{"text": "A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. However, there is very little known about how inputs across the body mid-line are processed within SII.Optical intrinsic signal imaging was used to evaluate the response of primary somatosensory cortex (SI and SII in the same hemisphere) to 25 Hz sinusoidal vertical skin displacement stimulation (\"skin flutter\") applied contralaterally, ipsilaterally, and bilaterally to the central pads of the forepaws. A localized increase in absorbance in both SI and SII was evoked by both contralateral and bilateral flutter stimulation. Ipsilateral flutter stimulation evoked a localized increase in absorbance in SII, but not in SI. The SII region that responded with an increase in absorbance to ipsilateral stimulation was posterior to the region in which absorbance increased maximally in response to stimulation of the contralateral central pad. Additionally, in the posterior SII region that responded maximally to ipsilateral stimulation of the central pad, bilateral central pad stimulation approximated a linear summation of the SII responses to independent stimulation of the contralateral and ipsilateral central pads. Conversely, in anterior SII , bilateral stimulation was consistently less than the response evoked from the contralateral central pad.The results indicate that two regions located at neighboring, but distinctly different A-P levels of the anterior ectosylvian gyrus process input from opposite sides of the body midline in very different ways. The results suggest that the SII cortex, in the cat, can be subdivided into at least two functionally distinct regions and that these functionally distinct regions demonstrate a laterality preference within SII. There is general agreement that in cats and monkeys (and presumably in humans) the spike discharge activity a mechanical stimulus sets up in rapidly adapting (RA), slowly adapting (SA), and Pacinian (PC) skin mechanoreceptors is projected centrally, at short latency and with relatively minor transformation, to primary somatosensory cortex (both SI and SII) in the contralateral hemisphere. There is no consensus, however, about the way in which the stimulus-evoked response in the ipsilateral hemisphere contributes to cerebral cortical somatosensory information processing and somatosensation. A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. Similar to SI, SII possesses a clear topographic organization , but unlFigure To more accurately characterize the spatial disparity between SII loci activated by contralateral vs. ipsilateral stimulation, the absorbance values were obtained and plotted along the posterior-anterior axis of SII. Figure A more comprehensive view of the SII response to the contralateral and bilateral stimulus conditions can be better appreciated with a multi-dimensional surface plot of the data. Figure To directly compare the time course of the response of the anterior and posterior regions of SII to the three stimulus conditions, we determined the time course of the absorbance changes in each region under each stimulus condition Figure . The ploCluster plots were used to directly compare the response of SII to the different conditions of ipsilateral and contralateral stimulation. In each plot in Figure 2 boxel surrounding the peak. In anterior SII, the majority of the responses to contralateral stimulation are stronger than the responses to bilateral stimulation \u2013 hence, the majority of the points plotted fall below the reference line (which has a slope of 1). In the posterior region, the majority of the points plotted are above the reference line \u2013 indicating that the response to the bilateral stimulus was greater than the response to the contralateral stimulus.The contralateral and bilateral stimulus conditions can also be compared using cluster analysis. However, because the difference between the contralateral and ipsilateral responses is more robust than the difference between the contralateral and bilateral responses, Figure Thus far, the results suggest that the anterior and posterior regions of SII are differentially activated by contralateral, ipsilateral and bilateral stimulation. To determine the across-subject consistency of these findings, the average absorbance values evoked by the 3 different stimulus conditions were determined for all 6 of the subjects Figure . ClearlyThe findings of this study demonstrated clearly that the anterior and posterior regions of SII process bilateral inputs very differently. At the locus of the maximal OIS response evoked in the posterior region by an ipsilateral stimulus, bilateral stimulation evoked a response that was, on average, about 25% larger than that evoked from the contralateral stimulus site. Conversely, at the locus of the maximal OIS response evoked by contralateral stimulation in the anterior region, bilateral stimulation evoked a response that was, on average, 35% lower than the activity evoked by a contralateral stimulus. This discrepancy between the optical responses of the anterior and posterior regions could be related to neurophysiological observations reported in earlier studies. For example, Carreras and Andersson  found inThe main, although not the only, route for ipsilateral input to SII is through the corpus callosum, from cells located in SI and SII of the opposite cerebral hemisphere ,7,8,11. The neurons in the distal limb regions of SII do receive substantial callosal connections, but these neurons have been reported to lack ipsilateral RFs , indicatA number of interactions between stimuli applied to both hands have been demonstrated in human psychophysical studies. Gilson  found thA recent report  demonstrOne question that the results suggest is whether or not SII can be segregated by laterality preference, in a manner similar to that observed in other sensory systems. Laterality has been demonstrated in the primary sensory cortex of both the visual system and the auditory system of both primates and cats, and the data in this report strongly suggest that there are cortical areas within SII that exhibit preference to ipsilateral or contralateral inputs. In terms of processing information from simultaneous contralateral and ipsilateral stimuli, there could be further similarities between the somatosensory, auditory and visual systems that have yet to be described. Future investigations will aim to further clarify the role of SII in integration of information from inputs across the body midline.The responses evoked by contralateral and ipsilateral flutter stimulation of the central pad of the cat forepaw define functional subdivisions in SII: the two modes of stimulation maximally activate cortical regions that are anterior and posterior to one another, respectively. Bilateral stimulation, or providing simultaneous contralateral and ipsilateral stimulation, reveals, additionally, that the two adjacent cortical areas process bilateral inputs differently. In the posterior region, where ipsilateral stimulation evokes a maximal response, bilateral stimuli evoke a response that is greater than the response evoked by either the individual ipsilateral or contralateral response. In the anterior region of SII, where the contralateral stimulus evokes a maximal response, bilateral stimuli evoke responses that are smaller in magnitude than the responses evoked by the contralateral stimulus.Adult cats  were subjects. All surgical procedures were carried out under deep general anesthesia . After induction of general anesthesia the trachea was intubated with a soft tube and a polyethylene cannula was inserted in the femoral vein to allow administration of drugs and fluids (5% dextrose and 0.9% NaCl). For each subject, a 1.5 cm diameter opening was made in the skull overlying somatosensory cortex, a chamber was mounted to the skull over the opening with dental acrylic, and the dura overlying anterior parietal cortex was incised and removed. Following the completion of the surgical procedures all wound margins were infiltrated with long-lasting local anesthetic, the skin and muscle incisions were closed with sutures, and each surgical site outside the recording chamber was covered with a bandage held in place by adhesive tape.2 between 3.0 \u2013 4.0%; EEG and autonomic signs  were monitored and titrated (by adjustments in the anesthetic gas mixture) to maintain levels consistent with light general anesthesia. Rectal temperature was maintained (using a heating pad) at 37.5\u00b0C.Subjects were immobilized with Norcuron and ventilated with a gas mixture  delivered via a positive pressure respirator 1\u20133 hours prior to the data acquisition phase of the OIS imaging experiments. Respirator rate and volume were adjusted to maintain end-tidal COEuthanasia was achieved by intravenous injection of pentobarbital (45 mg/kg) and by intracardial perfusion with saline followed by fixative . Following perfusion fiducial marks were placed to guide removal, blocking, and subsequent histological sectioning of the cortical region studied. All procedures were reviewed and approved in advance by an institutional committee and are in full compliance with current NIH policy on animal welfare.Results were obtained during stimulation of the contralateral central pad of the forepaw and/or the ipsilateral central pad of the forepaw. The stimuli always consisted of sinusoidal vertical skin displacements  and were applied using a servocontrolled transducer  that is capable of delivering sinusoidal stimuli in the range of 1\u2013250 Hz at amplitudes in the range of 0\u20131000 microns. The stimuli were delivered independently to the ipsilateral and contralateral skin sites, and also were applied simultaneously to both sites . The stimulus probes were positioned 500 microns beyond the point at which skin contact was detected (via force transducer on the Cantek). The bilateral stimulus protocols reported in this paper were synchronized to start and stop at the same time. The contralateral, ipsilateral and bilateral stimuli were interleaved on a trial-by-trial basis. This approach was used to control for temporal changes in cortical \"state\" unrelated to stimulus conditions which, if unrecognized, might obscure or modify any differences between the optical responses evoked by the contralateral, ipsilateral and bilateral stimulus conditions.Near-infrared  OIS imaging was carried out using an oil-filled chamber capped with an optical window . Images At the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 \u03bcm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 \u03bcm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries  identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria .A-P = anterior-posteriorRA = rapidly adaptingSA = slowly adaptingPC = PacinianRF = receptive fieldOIS = optical intrinsic signalIR = near infraredEEG = electro-encephalogramMEG = magneto-encephalogramBW and OF participated in the design of the experiments, the data collection, and drafting of the manuscript. SS, JC, and VT made significant contributions to the data collection and the analysis of the data. MT played a major role in all aspects of the development of the manuscript."}
{"text": "Previous studies have shown that spatio-tactile acuity is influenced by the clarity of the cortical response in primary somatosensory cortex (SI). Stimulus characteristics such as frequency, amplitude, and location of tactile stimuli presented to the skin have been shown to have a significant effect on the response in SI. The present study observes the effect of changing stimulus parameters of 25 Hz sinusoidal vertical skin displacement stimulation (\"flutter\") on a human subject's ability to discriminate between two adjacent or near-adjacent skin sites. Based on results obtained from recent neurophysiological studies of the SI response to different conditions of vibrotactile stimulation, we predicted that the addition of 200 Hz vibration to the same site that a two-point flutter stimulus was delivered on the skin would improve a subject's spatio-tactile acuity over that measured with flutter alone. Additionally, similar neurophysiological studies predict that the presence of either a 25 Hz flutter or 200 Hz vibration stimulus on the unattended hand  would decrease a subject's ability to discriminate between two points on the skin.improved spatial acuity by 20 to 25%, the two-point limen was not significantly affected by substantial changes in stimulus amplitude (between 100 \u2013 200 \u03bcm). In contrast, simultaneous stimulation of the unattended hand , impaired spatial acuity by 20% with flutter stimulation and by 30% with vibration stimulation.A Bekesy tracking method was employed to track a subject's ability to discriminate between two-point stimuli delivered to the skin. The distance between the two points of stimulation was varied on a trial-by-trial basis, and several different stimulus conditions were examined: (1) The \"control\" condition, in which 25 Hz flutter stimuli were delivered simultaneously to the two points on the skin of the attended hand, (2) the \"complex\" condition, in which a combination of 25 Hz flutter and 200 Hz vibration stimuli were delivered to the two points on the attended hand, and (3) a \"bilateral\" condition, in which 25 Hz flutter was delivered to the two points on the attended hand and a second stimulus (either flutter or vibration) was delivered to the unattended hand. The two-point limen was reduced  under the complex stimulus condition when compared to the control stimulus condition. Specifically, whereas adding vibration to the unilateral two-point flutter stimulus It was found that the addition of 200 Hz vibration to a two-point 25 Hz flutter stimulus significantly improved a subject's ability to discriminate between two points on the skin. Since previous studies showed that 200 Hz vibration preferentially evokes activity in cortical area SII and reduces or inhibits the spatial extent of activity in SI in the same hemisphere, the findings in this paper raise the possibility that although SI activity plays a major role in two-point discrimination on the skin, influences relayed to SI from SII in the same hemisphere may contribute importantly to SI's ability to differentially respond to stimuli applied to closely spaced skin points on the same side of the body midline. Recently, we reported the development of a semi-automated method for measuring a human subject's ability to discriminate between two points on the skin . In thatimprove a subject's ability to discriminate between two points. Alternatively, recent findings comparing the SI activity evoked by different conditions of contralateral, ipsilateral and bilateral stimulation in the cat show that the magnitude of response in SI evoked by contralateral stimulation is reduced in the presence of an ipsilateral stimulus [Mountcastle and Darian-Smith  proposedstimulus . Similarimproved with same-site vibration and degraded with the addition of either a flutter or vibration stimulus on the opposite, unattended hand.Bekesy tracking algorithms were used to find a subject's two-point limen at the dorsal surface of the right hand under four different stimulus conditions. Exemplary results for a single session (four runs) of a subject are shown in Figure reduced  for the complex condition \u2013 the two-point limen tracks at approximately 80% of the values measured under the flutter condition. In contrast, the two-point limen was larger  for both bilateral conditions. In the case in which the opposite or unattended hand was presented with a simultaneous 25 Hz flutter stimulus, the two-point limen tracks approximately 20% higher than the control (attended hand only) condition. Similarly, applying a 200 Hz vibration stimulus simultaneously to the unattended hand resulted in two-point limen values that were approximately 30% higher than the control condition.To determine subject consistency of the above findings, the tracking data collected under each condition for an individual subject were averaged. The data were normalized to the flutter condition since the primary objective of this study was to determine the effect of vibration on the response normally evoked by two-point flutter stimulation. Thus, the two-point limen for the flutter condition was defined as the value \"1\" and all other distances are plotted as a proportion of the values obtained under the flutter condition [To determine the across-subject consistency of the above findings, the data normalization process applied to the single subject case, as shown in Figure In order to more directly compare the responses measured under each of the stimulus conditions, the tracking values obtained from the last five trials across all subjects was averaged and normalized to the flutter condition Figure . Again, To ensure that the enhanced acuity of a subject under the complex stimulus condition was not due simply to the increased amplitude that resulted from adding vibration to a flutter stimulus (which resulted in a stimulus amplitude of 120 \u03bcm), the two-point limen was tracked on the attended hand at 25 Hz flutter of varying amplitudes. Specifically, a separate series of sessions were conducted to track and compare the two-point limen for the amplitudes of 100, 150, and 200 \u03bcm in the flutter-only condition. The results were normalized to the distances observed under the 100 \u03bcm condition and were plotted in the same manner as the previous results , the two-point limen was not affected. Simultaneous stimulation of the hand contralateral to the attended site, however, impaired or reduced spatial acuity by 20% with a flutter stimulus and 30% with a vibratory stimulus.In the present study, we observed stimulus-dependent effects on two-point tracking of a flutter stimulus at the dorsal surface of the attended hand. The two-point limen was reduced  with a complex stimulus that consisted of 25 Hz flutter and 200 Hz vibration components. Specifically, it was found that adding vibration to the unilateral two-point flutter stimulus Vega-Bermudez and Johnson , using gSummers and Chanter  reportedTommerdahl et al.  comparedThe finding in previous OIS imaging experiments in cats that high-frequency skin stimulation is accompanied by a contralateral absorbance increase in area SII and, simultaneously, by a decline in absorbance in SI in the same hemisphere led Tommerdahl et al.  to consiA recent report described that the ability to localize a stimulus on the fingertips of one hand may be impaired with the interference of a similar stimulus on a fingertip of the opposite hand , suggestIn a previously published report, Vierck and Jones  found thWhen stimuli consisting of two points are oscillated on the skin at low-frequency 25 Hz flutter at distant sites, the peaks of SI response are distinct and non-overlapping Figure . Thus, tIn this paper, we propose a model that predicts a correlation between SI cortical activity and spatial acuity. Spatial acuity, as measured by the two-point limen, can be modified by changing stimulus conditions that would be predicted to have an impact on the SI cortical response. In particular, while vibration has the effect of reducing the spatial extent of SI cortical response normally evoked by flutter, such as when a vibrotactile stimulus comprised of both flutter and vibration is delivered to the skin, it also has the effect of improving a subject's ability to discriminate between two points on the skin. Presumably, this occurs as a result of vibration decreasing the spatial extent of the SI cortical response. Alternatively, stimulus conditions that are known to reduce the magnitude of the SI cortical response without changing the shape of response, such as when a second and simultaneous stimulus is delivered to a homotopic skin site on the opposite unattended hand, result in a reduction in spatial discrimination. While SI is regarded as playing a major role in two-point discrimination, this study provides evidence that other cortical areas that are connected to SI (such as SII) contribute importantly to SI's ability to differentially respond to closely spaced tactile stimuli.Five na\u00efve subjects (21\u201332 years in age) participated in this psychophysical study. All procedures were reviewed and approved in advance by an institutional review board.Sinusoidal vertical skin displacement stimuli were delivered using the Cantek Metatron CS-525 vertical displacement stimulator . The stimulator made contact with the skin via the two tips of the Two-Point Stimulator (TPS) attachment  fitted to the terminal end of the moving shaft of the stimulator transducer. The TPS is described in detail in a separate report . An adjuThe subject was seated in a chair with arms placed comfortably on a table surface. Both arms were placed on X-ray bags filled with glass beads. The investigators molded the bags to fit the contours of the subject's arms, and when the subject was comfortable and the arms positioned appropriately to allow unimpeded access of the stimulator to the center of the dorsal surfaces of each hand, the bags were made rigid by evacuating them of air (achieved by connecting the bag to a vacuum line). In this way the arms were maintained in a comfortable but stable position for the full duration of the experimental session. The subject was unable to see either the experimenter or the stimulator and stimulus-control instrumentation. White noise presented via headphones eliminated potential auditory cues. A micrometer permitted the stimulator transducers and probe assembly to be lowered towards the predefined skin sites. The micrometer position at which the digital display on the stimulator controllers registered a 0.1\u20130.2 g change in resistive force was interpreted as the point at which the stimulator probes made initial contact with the skin.A tracking protocol was used to conduct a two-point limen test, which determines the \"least two-point separation at which the subject feels (has the subjective impression of) two points,\"  at the dVT conducted the experiments, analyzed the data and drafted the manuscript. RD had a role in the conduct and design of the experiments. MT was involved with the design of the experiments and the preparation of the manuscript."}
