, the lower and middle branches of the splenic artery in two of these four patients were selectively embolized using gelatin sponges and pushable coils. The remaining patients with pseudoaneurysm underwent selective embolization of the lower branch of the splenic artery using gelatin only.The initial CT scans carried out at the time of admission and the second follow‐up CT scans undertaken 1–2 days after admission did not show any significant findings that could help in predicting the undesirable results of delayed events such as pseudoaneurysm formation or delayed rupture.The values of laboratory data, lactate, volume of used blood products in the 24 h after admission, and Trauma and Injury Severity Score showed no significant differences between the two groups Table .P = 0.04). Other specific symptoms or complaints were not documented for the patients with delayed events. Vital signs like fever or heart rate did not change to an extent to detect the delayed events for these patients. There were no significant relationships between the starting time of per oral nutrition or early ambulation and the delayed events (Table Evaluation of the relationship between patients with continuous intolerable pain using pain control drugs, such as fentanyl, and those who experienced delayed events showed a tendency for longer continuous abdominal pain in those with delayed events than in event‐free patients (ts Table .All patients with delayed events were treated successfully with TAE and discharged to home with no complications at approximately 2 weeks after the intervention.et al.et al.et al.et al.The patients treated with NOM for their visceral organ injuries have been increasing, hence the incidence of delayed events should be predicted as much as possible. In this study, delayed events were detected in 11.4% of the patients within 7 days after injury. Previously published papers describing these delayed events are almost all limited to splenic pseudoaneurysm. Davis et al.Francisco et al.Although renal artery pseudoaneurysm is also rare, Yamaçake et al.The relationships between symptoms or the timing of starting per oral nutrition and delayed events remain unclear. Bukur et al.et al.Although activity level is one of the most important problems for NOM patients, there are few data on the activity level permitted following injury. London Although routine CT follow‐up has recently tended not to be recommended, especially in patients with a low‐grade organ injury score, the findings from this study and previous reports suggest that monitoring and the timing of follow‐up CT scan 6–7 days after injury could be a reasonable period to detect delayed undesirable events stemming from blunt visceral organ injuries.The present single‐center study is a small preliminary report, and we will definitely have to plan further multi‐institutional, prospective, randomized trials on the basis of this study to assess the appropriate timing of follow‐up CT scans.Delayed undesirable events were recognized to occur during hospital days 6–7 by follow‐up CT in 11.4% of patients treated with NOM. These events tended to be associated with high‐grade splenic injuries and continuous symptoms. We conclude that repeated screening of these patients approximately 6–7 days after injury may be warranted because of the potential risk of delayed events occurring within this time.Conflict of interest: None declared.


This study demonstrates that using LiST modelling and MIS data are viable options for evaluating programmes to reduce child mortality.Many interventions have attempted to increase vulnerable and remote populations’ access to ORS and zinc to reduce child mortality from diarrhoea. However, the impact of these interventions is difficult to measure. From 2010 to 15, Micronutrient Initiative (MI), worked with the public sector in Bihar, India to enable community health workers to treat and report uncomplicated child diarrhoea with ORS and zinc. We describe how we estimated programme’s impact on child mortality with Lives Saved Tool LiST modelling software to estimate programme impact on child mortality. Four scenarios estimated the effects of different rates of programme scale-up and programme coverage on estimated child mortality by measuring children’s lives saved.We used MI’s programme monitoring data to estimate coverage rates and The programme saved an estimated 806–975 children under-5 who had diarrhoea during five-year project phase. Increasing ORS and zinc coverage rates to 19.8% & 18.3% respectively under public sector coverage with effective treatment would have increased the programme’s impact on child mortality and could have achieved the project goal of saving 4200 children’s lives during the five-year programme.LiST modelling software to estimate coverage rates and programme impact on child mortality. This modelling approach may cost less and yield estimates sooner than directly measuring programme impact with population-based surveys. However, users must be cautious about relying on modelled estimates of impact and ensure that the programme monitoring data used is complete and precise about the programme aspects that are modelled. Otherwise, LiST may mis-estimate impact on child mortality. Further, LiST software may require modifications to its built-in assumptions to capture programmatic inputs. LiST assumes that mortality rates and cause of death structure change only in response to changes in programme coverage. In Bihar, overall child mortality has decreased and diarrhoea seems to be