{"text": "While SII cortex is considered to be the first cortical stage of the pathway that integrates tactile information arising from both sides of the body, SI cortex is generally not considered as a region in which neuronal response is modulated by simultaneous stimulation of bilateral (and mirror-image) skin sites.Optical intrinsic signal imaging was used to evaluate the response of SI and SII in the same hemisphere to 25 Hz sinusoidal vertical skin displacement stimulation (\"skin flutter\") applied contralaterally, ipsilaterally, and bilaterally (simultaneously) to the central pads of the forepaws. A localized increase in absorbance in both SI and SII occurred in response to both contralateral and bilateral flutter stimulation. Ipsilateral flutter stimulation evoked a localized increase in absorbance in SII, but little or no change in SI absorbance. In the forepaw representational region of SI, however, bilateral stimulation of the central pads evoked a response substantially smaller  than the response to flutter stimulation of the contralateral central pad.The finding that the response of SI cortex to bilateral central pad flutter stimulation is substantially smaller than the response evoked by a contralateral flutter stimulus, together with the recently published observation that a region located posteriorly in SII responds with a substantially larger response to a bilateral flutter stimulus than the response evoked from the contralateral central pad, lead us to propose that the SI activity evoked by contralateral skin stimulation is suppressed/inhibited  by the activity a simultaneous ipsilateral skin stimulus evokes in posterior SII. In contrast, SII has long been known to be activated at short latency by mechanical stimulation of skin sites on both sides of the body midline. Although this differential responsivity of SI and SII to stimulation of contralateral es; e.g. ), the coes; e.g. ,3.Recently, we reported the results from experiments in which we obtained simultaneous observations of the activity evoked in both SI and SII in the same hemisphere of cat cerebral cortex by a 25 Hz sinusoidal vertical skin displacement stimulus (\"skin flutter\") applied contralaterally, ipsilaterally, or bilaterally to the central pads of the forepaws . Brieflyvs. posterior components that comprise SII cortex in the same hemisphere.This report addresses the response of SI cortex to the same modes of stimulation used in the above-described study that focused on SII . The central finding is that flutter stimulation of the ipsilateral central pad exerts a suppressive/inhibitory influence on the SI response to a 25 Hz flutter stimulus to the contralateral central pad (forepaw). In addition, the temporal relationship between stimulus-evoked activity in selected locations in the responding regions of SI and SII is evaluated quantitatively (using the approach of correlation mapping), revealing a previously unrecognized, and presumably functionally important high degree of coordination between the activities evoked by forepaw stimulation in both SI and the recently identified  anteriorFigure vs. bilateral stimulation at 5 seconds after stimulus onset . The top left panel indicates the orientation of the sampled region of interest (ROI) in SI. The surface plots show the absorbance value at each pixel location in the imaged field, with absorbance defined both by elevation along the z-axis and by pseudocolor . Consistent with the difference images shown in Figure The SI response to each of the different stimulus conditions can be better appreciated when the imaging data are displayed as a multi-dimensional surface plot. Figure vs. contralateral stimulation appears to increase with increasing time after onset of stimulation.Figure vs. bilateral stimulus conditions. Additionally, the plots suggest that there is a time dependency in the development of the response to bilateral stimulation \u2013 that is, there is little difference between the responses to the two conditions at 1 sec after stimulus onset , yet at a later time (at t = 5 in each subject many of the pixels are shifted to a position below the reference line). Points located below (or to the right of) the reference line (slope = 1) represent pixels, or spatial locations in SI, where the response to contralateral stimulation is greater than the response to bilateral stimulation. It should be emphasized that this type of graphic does not reflect spatial differences in the responses to the different stimulus conditions, but rather, emphasizes whether or not different members of a set respond differently to the different stimulus conditions. Accordingly, the plots in Figure Scatter plots were used to directly compare the response of SI to the different conditions of contralateral and bilateral stimulation. In each plot in Figure o was that the ratio = 1).The across-subject consistency of the results was evaluated by determining the average absorbance values evoked by the 3 different stimulus conditions (with a 5 sec stimulus duration) for the 5 subjects at 5 sec after stimulus onset . In this method, rather than correlating each pixel with a standard time course observed at one particular cortical region for the same stimulus condition, performs a cross correlates the time courses of absorbance values obtained at the same spatial location under different stimulus conditions. Thus, the correlation coefficient generated at each pixel location represents how similar (indicated by a positive correlation) or how different (indicated by a negative correlation) are the temporal responses of a cortical region recorded under 2 different stimulus conditions. The map in Figure A second method of correlation mapping was used to directly compare the responses evoked by contralateral The findings of this study demonstrate that bilateral flutter stimulation of the central pads of the forepaws evokes an SI response significantly smaller than the response evoked when the stimulus is applied contralaterally. At the locus of the maximal OIS response evoked in the SI region by a contralateral stimulus, bilateral stimulation evoked a response that was, on average, 35% smaller than that evoked by the contralateral stimulus. Concurrently, at the locus of the maximal OIS response evoked by contralateral stimulation in anterior SII , bilateAlthough the neural mechanisms responsible for the above-described effect remains uncertain, some workers have reported that callosally-transmitted inputs exert modulatory, but inconsistent effects on SI neurons. Schnitzler et al , using MAlthough an influence mediated via direct callosal projections from ipsilateral SI to contralateral SI  should nAn extensive literature addresses the neuroanatomical routes by which information about the status of skin mechanoreceptors accesses SI and SII  is negatively correlated with the activity in SI. Additionally, correlation mapping of the results obtained from the contralateral vs. bilateral stimulus conditions demonstrated that the time course of the activities evoked in SI and anterior SII by these conditions are similar. The data lead to the proposal that increasing levels of activity in posterior SII evoked by an ipsilateral skin stimulus suppress/inhibit the responses normally evoked in both anterior SII and SI by contralateral stimulation.Adult cats  were subjects. All surgical procedures were carried out under deep general anesthesia . After induction of general anesthesia the trachea was intubated with a soft tube and a polyethylene cannula was inserted in the femoral vein to allow administration of drugs and fluids (5% dextrose and 0.9% NaCl). For each subject, a 1.5 cm diameter opening was made in the skull overlying somatosensory cortex, a chamber was mounted to the skull over the opening with dental acrylic, and the dura overlying anterior parietal cortex was incised and removed. Following the completion of the surgical procedures all wound margins were infiltrated with long-lasting local anesthetic, the skin and muscle incisions were closed with sutures, and each surgical site outside the recording chamber was covered with a bandage held in place by adhesive tape.Subjects were immobilized with Norcuron and ventilated with a gas mixture  delivered via a positive pressure respirator 1\u20133 hours prior to the data acquisition phase of the OIS imaging experiments. Respirator rate and volume were adjusted to maintain end-tidal CO2 between 3.0 \u2013 4.0%; EEG and autonomic signs  were monitored and titrated (by adjustments in the anesthetic gas mixture) to maintain levels consistent with light general anesthesia. Rectal temperature was maintained (using a heating pad) at 37.5\u00b0C.Euthanasia was achieved by intravenous injection of pentobarbital (45 mg/kg) and by intracardial perfusion with saline followed by fixative . Following perfusion fiducial marks were placed to guide removal, blocking, and subsequent histological sectioning of the cortical region studied. All procedures were reviewed and approved in advance by an institutional committee and are in full compliance with current NIH policy on animal welfare.Results were obtained during stimulation of the contralateral central pad of the forepaw and/or the ipsilateral central pad of the forepaw. The stimuli always consisted of sinusoidal vertical skin displacements  and were applied using a pair of servocontrolled transducers  that is capable of delivering sinusoidal stimuli in the range of 1\u2013250 Hz at amplitudes in the range of 0\u20131000 microns. The stimuli were delivered independently to the ipsilateral and contralateral skin sites, and also were applied simultaneously to both sites . The stimulus probes were positioned 500 microns beyond the point at which skin contact was detected (via force transducer on the Cantek). The bilateral stimulus protocols reported in this paper were synchronized to start and stop at the same time. The contralateral, ipsilateral and bilateral stimuli were interleaved on a trial-by-trial basis. This approach was used to control for temporal changes in cortical \"state\" unrelated to stimulus conditions which, if unrecognized, might obscure or modify any differences between the optical responses evoked by the contralateral, ipsilateral and bilateral stimulus conditions.Near-infrared  OIS imaging was carried out using an oil-filled chamber capped with an optical window  Images oij between the absorbance value of each pixel  and the average absorbance value within the reference region over the time from stimulus onset to stimulus offset. The region selected as the reference was defined by a boxel (\u03c0 mm2 area) centered on the region of interest (ROI). Each pixel  on the correlation map is represented by a coefficient of determination r2ij  for different stimulus conditions. Thus, each coefficient of determination r2ij represents how similar (positive correlation) or how different (negative correlation) the response in a region is to a change in stimulus condition.Correlation maps were constructed for comparison of spatio-temporal characteristics of the OIS response. One aspect of this method of analysis has been previously described in detail ,29. BrieAt the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 \u03bcm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 mm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries  identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria -38.BW and OF participated in the design of the experiments, the data collection, and drafting of the manuscript. SS and JC made significant contributions to the data collection and the analysis of the data. MT played a major role in all aspects of the development of the manuscript."}
{"text": "It is established that increasing the amplitude of a flutter stimulus increases its perceived intensity. Although many studies have examined this phenomenon with regard to the responding afferent population, the way in which the intensity of a stimulus is coded in primary somatosensory cortex (SI) remains unclear.Optical intrinsic signal (OIS) imaging was used to study the evoked responses in SI of anesthetized squirrel monkeys by 25 Hz sinusoidal vertical skin displacement stimulation. Stimuli were 10 sec duration with a 50 sec inter-stimulus interval. Stimulus amplitude ranged from 50 to 400 microns and different amplitudes were interleaved. Control levels of activity were measured in the absence of stimulation, and used to compare with activation levels evoked by the different stimulus amplitudes. Stimulation of a discrete skin site on the forelimb evoked a prominent increase in absorbance within the forelimb representational region in cytoarchitectonic areas 3b and 1 of the contralateral hemisphere. An increase in stimulus amplitude led to a proportional increase in the magnitude of the absorbance increase in this region of areas 3b and 1 while surrounding cortex underwent a decrease in absorbance. Correlation maps revealed that as stimulus amplitude is increased, the spatial extent of the activated region in SI remains relatively constant, and the activity within this region increases progressively. Additionally, as stimulus amplitude is increased to suprathreshold levels, activity in the surround of the activated SI territory decreases, suggesting an increase in inhibition of neuronal activity within these regions.Increasing the amplitude of a flutter stimulus leads to a proportional increase in absorbance within the forelimb representational region of SI. This most likely reflects an increase in the firing rate of neurons in this region of SI. The relatively constant spatial extent of this stimulus-evoked increase in absorbance suggests that an increase in the amplitude of a 25 Hz skin stimulus does not evoke a larger area of SI neuronal activation due to an amplitude-dependent lateral inhibitory effect that spatially funnels the responding SI neuronal population. The way in which stimulus intensity is represented in primary somatosensory (SI) cortex has remained an intriguing question in the study of the cortical correlates of perception. Although there have been numerous studies of the SI response to changes in stimulus intensity, few have focused on the response at the population level of analysis. Thus, a dearth of information about the global SI response to changes in stimulus intensity exists \u2013 current knowledge of the subject depends almost exclusively on reconstruction of predictions of the SI neuronal population response from afferent recordings -5 and si2 [A number of studies have examined the global SI response using imaging techniques such as fMRI  -10 and M2 ,11.Recently, Chen and colleagues used the optical intrinsic signal (OIS) to demonstrate that a proportionally greater (larger magnitude) response is evoked in SI of squirrel monkeys as the amplitude (as measured by force) of a skin stimulus is increased. HoweverFigure To identify the boundaries of the regions of increased absorbance spatial histograms were constructed. Figure Figure evoked was used. \u0394Absorbanceevoked was defined as the difference between the absorbance measured at 1 sec (prior to stimulus onset) and 11 sec (point of stimulus offset), and is shown in the plot at the bottom of Figure evoked vs. amplitude is well described (coefficient of determination R2 = 0.9921) by the linear function (solid line) \u0394Absorbanceevoked = (4 \u00d7 10-6)*d + 0.0005. This type of analysis, however, gives little or no information about the spatial properties of the response.The analysis approach illustrated in Figure Radial histograms were constructed to better visualize the spatiotemporal relationship of the OIS response at different amplitudes. The radial histograms shown in Figure max was defined as the difference between the minimum absorbance and the maximum absorbance value obtained at any point during the recording. Interestingly, the relationship between stimulus amplitude and \u0394Absorbancemax in the surround is not linear  to the location of the pixel being compared. This gives a fairly good approximation of the signal at all locations in the image. Since there is no significant difference in the spatial properties between stimuli at intermediate amplitudes  only the 50 and 400 \u03bcm amplitudes will be compared with this technique. Figure Correlation maps were constructed to further characterize the spatial properties of the SI response to 25 Hz flutter. A correlation map compares every pixel in the image with the signal referenced from the ROI, and assigns a correlation coefficient (2 (4 \u00d7 4) area centered around the ROI. Figure To examine the spatial dynamics of the SI response in more detail we examined the patterns of activity generated by low- and high-amplitude stimulation in a 16 mmThis study evaluated the global SI response to different amplitudes of flutter stimulation by imaging the optical intrinsic signal (OIS). The OIS indirectly reflects both cortical neuronal spike discharge activity and the local, subthreshold changes in neuronal membrane potential evoked by sensory stimulation -16. As a+ and neurotransmitter [One important distinction between previous work done using the OIS and the present study is our use of near-infrared wavelengths for illumination during acquisition of the OIS. The OIS obtained using infrared light has been shown to be highly correlated with light scattering effects that accompany astrocyte swelling subsequent to the clearance of extracellular Knsmitter ,15,18 annsmitter ,20. Althnsmitter  but the nsmitter ,15,19.Previous studies conducted by this laboratory have reported that the SI optical response evoked by an extended period (>1 sec) of flutter stimulation not only consists of an increase in absorbance in the region that receives its input from the skin site that was stimulated, but also decreases in absorbance (frequently to levels well below-background) that occur in the surrounding cortex . The preThe correlation maps shown in figure A receptor mediated inhibition (barbiturates), significantly reduce the dimensions of the receptive field of individual SI neurons; actions that would reduce the size of the responding SI neuronal population [Examination of spatial histograms figure  and the pulation . Chen etpulation .It has been suggested that the amplitude of skin flutter stimulation is coded by both the number of activated SI neurons as well as by their level of spike discharge activity . This suThis study investigated the SI response to flutter stimulation of the skin using the OIS. An increase of the amplitude of the flutter stimulus was associated with an increase in absorbance within the region of SI cortex that receives its input from the stimulated skin field. The relationship between the maximal change in absorbance and stimulus amplitude is well characterized by a linear function within the range of amplitudes studied. Measurement of the spatial extent of the activated SI region showed that higher amplitudes of stimulation do not produce a more extensive region of SI activation. Instead, as amplitude is increased, while average peak absorbance within the same ~2 mm diameter SI region increases with amplitude of stimulation, the region of surrounding cortex undergoes a prominent decrease (frequently to levels well below background) in absorbance. Further studies are required to establish the relationship between the effect of different amplitudes of skin flutter stimulation on SI absorbance and SI neuroelectrical activity.2O) and oxygen, the trachea was intubated. A veterinary anesthesia machine (Forreger Compac-75) provided an anesthetic gas mix whose composition could be adjusted  to maintain a stable level of surgical anesthesia. Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to lessen the probability of halothane-induced cerebral edema and prevent bacterial septicemia, respectively. Placement of a valved catheter into a superficial hindlimb vein enabled administration of 5%glucose, 0.9%NaCl, and drugs.All methods and procedures are consistent with USPHS policies and guidelines on animal care and welfare in biomedical research. They were reviewed and approved by an institutional committee prior to initiation of the experiments. Experiments were conducted in 10 squirrel monkeys. Following induction of anesthesia with 4% halothane in a 50/50 mix of nitrous oxide  to maintain heart rate, blood pressure, and the EEG at values consistent with general anesthesia. Rate and depth of ventilation were adjusted to maintain end-tidal CO2 between 3.0 and 4.5%. Under these experimental/anesthetic conditions both SI neuron spontaneous and stimulus-evoked spike discharge activity patterns are highly reproducible over even prolonged (>1 hr) time periods.A 1.5 cm opening was trephined in the skull overlying SI cortex. A recording chamber (25 mm i.d.) was placed over the opening and cemented to the skull with dental acrylic. Wound margins were infiltrated with local anesthetic, closed with sutures and bandaged, and the dura overlying SI was resected. After the completion of all surgical procedures subjects were immobilized with norcuron . From this point on, the animal was ventilated with a 50/50 mix of NAfter obtaining a photograph of the exposed cortical surface, the recording chamber was filled with artificial cerebrospinal fluid, and hydraulically sealed using a clear glass plate. In each of five experiments, the OIS evoked in SI by cutaneous flutter stimuli on the thenar region of the forelimb was recorded. The flutter stimulus was delivered at 50, 100, 200 and 400 \u03bcm and was interleaved in order to prevent conditioning of the response. The imaging system consisted of a computer-interfaced CCD camera (Quantix 540 from Roper Scentific), light source, guide and filters required for near-infrared (833 nm) illumination of the cortical surface, a focusing device, and a recording chamber capped by an optical window . Images of the exposed anterior parietal and surrounding cortical surface were acquired 200 ms before stimulus onset (\"reference images\") and continuously thereafter for 22 s after stimulus onset (\"post-stimulus images\") at a rate of one image every 0.9\u20131.4 s. Exposure time was 200 ms. Difference images were generated by subtracting each pre-stimulus image from its corresponding post-stimulus image. Averaged difference images typically show regions of both increased light absorption (decreased reflectance) and decreased light absorption (increased reflectance) which are believed widely  to be accompanied by increases and decreases in neuronal activation, respectively.Although onset of the OIS is delayed at longer wavelengths ,20, use ij between the reflectance value of each pixel  and the average reflectance value within the reference region over the time from stimulus onset to stimulus offset. The region selected as the reference was defined by a boxel (\u03c0 mm2 area) centered on the region of interest (ROI). Each pixel  on the correlation map is represented by a correlation coefficient ij r identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria -31.SS participated in acquisition of optical data, performed analysis of the data and drafted the manuscript. VT and JC assisted in the data collection and the analysis of the optical imaging data. OF and BW participated in the design of the study, the conduct of the experiments and the drafting of the manuscript. MT participated in the design, conduct, and analysis of the experiments, and in the manuscript preparation."}
{"text": "Recently we reported that vibrotactile flutter stimulation of a skin locus at different amplitudes evokes an optical response confined to the same local region of the primary somatosensory cortex (SI), where its overall magnitude varies proportionally to the flutter amplitude. In this report, we characterize the impact of the flutter amplitude on the spatial patterns of activity evoked within the responding SI region.In order to characterize the spatial pattern of activity within the responding SI region, images of the flutter-evoked SI optical response were segmented and analyzed with spatial frequency analysis. The analysis revealed that: (1) dominant spatial frequencies in the optical intrinsic signal emerge within the responding SI region within 3\u20135 sec of stimulus onset; (2) the stimulus-evoked activity is spatially organized in a form of several roughly parallel, anterior-posteriorly extended waves, spaced 0.4\u20130.5 mm apart; (3) the waves themselves exhibit spatial periodicities along their long axis; and (4) depending on the flutter stimulus amplitude, these periodicities can range from fine 0.15 mm \"ripples\" at 50 \u03bcm amplitude to well-developed 0.5 mm fluctuations at the amplitude of 400 \u03bcm.The observed spatiointensive fractionation on a sub-macrocolumnar scale of the SI response to skin stimulation might be the product of local competitive interactions within the stimulus-activated SI region and may be a feature that could yield novel insights into the functional interactions that take place in SI cortex. In this map, a skin locus provides afferent input to an extensive cortical region in SI ,2. In pacortex \u2013 ,4). The cortex \u2013 . These ccortex \u2013 -10.2 of cortical territory in area 3b of SI [Such spatially extensive cortical regions are not functionally homogeneous. For example, using Optical Intrinsic Signal (OIS) imaging in near-infrared 830 nm) range, we find that in squirrel monkeys a small-diameter stimulus probe oscillating on the skin at 25 Hz activates more than 3 mm3b of SI ,8,11. Su30 nm ran3b of SI ) organiz3b of SI . Chen et3b of SI  reported3b of SI ,14-18.Together these considerations suggest that the spatial pattern of activity evoked in SI by even the smallest stimuli might be structurally more complex than a typically envisioned basic bell-shaped pattern. A closer inspection of such patterns might reveal certain spatial formations within them with significant functional implications. Recently, we investigated the response of SI cortex to varying amplitudes of flutter stimulation. Regardless of the amplitude of stimulation (in the range of 50 to 400 \u03bcm), we found that the spatial extent of the response of SI cortex remained the same . InsteadFigure Comparable results were obtained from the 4 other subjects,  of mechanical skin stimulation. Similarly, Bruno and colleagues recently demonstrated that individual barrels in rat SI contain minicolumns of neurons preferring the same whisker deflection angle and that these angular tuning domains could be the result of convergent inputs from thalamocortical cells with corresponding angular preferences . It is pFinally, considering that spatial periodicities in SI OIS response patterns emerge gradually, and evolve with time after the stimulus onset, the fine sculpting of the SI response might be the result of a network-level neurocomputational process that involves competitive and cooperative interactions among local neuronal aggregates. That is, SI sensitivity to the stimulus attribute(s) responsible for the observed fractionation of activity within the responding SI region might be an emergent property of the SI network , rather than a simple outcome of selective convergence of thalamocortical afferents on SI neurons.Observations of the spatial patterns of SI cortical response within an activated region, such as those evoked by flutter stimulation of the skin, suggest that evoked cortical activity within such a territory is not evenly distributed. Furthermore, the cortical activity patterns change in a manner that appears to be dependent upon stimulus conditions. The observed spatiointensive fractionation on a sub-macrocolumnar scale of the SI response to skin stimulation might be the product of local competitive interactions within the stimulus-activated SI region, and as such can lead to new insights about the functional interactions that take place in the SI cortex.All methods and procedures are consistent with USPHS policies and guidelines on animal care and welfare in biomedical research, and were reviewed and approved in advance by an institutional animal use committee (IACUC). Experiments were conducted in 5 squirrel monkeys. Surgical procedures were carried out under deep general anesthesia . After induction of anesthesia the trachea was intubated to facilitate positive pressure ventilation and delivery of the gaseous general anesthetic. A catheter was inserted into a branch of the femoral vein of the hindlimb ipsilateral to the hemisphere to be imaged, allowing intravenous (IV) administration of drugs and fluids (5% dextrose and 0.9% NaCl). Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to lessen the probability of halothane-induced cerebral edema and prevent bacterial septicemia, respectively.A 1.5 cm diameter opening in the skull exposed the forelimb region of SI cortex. A recording chamber was positioned over the opening and cemented to the skull with dental acrylic. The chamber was filled with artificial cerebrospinal fluid, the dura mater overlying SI cortex incised and removed, and all wound margins outside the chamber dressed with long-lasting local anesthetic in oil (Cetacaine). All skin and muscle incisions were closed with sutures and bandaged.After the completion of all surgical procedures subjects were immobilized with Norcuron  and ventilated with a gas mixture . At these concentrations and under normocapnic conditions, halothane has no effect on brain energy metabolism ,37, and The recording chamber was filled with artificial cerebrospinal fluid and hydraulically sealed using a clear glass plate. Vibrotactile stimuli were delivered to selected loci on the hand using a servocontrolled vibrotactile stimulator , capableThe optical imaging system consisted of a computer-interfaced CCD camera, light source, guide and filters required for near-infrared (830 nm) illumination of the cortical surface, a focusing device, and a recording chamber capped by an optical window . ImaCortical images were taken at light/time exposures that place the region of interest in the middle of the range of the recorded pixel values. Histogram analysis was used during experimental setup to make sure to avoid any nonlinearities that may arise from overexposure. In some of the experiments the camera was rotated by 90\u00b0 relative to the SI orientation to better capture the responding cortical field. These rotations did not have any noticeable effect on spectral power distributions along the anterior-posterior and medial-lateral cortical dimensions.vs. distance plot . Power spectra of the spatial histograms were then computed using Discrete Fourier Transform (DFT) algorithm and plotted as a periodogram. Fourier analysis was always performed on raw, unfiltered images.The spatial organization of the stimulus-related light absorbance changes in SI was evaluated using linear image segmentation. This involved segmentation of the relevant region of the image into a linear series of bins and computation of the average absorbance value of the pixels in each bin. The sequence of average absorbance values obtained in this way was plotted as a function of distance (mm) along the cortical path traced by the central points of the series of bins \u2013 yielding an absorbance At the end of the experiment the subject was euthanized by overdose of pentobarbital (50 mg/kg/IV), followed by intracardial perfusion with saline and 10% formalin.JC participated in acquisition of optical data, performed analysis of the data and drafted the manuscript. OF and BW participated in the design of the study, the conduct of the experiments and the drafting of the manuscript. MT participated in the design, conduct, and analysis of the experiments, and in the manuscript preparation."}
{"text": "To compare the diagnostic accuracy of two continuous screening tests, a common approach is to test the difference between the areas under the receiver operating characteristic (ROC) curves. After study participants are screened with both screening tests, the disease status is determined as accurately as possible, either by an invasive, sensitive and specific secondary test, or by a less invasive, but less sensitive approach. For most participants, disease status is approximated through the less sensitive approach. The invasive test must be limited to the fraction of the participants whose results on either or both screening tests exceed a threshold of suspicion, or who develop signs and symptoms of the disease after the initial screening tests.paired screening trial bias. This bias reflects the synergistic effects of inappropriate reference standard bias, differential verification bias, and partial verification bias. The absence of a gold reference standard leads to inappropriate reference standard bias. When different reference standards are used to ascertain disease status, it creates differential verification bias. When only suspicious screening test scores trigger a sensitive and specific secondary test, the result is a form of partial verification bias.The limitations of this study design lead to a bias in the ROC curves we call paired screening trial bias on a paired comparison of area under the curves. We fix the prevalence of disease, and the chance a diseased subject manifests signs and symptoms. We derive the formulas for true sensitivity and specificity, and those for the sensitivity and specificity observed by the study investigator.For paired screening tests with bivariate normally distributed scores, we give formulae and programs to quantify the effect of The observed area under the ROC curves is quite different from the true area under the ROC curves. The typical direction of the bias is a strong inflation in sensitivity, paired with a concomitant slight deflation of specificity.In paired trials of screening tests, when area under the ROC curve is used as the metric, bias may lead researchers to make the wrong decision as to which screening test is better. Paired trials designed to compare the diagnostic accuracy of screening tests using area under the receiver operating characteristic (ROC) curve may fall victim to a strong bias that renders the conclusions of the trial incorrect. In English, \"bias\" often has a pejorative connotation, implying that those who conduct the study prefer one scientific conclusion, rather than another. We use the term \"bias\" in the epidemiological and statistical sense, as the difference between the results obtained in a study, and the true results.The bias occurs because limitations in the trial design may differentially affect the area under the ROC curve for each screening test. Many competing statistical approaches have been suggested for comparing the diagnostic accuracy of two continuous tests. We consider area under the ROC curve, because it continues to be used as the standard in prominent medical journals -3.true disease status is not known correctly for all participants, the observed disease status is used for calculations of diagnostic accuracy.A common design for the comparison of two continuous screening tests is to evaluate participants with both screening tests. The disease status is then determined by either an invasive secondary test, or by a less invasive, but less sensitive approach. Ethically and practically, the invasive secondary test must be reserved only for those participants who have a suspicious result on one or both screening tests, or for those who have signs and symptoms of disease. For those who have a normal result on both screening tests, a less sensitive process is used to approximate the disease status. As the For potentially lethal diseases like cancer, where the invasive test is biopsy, this design is the best possible available design. The imperfections of the study design occur because the disease is difficult to diagnose since it is clinically occult, and the study designers must keep the risk of potential harm to subjects as low as possible.paired screening trial bias. This bias results from the synergistic effects of inappropriate reference standard bias, differential verification bias, and partial verification bias , standard deviations both 1 and correlation \u03c1, evaluated at the points x and y. That is, if X and Y have a bivariate normal distribution, we write \u03a6 to indicate Pr (X \u2264 x and Y \u2264 y|\u03c1)The prevalence of disease in the population is \u03b8, the threshold value for referral to the invasive, yet sensitive and specific secondary test. Thus, for each value of x, we can describe a series of events cross-classified by the Test 1 score, the Test 2 score, the true disease status of the subject and the presence of signs or symptoms. We classify each event both as it truly occurs, and how it is observed by the study investigator. There are 22 possible situations when x <\u03b8 , and the truth (the presence or absence of disease). The cell and marginal probabilities for this cross-classification are shown in Tables For each screening test and each value of the test cutpoint true sensitivity for each test as the number of true positives identified by that screening test divided by the total number of true cases. The true specificity for each test is the number of true negatives correctly identified as negative by that screening test divided by the total number of true non-cases. The true ROC curve is generated by plotting the true sensitivity on the vertical axis versus one minus the true specificity on the horizontal axis.We then calculate the observed sensitivity and observed specificity. In order to generate the observed ROC curves, for each test and each value of the cutpoint x, we define a table that cross classifies the response of the test (positive or negative), and the observed disease status (the presence or absence of observed disease). The cell and marginal probabilities for this cross-classification are shown in Tables observed sensitivity and the observed specificity. The observed sensitivity is the fraction of participants observed to have disease who have a positive screening test result. The observed specificity is the fraction of participants who apparently have no disease who have a negative screening test result. Some participants actually may have disease, but the disease is not detected in the trial.We use a similar technique to calculate the observed ROC curve is generated by plotting the observed sensitivity on the vertical axis versus one minus the observed specificity on the horizontal axis. Simpson's rule numerical integration methods We calculate the theoretically correct ROC curves and AUCs (ignoring the error of integration), using our mathematical derivations. In a real trial, the study investigator would use a hypothesis test and a p-value to compare the difference in AUCs. Depending on the sample size chosen for the trial, the precision of the estimates and the accuracy of the decision may change.true state of disease, and the observed state of disease, we record the magnitude of the differences in AUCs, and the decision whether to reject the null. The proportion of rejections for the true and observed data is estimated by the number of rejections, divided by 10,000. Ten thousand is chosen so the maximum half width for the confidence interval for the proportion rejected is no more than 0.01.To illustrate the effect of the bias, we present the theoretical results. To illustrate the effect of sample size on the precision of the estimates, we conduct a simulation. For the simulation, we suppose that the study investigator decided to test the null hypothesis of no difference between the areas under the ROC curves, using a non-parametric AUC test for paired data , and fixobserved ROC curve differs from the true ROC curve, with the amount of bias depending on the correlation between the screening tests for participants with disease, \u03c1C, the rate of signs and symptoms, \u03c8, and the threshold for recall, \u03b8. In some cases, the bias equally affects the observed ROC curve for both screening tests, and the scientific conclusion is the same as it would have been had the true disease state been observed. In other cases, the bias causes a change in the direction of the scientific conclusion. The scientific conclusion only changes direction when for one screening test, for participants with disease, a higher proportion of the scores lead to recall than for the other screening test. Thus, for that screening test, a larger percent of participants with true disease go on to have their disease status correctly ascertained, and observed in the study, than for the other screening test.Our derivations demonstrate that the Figure observed curves have inflection points, where the slope changes. There is no inflection point in the true ROC curves for either test, because the formulae that govern the sensitivity and specificity for the true curves are the same no matter what the ROC cutoff points are . However, in Figure observed difference in AUC between Test 2 and Test 1 is negative (Test 2 observed AUC \u2013 Test 1 observed AUC = 0.75 \u2013 0.82 = -0.07). If the study investigator were to observe these exact theoretical results, the study investigator would conclude that screening Test 1 has better diagnostic accuracy than Test 2, when in fact the opposite is true.Since Test 2 truly has better diagnostic accuracy than Test 1, the true and observed disease status, we conducted a simulation. For the parameters of Figure Study investigators never observe the true state of nature. They observe data, and make estimates, the precision of which depends on the sample size. They decide which screening test is better using hypothesis tests. To see which conclusion the hypothesis tests would suggest, both for the observed state of disease. In that case, the study investigator will reject the null hypothesis only 71% of the time. The remaining 29% of the time, the study investigator will conclude that there is no difference in AUC between Test 1 and Test 2. The lower power is due to more variance in the observed data, compared to the true data. When the study investigator rejects the null, every time, she concludes incorrectly that Test 1 is better than Test 2.If we conduct the same simulation experiment from the point of view of the study investigator, for the experimental situation of Figure observed sensitivity for Test 1 is inflated more than the observed sensitivity for Test 2. The increase in observed sensitivity makes the observed ROC curve higher for Test 1 than for Test 2. A higher observed ROC curve means a higher observed AUC for Test 1 than for Test 2.The incorrect conclusion in Figure true and observed ROC curves shown in Figure observed sensitivity relative to true sensitivity, for Test 1. This occurs when specificity is 0.82. For a hypothetical study of 10,000 participants, and specificity of 0.82, the observed and true 2 \u00d7 2 tables for Test 1 and Test 2 are shown in Figure To understand how and why paired screening trial bias occurs, consider a single specificity value on the observed table, Test 1 is positive if it exceeds 2.511; for the true table, Test 1 is positive if it exceeds 2.515. For the observed table, Test 2 is positive if it exceeds 1.269; for the true table, Test 2 is positive if it exceeds 1.265. The tables have different ROC cutpoints because they were chosen to have the same specificity, not the same cutpoint.Each one of the four tables uses a slightly different ROC cutpoint. For the observed number of cases of disease is smaller than the true number because not every participant undergoes the invasive, yet sensitive and specific secondary test, and thus some cases of disease are missed. The observed number of cases of disease is the denominator of the observed sensitivity. Because the denominator is smaller for observed sensitivity than for true sensitivity, the observed sensitivity is strongly inflated for both tests. When specificity is 0.82, the observed sensitivity of Test 1 is 0.72, with true sensitivity of 0.33. For Test 2, the observed sensitivity is 0.52, with true sensitivity of 0.43.Also, the number of cases of disease observed in the study, 45, is much smaller than the true number of cases of disease in the population, 100. The Yet if the bias only affected the denominator, the inflation in sensitivity would be the same for both tests. After all, the same number of observed cases is used as the denominator for both tests. The differential inflation for Test 1 compared to that for Test 2 must be due to the numerator of the observed sensitivity.For Test 2, the numerator of the observed sensitivity is the number of study participants who are positive on Test 2, and who are observed to have disease in the study. For Test 2, the numerator for observed sensitivity, 23, is smaller than the true numerator, 43. The difference occurs because disease can only be observed if the invasive, yet sensitive and specific secondary test is used. Even though the participants have a score that exceeds the ROC cutpoint for Test 2, they do not all undergo the invasive, yet sensitive and specific secondary test. Thus, they do not yield observed cases of disease. By contrast, for Test 1, because the ROC cutpoint is higher than the threshold which leads to the invasive, yet sensitive and specific secondary test, every participant positive on Test 1 undergoes the secondary test, and is shown to have disease. For each test, there is a different proportion of participants who exceed the cutpoint, who truly have disease, and who proceed to secondary testing. This is the source of the differential bias that causes the curves to reverse order in Figure Paired screening trial bias also increases as the proportion of participants with disease who have signs and symptoms (\u03c8) decreases. If all the cases of the disease were observed during the trial, there would be no difference between true and observed disease status, and no bias. Yet, in every screening trial, some cases of diseases are not identified by either screening test, and never present with signs and symptoms. As the proportion of participants presenting with signs and symptoms (\u03c8) decreases, fewer cases of disease are discovered during the trial in the interval after screening, and the difference between observed and true disease status grows.Paired screening trial bias increases with the increase in correlation between the results of the screening tests for participants with disease, \u03c1C. The bias in the observed ROC curves increases because as the two index tests become more highly correlated, the number of observed cases of disease becomes smaller relative to the number of true cases of disease. When the two index tests are highly correlated, they essentially produce the same information as to whether a participant has disease. When the index tests are independent, each test makes diagnoses on its own that the other test misses. Thus, when the tests are independent, and \u03c1C is 0, the number of observed cases is highest, relative to the number of true cases. The percentage of participants receiving the infallible secondary test increases as \u03c1C decreases. The bias lessens as the true disease status is ascertained for more participants.paired screening trial bias tends to strongly increase the sensitivity, while slightly decreasing the estimate of specificity. The increase in observed sensitivity compared to true sensitivity is expected with verification bias [In general, ion bias .In this paper, we define a new type of bias that is a result of the interaction between a particular design for a paired screening trial, and the choice of a particular statistical test. Specifically, the bias occurs when the diagnostic accuracy of two continuous tests are compared using area under the ROC curve in a design with two limitations. First, different methods are used to ascertain disease status, depending on the results of the initial screening tests. Secondly, only some subjects undergo an invasive, yet sensitive and specific secondary test. Thus, some cases of disease are missed because the method used to ascertain disease status for those who test negative on both initial screening tests may not be 100% sensitive.Both the statistical test and the trial design we considered were modeled closely after recently completed and published trials -3. TheseAlthough we modeled our trial design on real trials, we made simplifying assumptions, which may not accurately reflect reality. We assumed that there was a method for determining disease status which was infallible. In reality, all methods of determining disease status may be fallible. In breast cancer, for example, diagnostic mammography, biopsy and follow-up all make errors. Too short a follow-up time may miss cases of disease. While longer follow-up time will reveal a larger fraction of occult disease, it may also reveal increasing numbers of cases of disease that developed after the initial screening period, thus confusing the results. We assumed that all cases of disease are harmful. In screening studies, cases of disease may resolve, or proceed so slowly as to be considered harmless.We assumed that a test to determine disease status would be conducted any time a screening test result exceeded a given threshold. However, in cancer screening, because other factors may be taken into consideration when deciding a course of clinical action, there is a range of scores that may result in further testing.We also made the simplifying assumption that the scores of the screening tests followed a bivariate normal distribution. In real paired cancer trials, the scores have a conditional probability structure driven by the fact that real observers miss cancers (and score a screening test as if no disease were present), and see cancer where there is none . The resulting distribution of scores is far from the bivariate normal distribution we assumed.There is some theoretical justification that our results will still hold even if the data are non-normal. Hanley  points opaired screening trial bias.The previous literature on bias provides some hint of the plethora of possible designs and tests used for statistical analysis. Most previous statistical literature dealt with biases that occur for single, as opposed to paired, tests. A complete summary of biases is given in . Extremepaired screening trial bias, because the secondary test is typically biopsy. Negative screening results cannot lead to biopsy because there is no visible lesion to be biopsied. Because biopsy is painful and invasive, it is infeasible and unethical to do a biopsy unless there are suspicious screening test results. Also, one can only biopsy what one can see: one cannot put a needle in an invisible lesion. Negative screening test results are verified, but typically by follow-up, which has lower sensitivity than biopsy.Cancer screening trials in particular are susceptible to et al., [Our research suggests that in many published paired screening trials, bias did not affect the scientific conclusion. For example, in Pisano et al., , digitalWhy criticize a trial design, that though imperfect, cannot be improved, because of ethical constraints? It is our philosophy that it is preferable to understand all the causes of bias. With mathematical formulae for bias, we can defend trials that are fundamentally correct, and reserve doubt for those trials that may be subject to incorrect conclusions. In addition, models for bias are the necessary first step toward mathematical corrections for bias in sensitivity and specificity, and toward designing new clinical trial methodologies.paired screening trial bias has the potential to subvert the results of paired screening trials, especially when the fraction of the population recalled for secondary testing differs for each screening test. The bias is affected by the rate at which diseased participants experience signs and symptoms of disease, and the chance of recall for a sensitive secondary test. The bias is also influenced by the distributions of the scores for the cases and non-cases for each screening test, and by the correlation between the screening tests. Further research on this bias is needed, so that mathematical corrections for paired screening trial bias can be developed.Using a simplified paradigm, we have shown that Programs implemented in SAS and Mathematica to calculate the true and observed sensitivity, specificity, ROC curves, and areas under the curves are available by request from the authors.ROC: Receiver operating characteristic; AUC: Area under the receiver operating characteristic curve.Financial support for this study was provided by NCI K07CA88811, a grant from the National Cancer Institute to the Colorado School of Public Health, Deborah Glueck, Principal Investigator. The funding agreement ensured the authors' independence in designing the study, interpreting the data, writing, and publishing the report. The authors declare that they have no competing interests.DHG conceived of the idea and derived the mathematics. The first draft of the manuscript was a collaborative effort of DHG and MML. MML provided epidemiological expertise. CIO programmed the formulae and produced graphs. KEM provided advice about the mathematics. BMR and JTB aided in a thorough revision of the manuscript. JML and EDP provided clinical expertise and suggestions for how to relate the topic to medical studies. TAA assisted in the literature review, and in checking the math, and collaborated on the revision of the manuscript. All authors read and approved the final manuscript.For those readers not familiar with ROC analysis, we give a short tutorial. For a complete discussion, see ;Chapter ; ChapterThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/9/4/prepub"}