less lethal than previously, but at present LiST does not adjust its estimates for these sorts of changes.Programme monitoring data can be used with  In 2013 alone more than 0.57 million children under five years of age died from diarrhoea .LiST [From 2010 to 2015, Micronutrient Initiative (MI), now Nutrition International conducted the Childhood Diarrhoea Management Programme (CDMP), also called the Diarrhoea Alleviation through Zinc and ORS Therapy (DAZT) project, in Bihar, Gujarat, and Uttar Pradesh. Children’s Investment Fund Foundation (CIFF) supported the Bihar programme . JHSPH aLiST , 8 modelLiST has evolved to model more types of interventions and outcomes [LiST modelling to measure the potential effectiveness of an intervention or set of interventions related to mother and child health under scale up scenarios by estimating the number of lives saved [LiST has been used to identify high impact health interventions to end preventable deaths in mothers, newborns and stillbirths and to estimate their associated costs [LiST estimates deaths averted or lives saved by public sector interventions to reduce diarrhoeal deaths. Their findings demonstrate that LiST can be used as an alternative to large and expensive mortality impact studies.Over the years outcomes . Studieses saved –12. It hes saved . More reed costs . Furthered costs  detail hLiST software, MI supported the state government to manage a robust management information system (MIS) to estimate the cases of diarrhoea and forecast the supply of ORS and zinc needed at different levels of service provision. Our study incorporates MIS data in LiST modelling to estimate the number of lives saved due to the ORS and zinc programme coverage. There are currently no studies of MIS data being utilized in LiST modelling, therefore our innovative approach has implications for scaling up cost-effective and impactful programmes.Along with using LiST) modelling software and MIS data to estimate the Bihar programme’s effect on child mortality. Through this study, we further highlight the possibilities and challenges of using LiST and MIS data to measure programmes.This paper describes how we used the Lives Saved Tool  measures of population-level health status including mortality and causes of death (available by default in LiST); (2) effect sizes [We used ct sizes , 17 of iLiST modelling did, our 2015 LiST modelling exercise used available data from MI’s monitoring data to estimate cases covered at the district level, and used other data sources to estimate the population affected.Rather than use primary data for both numerator (cases covered) and denominator (population affected) as the 2013 JHSPH The target population of the intervention and the modelling is children aged 2–59 months from 15 demonstration districts.The lower bound of diarrhoea incidence was estimated as 1.81 (diarrhoea incidence of 1.81 episodes/child/year) and a higher bound of 2.20 .In our case the programme intervention for children with diarrhoea was those who received both ORS and zinc from a public-sector service provider. In the previous modelling exercise, JHSPH conducted two cross-sectional studies – baseline (2011) and a follow-up (2013) survey – and used coverage information from these two surveys to estimate the number of lives saved (or deaths averted) due to programme impact.To estimate public sector coverage, we used three data sources i) MI’s MIS data, ii) JHSPH’s 2011 baseline and 2013 follow-up coverage data for ORS and zinc treatment from public health sector functionaries, and iii) the 2011 India Census, for estimates of population affected by childhood diarrhoea. We estimated the coverage rate numerator from MI’s MIS data and from JHSPH’s 2013 follow-up data, and the denominator from the 2011 India Census.Anganwadi workers (AWWs), Auxiliary Nurse Midwives (ANMs), Primary Health Centres and government hospitals. Front line health workers (ASHAs and AWWs) at the village level reported each diarrhoeal case and those treated with ORS and zinc within their respective geographies. Auxiliary Nurse Midwives reported cases and treatment at the sub-centre level. Monthly consolidated data was shared at a district and state level.MI’s intervention was completed solely through public health functionaries in the intervention districts: Accredited Social Health Activists (ASHAs), LiST modelling as listed in Table In 2011 and 2013, JHSPH and SAS surveyed caregivers of children who had had a diarrhoeal episode in the 14 days before the survey. The caregivers who reported having given their children ORS and/or zinc to treat the diarrhoea were asked about the source of the ORS and/or zinc. In addition to coverage estimates obtained from surveys, we also used coverage estimates inputs obtained from MIS. MIS coverage estimates are bound to overestimate since they are not based on cross-sectional data. To nullify such error, we used a multiplication factor (0.68) that was obtained by dividing coverage rates obtained from the JHSPH’s follow-up survey (2013) with the coverage estimate obtained from MIS in the same year. This adjustment was carried out for the coverage estimates calculated from MIS in subsequent years and inputted into the Similar adjustments were made while calculating zinc coverages.LiST’s default values for intervention efficacies and population affected fractions.The denominator of the coverage rate was estimated based on the number of children (2–59 months) who are affected with diarrhoea in the intervention areas. This was estimated