{"text": "The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function.AtAPY1 fused to the SNAP--tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea  were able to release the AtAPY1 protein from microsomal membranes.Transgenic Arabidopsis lines expressing AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi. Tagging AtAPY1 was chosen over raising antibodies against it because AtAPY1 and AtAPY2 are so identical in their amino acid (aa) sequence: There is only one six-aa-stretch in AtAPY1 (aa 44\u201349) that has four different and two similar aa to the corresponding sequence in AtAPY2. All oth6-alkylguanine-DNA alkyltransferase, SNAP binds covalently to benzylguanine-based substrates. There are a large number of substrates coupled to different fluorescent dyes and other labels commercially available making the SNAP-tag a versatile tool for localization studies. The expression of AtAPY1-SNAP was placed under the control of the native promoter region, because overexpression can lead to localization artifacts. Despite this risk, another tagged AtAPY1 version, AtAPY1-GFP, was fused to the strong cauliflower mosaic virus 35S promoter because expression levels of NTPDases are generally low[Among the tags available, the SNAP-tag,45 seemeally low.AtAPY1 and AtAPY2 (DKO) is seedling-lethal[35S promoter made the rescue of the DKO mutant with AtAPY1-GFP impossible as confirmed experimentally, because this promoter is turned off in pollen[apy2 SKO plants  were used as the genetic background for transformation with each tagged AtAPY1 construct. These plants carried AtAPY2 under the control of the pollen-specific promoter SPIK which ensured the survival of the DKO pollen.The SNAP- or GFP-tag was fused to the C-terminus of AtAPY1 to avoid losing the tag by a possible N-terminal cleavage in a subcellular targeting process. Since tags can impair protein function and lead to mislocalization, a complg-lethal. A DKO sn pollen. Withoutn pollen. In ordeAtAPY1::AtAPY1-SNAP or 35S::AtAPY1-GFP and used for genotyping by PCR. Several DKO plants without a WT AtAPY1 and AtAPY2 gene, but with a tagged apyrase construct were identified hereafter called DKO-SNAP and DKO-GFP, respectively. The PCR analysis of two such mutants is shown in FigureSPIK::AtAPY2 construct was always present in the DKO-GFP mutants as expected, but interestingly also in the DKO-SNAP mutants. One possible explanation is that some regulatory elements necessary for optimal expression in pollen were missing in the chosen promoter region. The promoter region used previously for AtAPY1::GUS analyses[AtAPY1. Since the gene At3g04090 was deemed unnecessary for successful complementation, it was mostly excluded in the AtAPY1::AtAPY1-SNAP construct.DNA was isolated from progeny of the partially complemented SKO plants containing either analyses,17,18 wa+/+; apy2/apy2; SPIK::AtAPY2; AtAPY1::AtAPY1-SNAP) and SKO-GFP  plants were included in the study to check for possible dominant negative effects of the tagged apyrase on the WT phenotype. DKO seedlings without a construct coding for a tagged AtAPY1 had an abnormal phenotype with fleshy cotyledons and no root  could be rescued by transformation with AtAPY1-SNAP or AtAPY1-GFP making the DKO-SNAP and DKO-GFP plants suitable tools for localization studies.The lethal DKO . Although specific labeling of fusion proteins in yeast and animin yeast, a high AtAPY1 is expressed strongly in root hairs as well as in guard cells among other cell types[Therefore, indirect immunofluorescence was chosen as a different approach. DKO-SNAP seedlings were fixed. After cell wall digestion and plasma membrane permeabilization, they were incubated with primary antibodies against the SNAP-tag. Following several washing steps, FITC-labeled secondary antibodies were added to visualize AtAPY1-SNAP for the CLSM. The background was low, but no specific signals could be detected (data not shown). To increase the fluorescent signal, the tyramide signal amplification (TSA) technique was applied. This tell types,18.AtAPY1, the indirect immunofluorescence approach was repeated with transgenic plants expressing AtAPY1-GFP under the control of the strong 35S promoter. We used primary antibodies against GFP and secondary Alexa Fluor 488-coupled antibodies in two different approaches: 1. Post-embedding labeling of 200-nm-thin Tokuyasu cryo-sections .The method of live imaging of GFP-tagged proteins is suitable to detect apyrase in the cell wall as shown for apoplastic apyrases in other plant species,54. But d FigureG which dd FigureH, but thd FigureH. This re FigureI, J. Seeapoplast. Howeverin vivo imaging, revealed the same punctate structures, but no signals at the plasma membrane or in the extracellular space.Both detection methods, immunofluorescence and trans-Golgi network, but not the Golgi apparatus[35S::AtAPY1-GFP seedlings did not reveal any co-localization of the fluorescent dye and the GFP signal, even 120\u2009min after the infiltration  and SYP32 (Syntaxin of plants 32) are SNARE proteins localized in the Golgi apparatus. Got1p (erevisiae and its r) of the two fluorescent signals  and 12 nuclei (= 27\u2009\u03bcm2), respectively. Multivesicular bodies (MVB) were also positively immunolabeled by gold particles [Acer pseudoplatanus)[If the Golgi localization of AtAPY1 were correct, AtAPY1 should exhibit the substrate specificity typical of Golgi apyrases. Therefore, the activity of AtAPY1-GFP was tested in the presence of known apyrase substrates at pH 6.5 which equals the pH found in the Golgi. AMP is  sativa) and sycalatanus).In order to investigate the possibility that the substrate specificity was pH-dependent and that it would change in favor of ATP and ADP once AtAPY1 reached the apoplast, the activity assay of AtAPY1-GFP was repeated at pH 5.5, the pH typically found in the cell wall. HoweverOne objective was to determine if AtAPY1 was a soluble protein in the lumen of the Golgi or, as implied by the TMHMM prediction program, a GolgiAtAPY1-GFP seedlings and their purity was verified with antibodies against marker proteins for cytosolic and insoluble proteins  and denaturing (4\u2009M urea) conditions. Peripheral proteins are removed from membranes by urea which disturbs protein-protein interactions or by high salt and alkaline treatments which disrupt electrostatic and hydrophobic interactions, respectively. Except for trace amounts, the salt and Na2CO3 treatment did shift AtAPY1 from the pellet fraction to the supernatant  1.0 suggesten medium. This exn medium. This ren medium. The simn medium.in vitro germinated pollen also showed that the protein exhibiting apyrase activity in the pollen germination medium was soluble because it was measureable in siphoned off aliquots[The experiment with the aliquots. Since AAtAPY1 and AtAPY2 have been implicated to be the regulators of eATP signals in Arabidopsis,15,77-79. [AtAPY2 that could result in different localizations of the corresponding proteins. The fact that transgenic Arabidopsis plants overexpressing AtAPY2 show less eATP-mediated superoxide production than WT[Even though AtAPY1 did not agree with the profile of an ecto-apyrase, AtAPY2 remains a candidate. However, Dunkley et al.  noted a . . Neverth than WT points tSince AtAPY1 was not detected in the cell wall by three subcellular detection methods and did not exhibit the substrate specificity of an ecto-apyrase, we hypothesize that some apyrase other than AtAPY1 is the regulatory enzyme observed in eATP signaling of Arabidopsis.Pisum sativum[i by an integral membrane protein facing the Golgi lumen with its active site. It was also shown in P. sativum that UDP inhibits glycosyltransferases[A diphosphatase activity was described in the Golgi of m sativum. UDP wassferases, which fsferases in a feesferases. A role sferases, no rootsferases could beSaccharomyces cerevisiae mutant \u0394gda1. This yeast mutant lacks the Golgi apyrase guanosine diphosphatase 1 (GDA1) leading to a glycosylation defect[AtAPY1[xMore evidence for a function in glycosylation comes from a complementation experiment with the yeast n defect which ist[AtAPY1.It is also believed that Golgi apyrases provide the nucleoside monophosphates which serve as the substrates in exchange for nucleotide sugars from the cytosol in an antiport mechanism. TherefoAtAPY1-SNAP or -GFP allowed specific labeling of AtAPY1 at the subcellular level. The cell organelles exhibiting the AtAPY1-specific fluorescent signals were identified as the Golgi apparatus by the following criteria: (i) morphology, (ii) size, (iii) lack of co-localization with the endocytic marker stain FM4-64 and trans-Golgi marker protein Rab E1d, but (iv) co-localization with the three Golgi marker proteins MEMB12, SYP32 and Got1p homolog. In addition, Golgi stacks were immunolabeled with \u03b1-GFP antibodies. While this paper was under review, the Golgi localization of AtAPY1 was independently confirmed by proteome analysis of Golgi membranes and by co-localization of AtAPY1-YFP with the CFP-labeled cis-Golgi marker \u03b1-mannosidase I in transiently transformed onion peels[The use of two different transgenic lines expressing either on peels.Although it can never be ruled out that the missing detection of extracellular AtAPY1 is a matter of methodological sensitivity, our results show that AtAPY1 is primarily present in the Golgi. Therefore, AtAPY1 is highly unlikely to represent the regulatory enzyme of eATP levels,15,77,78i: Inorganic phosphate; PM: Plasma membrane; PPT: Phosphinothricin; RFP: Red fluorescent protein; RT: Room temperature; S: Supernatant; SNAP: O6-alkylguanine-DNA alkyltransferase; SPIK: Shaker pollen inward K+ channel; SYP32: Syntaxin of plants 32; SKO: Single knockout; T-DNA: Transfer DNA; TBS: Tris buffered saline; TEM: Transmission electron microscopy; TM: Transmembrane domain; TSA: Tyramide signal amplification; UA: Uranyl acetate; UDP: Uridine diphosphate; v: Version; WT: Wild type or wild-type; YFP: Yellow fluorescent protein.aa: Amino acid(s); ACR: Apyrase conserved region; AEBSF: 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride; apyrase: Adenosine pyrophosphatase; CLSM: Confocal laser scanning microscopy; DKO: Double knockout; eATP: Extracellular ATP; ECM: Extracellular matrix; FITC: Fluorescein isothiocyanate; FM4-64: N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide; GDP: Guanosine diphosphate; GFP: Green fluorescennt protein; Got1p: Golgi transport 1 protein; HeNe: Helium-neon; HRP: Horseradish peroxidase; IDP: Inosine diphosphate; MC: Methyl cellulose; MEMB12: Membrin 12; MS: Murashige and Skoog; MVB: Multivesicular body; NTPDase: Nucleoside triphosphate diphosphohydrolase; ORF: Open reading frame; P: Pellet; PB: Phosphate buffer; PBS: Phosphate buffered saline; PFA: Paraformaldehyde; PAtAPY1-GFP expressing seedlings. IS conceived of the study, generated the AtAPY1-SNAP transgenic lines and the lines co-expressing AtAPY1-GFP and a marker gene, performed the genetic complementation of the DKO-GFP mutants and drafted the manuscript. All authors read and approved the final manuscript.MS carried out the molecular genetic studies of the DKO-SNAP mutants, performed the localization studies including confocal laser scanning microscopy and image analysis, sample preparations and treatments, analyzed the solubility of the AtAPY1-GFP and participated in the drafting of the manuscript. CM performed the substrate specificity analyses of AtAPY1-GFP. TK conducted the localization studies by immunogold labeling and by indirect immunofluorescence with AtAPY1-SNAP DNA sequence. The AtAPY1-SNAP DNA sequence present in the AtAPY1::AtAPY1-SNAP transgenic lines is shown. Click here for fileAtAPY1-GFP DNA sequence. The AtAPY1-GFP DNA sequence present in the 35S::AtAPY1-GFP transgenic lines is shown. Click here for fileImmunofluorescence of pre-imbedding labeled AtAPY1-GFP in root hair. Roots from AtAPY1-GFP expressing seedlings were immunostained whole mount, embedded in resin and sectioned. The sections were successively incubated with \u03b1-GFP and secondary \u03b1-rabbit Fab fragments coupled with Alexa Fluor 488. A 3-\u03bcm cross-section of a root hair is shown. The Alexa Fluor 488 fluorescence is shown in green. Scale bar equals 20\u2009\u03bcm. Click here for fileLive imaging of AtAPY1-GFP in various cell types. CLSM images of various tissues in AtAPY1-GFP expressing seedlings show the GFP signals in green overlaid with bright field view. (A) Cotyledon epidermis, (B) hypocotyl and (C) root tip. Scale bars\u2009=\u200920\u2009\u03bcm. Click here for fileSpecificity of the imaging settings for the detection of GFP and YFP fluorescence. The epidermis of cotyledons from transgenic plant lines was imaged with the GFP and YFP settings outlined under Methods for the \u201cCLSM\u201d. The GFP and YFP fluorescence is shown in green and magenta, respectively. Scale bars\u2009=\u200920\u2009\u03bcm. (A) The identical epidermal section with two guard cells from plants expressing AtAPY1-GFP only was imaged sequentially with the GFP and YFP settings. Dot-like signals appeared in the GFP detection channel only. Only weak autofluorescence of the thickened cell wall around the stomate was visible in the YFP detection channel. (B) The identical epidermal section with two guard cells from plants expressing YFP-SYP32 only was imaged sequentially with the GFP and YFP settings. Here, only very weak signals were detectable in the GFP detection channel, but strong YFP fluorescence appeared with the YFP-specific excitation and detection. Click here for fileCo-localization analysis of AtAPY1-GFP and YFP-Got1p homolog. CLSM images of epidermal cells of cotyledons from transgenic lines co-expressing AtAPY1-GFP and YFP-Got1 phomolog were taken. The GFP fluorescence is shown in green and the YFP fluorescence in magenta. The bright field-type image was acquired with the transmitted light detector. The fluorescence signals for AtAPY1-GFP and YFP-Got1p homolog were detected separately and merged for co-localization with the \u201cCo-localization Finder\u201d plugin of ImageJ. Co-localization of the two proteins is depicted as white signals. The corresponding scatterplot was analyzed with the ImageJ \u201cColocalization Threshold\u201d and \u201cColoc2\u201d tool from ImageJ. The x-axis represents the pixel intensities from the GFP channel and the y-axis from the YFP channel. Rr\u2009=\u2009Pearson\u2019s correlation coefficient. Scale bar\u2009=\u200920\u2009\u03bcm. Click here for fileSubstrate specificity of AtAPY1-GFP at pH 5.5. The activity of AtAPY1-GFP at pH 5.5 in the presence of various substrates was measured. No activity was detectable with AMP as substrate (data not shown). Different letters above the columns indicate mean values that are significantly different from one other . Error bars represent standard deviations of two phosphate measurements from one reaction (see Methods). Abbreviation: Pi, inorganic phosphate. Click here for fileAnalysis of the purity of microsomal membrane and soluble protein fractions. Protein extracts from transgenic plants expressing 35S::AtAPY1-GFP were treated with either extraction buffer, 0.2% SDS, 2\u2009M NaCl, 0.2\u2009M Na2CO3 or 4\u2009M urea and then centrifuged at 100,000\u2009g to obtain microsomal membrane fractions (P100) and supernatants (S100). Proteins from each fraction (40\u2009\u03bcg each) were subjected to Western blot analysis. The enrichment of microsomal and insoluble proteins in the P100 fractions and of soluble proteins in the S100 fractions was confirmed with antibodies against marker proteins. The 37-kDa cytosolic fructose-1,6-bisphosphatase (cFBPase) served as a marker protein for soluble proteins. Actin (45\u2009kDa) was used as a marker protein for insoluble proteins under actin polymerizing conditions found in the extraction buffer and 2\u2009M NaCl[2CO3, 0.2% SDS and 4\u2009M urea[2\u2009M NaCl and as a4\u2009M urea,90. Click here for file"}
{"text": "Birth weight (BW) predicts many health outcomes, but the relative contributions of genes and environmental factors to BW remain uncertain. Some studies report stronger mother-offspring than father-offspring BW correlations, with attenuated father-offspring BW correlations when the mother is stunted. These findings have been interpreted as evidence that maternal genetic or environmental factors play an important role in determining birth size, with small maternal size constraining paternal genetic contributions to offspring BW. Here we evaluate mother-offspring and father-offspring birth weight (BW) associations and evaluate whether maternal stunting constrains genetic contributions to offspring birth size.Data include BW of offspring  born to female members (n\u200a=\u200a382) and spouses of male members (n\u200a=\u200a275) of a birth cohort (born 1983\u201384) in Metropolitan Cebu, Philippines. Regression was used to relate parental and offspring BW adjusting for confounders. Resampling testing was used to evaluate whether false paternity could explain any evidence for excess matrilineal inheritance. In a pooled model adjusting for maternal height and confounders, parental BW was a borderline-significantly stronger predictor of offspring BW in mothers compared to fathers (sex of parent interaction p\u200a=\u200a0.068). In separate multivariate models, each kg in mother\u2019s and father\u2019s BW predicted a 271\u00b153 g (p<0.00001) and 132\u00b155 g (p\u200a=\u200a0.017) increase in offspring BW, respectively. Resampling statistics suggested that false paternity rates of >25% and likely 50% would be needed to explain these differences. There was no interaction between maternal stature and maternal BW (interaction p\u200a=\u200a0.520) or paternal BW (p\u200a=\u200a0.545).Each kg change in mother\u2019s BW predicted twice the change in offspring BW as predicted by a change in father\u2019s BW, consistent with an intergenerational maternal effect on offspring BW. Evidence for excess matrilineal BW heritability at all levels of maternal stature points to indirect genetic, mitochondrial, or epigenetic maternal contributions to offspring fetal growth. Individuals with lower birth weights (BW) have heightened risk of mortality in infancy and early childhood in utero. In this model, paternal factors were more important as influences on postnatal growth and final size.Because fetal growth serves as a marker of prenatal conditions that influence an array of later biological functions and health outcomes, there is a need to understand the causes of variation in fetal nutrition, growth and birth outcomes. Estimates of heritability for BW typically range between 0.2 and 0.4 In humans, evidence for a disproportionate maternal contribution to birth outcomes comes from several sources. Studies have compared the strength of father-offspring and mother-offspring BW correlations e.g.  and mostin utero, could override genetic influences on the prenatal nutritional environment experienced by the next generation and be reflected in fetal growth rate and birth size. This idea has gained limited support in some In addition to evidence for stronger intergenerational BW correlations through the matriline, a related hypothesis posits that genetic contributions to BW are constrained in women who experienced early life histories of nutritional stress in utero, and whether the intergenerational BW correlations are reduced among women who were shorter as adults.Here we contribute to the literature on genetic and environmental contributions to BW by reporting the strength of mother-offspring and father-offspring BW associations in a well-characterized birth cohort in Cebu City, the Philippines. As part of this analysis, we run a resampling analysis in an effort to determine the false-paternity rate that would be needed to account for any differences in matrilineal and patrilineal intergenerational correlations in this sample. Finally, we test for maternal constraint on fetal growth rate by evaluating whether the strength of parental-offspring BW correlations is stronger among heavier babies that were less likely growth-restricted Data come from the Cebu Longitudinal Health and Nutrition Survey (CLHNS), a population-based study that originally enrolled 3327 pregnant mothers and has since followed their offspring into adulthood, many of whom are now parents with offspring of their own Offspring BW information was obtained through recall among female birth cohort members and among the current spouses of male cohort members. Each mother was asked to recall, among other factors, the status of each pregnancy , birth date, whether the baby\u2019s weight was weighed and if so the BW, gestational age at parturition, and sex of each of her offspring. Because BW is reduced in twin pregnancies, and twin pregnancies were rare, analyses were limited to singleton liveborn pregnancies. In addition to birth outcome characteristics, we obtained information on characteristics of each pregnancy, including whether the woman worked while pregnant and whether she obtained prenatal care. Current height and weight were obtained using standard anthropometric techniques Of the 1235 singleton liveborns for which all maternal and birth control variables were available, the baby was not weighed at 134 of these births (10.9%) and these pregnancies were therefore excluded from analysis. We compared women whose babies were weighed at birth to those whose baby was not. Women who did not have their baby weighed at birth came from lower income households , they were slightly less likely to receive prenatal care , and they were also less likely to report having worked during pregnancy . However, women whose baby was not weighed had a similar adult stature to women whose baby was weighed at birth .All analyses were performed with Stata version 10 , except the paternal uncertainty analyses which were conducted in R . In a model predicting birth outcomes in female cohort members and spouses combined, there was a borderline sex of parent by parental BW interaction (p\u200a=\u200a0.068), showing that maternal and paternal BW relate to offspring BW with (borderline) different slopes. All subsequent models were stratified on gender of the parent for whom BW was measured.While we can say with high certainty that the mothers in this study are the biological mothers of the children, paternity is less certain. False paternity would tend to cause an attenuation of father-offspring BW associations that could imitate a maternal effect in utero, the hypothesis of maternal constraint leads to the expectation that genetic contributions to BW will be stronger in larger newborns. Because prior work on maternal constraint in humans and other non-human primates reported evidence for an effect specific to the matriline We next tested for evidence of maternal constraint in the sample using several strategies. First, following Veena et al Finally, we tested whether maternal and paternal BW were stronger predictors of offspring BW when the mother was taller ; Model 5In this sample of Filipino young adults, we find evidence for stronger intergenerational BW correlations through the matriline than through the patriline, consistent with disproportionate maternal contributions to offspring BW. However, contrary to the concept of maternal constraint, there was no evidence that either patrilineal or matrilineal intergenerational BW correlations were stronger among taller women. Thus, there was no evidence that a woman\u2019s history of nutritional stress, as reflected in her adult stature, constrain hereditary contributions to birth weight in this sample.The finding of stronger maternal-offspring than paternal-offspring BW correlations has been described in most prior human studies. In a study of more than 67,000 Norwegian births, the correlation between mothers\u2019 and offspring BW (r\u200a=\u200a0.226) was stronger than that of the paternal BW-offspring BW correlation (r\u200a=\u200a0.126) Although the pattern that we find is generally consistent with most past studies, little is currently known about the causes of the difference in strength of mother-offspring versus father-offspring BW correlations. Higher false paternity rates will tend to decrease the correlation between putative father and offspring for all traits with a genetic component in utero also tends to influence her child in utero. While these explanations are difficult to disentangle, here we consider some of the prime candidates in hopes of guiding future studies aimed at clarifying underlying mechanisms.Other possible explanations for this pattern include sex-linked genetic effects, indirect genetic effects, epigenetic effects, and shared environmental or cultural effects. Although varying widely in mechanism, these explanations all share an intergenerational character. They all require that some environmental, physiological, or genetic factor which influences the mother Fathers do not pass on their X chromosomes to sons but only to daughters, which results in a distinctive inheritance pattern for X chromosome-linked traits which in theory could in result discrepancies in patrilineal and matrilineal inheritance. However, consistent with previous large studies Macaca nemestrina) suggested that mitochondrial inheritance could account for 9% of the variance in BW Another well-established sex-linked genetic effect which might explain excess matrilineal inheritance, mitochondrial inheritance, cannot be ruled out so readily. Mitochondria are present in the cytoplasm of cells, and contain their own 16,569 base-pair circular genome Indirect genetic effects are another plausible contributor to excess matrilineal BW heritability. In contrast to mitochondrial effects or non-genetic maternal effects, indirect genetic effects occur because the presence of alleles in the offspring predicts the alleles of the mother and some of these alleles alter maternal physiology or metabolism, thereby influencing offspring phenotypes such as BW. As one concrete example, maternal alleles associated with type 2 diabetes influence the BW of offspring in part by influencing maternal fasting glucose and insulin levels during pregnancy Environmental or cultural factors might also help explain the excess matrilineal BW heritability. Mothers and grandmothers often provide advice to their daughters and granddaughters about ideal behaviors during pregnancy , which could contribute to similarities in the gestational environments and birth weights of successive generations (shared family effects). Similarly, wealth is transmitted across generation, which could lead to similarities across generations in environmental factors like nutrition and access to health care Finally, there is also increasing evidence that the fetal growth conditions experienced by the mother, as reflected in her own gestational nutrient or hormonal milieu, can lead to durable epigenetic or developmental changes that influence offspring through several possible pathways. This can occur when the mother\u2019s early life experiences influence her own fetal growth and also have durable effects on her adult physiology and metabolism that modify fetal growth rate of her offspring Regardless of the specific causes of the differences in matrilineal and patrilineal BW inheritance observed here, our findings, and those of previous investigators, clearly show that maternal contributions are more important than paternal contributions in determining offspring BW. Because prenatal nutrition and growth rate predict an expanding array of long-term functional and health outcomes Some limitations of this study warrant consideration. Most notably, offspring BW information was recalled by women during interviews, rather than being prospectively collected after each pregnancy. In Cebu, most women have birth records, and analyses were limited to births that were weighed. Although this will minimize recall error, female cohort members were asked about their reproductive histories during several survey rounds, while the spouses of male participants were asked only once in 2009. Thus, it is possible that female participants were better able to recall, or have records of, their offspring\u2019s BW, which could reduce measurement error among the offspring of female participants. However, we found that maternal socioeconomic and pregnancy characteristics were as strong or often stronger as predictors of offspring BW among spouses of male participants, suggesting that the apparently weaker relationship between paternal BW and offspring BW was not simply an artifact of poor measurement reliability of BW among offspring of male participants.In conclusion, mother\u2019s BW is a stronger predictor of offspring BW than is the father\u2019s BW in this population, independent of the mother\u2019s adult size. This suggests that some combination of maternal indirect genetic, environmental or epigenetic factors influence the intrauterine environment or fetal growth above and beyond any direct genetic effects. Although this contributes to an excess of BW heritability through the matriline, we found no evidence that the slope of intergenerational BW relationships were steeper or stronger in heavier offspring or among newborns born to taller women, as would be predicted if maternal nutritional stress or stunting constrained genetic contributions to birth size. These findings suggest that maternal effects on fetal growth are present across the full range of maternal stature in this population. By extension, they suggest that maternal environmental, epigenetic or indirect genetic factors influence the diverse array of offspring phenotypes that are downstream of fetal nutrition and growth.File S1Non-paternity analysis.(DOC)Click here for additional data file."}
{"text": "Materials with strong spin-orbit coupling, which competes with other particle-particle interactions and external perturbations, offer a promising route to explore novel phases of quantum matter. Using LDA\u2009+\u2009DMFT we reveal the complex interplay between local, multi-orbital Coulomb and spin-orbit interaction in elemental bismuth. Our theory quantifies the role played by collective dynamical fluctuations in the spin-orbit Kondo state. The correlated electronic structure we derive is promising in the sense that it leads to results that might explain why moderate magnetic fields can generate Dirac valleys and directional-selective magnetoresistance responses within spin-orbit Kondo metals. EF. Beyond the inherent importance of exploring complex phases of quantum matter5T)Elemental bismuth, a material with a strong spin-orbit (SO) interaction, plays a key role in solid-state physics235678911P-band semimetals like bulk bismuth and graphite undergo a metal-insulator-like transition upon exposure to modest magnetic fieldsT2 dependence works quite well down to 4\u2009KT study showed significant deviation from canonical FL systems, where the T2 behaviour changes smoothly into a T5-like form at very low temperaturesT behavior. Complex low-energy physics in bismuth is also unveiled by optical measurements, showing large changes in plasma frequency and anomalous midinfrared features23During the 20th century, transport properties of bismuth were extensively studied12p-band topological insulators, we have recently shown that Kondo- and Mott-like features are well accounted for by the diagrammatic multi-orbital (MO) iterated-perturbation-theory27Ueff\u2009\u2009\u2261\u2009U/WLDA\u2009\u2248\u20091, U and WLDA are, respectively, the on-site Coulomb repulsion and the LDA band width) good semiquantitative accord with experimental data can be obtained. MO-Hubbard plus SO interaction qualitatively account for dynamical signatures of bulk correlation effects in photoemission (PES)611Deviations from FL-like behaviour61118p31323334sp, s-carriers) is appreciable compared to the electron-electron interactions, as distinct from d-band systems, where the d electrons reside in much narrower bands (hence the effective U/WLDA is sizable). However, we recall here that in canonical s metals like elemental lithium evidences for correlated electron physics have been reported by Stutz et al.97Li3p or s bands is undoubtedly an issue of great contemporary interest. In light of the discussion above, we study how an orbital-selective interplay between appreciable p-band itinerance and on-site Coulomb repulsion, U, plays a central role in this unique spin-orbit Kondo state of elemental bismuth.The possibility of correlated electron physics in purely A7, \u03b1-arsenic type structure (1EF), see 41EF. Elemental bismuth has a formal +3 oxidation state, which implies an equal number of electrons and holes in the p sector. As pointed out, all electronic 6p states acquire some itinerance, providing valence and conduction band states in all active 6p- orbitals. The situation encountered here with three half-filled bands provides the underlying microscopic one-band seeds of a quantum correlated scenario in elemental bismuth.Bismuth crystallises in the tructure . In the a\u2009=\u2009x, y, z labels the diagonalised 6p-bands. In light of the sizable correlation effects within the Bi-channel of bulk Bi2Se3 and Bi2Te2Se topological insulators27U\u2032\u2009\u2261\u2009U\u2009\u2212\u20092JH and U(U\u2032) is the intra- (inter-) orbital Coulomb repulsion. JH and V are, respectively, the Hund\u2019s rule and the local SO coupling. Within our formulation, the SO interaction acts as a transverse magnetic-fieldpa spin states of bismuth. The DMFT self-energy, \u03a3a(\u03c9), requires a solution of the MO quantum impurity problem self-consistently embedded in an effective mediumWithin LDA, the one-electron part of the many-body Hamiltonian for bismuth is U promotes a gradual reduction of the bulk band gap size and coherent Kondo clouds are created near EF in Bi-chalcogenide topological insulators27U the energy gap is suppressed by dynamic transfer of spectral weight characteristic of correlated electron systems502Y2X , X\u2009=\u2009Se) topological materials. In JH\u2009=\u20090.5\u2009eV27nt\u2009=\u20093.0, showing that a quantum correlated scenarioU, U\u2032 and JH leads to spectral weight redistribution over large energy scales and the formation of an electronic structure similar to topological Kondo systems27spin-orbit Kondo state is characterised by the presence of sharp in-gap states near the Fermi energy, EF(=\u2009\u03c9\u2009=\u20090). As a first testing ground to our proposal, in Electron-electron interactions may induce topologically nontrivial electronic phases that can change the nature of single-particle excitation of topological insulators48A7 element, we show in the main panel of T dependence of dc resistivity computed using the orbital resolved, LDA\u2009+\u2009DMFT spectral functions, x, y, z) 6p-bands, f(\u03c9) is the Fermi function. As in ref. k-dependence of electron\u2019s velocity, vk,a. In this situation, following Saso et al.54vk,a by a single average carrier velocity (v) for all orbitals. In fact, Saso et al. and Baldassare et al.\u03c3ac(\u03c9) for Kondo insulators (FeSi and YbB12) as well as for V2O3, supporting our approximation in \u03c3dc(T) above. The observed features in resistivity \u03c1dc(T)\u2009\u2261\u20091/\u03c3dc(T) originate from SO and Zeeman-field induced spectral changes: Showing how this provides a compelling description of extant experimental data is our focus here.To elucidate the crucial role played by local quantum correlations on electric transport in this V\u2009=\u20091.05\u2009eV we obtain a quasilinear T-dependence above 30\u2009K which is close to that observed in bulk crystalsT. Interestingly, this drop in resistivity at low-T is enhanced with increasing the SO interaction V, see bottom-right panel of V\u2009\u2264\u20091.25\u2009eV. In our theory this robust phenomenon is the fingerprint of SO-induced local quantum fluctuationsp-band semimetals like bismuth and graphiteU\u2009=\u200910\u2009eV supports our proposal of non negligible electron-electron interactions in bismuth. In 0.9Sb0.10.9Sb0.1 system might exhibit some sort of lattice disorder effects induced by antimony. While PES experiment resolves a low-energy bump which was interpreted as gapless surface states coexisting with an insulating bulkpz DOS unveil a quasiparticle peak structure near EF, which is consistent with observation of out-of-plane surface states in (AR)PES6pz orbital symmetry in surface-like states would place our theory in a solid ground.The dc resistivity in \u03bcB is the Bohr magneton, g is the Land\u00e9 factor, and Ba is the external (Zeeman) magnetic field applied along the a directionha, keeping the total band filling (nt\u2009=\u20093) fixed to simulate the electronic changes upon Zeeman field splitting. As seen in ha drives appreciable SWT, producing drastic orbital-selective renormalisations of the one-particle spectral functions: The out-of-plane pz band is most severely affected. As expected, ha shifts the relative positions of the spin polarised bands at which the magnetism changes. The resulting magnetisation can be directly calculated from ma\u2009=\u2009\u2329na\u2191\u232a\u2009\u2212\u2009\u2329na\u2193\u232a, with the particle\u2019s number \u2329na\u03c3\u232a being computed using orbital and spin resolved spectral functions. According to our results a trial field applied along the hexagonal plane, i.e hx,y\u2009=\u2009h  gives negligibly small magnetisation values, mx,y\u2009\u2248\u20090.01. This is an indication of reduced magnetic moments in the frustrated hexagonal plane. Interestingly, in this regime the in-plane Zeeman field drives the system towards to a Kondo insulating state with emergence of a narrow semiconducting band gap in the spin resolved spectral functions. Our results are thus consistent with a Dirac valley scenario in bulk bismuth12V\u2009=\u20091.25\u2009eV compared to V\u2009=\u20091.05\u2009eV can be regarded as the manifestation of SO induced weak antilocalisation effectspz spectral functions. Here, we obtain mz\u2009=\u20090.14 which arises from the formation of local moments in the majority (spin-\u2191) channel. As seen in the right panels of pz-band is pushed towards to a Mott insulating state with appearance of a lower Hubbard band , the minority (\u2193) channel undergoes to a Kondo-like metallic state characterised by a broad quasiparticle peak at EF. This unexpected field-induced phase transition is apparently consistent with recent DMFT studyWe now turn to the interplay between local transverse-field fluctuations induced by SO coupling and intra-orbital Zeeman level splittings upon application of an external magnetic field in bismuth. To proceed, we consider the orbital-dependent on-site energy term, p-band semimetals is mandatory for designing novel MR materials. An affirmative theoretical answer to the MR effect in elemental bismuth is shown in \u03c1dc(T)] computed using LDA\u2009+\u2009DMFT spectral functions within the Kubo formalismha: Showing how this provides a compelling explanation for the MR effect for in-plane  and out-of-plane (hz) magnetic fields is our focus here. Various interesting features immediately stand out in T\u2009\u2192\u20090 the resistivity curve for a trial field \u03bcBT-dependence, in accordance with the insulating behaviour at small fields seen in experimentha the dc resistivity in \u03c1dc(T) increases as T\u2009\u2192\u20090 at finite in-plane field, hx,y, which is consistent with a semiconducting like behaviour due to field-induced Dirac valley spectrumT metallicity accompanying an insulator-to-metal crossover. The interplay between local spin fluctuations27T. This is our mechanism for the experimentally observed reduction of \u03c1dc(T) at low T in bismuth, where a crossover from a metal to an insulating-like behaviour above T\u2009=\u200910\u2009K is obtained, in good semiquantitative agreement with extant data11p-band semimetal.An understanding of the correlated nature of p electrons differs because of various competing local quantum fluctuations. For fields applied parallel to the hexagonal plane the emergent Dirac valley reflects the competition between Zeeman field splitting and screening of local moments. On the other hand, for fields perpendicular to the basal plane spin-selective metallic and insulating states similar to that of correlated half-metalsp-band semimetals1864In conclusion, using LDA\u2009+\u2009DMFT for a multi-band model, we resolve the nature of the metallic regime in bismuth. Good semiquantitative accord with key spectral6The local-density-approximation plus dynamical-mean-field-theory (LDA\u2009+\u2009DMFT) by construction takes into consideration the most relevant multi-orbital correlation effects and all-electron degrees of freedom. It is an ideal starting point towards the description of Coulomb-driven metal-to-insulator transitions, Fermi and non-Fermi liquid metallic states and in general grounds the role played by dynamical correlations in idealised many-particle models as well as in real multi-orbital systemsp-band electronic states in elemental bismuth, we employ our state-of-the-art implementation of LDA\u2009+\u2009DMFTk-points. The radii of the atomic spheres were chosen as r\u2009=\u20093.35 a.u. in order to minimise their overlap. To incorporate the effects of electronic correlations in this p-band semimetal, we use the multi-orbital iterated-perturbation-theory (MO-IPT) as an impurity solver of the many-particle problem in DMFT, as described in detail in refs. In order to prove the orbital-selective nature of the U\u2009=\u200910\u2009eV was necessary for a good semi-quantitative agreement with experiments within LDA\u2009+\u2009DMFT, see U larger than typical values for transition metals are the consequence of orbital hybridisation and covalent bonds, which localise electrons between atoms35U\u2009\u2264\u200912), orbital occupancy yields nx,y\u2009=\u2009nz\u2009=\u20091.0. Biasing nx,y, LDA\u2009+\u2009U calculations (U\u2009\u2248\u200910\u2009eV) converge towards the ground state with nx,y\u2009=\u20091.05, nz\u2009=\u20090.9 in good agreement with LDA\u2009+\u2009DMFT.A Hubbard How to cite this article: Craco, L. and Leoni, S. Magnetoresistance in the Spin-Orbit Kondo State of Elemental Bismuth. Sci. Rep.5, 13772; doi: 10.1038/srep13772 (2015)."}
{"text": "Here, we use the density functional dynamical mean-field theory (DFDMFT) scheme to comprehensively explain why tetragonal FeS shows both semiconducting and metallic responses in contrast to tetragonal FeSe which is a pseudogaped metal above the superconducting transition temperature. Within local-density-approximation plus dynamical mean-field theory (LDA+DMFT) we characterize its paramagnetic insulating and metallic phases, showing the proximity of mackinawite to selective Mott localization. We report the coexistence of pseudogaped and anisotropic Dirac-like electronic dispersion at the border of the Mott transition. These findings announce a new understanding of many-particle physics in quantum materials with coexisting Dirac-fermions and pseudogaped electronic states at low energies. Based on our results we propose that in electron-doped FeS substantial changes would be seen when the metallic regime was tuned towards an electronic state that hosts unconventional superconductivity.Transport properties of tetragonal iron monosulfide, mackinawite, show a range of complex features. Semiconductive behavior and proximity to metallic states with nodal superconductivity mark this  On the other hand, iron-based superconductors are materials in which the electronic states near EF are derived from Fe-3d orbitals. Given the fact that the spin-orbit interaction on iron is rather weak compared to Bi-based p-band TIs might look as if these two systems belonged to a different material class. However, a perusal of extant experimental567891012139Over the past few years iron-based superconductors122As2 was the first material to reveal an anisotropic Dirac point near EF2A2 until SDW ordered phase vanishes73 substrate where nontrivial (Z2) topology can be tuned through band inversion at M point of the Brillouin zoneIn the recent history of Dirac-fermion physics in iron-based superconductors0.88Tc\u2009\u2248\u20095\u2009K) in tetrahedral FeS single crystals were recently reported by different groups272831282731\u03bcSR data\u03bcSR measurements indicate coexistence of low-moment magnetism and bulk superconductivityTetragonal FeS is found naturally as the mineral mackinawite, which was identified as the initial product from corrosion of metallic iron on sulphur202425272425d bandwidth in PES spectra is 25-to-30% narrower than that predicted by first-principles band-structure calculations. The one-particle density-of-states (DOS) was shown to be accompanied by an intense tail at high binding energies, consistent with the correlated fingerprints found in iron-superconductorsd band narrowing at low energies and the satellite features at high-binding energies can be explainedThe localized nature of hexagonal FeS (troilite) and its correlated electronic structure has been investigated by photoemission (PES) and inverse-photoemission spectroscopy experimentsab initio density functional calculations for FeX  demonstrate that the chalcogen p states lie well below EF and are weakly hybridized with Fe 3d states2+ (with d6 electronic configuration) layers with almost direct Fe-Fe hopping. In previous work we undertook systematic local density approximation plus dynamical-mean-field-theory (LDA+DMFT)40Currently, the theoretical understanding of tetragonal FeS is restricted to one-electron band structure calculations41A proper microscopic description of localization-delocalization transition26P4/nmm) structure  is seen in the orbital-resolved DOS of FeS. While the x2\u2009\u2212\u2009y2 and xz, yz orbitals show less pronounced effects due to smaller ionic radii of sulphur compared to selenium, the 3z2\u2009\u2212\u2009r2 and the xy channels display appreciable one-particle band narrowing and small changes in the bonding/antibonding splitting of the electronic states close to EF, characteristic of the tetragonal unit cell. As seen in the inset (right-lower panel) of xy orbital shows tendency towards the formation of a V-shaped electronic spectrum, which is