based on the total number of children in that age group multiplied by the estimated incidence of diarrhoea in the age group. The total number of children aged 2–59 months was projected from a base population in 2011 using Census population [We developed four scenarios to establish appropriate mixes of coverage information. The following Table LiST to achieve the target of 4200 deaths averted cumulatively.During the programme design phase, in 2010, CIFF estimated that the programme could save at least 4200 children’s lives. Scenario 3 of the 2015 modelling is a hypothetical as we estimated what programme coverage would have been necessary to reach this level of impact. This was worked out “backwards” by attempting several iterations in LiST estimated that a total of between 965 (Scenario 1) and 975 (Scenario 2) additional lives were saved in children under 5, as a result of scaling up ORS and zinc treatment for diarrhoea through public sector providers  combined with the best possible programme coverage  in 2010 to 25.9% for ORS and from 3.7% in 2010 to 14.3% in 2015 for zinc – by 2015, more than 3400 diarrhoea related deaths among under-5 children in 15 demonstrated districts of Bihar would have been averted.JHSPH’s 2013 projections based on LiST modelling results reported above, based on Scenarios 1–4, focus on public sector service coverage and are consistent with the 2013 JHU LiST modelling results. To have greater impact on child mortality, public sector coverage levels would have needed to increase quite ambitiously and remain increased as compared to the 2010–11 baseline levels. Had the programme been able to increase public sector coverage by 40%, more than 2100 children’s lives would have been saved, and had public sector coverage increased by 60%, more than 2300 lives would have been saved. To save 4200 children’s lives, the intervention would have had to increase its public sector ORS coverage from 1.8% in 2010–11 to 19.8% in 2014–15, and also its public sector zinc coverage from 1.4% in 2010–11 to 18.3% by 2014–15. These modelled coverage rates for ORS and zinc are ambitious given the field realities such as persistent stock outs of zinc and ORS in Bihar’s public sector made this sustained increase unfeasible [The 2015 LiST module was suitable for use at a sub-state level such as district in Bihar. However, the current LiST software present several limitations to our study as well.Throughout our study, the LiST does not provide any guidelines or modules on using MIS data to compute coverage rates for intervention programmes or extracting coverage rates separately from MIS data. Secondly, LiST does not include a joint coverage of both ORS and zinc though it is the recommended treatment for diarrhoea. Since LiST allows inputs for coverage rates of “ORS” (only) or “Zinc” (only) we had to accommodate the ORS + Zinc data into the LiST analysis by clubbing information pertaining to ORS + Zinc with ORS (only) and made two categories – ORS and zinc.Firstly, Thirdly, for the zinc (only) category, the field reports suggest that when health workers had no stock of ORS, they generally advised caregivers to give children oral rehydration therapies (ORTs) along with zinc, or get ORS from other sources. The potential impact of ORTs in combination with zinc is not captured in the model results, which may imply some level of additional effectiveness in treating diarrhoea cases.LiST is that any changes in the mortality rates and the cause of death structure is solely in response to changes in intervention coverages [LiST model to short-term projection purposes not suitable for long-term projection as child mortality rates and cause of death distribution are rapidly changing.Lastly, the overarching assumption in overages . This asLiST software to model a programme’s impact on deaths from diarrhoea among children under-5 in Bihar. Our modelling assessed the impact of MI’s public sector ORS and zinc programme in 15 demonstration districts in Bihar using a population survey and programme MIS reporting data. We demonstrated that even with the limited data available in resource-poor settings, LiST software and utilization of MIS data can be a useful tool for programme evaluators and planners to estimate a programme’s impact on child mortality. For example, CIFF used LiST modelling to estimate the programme’s potential for impact on child mortality. The modelled impact results reported here (806 to 975) fell short of the 2010 CIFF projections which may be attributable to the challenges of the LiST software, programme execution, and data quality.This article reports on the authors’ experience with using LiST software. For example, LiST developers can provide appropriate guidelines on how to use MIS data to compute coverage rates for different intervention programmes and extract coverage rates from MIS data. The developers can also incorporate data collection for joint interventions such as treatment with both ORS and zinc. Finally, the software could expand the use of LiST model to long-term projection purposes as child mortality rates and cause of death distribution are rapidly changing.Our study also highlights the need to improve the LiST, the software and utilization of MIS data should be seen as a complement rather than a replacement for household surveys. LiST and MIS data are viable options for estimating programme impact without the time and expense required to measure programme impact with a household coverage survey.Despite the improvements that need to be made to