the one-particle seed towards a reconstructed Dirac-like linear band dispersion in tetragonal FeS, as shown below. As comprehesivelly described in this work, due to one-particle band narrowing correlations effects can increase dramatically in FeS compared to FeSe, leading to an orbital blocking mechanism and appearance of marginal Dirac fermions in the d-band electronic states of FeS.Within the tetragonal  denotes the diagonalized 3d orbitals of FeS and \u03b5a(k) is the one-electron band dispersion, which encodes details of the actual one-electron (LDA) band structure. We evaluated the many-particle Green\u2019s functions of the Hamiltonian above within LDA+DMFTwhere U\u2009=\u20094.0\u2009eV (and JH\u2009=\u20090.7\u2009eV)40r2\u2009\u2212\u2009z2 orbital shows strong correlation effects with pronounced charge gap and Hubbard bands, while the x2\u2009\u2212\u2009y2 orbital has less tendency towards local moment formation (LHB). In this figure we also display our previous LDA+DMFT results for FeSe superconductorU\u2009=\u20093.6\u2009eV and that of stoichiometric d6 FeSe. Clearly, the two spectral functions show similar behavior at low energies, indicating that FeS is able to host an unconventional superconducting state at low T if its electronic structure is properly tuned via suitable sample preparation conditions2728U in metallic FeS is at least 0.4\u2009eV smaller than that of tetragonal FeSe as in U/W), driving the electronic states of stoichiometric FeS close to that of tetragonal FeSe superconductor.We now discuss our LDA+DMFT results. In z2\u2009\u2212\u2009r2, xz, yz) and quasi-linear  spectral functions near EF can be tuned in the bulk. Also, the Dirac cones in tetragonal FeS appears to be anisotropic, consistent with observations for BaFe2As2EF. This result can be taken as an evidence that the interplay between band narrowing and Coulomb perturbationsz2\u2009\u2212\u2009r2, xz, yz) states coexisting with Dirac-like fermions at the x2\u2009\u2212\u2009y2, xy orbitals. Thus, within the hidden Dirac-liquid phase the reconstructed Fermi surface is expected to be composed of two distinct components, and future angle-resolved photoemission (ARPES) experiments could verify this aspect.To characterize the hidden Dirac-liquid regimeV-shaped liner dispersions with Dirac-cone like carriers4EF and the contribution of the psedogaped bands must be suppressed to extract the unique carrier transport intrinsic to Dirac cones6y1+Tex1\u2212Sex single crystalsUc\u2009<\u20094.0\u2009eV. Electron-electron interactions renormalizes the LDA+DMFT results in two steps. First, the MO self-energy [\u03a3a(\u03c9)] renormalizes the relative band positions depending upon their orbital occupations. The frequency-dependent self-energy . Below the Mott transition only the degenerated xz, yz orbitals show characteristic FL like features, while the more correlated orbitals display deviations from the \u2212\u03c92 FL form at small \u03c9, being consistent instead with (sub-) linear \u03c9-dependence of marginal Fermi liquidsmarginal Dirac-liquid.Our main result in ergy see  causes SV-shaped \u03c9-dependence in \u03c9\u2009=\u20090. This implies that, close to the Mott insulator-metal transition, we have a renormalized (by DMFT) picture where effectively Dirac-like fermions co-exist with incoherent electronic states from the remaining  bands. In our LDA+DMFT, the FL quasiparticle coherence would be destroyed near the Mott transition in the disorder-free case of FeS, as evidenced by the fact that EF while \u03c9 dependence could result in experiments: recall that our DMFT self-energies are not inconsistent with a weak power-law (in \u03c9) dependence at low energy. In this case, V-shaped spectral functions were also obtained within LDA+DMFT for tetragonal FeSe superconductorAs shown in U, U\u2032 increases a subset of d-orbitals gets selectively Mott localized. The metallic phase is then the orbital-selective metal found in various contexts59EF) is pinned to the renormalized orbital energy of the localized orbital(s). This behavior is understood from the mapping of the corresponding impurity model to that of the \u201cX-ray edge\u201d, where the orthogonality catastrophe destroys FL coherence. The spectral functions then exhibit asymmetric continuum at low energy, instead of symmetric Abrikosov-Suhl Kondo resonance features at low energies, and the metallic phase is non-Fermi liquid. As shown here, the frequency-dependence of the self-energy imaginary parts is important in stabilizing the marginal Dirac liquid state, and consideration of the instability of such an orbital-selective state to a superconducting (SC) state should lead to a strongly frequency-dependent superconducting gap function.  As expected, in a correlated electron picture, the physical response functions in the normal state are controlled by both, intrinsic orbital degrees of freedom as well as the \u03c9 dependent damping originating in the incoherent normal state. Future observation of features such as the presence or absence of an incoherent low-energy peak in X-ray emission spectroscopy (XES)What is the microscopic origin of the orbital-selective features found in the LDA+DMFT solution of tetragonal FeS system near the Mott transition? On general grounds, as d bands are usually shifted relative to each other because of the action of structural induced changes in the crystal-field splittings: the six d electrons found in iron-based superconducting materials are distributed among all d orbitals and, in the case of a system with sizable MO interactions, this might induce orbital polarization and SWT. In this situation, MO correlations cause various intimately linked changes: the static MO Hartree shifts, which depends upon the occupations of each orbital as well as on the inter-orbital correlations [U\u2032 and JH] renormalizes the on-site energies of each orbital in different ways. In particular, it causes inter-orbital charge transfer between the different d-orbitals, self-consistently, modifying their energies and occupationsxy, xz, yz orbitals in LDA will be lowered in energy while the 3z2\u2009\u2212\u2009r2, x2\u2009\u2212\u2009y2 will be pushed further up by the MO-Hartree shifts. The amount of this electronic modification is normally determined by the orbital occupation(s) and by the values of U, U\u2032, JH relative to their respective LDA band width(s), and to the bare crystal-field splittings. As common to correlated electron systems, the dynamical correlations associated with U, U\u2032, JH results in a large-scale dynamical SWT. Here, small changes in the bare (LDA) band structure induced by MO electronic correlations  are sensitive functions of orbital orientation in the real crystal structure. Moreover, the 3U\u2009=\u20093.6\u2009eV and that of FeSe (see T) dependence of the dc resistivity computed using the orbital resolved LDA+DMFT spectral functions, Aa. Within the Kubo formalismdc-conductivity can be expressed asFinally, to elucidate close similarities between the metallic state found for FeS at FeSe see  as well d-bands, V is the unit cell volume, and f(\u03c9) is the Fermi function.where U\u2009=\u20093.6\u2009eV and JH\u2009=\u20090.7\u2009eV) derived using the LDA+DMFT spectral functions. In the same figure we also display our theory-experiment comparison of the normalized resistivity n\u2009>\u20096.0) shows the S-like shape characteristic of a pseudogaped metal similar to FeSe superconductorT, the FL-like T2 behavior is not seen in n\u2009=\u20096.0, \u03c1dc(T) becomes more metallic-like at low temperatures, smoothly suppressing the S-like form below n\u2009=\u20096.1. At low T, T2 form is also not observed upon partially electron doping the parent compound. The underlying reason for this behavior is that at small U, JH, the moderately correlated FL behavior cannot be destabilized by a small JH. For large U and sizable JH, however, the correlated-Landau Fermi-liquid scale, already driven to small values by sizable U, can be readily destroyed by U\u2032. As seen, with increasing total electron concentration n\u2009>\u20096.0 of the iron d shell, \u03c1dc(T) becomes more bad-metallicX  systems, showing similar metallic behavior in both cases . In order to highlight the possibility of a correlation induced proximity to an unconventional superconducting state in undoped FeS, in this figure we also display the experimental resistivity data of tetragonal FeSe superconductorn\u2009=\u20095.6 and U\u2009=\u20094.0\u2009eV, with decreasing the on-site Coulomb interaction \u03c1dc(T) of metallic FeS becomes more incoherent-like below T-dependence resembles that of the FeSe system.We now describe our electric transport results for metallic FeS. In n\u2009>\u20096.0) metallic FeS parent compound. An intriguing observation is that a strong orbital reconstruction occurs at small electron doping. Hence, based on our previous LDA+DMFT results for iron-chalcogenide systemsEF are visible for n\u2009=\u20096.0 and n\u2009=\u20096.1. Also interesting is low-energy electronic reconstruction near the Fermi energy with further increasing the averaged occupation number of the iron d shell, where a more quasicoherent normal state behavior sets in for electron-doping above n\u2009=\u20096.2. We propose that future photoemission and X-ray absorption spectroscopy results, which probe one-electron subtraction and addition spectra, can be directly compared with these: In particular, a broad incoherent peak below \u22120.4\u2009eV should be seen in both pure and doped cases. Additionally, drastic modification of the LDA+DMFT spectra at high binding energies with n is visible, notwithstanding large-scale dynamical spectral weight transfer common in all cases. These are stringent tests for our proposal, and experimental verification should place it on solid ground.In To summarize, we have used LDA+DMFT for a five-band Hubbard model to derive a correlation-induced orbital reconstruction in layered iron-chalcogenides. In particular, considering FeS as a suitable template, we have analyzed its paramagnetic insulating and metallic behavior, unraveling it as an effect of multi-orbital dynamical correlations. Also interesting is the orbital-selective electronic behavior obtained at the border of the Mott metal-insulator transition, where pseudogaped and Dirac fermions coexist at low energies. This coexistence arrises from charge-carrier scatterings due to interplay between multiband itinerance and electron-electron interactions, and this could be tested by a combination of spectral and transport measurements on metallic samples. Such studies are called for, and should confirm or refute our proposal of a marginal Dirac liquid for tetragonal FeS, which we ascribe to a lattice orthogonality catastrophed transition-metal chalcogenide, we use the multi-orbital iterated-perturbation-theory (MO-IPT) as an impurity solver of the many-particle problem in DMFT, as described in detail in refs To reveil insulating and metalic phases probed in electric transport experiments as well as the coexistence of pseudogaped and anisotropic Dirac-like electronic dispersion at the border of the Mott transition in tetragonal FeS, we employ the local-density-approximation plus dynamical-mean-field-theory (LDA+DMFT) which by construction takes into consideration the most relevant multi-orbital correlation effects and all-electron degrees of freedom. The state-of-the-art LDA+DMFTHow to cite this article: Craco, L. and Leoni, S. Selective orbital reconstruction in tetragonal FeS: A density functional dynamical mean-field theory study. Sci. Rep.7, 46439; doi: 10.1038/srep46439 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
{"text": "Finding of superconductivity in solid O2 on the border of an insulator-metal transition at high pressures close to 96\u2009GPa is thus truly remarkable. Neither the insulator-metal transition nor superconductivity are understood satisfactorily. Here, we undertake a first step in this direction by focussing on the pressure-driven insulator-metal transition using a combination of first-principles density-functional and many-body calculations. We report a striking result: the finding of an orbital-selective Mott transition in a pure p-band elemental system. We apply our theory to understand extant structural and transport data across the transition, and make a specific two-fluid prediction that is open to future test. Based thereupon, we propose a novel scenario where soft multiband modes built from microscopically coexisting itinerant and localized electronic states are natural candidates for the pairing glue in pressurized O2.Unusual metallic states involving breakdown of the standard Fermi-liquid picture of long-lived quasiparticles in well-defined band states emerge at low temperatures near correlation-driven Mott transitions. Prominent examples are ill-understood metallic states in  Particularly interesting examples of intriguing physics in solidized molecular phases of gases are dense hydrogen3 and solid oxygen5, as well as the most recent report of very high-Tc superconductivity in solid H2S under very high pressure6. H2 is predicted to metallize under high pressure, while solid O2 even shows a superconducting phase (Tc\u2009=\u20090.6\u2009K) at the border of a pressure-driven transition from a non-magnetic insulator to paramagnetic metal, joining the long list of materials exhibiting superconductivity proximate to metal-insulator transitions.The unique properties of high-pressure induced solid phases of molecular gases continue to evince keen and enduring interest in condensed matter physics. Beginning with early ideas of Mott\u03b1, \u03b4, \u03b5 and \u03b6 phases7. At lower pressure, the antiferromagnetically ordered \u03b1 phase transforms into another antiferromagnetically ordered \u03b4 phase at 5.4\u2009GPa, followed by a non-magnetic \u03b5 phase at 8\u2009GPa. Higher pressure, 8, followed by emergence of superconductivity below 9. This astounding behavior in a molecular system, reminiscent of strongly correlated, doped Mott insulators in d-band oxides like cuprates, presents a significant challenge for theory. The high-P \u03b5\u2013\u03b6 phase transition is also accompanied by significant volume reduction10, with a contraction of about 10% of the lattice parameter along the b direction. The \u03b5 phase retains the layered nature of the lower pressure phases4, and the monoclinic (C2/m) structure11 as shown in Fig.\u00a0Pressurized molecular oxygen forms various low-temperature solid phases under pressure, labelled mentclass2pt{minim\u03b1\u2013\u03b2 transition at moderate T is dominantly magnetic has been established in a series of careful studies14. Indeed, early work of da Silva and Falicov15 already explained the measured heat of formation at the \u03b1\u2013\u03b2 transition in terms of the entropy difference computed from cluster analysis of a multi-orbital Hubbard model . Observation of very different magnetic orders in the \u03b1, \u03b2 phases, correlation between magnetic and structural changes along with ferromagnetic coupling between the off-plane near neighbors in the \u03b4 phase are reminiscent of those found in classic multi-band systems like V2O316, taken together with the above, favor a multi-orbital description. Additional evidence for multi-orbital effects is provided by the anisotropic and partially discontinuous pressure-induced changes in the lattice parameters in the different phases17. In such a scenario, increasing pressure is expected, in the simplest approximation, to decrease lattice spacings and increase the carrier itinerance. The result would then be to suppress antiferromagnetic order along with insulating behavior, and to induce metalization. In solid O2, antiferromagnetic order is destroyed well before metalization occurs18, and so, within the p4 configuration of oxygen, the insulator-metal transition across the \u03b5\u2013\u03b6 transition must be regarded as a Mott metal-insulator transition. This suggests that on the one extreme, a Heisenberg model description is only valid in the insulating \u03b1, \u03b2, \u03b4 phases, and that a more general multi-orbital Hubbard model must be used, at least for the \u03b5 phase. At the other extreme, one-electron band structure calculations for the antiferromagnetically ordered phases do provide qualitatively correct ground states19. In addition, electronic structure calculation based on generalized gradient approximation (GGA) shows that the nonmagnetic insulating state is energetically favored at pressures corresponding to the \u03b5-phase21. However, by construction, ab initio density-funtional calculations have intrinsic difficulties in describing non-magnetic insulating phases, and in particular the \u03b5 phase23, for reasons described in detail in ref. That the driving force for the e.g., by holes, creates two available states at the Fermi energy. This is at the heart of spectral weight transfer, a phenomenon ubiquitous to Mott, as opposed to band, insulators. In both cases, electron hopping might still be prevented by inter-orbital Coulomb interactions in a multiband system. The resulting metallic state upon doping can vary from a Fermi liquid at weak coupling to an exotic orbital-selective, non-Fermi liquid metal for stronger electron-electron interactions, as doping and temperature25 are varied. This fundamental difference between band and multi-orbital Mott-Hubbard insulators is of basic and practical interest. Below we show that sizable multiband electronic interactions are the clue to the insulating state of the \u03b5-phase of solid oxygen and its evolution to a non-Fermi liquid metallic state at high pressures.Before presenting our local-density-approximation plus dynamical-mean-field (LDA\u2009+\u2009DMFT) results, we point out essential differences between band and Mott insulators. In conventional semiconductors (or band insulators) all bands below the Fermi energy are filled and, therefore, inert. Removing an electron leads to an empty state which can be thought of as a hole moving freely through the solid. The same is true for an added electron, which occupies the first empty band. In a multi-orbital Mott-Hubbard insulator, the insulating state arises because electron hopping from one site to another is inhibited by intra- and inter-orbital Coulomb repulsions. In these systems, when the band filling is slightly reduced from its commensurate value, a small number of unoccupied states are created; similarly adding electrons creates locally doubly occupied electronic states. The crucial difference in this case is that since the doped carriers can have either spin  with equal probability, doping a Mott insulator, p29 or s30 band systems is very intriguing, since the naive expectation dictates that the itinerance  is appreciable compared to the electron-electron interactions, as distinct from d-band systems, where the d electrons reside in much narrower bands 31. Thus, understanding Mottness (or the proximity to a Mott-Hubbard insulating state) in materials with active p or s bands is undoubtedly an issue of great contemporary interest32. In light of the discussion above, we study how an orbital-selective interplay between appreciable p-band itinerance and sizable, on-site Coulomb repulsion, U, plays a central role in this unique Mott transition in solid O2.Possibility of Mott-Hubbard physics in purely 2, we start with the C2/m structure 35 scheme in the atomic sphere approximation36. The corresponding LDA density-of-states of the three (symmetry) inequivalent atoms11 is shown in Fig.\u00a0pz, i.e., the \u03c3-orbital37 in the energy level diagram of O2. However, due to inter-molecular orbital overlap in the monoclinic structure, the pz states acquire some itinerance, explaining the small amount of pz states found at the Fermi energy. As seen in Fig.\u00a0\u03c0-bands cross the Fermi energy, providing a metallic state within LDA.To quantify the correlated electronic structure of solid Oure Fig.\u00a0 with lata\u2009=\u2009x, y, z label the three diagonalized p orbitals and the \u0394a are on-site orbital energies in the real structure of solid O2. In light of antiferromagnetic insulator15 phases and the non-magnetic Mott transition, local multi-orbtial interactions are mandatory to understand O2. These constitute the interaction terms JH is the Hund\u2019s rule term. Following da Silva and Falicov15, we use U\u2009=\u200911.6\u2009eV and JH\u2009=\u20090.45\u2009eV, along with the LDA bands of the three inequivalent oxygen atoms described above. In this work, the correlated multi-orbital problem of solid O2 encoded in H\u2009=\u2009H0\u2009+\u2009Hint is treated within the state-of-the-art local-density-approximation plus dynamical-mean-field-theory (LDA\u2009+\u2009DMFT) scheme31. The DMFT self-energy, 31. We use the multi-orbital iterated-perturbation-theory (MO-IPT) as an impurity solver for DMFT38: This analytic solver has a proven record of successes in describing finite temperature Mott transitions39 as well as unconventional behavior in correlated p-band systems42.Within LDA, the one-electron part of the many-body Hamiltonian for solid oxygen is now p-bands in different ways. Generically, one expects partial (Mott) localization of a subset of bands, leading to orbitally selective Mott transitions, and bad metallic states43. Within LDA\u2009+\u2009DMFT, this orbital-selective mechanism involves two renormalizations: static  renormalization shifts the p-bands relative to each other by amounts depending upon their bare on-site orbital energies (\u0394a) and occupations (na). In addition, dynamical effects of U, 43. The large, anisotropic changes in dynamical spectral weight transfer in response to small changes in bare one-particle (LDA) parameters 39 are known to drive the orbital-selective Mott transition in real multi-orbital systems. As we show below, precisely such an orbital-selective Mott transition, accompanied by an incoherent metallic phase in solid O2, occurs at very high pressures.With orbital orientation-induced anisotropic LDA one-particle energies and hoppings, multi-orbital correlations renormalize various U, U\u2032, JH as obtained in ref. \u03b5 phase is a Mott insulator, as shown in Fig.\u00a0px band compared to the other two. Large spectral weight transfer, characteristic of dynamical local correlations, is explicitly manifested in the qualitative difference between LDA and LDA\u2009+\u2009DMFT spectra. At first sight, derivation of a Mott insulator with U\u2009<\u2009W in solid O2 seems a bit puzzling. The reason, however, is that, in this multi-orbital system, both U, U\u2032 are appreciable, and the combined effect of both acting in tandem is to (i) reduce the band-width of each band , and (ii) the dominant effect of U\u2032 on a reduced bandwidth is to split the bands via the Mott mechanism. In the actual multi-orbital problem, both effects are simultaneously operative, and reinforce each other.Using \u03b4a) and occupations (na) within LDA and LDA\u2009+\u2009DMFT. Within LDA, we find 44, is an interesting manifestation of correlation-induced orbital rearrangement, and controls structural changes across the Mott transition (see below).From our results in Fig.\u00a02 at high P, and adopt the following strategy to derive this transition. Instead of reverting back to the LDA to use a different LDA density-of-states corresponding to the metallic \u03b6-phase, we search for an instability of the insulating, \u03b5 phase to the paramagnetic-metal by varying \u03b4a, found for the Mott insulator above. To proceed, consider the orbital-dependent on-site energy term, x\u2009=\u2009\u2212\u0394, \u0394y\u2009=\u2009\u0394, to simulate the structural  changes upon pressure. Here, \u0394a acts like an external field in the orbital sector , sensitively controlling the occupations of each orbital in much the same way as the magnetization of a paramagnet as function of an external magnetic, Zeeman field. However, in order to draw a qualitative interpretation of H\u0394 in our total model Hamiltonian 2, we recall that the pressure derivative of crystal-field splitting is given by K is the bulk modulus and \u03be is a constant value45. This in turn suggests that in the pressure range of interest in this work solid O2 could be in a linear regime as observed in other materials under external pressure conditions47. As for V2O339, YTiO343 and, more recently, for FeS48 we search for the second self-consistent LDA\u2009+\u2009DMFT solution by solving the multi-orbital DMFT equations for each trial value of \u0394 keeping U, U\u2032 fixed. As seen in Fig.\u00a0px and py are most severely affected. At a critical \u0394c\u2009=\u20090.3 eV, the px density-of-states remains Mott insulating, while the py band undergoes an insulator to bad-metal  transition. Thus, our results imply that the paramagnetic, metallic phase of \u03b6-oxygen is an orbital-selective incoherent metal without Landau quasiparticles, characterized by a pseudogap at EF in the py, and hence, in the total spectral function, at EF. Our simulations (not shown) indicate that Kondo-like resonance found below EF for \u0394\u2009=\u20090.4\u2009eV will cross the Fermi level at extremely high pressures, driving solid O2 into (quasi)coherent Fermi liquid-like metallic state at even higher  pressures. The underlying theoretical reason for this is as follows: At \u0394c, strong scattering between the effectively (Mott) localized and itinerant components of the matrix DMFT propagators produces an incoherent metal because strong interband scattering indeed operates in a sizably orbitally polarized metallic system. However, at very high pressure (large \u0394\u2009>\u2009\u0394c), the px band becomes almost fully polarized  localized and itinerant components of the full DMFT matrix propagators. This behavior is intimately linked to orbital-selective Mott-like physics within DMFT50.Our results in Fig.\u00a0\u03b4a across the insulator-metal transition. The features are well understood as follows. In a multiband situation, \u0394 acts like an external \u201cZeeman\u201d field43 in the orbital sector. The insulator-metal transition is characterized by a sudden jump in the renormalized \u03b4z, and in the px and py populations as a consequence, suggesting that anisotropic structural (and volume) changes will accompany the orbital-selective Mott transtion. Here, we propose that these changes in na control anisotropic changes in lattice parameters (a\u2013c) across the insulator-metal transition: indeed, the changes in a\u2013c are expressible in terms of na as g is the electron-phonon coupling constant, M the ion mass, and vsa is the velocity of sound along a. Changes in \u03b3a across the insulator-to-metal transition thus follow those in the na. Though values of vsa and g in the \u03b5 phase are unknown, we deduce that the lattice parameter a increases, while b,c descrease across the orbital-selective Mott transition as in Fig.\u00a010 provides further support for our Mottness scenario in solid O2.In Fig.\u00a051. In our theory, the observed features in \u03c1dc(T) originate from changes in the correlated spectral functions with \u0394. Showing how this provides a compelling description of the admittedly limited available data is our focus in what follows. In Fig.\u00a0\u03c1dc(T) for three values of \u0394 in solid O2, computed using the LDA\u2009+\u2009DMFT orbital resolved spectral functions . Various interesting features immediately stand out. First, \u03c1dc(T\u2009\u2192\u20090) in the \u03b5 phase (\u0394\u2009=\u20090) shows semiconducting behavior, in accord with the insulating classification at lower pressures, when \u0394\u2009<\u2009\u0394c. Secondly, at all T, no Fermi liquid T2-like contribution is detectable in the metallic phase with \u0394\u2009=\u20090.4\u2009eV: instead, \u03c1dc(T) is approximately constant up to 10\u2009K. For intermediate pressure , \u03c1dc(T) crosses over from semiconductor-like (at high T) to bad-metallic (at low T) behavior. Remarkably, the detailed T-dependence closely resembles that seen in experiment9, in the normal state up to 2\u2009K  for both values of \u0394\u2009=\u20090.2, 0.4\u2009eV is characteristic for carriers scattering off dynamically fluctuating and coupled, short-range spin and charge correlations. On general grounds, we expect this effect to be relevant near a correlation-driven Mott transition. Since an external magnetic field will generically quench spin fluctuations, we predict that destroying the \u03b5 Mott insulating state by a magnetic field52 might reveal this behavior. We emphasise that resistivity measurements as a function of pressure over extended T scales is a smoking gun for our proposal, as would be the study of the T-dependence of the dc Hall constant. These can distinguish a band-versus-Mott scenario for the pressure-induced insulator-metal transition: in the band-insulator-to-metal transition, there is no reason why, e.g., \u03c1dc(T) should show the above form, since neither orbital selectivity nor local antiferromagnetic spin fluctuations are operative there. More detailed transport work to corroborate our prediction are thus called for in future.To illustrate the importance of correlation-induced changes in the orbital \u201cZemman\u201d field \u0394 under high pressure in our theory, we now discuss our results for the normal state resistivity computed within the Kubo formalismpx sector, this electronic system is inherently unstable to soft two-particle instabilities53. In solid O2 at high pressure, lack of conditions supporting magnetic and charge-density instabilities in the correlated electronic structure near the insulator-metal transition opens the door to superconductivity as the only two-particle instability that can quench this finite normal state entropy. In fact, the situation is quite similar to that considered by Capone et al.54 in the fulleride context, where the pseudogapped, bad metal arose from an unstable (intermediate coupling) fixed point in the impurity problem, corresponding to the Kondo unscreened phase: in our case, precisely the same effect results from selective localization of the px band at the orbital-selective Mott phase. In fact, in the orbital-selective metal, the low-energy physics is selfconsistently controlled by strong scattering between quasi-itinerant py,z and Mott localized px orbital states, implying low-energy singularities in one- and two-particle propagators55. This suggests soft, multi-orbital electronic modes at low energy, which can potentially act as a pair glue. In a way similar to the fulleride case, we then expect that multiband spin-singlet s-wave superconductivity (notice that S\u2009=\u20090), driven by such soft inter-orbital electronic fluctuations in this unstable phase, will cut off the incoherent metal found above, and that the superconducting transition temperature Tc will rise to values larger than those obtained for the weakly correlated case54. Interestingly, the variation of Tc with decreasing U/W (increasing pressure) found by Capone et al. does bear uncanny resemblance  observed in solid O2 under high pressure. This is suggestive, but out of scope of the present work. We leave details for the future.Insofar as superconductivity arises at the boundary of the Mott transition, our analysis provides tantalizing insight into sources of the pairing glue. Since the incoherent metal has a finite residual entropy (2. In view of the complexity of the problem (as discussed below), we only restrict ourselves to a qualitative discussion. Given selective incoherent metallic state within DMFT in our case, residual, inter-site and inter-orbital (in multiband systems) two-particle interactions can generate ordered states directly from the bad metal. Following the philosophy used earlier53 for the iron-pnictides superconducting systems we restrict ourselves to the y, z orbital sector. In this situation the interaction in the Cooper channel reads a, b\u2009=\u2009y, z and the scattering vertex is Hpair in the particle-particle channel using Gorkov\u2019s formalism56 gives 2. A plausible assuption to be made here is that the superconducting gap function has no nodes and therefore it can be taken to be momentum independent, i.e., s-wave case of solid O2. However, as shown below this assumption has profound effects in the multiband excitation spectrum withing the s-wave superconducting phase.Our findings put constraints on mechanisms of superconductivity in solid O2 across the superconducting phase transition we have extented our normal state electronic structure calculation to treat Hpair above within LDA\u2009+\u2009DMFT formalism for the superconducting state53. In fact, using our assumption for the s-wave superconducting gap function \u0394SC the LDA\u2009+\u2009DMFT equations are readily extendable to the superconducting regime. As in ref. Gab) have normal and anomalous components yielding remormalized Gaa propagators, which are solved by extending the normal state LDA\u2009+\u2009DMFT solution to include an explicit pair-field term. Including the pair-field, the DMFT propagators are writen as ref. p-orbitals of oxigen, the opening up of a superconducting gap in a particular p-band might induce secondary gaps in the remaining p orbitals, in a way reminiscent of the inter-band proximity effect induced by U\u2032 and JH in multiband supeconductors. With this caveats in mind, we now describe our results within the superconducting state of solid O2. Using the normal state LDA\u2009+\u2009DMFT solution for \u0394\u2009=\u20090.4\u2009eV ] prevents opening up of a clean superconducting gap in the electronic spectrum. Remarkable as well is the fact that the insulating px channel is not affected at low energies by the superconducting instability, and this is strictly related to the fact that in this electronic channel the excitation spectrum is already gapped in the normal state. Moreover, weak spectral weight transfer from low- to high-energies occurs in all p bands as seen in Fig.\u00a057. Future tunneling spectroscopy (dI/dV) measurements are also called for to corroborate our predition for the changes in the total one-particle spectral function  and Mott localized (px) states arising at this orbital-selective Mott transition act as the pairing glue for the superconducting state found at low T in solid O2. Our work underlines the importance of local dynamical correlations in this molecular-solid system, and holds promise for understanding similar physics in other solidified gases.In conclusion, we have theoretically studied the insulator-metal transition in highly pressurized solid O31, which correctly takes disorder, temperature and pressure effects into account in numerous strongly correlated multiband systems39. Within our scheme, the LDA band structure calculations were performed for the experimental crystal (C2/m) structure of solid O2 using the non-fully relativistic version of the PY-LMTO code35. To incorporate the effects of dynamical electronic correlations in solid O2, we use the multi-orbital iterated-perturbation-theory (MO-IPT) as an impurity solver of the many-particle problem in DMFT58. This method has recently been benchmarked by comparison with merically exact continuous time quantum Monte Carlo method (CTQMC) as an impurity solver in DMFT58. These authors find very good accord between MO-IPT and CTQMC for both one-band and multi-band Hubbard models when realistic, non-negligible crystal field splittings are included. Thus, with sizable effects of the real crystal field splitting in solid O2, our IPT solver is sufficiently accurate for quantifying the effects of sizable electronic correlations in solid O2. LDA\u2009+\u2009DMFT is thus to be viewed as a combined density functional plus dynamical mean-field (DMFT) scheme31, and adequately describes the effect of local dynamical interactions in the limit of large lattice dimensions (DMFT)59. Thereby, practical calculations become feasible even for complex multiband systems like solid O2. The basic idea in a DMFT solution involves replacing the lattice model by a self-consistently embedded MO-Anderson impurity model, and the self-consistency condition requiring the local impurity Green\u2019s function to be equal to the local Green\u2019s function for the lattice. The full set of equations for the MO case can be found in refs ansatz that connects the two exactly soluble limits of the one-band Hubbard model60, namely, the uncorrelated (U\u2009=\u20090) and the atomic (\u03b5k\u2009=\u20090) limits. At intermediate U, it ensures the correct low-energy behavior of the self-energies and spectral functions by strict observance of the Friedel-Luttinger sum rule in the impurity solver (we have used MO-IPT). It thus accounts for the correct low- and high-energy behavior of the one-particle spectra by construction. It ensures the Mott-Hubbard metal-insulator transition from a correlated FL metal to a Mott-Hubbard insulator occurs as a function of the Coulomb interaction U. Compared to numerically expensive QMC solvers, IPT-based schemes are known to be computationally very efficient, and readily yield real frequency data at zero and finite temperatures without the need to perform numerical analytic continuations, which are known to be a very delicate matter in QMC schemes. Finally, we have carried out the computation of electrical transport within the Kubo formalism51.To unveil the electronic reconstruction at the border of the Mott metal-insulator transition in solid Oxygen, we employ an implementation of the local density plus dynamical mean-field (LDA\u2009+\u2009DMFT) scheme"}
{"text": "The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity. Restriction factors are cellular proteins that inhibit viral replication and represent a first line of defense against viral pathogens. The APOBEC3 (A3) family of restriction factors plays an important role in blocking retroviral infections. HIV-1 encodes the Vif protein to antagonize A3, which allows spread of virus in host and ultimately development of AIDS. Prior studies indicate that Vif hijacks host proteolysis pathways to degrade A3 restriction factors; however, our work demonstrates that Vif can neutralize A3s in a degradation-independent manner. These findings suggest viral suppressors of innate immunity work by multiple mechanisms to ensure robust replication. Knowledge of such mechanisms is critical for development of therapeutic strategies to restore the ability of the immune system to cripple viral infections. Despite tremendous advances in control and prevention, HIV has remained a persistent worldwide health concern. Current anti-retroviral treatments often use a combination of drugs that target key viral enzymes such as the protease, integrase, and reverse transcriptase . The exiThe HIV protein Viral Infectivity Factor (Vif) has become recognized as a promising therapeutic target \u20133. The pin vivo, inhibition of Vif is an attractive strategy to restore the restrictive function of A3 family members and in turn cripple HIV infection. We and others have demonstrated that pharmacological inhibition of Vif unleashes the restriction potential of A3 family members [As Vif is required for viral replication both in cell culture and  members \u201320; howe members ,22. TherVif, the transcription co-factor CBF\u03b2, and adapter proteins ELONGIN B and C  to the minimal Vif complex capable of binding A3 family members. This complex, termed VCBC, consists of HIV-1  B and C . Through10 to identify Fabs against VCBC [D) in the low nanomolar range as determined by BioLayer Interferometry (BLI)  , S2 Fig.in vitro using either the Vif-binding, C-terminal domain of A3F (A3F-CTD) or full-length A3G  were generated for intra-cellular expression and tested for their ability to disrupt Vif-mediated degradation of A3C, A3F, and A3G  assays and found that the presence or absence of 3C9 had no affect on the VCBC/A3F-CTD interaction. For the direct binding assay, fluorescently labeled A3F-CTD was titrated with either VCBC or VCBC-3C9, and the Kdentical . For thedentical . Second,dentical .Cullin-RING E3 ligases catalyze ubiquitin transfer by orienting substrate lysines toward an activated E2~Ub thioester that is bound by the RING-Box subunit ,28. One Taking a similar approach as we did with 3C9, we assessed the Vif-mediated ubiquitination of Myc-tagged A3F-CTD in the presence of Flag-tagged 1D1. In contrast to 3C9, 1D1 was not ubiquitinated even though it inhibited A3F-CTD ubiquitination . 1D1 mayMultiple groups have shown that disrupting A3 ubiquitination by mutating the A3 binding surface on Vif, by expressing a dominant negative CUL5 variant, or by pharmacologically inhibiting the E3-ligase restores cellular A3 levels and results in A3 packaging ,29. Simi2+ to help stabilize this inter-domain region of Vif.To determine the structural basis for VCBC antagonism by 3C9 and 1D1, we used negative stain electron microscopy (NSEM) to define the VCBC-3C9-1D1 complex at low resolution . InitialIn constructing a molecular model, the crystal structure of VCBC (PDB: 4N9F) and homology models of 3C9 and 1D1 were fitted into the EM map . The speThe NSEM data revealed the 3C9 and 1D interacting patches on the surface of VCBC, and both Fabs appear to bind VCBC across composite protein-protein interfaces . The 3C9To identify structural regions that may contribute to 3C9 \u201cshielding\u201d we modeled full-length CUL5/RBX2 on the VCBC-3C9-1D1 structure and highlighted Vif residues known to be important to A3F binding . Indeed,Several studies have indicated that inhibition the Vif E3-ligase complex can restore A3 restriction and block viral replication, but there are no known drugs to date that directly target Vif. In this study, we developed Fabs that bind to the HIV-1 VCBC complex and demonstrated the utility of these Fabs as selective reagents to probe Vif function. Our panning campaign resulted in six Fabs that bound to VCBC with high affinity, of which 3C9, 3F12, and 1D1 were selected for additional studies as they recognize unique epitopes on VCBC. We initially categorized the Fabs based on their ability to disrupt Vif-mediated A3 ubiquitination, as Fabs that disrupt A3 ubiquitination would provide valuable information regarding Vif inhibition strategies, and Fabs that do not perturb A3 ubiquitination may be useful reagents for structural studies. Indeed, we have isolated Fabs that have no effect on A3 ubiquitination (3F12), inhibit A3F/A3C ubiquitination (3C9), or inhibit all A3 ubiquitination (1D1).We show that 3C9 is able to selectively inhibit the ubiquitination of A3F/A3C, but not A3G, indicating the feasibility of targeting specific A3 sites on Vif . ImportaWe identified a second biologically relevant Fab, 1D1, which acts as a pan-inhibitor of A3 ubiquitination. Structurally and biochemically, we have shown that 1D1 disrupts the VCBC-CUL5 interaction, thereby preventing A3 ubiquitination and degradation. Initially, this seemed like a promising strategy to antagonize Vif as it would 1) restore the levels of all A3 proteins degraded by Vif, 2) be potentially amenable to small molecule inhibition as the Vif-CUL5 interface is the smallest protein-protein interface in the Vif complex, and 3) may avoid off-target effects as Vif uses a non-canonical Cullin-binding motif to interact with CUL5 \u201333. To oThe prevalent mechanism for Vif-mediated antagonism of A3 is through ubiquitination and proteasomal degradation. Previous studies investigating A3 mutants and cell-to-cell variability have suggested that a degradation-independent mechanism exists ,34\u201337. DIn summary, we have developed a diverse panel of Fabs for use as biochemical probes to better understand Vif antagonism of the innate immunity provided by A3 restriction factors. These studies support the feasibility of pursuing Vif as a therapeutic target, but indicate that inhibition of ubiquitination by the Vif E3-ligase alone may not be sufficient to rescue A3 packaging. Importantly, our data establish an degradation-independent mechanism by which Vif prevents A3 packaging, and this together with previous studies show that Vif repression of A3 packaging is a multilayered process involving both degradation dependent and independent mechanisms ,29,38,39All constructs were generated by standard PCR and restriction-based cloning methods unless otherwise noted. For mammalian expression, scFv3C9 and scFv1D1 with C-terminal 3x-Flag tags were cloned into pcDNA4 (Invitrogen). Human A3C, A3F, and A3G expression constructs with C-terminal 3xHA tags have been previously reported . Env-defE. coli. Recombinant Fabs were expressed from phagemid as described [6-thioredoxin (Trx)-tagged CBF\u03b2/Vif and ELOC/ELOB were co-expressed from pET-Duet and pCDF-Duet plasmids, respectively. HIS6-GB1-tagged CUL5-RBX2 was co-expressed from a pRSF-Duet plasmid. SOCS4 and ELOC/ELOB were co-expressed from pET28a and pCDF-Duet plasmids, respectively. HIS6-Trx-myc-tagged A3F-CTD was expressed from a pHisTRX plasmid. For E. coli expression, plasmids were transformed into E. coli BL21(DE3) (Invitrogen) cells and grown at 37\u00b0C to an optical density of 0.6\u20130.8 and induced with 0.5 mM IPTG overnight at 18\u00b0C (or 1mM IPTG at 20\u00b0C for Fabs).All proteins, except A3G, were expressed in escribed . HIS6-thVif-CBF\u03b2 complex, and the SOCS4-ELOBC complex were obtained as described previously [Purified VCBC, 6-protein CRL5eviously ,22. Fabseviously . BrieflyA3G was C-terminally myc-tagged followed by a TEV cleavage site and GFPII and expressed in baculovirus infected Sf9 cells. Purification was carried out as previously described . Minor m10 to identify Fabs against VCBC (23). Fabs were isolated using a phage display panning protocol descirbed in detail by Kim et al, 2011 [We used a previously published human na\u00efve B cell phage library with a diversity of 4.1 x 10al, 2011 . Brieflyal, 2011 . A fullynst VCBC . Fabs weal, 2011 . The panal, 2011 . Clones The ELISA experiments were done as described previously . All theKinetic constants for VCBC Fabs were determined using an Octet RED384 instrument (ForteBio). Four concentrations of each Fab were tested for binding to the biotinylated VCBC complex immobilized on ForteBio streptavidin SA biosensors. All measurements were performed in 20 mM HEPES pH 7.4, 0.5 M NaCl with 0.1% (w/v) bovine serum albumin (BSA) and 0.02% (v/v) Tween 20 at room temperature in 384-well microplates. Data were analyzed using a 1:1 interaction model on the ForteBio data analysis software 8.2.in vitro ubiqutination of A3F-CTD and A3G were done as previously reported [NL4-3 E3 holoenzyme, 2\u20134 \u03bcM myc-tagged A3F-CTD or myc-tagged A3G, and the ubiquitin activating system containing: 1.2 \u03bcM ATP, 1.2 \u03bcM ubiquitin activating enzyme (UBE1), 45 \u03bcM ubiquitin, 4.8 \u03bcM E2 (CDC34). Ubiquitination reactions done in the presence of Fabs contained 0.1 \u03bcM, 0.5 \u03bcM, or 1\u03bcM of indicated Fab. Ubiquitinated A3 proteins were detected using a monoclonal anti-c-myc antibody (Sigma).Vif-mediated reported . BrieflyHuman Embryonic Kidney (HEK) 293T cells (ATCC) were maintained in Dulbecco\u2019s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin (P/S).At 50% confluency, HEK293T cells were transfected  with 1 \u03bcg Env-deficient HIV-1 NL4-3 proviral construct in the presence or absence of 25 ng of the indicated C-terminal HA-tagged APOBEC3 expression construct, and either 0, 50, 100, or 250 ng of the Flag-tagged scFv expression construct. Alternately, cells were transfected with 1 \u03bcg Env-deficient HIV-1 NL4-3 proviral construct in the presence or absence of 50 ng of the C-terminal HA-tagged APOBEC3G expression construct and in the presence or absence of 100 ng of the Flag-tagged CUL5 or ELOC expression construct. After 48 hours, viral supernatants and the cells were processed for immunoblotting. Cell lysates were prepared by resuspension of washed cell pellets directly in 2.5x Laemmli Sample Buffer , and homogenization at 95\u00b0C for 30 minutes. Virus-like particles were isolated from culture supernatants by purification through 0.45\u03bcm PVDF filters (Millipore) followed by centrifugation  through a 20% sucrose, 1x PBS cushion and lysis directly in 2.5x Laemmli Sample Buffer.Samples were run on 4\u201320% Tris-HCl SDS-PAGE gels (BioRad Criterion) at 150V for 90 minutes. Proteins were transferred to PVDF membranes by methanol-based electrotransfer (BioRad Criterion Blotter) at 90V for 2 hours. Membranes were blocked in 4% Milk in PBS, 0.1% Tween-20 overnight prior to overnight incubation with primary antibody against HA , Vif (NIH ARRRP 809), Flag (Sigma F7425), GAPDH (Sigma G8795), p24/capsid (NIH ARRRP 3537 courtesy of B. Chesebro and K. Wehrly). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (BioRad) were detected using Hyglo HRP detection reagents (Denville Scientific). Blots were incubated in a 1xPBS, 0.2M glycine, 1.0% SDS, 1.0% Tween-20, pH 2.2 stripping buffer before reprobing.A3F-CTD was labeled with maleimide\u2013conjugated fluorescein at 4\u00b0C for 12 hrs in 20 mM HEPES pH 7, 300 mM NaCl, 1 mM TCEP. For the direct binding assays, labeled A3F-CTD was diluted to 20nM and incubated with increasing amounts of VCBC or VCBC/3C9 for 20min at 25\u00b0C in 20 mM HEPES pH 8, 300 mM NaCl, 5% glycerol, 1 mM DTT. For the competition assays, VCBC was pre-bound with labeled A3F-CTD, and unlabeled A3F-CTD or 3C9 were titrated into the pre-assembled complex. Fluorescence polarization measurements were made using an Analyst plate reader (LJL Biosystems) with excitation and emission wavelengths of 485 nm and 530 nm, respectively. Titration assays were performed in duplicate, and all data were analyzed in SigmaPlot using equations derived as previously described .\u22129 M. For all experiments, 3 \u03bcL of the sample was applied to a glow-discharged, carbon-coated, 400 mesh Cu grid and stained with 2% (wt/vol) uranyl formate as previously described [1D13C9-VCBC was diluted to a final concentration of 50 \u00d7 10escribed . Images escribed  at a nomData was processed in one of two ways. For initial 2D class averages of VCBC fab combinations images were binned by a factor of two for particle picking and subsequent image processing. Individual particles were manually picked, extracted at a box size of 64 pixels and normalized to have a mean of 0 and a standard deviation of 1. Particles were subject to two-dimensional classification using Relion . For 3D The PIGS server for the automatic prediction of antibody structures was used to identify fab structures with the highest sequence homology to 3C9 and 1D1 and to generated a predicted structure for the variable regions of 3C9 and 1D1 . To obtaPulldown assays were performed in buffer containing 20 mM HEPES pH 8, 300 mM NaCl, and 10% glycerol at 25\u00b0C. MBP-tagged VCBC WT or mutant proteins were immobilized on amylose resin. Resin bound MBP-tagged VCBC was incubated with excess purified CUL5-NTD and/or 1D1 protein and subsequently washed. Pulldown samples were separated on a SDS\u2212PAGE gel and stained with coomassie blue dye.S1 Fig(A) Sequence alignment of HIV-1 NL4-3, HXB2, and consensus Vif. (B-C) HIV-1 infectivity data comparing WT and consensus Vif in the absence or presence of (B) A3G and (C) A3F.(TIF)Click here for additional data file.S2 FigPrimary sequences for the (A) light and (B) heavy chain CDRs for Fabs 1D1 and 3C9.(TIF)Click here for additional data file.S3 FigIn vitro ubiquitination of Myc-tagged A3F-CTD or A3G in the absence or presence of increasing amounts of (B) 3C9 and scFv3C9 or (C-D) 1D1 and scFv1D1.(A) Cartoon depiction of an antibody, a fab, and a scFv. The heavy (blue) and light (orange) chain constant and variable regions are highlighted in the fab and scFv. (B-D) (TIF)Click here for additional data file.S4 FigEnv-deficient NL4-3 HIV (either Vif+ or Vif-) was co-transfected with A3C and a gradient of (A) scFv3C9 or (B) scFv1D1.(TIF)Click here for additional data file.S5 Fig(A) Fluorescence polarization competition assay. Pre-bound fluorescently labeled A3F-CTD with VCBC was titrated with either 3C9 (red) or unlabeled A3F-CTD (black). (B) Simulated competition curves establish that 10 \u03bcM of 3C9 and 100 \u03bcM of A3F-CTD are sufficient to compete with the labeled A3F-CTD bound to VCBC. (C) SEC elution profile for VCBC/3C9/Trx-A3F-CTD (black), VCBC/3C9 (red), VCBC/Trx-A3F-CTD (blue), VCBC (green), Trx-A3F-CTD (orange). SDS-PAGE gel of peak fraction for VCBC/3C9/Trx-A3F-CTD.(TIF)Click here for additional data file.S6 Fig(A) SEC profile and corresponding SDS-PAGE gel for VCBC-3C9-1D1. (B) Representative NSEM micrograph and (C) 2D class averages for VCBC-3C9-1D1.(TIF)Click here for additional data file.S7 FigEM maps aligned on (A) 1D1 or on (B) 3C9 reveal that the two conformations arise due to flexibility in VCBC. (C) The CBF\u03b2 and ELOC binding regions of Vif are boxed off. Postulated \u201chinge\u201d connects these two regions and is comprised of the Zn+ binding motif and three interdomain loops.(TIF)Click here for additional data file.S8 Fig(A) NSEM 2D class averages and corresponding cartoon depictions of VCBC-Fab complexes show that 3F12 and 1D1 bind the same side of VCBC, and that 3C9 binds the opposite side of VCBC from 1D1 and 3F12. (B) SD200 elution profile and corresponding SDS-PAGE gels show that 3F12 is able to form a stable complex with SOCS4/ELOBC. (C) Purified VCBC complex resolved by SDS-PAGE gel. Myc-tagged Fabs were used as a primary antibody to WB for individual VCBC components. 3F12 is able to detect ELOB in a WB. The asterisk indicates a non-specific protein.(TIF)Click here for additional data file.S9 Fig(A) SD200 elution profile and corresponding SDS-PAGE gels for (B) SOCS4/ELOBC and SOCS4/ELOBC in the presence of (C) 3C9 or (D) 1D1. (E) SD75 elution profile and corresponding SDS-PAGE gels for (F) CBF\u03b2 and CBF\u03b2 in the presence of (G) 3C9 or (H) 1D1.(TIF)Click here for additional data file."}
{"text": "Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.The repeat region of the  Vaccines aim to protect by inducing the immune system to make molecules called antibodies that can recognize molecules on the surface of invading pathogens. In the case of malaria, our most advanced vaccine candidates aim to promote the production of antibodies that recognize the circumsporozoite protein (CSP) molecule on the surface of the invasive parasite stage called the sporozoite. In this report we use X-ray crystallography to determine the structure of CSP-binding antibodies at the atomic level. We use other techniques such as isothermal titration calorimetry and structural modeling to examine how this antibody interacts with the CSP molecule. Strikingly, we found that each CSP molecule could bind 6 antibodies. This finding has implications for the immune response and may explain why high titers of antibody are needed for protection. Moreover, because the structure of the CSP repeat is quite simple we determined that the number of different kinds of antibodies that could bind this molecule are quite small. However a high avidity interaction between those antibodies and CSP can result from a process called affinity maturation that allows the body to learn how to make improved antibodies specific for pathogen molecules. These data show that while it is challenging for the immune system to recognize and neutralize CSP, it should be possible to generate viable vaccines targeting this molecule. Plasmodium falciparum causes the deaths of around 430,000 people each year [Malaria caused by ach year . The mosach year . Phase IPlasmodium species are separated by a repeat region, which was the target of the original mAbs [P. falciparum, the CSP repeat has 38 repeats: 34 asparagine-alanine-asparagine-proline (NANP)-repeats interspersed with 4 asparagine-valine-aspartate-proline (NVDP) repeats that are concentrated towards the N-terminus [P. falciparum sporozoite neutralizing antibodies identified in these early studies was 2A10 which can block sporozoite infectivity in vitro [in vivo mouse models utilizing rodent P. berghei parasites expressing the P. falciparum CSP repeat region [Antibodies to CSP were first identified as potential mediators of protection following seminal studies that showed that immunization with irradiated sporozoites could induce sterile protection against live parasite challenge ,6. In thnal mAbs \u201311. In tterminus  though dterminus . One of in vitro  and in it region ,15.P. falciparum CSP mouse monoclonal antibodies (mAbs) identified some shared sequences [While CSP binding antibodies have been shown to be able to neutralize sporozoites and block infection, it has also been proposed that CSP is an immunological \u201cdecoy\u201d that induces a suboptimal, but broad, T-independent immune response due to the CSP repeat cross-linking multiple B cell receptors (BCRs) . Howeverequences . In humaequences .We therefore set out to test the hypothesis that the CSP repeat can bind multiple antibodies or BCRs and drive a T-independent immune response. To do this we undertook a comprehensive biophysical characterization of the 2A10 sporozoite-neutralizing antibody that binds to the CSP repeat. We found that this antibody binds with an avidity in the nano-molar range which was unexpected as previous studies using competition ELISAs with peptides predicted a micro-molar affinity ,23. StriAB) fragment to test the thermodynamic basis of the affinity of 2A10 FAB towards CSP. Experiments were also performed on the 2A10 FAB fragment with the synthetic peptide antigen (NANP)6, which is a short segment of the antigenic NANP-repeat region of CSP (KD) were found to be -49.0 kJ/mol and 2.7 nM for the full 2A10 antibody with CSP, -40 kJ/mol and 94 nM for the 2A10 FAB with CSP, and -36.4 kJ/mol and 420 nM for the 2A10 FAB with the (NANP)6 peptide.We began our analysis by performing isothermal titration calorimetry (ITC) to understand the interaction between 2A10 and CSP. For ease of expression we used a recombinant CSP (rCSP) construct described previously which was slightly truncated with 27 repeats compared to 38 in the 3D7 reference strain ,24. ITC n of CSP ; Fig 1. AB domain:antigen binding stoichiometry (6 peptide was bound to by ~2 FAB fragments (2.8 repeats per FAB domain). With the rCSP protein we observed that ~11 FAB fragments could bind to each rCSP molecule, , we observe binding of 5.8 antibodies per rCSP molecule (4.7 repeats per antibody). Therefore all complexes exhibit approximately the same binding stoichiometry of two FAB fragments/domains per ~5 repeat units. These results suggest that the antigenic region of CSP constitutes a multivalent antigen and that repeating, essentially identical, epitopes must be available for the binding of multiple FAB domains.Surprisingly, we did not observe a typical 1:1 antibody/Fhiometry . We founAB domains. The FAB:rCSP complex and the 2A10:rCSP complex had similar enthalpy and entropy of binding (vs. 94 nM for FAB:rCSP). The observation that this antibody-antigen (Ab-Ag) interaction is primarily enthalpically driven is consistent with the general mechanism of Ab-Ag interactions [Kd) of a single FAB domain to the (NANP)6 peptide is substantially higher (420 nM), and that the avidity, the accumulated strength of the multiple binding events between the FAB domains of the antibody and the CSP repeat, is the basis for the lower Kd value observed in the 2A10:rCSP complex. Thus, the characteristic repeating pattern of the epitope on the CSP antigen allows multiple weak interactions with 2A10 FAB domains to accumulate, which yields a complex with a high avidity dissociation constant in the low nM range.It is not possible to separate affinity from avidity in this system, although it is apparent that there is a substantial benefit to the overall strength of binding between the antibody and antigen through the binding of multiple F binding , but theractions . It is c6 peptide. These results were inconsistent with a disordered random coil structure lgorithm . The onllgorithm , and whilgorithm . Thus, tAB fragment in two conditions -repeat region, we solved the crystal structure of the 2A10 Fnditions , yieldinnditions , except  binding . Indeed, binding .AB and the (NANP)6 peptide were unsuccessful; unlike binary Ab-Ag interactions, in which the Ab will bind to a single epitope on an antigen and produce a population of structurally homogeneous complexes that can be crystallized, in this interaction we are dealing with an intrinsically-disordered peptide, the presence of multiple binding sites on the peptide, and the possibility that more than one 2A10 FAB domain can bind the peptide. Therefore it is difficult to obtain a homogeneous population of complexes, which is a prerequisite for crystallization. Attempts to soak the (NANP)6 peptide into the high-solvent form of 2A10 FAB, in which there were no crystal packing interactions with the binding-loops, caused the crystals to dissolve, again suggesting that the heterogeneity of the peptide and the presence of multiple epitopes produces disorder that is incompatible with crystal formation.Attempts to obtain a crystal structure of a complex between 2A10 F6 peptide complex, the accurate structures of the 2A10 FAB fragment, the (NANP)6 peptide, and the knowledge that antibodies seldom undergo significant conformational changes upon antigen binding [AB fragment (AB:(NANP)6 peptide interaction was plausible, we performed site directed mutagenesis of residues predicted to be important for binding. Our model predicted that the interaction with (NANP)6 would be mainly between CDR2 and CDR3 of the light chain and CDR2 and CDR3 of the heavy chain  binding , allowed binding . Using t binding , we obtafragment . We thenfragment , S3 Fig.vy chain .via its hydroxyl group, to a number of backbone and side-chain groups of the peptide. Loss of this side-chain abolished binding. Y56 also forms part of the same proline-binding pocket as Y38, and loss of this side-chain also resulted in an almost complete loss of binding. R109 forms a hydrogen bond to an asparagine residue on the side of the helix; mutation of this residue to alanine results in a partial loss of binding. Y116 is located at the center of the second proline-binding pocket; since loss of the entire side-chain through an alanine mutation would lead to general structural disruption of the FAB fragment, we mutated this to a phenylalanine (removing the hydroxyl group), which led to a significant reduction in binding. Finally, S36A was selected as a control: the model indicated that it was outside the binding site, and the ELISA data indicated that had no effect on (NANP)n binding.In the light chain , Y38 is Within the heavy chain , mutatioAB fragment to the (NANP)6 peptide is centered on two proline residues from two non-adjacent NANP-repeats 6 repeat, the binding epitope of the peptide is 2.5\u20133 alternate NANP repeats, with a symmetrical epitope available for binding on the opposite face (AB fragments per (NANP)6 peptide. To investigate whether this binding mode was also compatible with the indication from ITC that ~10.7 2A10 FAB fragments, or six antibodies (containing 12 FAB domains) could bind the CSP protein  [9 repeats with streptavidin conjugated phycoerythrin (PE) or allophycocyanin (APC). To validate our tetramer approach, mice were immunized with either P. berghei CSPf or another line of P. berghei with a mutant CSP (P. berghei CS5M) that contains the endogenous (P. berghei) repeat region, which has a distinct repeat sequence (PPPPNPND)n. (NANP)n-specific cells were identified with two tetramer probes bound to different conjugates to exclude B cells that are specific for the PE or APC components of the tetramers which are numerous in mice [P. berghei CSPf sporozoites developed large tetramer double positive populations, which had class switched (n-specific cells (+ CD38- indicating that they are GC B cells in agreement with results from a recent publication [We next set out to determine the implications of our structure for the B cell response to CSP. Because the CSP protein could conceivably cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T-independent. As a tool to test this hypothesis we used (NANP)ei CSPf) . The tet in mice . We founswitched . In contic cells . Furtherlication   and measured serum (NANP)n specific antibody by ELISA and the B cell response using our Tetramers. CD28-/- mice have CD4+ T cells but they are unable to provide help to B cell responses [n responses in the CD28-/- mice and control animals n specific B cell responses using our tetramers, in particular examining the number and phenotype (plasmablast vs GC B cell) of activated IgD- Tetramer+ cells esponses . Interes animals , indicatr+ cells . In agremablasts . However-Tetramer+ responses 4 days post immunization in control and CD28-/- mice n by sequencing the BCRs of the identified cells. While the repeat structure of CSP has been hypothesized to induce a broad polyclonal response based on data that the CSP repeat can absorb most of the sporozoite binding activity of human sera from immune individuals [n-specific cells 35 days post immunization of BALB/C mice with sporozoites. We performed this analysis in BALB/C mice as this is the background of mice from which the 2A10 antibody was derived. We then prepared cDNA from the cells and amplified the heavy and kappa chain sequences using degenerate primers as described previously [n specific cells and the bulk B cells from na\u00efve mice we calculated the Shannon entropy for these populations. This analysis formally demonstrated that the diversity of the antigen specific B cells was significantly lower than the diversity of the repertoire in na\u00efve mice (Our ability to identify and sort (NANP)ividuals ,38, an aeviously ,40. Heav\u00efve mice . We furt\u00efve mice . Similar\u00efve mice . The V r\u00efve mice , for exa\u00efve mice  Tables tn specific cells in immune mice. This analysis showed that while, as expected, sequences from na\u00efve mice contained few mutations, the sequences from immune mice had much higher levels of SHM. Importantly mutations were found to be concentrated in the CDR loops, and were frequently shared by immunized mice providing strong circumstantial evidence for affinity maturation  that would be indicative of B cells specific for CSP entering the GC. Taking advantage of the fact that our kappa chain primers capture the entire V-J sequences of the antibodies we sequenced we asked: 1) if the kappa chains shared between immune animals differed from the germline (providing evidence of SHM) and 2) if the mutations were conserved between different mice indicative of directed selection. Analysis of the reads from the kappa chains of the three immune mice showed that these had a much higher degree of mutation than bulk B cells from na\u00efve mice, demonstrating SHM in the CSP-specific antibodies . We furtTo directly test if CSP-binding antibodies undergo affinity maturation we expressed the predicted germline precursor to the 2A10 antibody (2A10 gAb) in HEK293T cells. We identified the predicted germline precursors of the 2A10 heavy and light chains using the program V-quest  S5 and  Figs. ThTo identify the specific mutations that were important we introduced the mutations individually into the gAb light chain construct. We prioritized mutations that were shared with the 27E antibody which has previously been found to be clonally related to 2A10 having been isolated from the same mouse and which shares the same germline heavy and light chains as the 2A10 mAb . We founPlasmodium falciparum sporozoite-neutralizing antibody (2A10). Having obtained this structure we further modeled the binding 2A10 with its antigen target, the repeat region of CSP, and provide a thermodynamic characterization of this interaction. Finally, we used novel tetramer probes to identify and sort antigen specific B cells responding to sporozoite immunization in order to measure the diversity and maturation of the antibody response. We found that the avidity of 2A10 for the rCSP molecule was in the nanomolar range, which was much higher than the affinity previously predicted from competition ELISAs with small peptides [Here we provide an analysis of the structure of a peptides ,23. ThisAB fragments we too obtained a dissociation constant in the micro-molar range (0.42 \u03bcM). The difference between the FAB binding to the peptide and the tight interaction of the antibody binding to full length CSP appears to be driven by a high avidity, multivalent interaction. There is also additional enthalpic stabilization (per FAB domain) in the 2A10:CSP complex, although this is partially offset by the increased entropic cost associated with combining a large number of separate molecules into a single complex. One caveat of these data is that we used a slightly truncated repeat in our recombinant CSP, however it is likely that longer repeats will have further stabilization of the interaction that could result in even higher affinity interaction between CSP and binding antibodies.Using ITC we determined the dissociation constant of 2A10 for rCSP to be 2.7 nM, which is not unusual for a mouse mAb. However it is a tighter interaction than that predicted from competition ELISAs, which predicted a micro-molar affinity ,23. HoweAB fragments alone are sufficient to block invasion [The mechanism of sporozoite neutralization remains unclear, however our structural data may provide some insights. Repeat specific antibodies can directly neutralize sporozoites (without complement or other cell mediators) in the circumsporozoite reaction ,42. Moreinvasion . Howeverinvasion ,45. It hinvasion . Cleavaginvasion . AntibodOur results uncovering how neutralizing antibodies bind to CSP has several implications for understanding the development of the immune response to CSP. Notably the finding that the CSP molecule can be bound by multiple antibodies/B cell receptors raises the possibility that this molecule can indeed crosslink multiple BCRs and potentially act as a type-II T independent antigen . We findn repeat contradicts previous suggestions that the response to CSP might be broad and polyclonal [n repeat shares structural similarity with a self-antigen as is speculated to happen with meningococcus type B antigens [The finding of a limited repertoire in the BCR sequences specific for the (NANP)lyclonal . One explyclonal . The replyclonal . Anotherantigens , howeverOne area for future investigation is to determine the binding modes and sporozoite neutralizing capacities of other antibodies in the response. It is clear that not all CSP-repeat binding antibodies have the same capacity for sporozoite neutralization . As suchWhile our work has been performed with mouse antibodies, there are major similarities between mouse and human antibody loop structure . The maiOverall our data provide important insights into how the antibody response to CSP develops. Our results also help explain why relatively large amounts of antibodies are required for sporozoite neutralization and suggest that the ability to generate an effective B cell response may be limited by the very simplicity of the repeat epitope. These data support previous suggestions that CSP may be a suboptimal target for vaccination. However, we also find that CSP binding antibodies can undergo somatic hypermutation and reach high affinities. This suggests if we can develop vaccination strategies to diversify the repertoire of responding B cells and favor the GC response it may be possible to generate long-term protective immunity targeting this major vaccine candidate antigen.All animal procedures were approved by the Animal Experimentation Ethics Committee of the Australian National University (Protocol numbers: A2013/12 and A2016/17). All research involving animals was conducted in accordance with the National Health and Medical Research Council's (NHMRC) Australian Code for the Care and Use of Animals for Scientific Purposes and the Australian Capital Territory Animal Welfare Act 1992.-/- [4P. berghei CS5M sporozoites expressing mCherry [4P. berghei CSPf sporozoites dissected by hand from the salivary glands of Anopheles stephensi mosquitoes. Mice were either infected with live sporozoites and then treated with 0.6mg choloroquine IP daily for 10 days or immunized with irradiated sporozoites (15kRad). For immunization with rCSP, 30ug rCSP was emulsified in Imject Alum according to the manufacturer\u2019s instructions (ThermoFisher Scientific) and delivered intra-peritoneally. All mice received only a single immunization in these experiments. To deplete CD4+ T cells mice were treated with two doses of 100ug GK1.5 antibody on the 2 days prior to immunization (BioXCell); control mice received an irrelevant isotype control antibody .BALB/C, C57BL/6 or CD28-/-  mice (br mCherry  or 5 x 19 tetramers conjugated to PE or APC. Antibodies were purchased from Biolegend while tetramers were prepared in house by mixing biotinylated (NANP)9 peptide with streptavidin conjugated PE or APC (Invitrogen) in a 4:1 molar ratio. Flow-cytometric data was collected on a BD Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo). Where necessary cells were sorted on a BD FACs Aria I or II machine.Single cell preparations of lymphocytes were isolated from the spleen of immunized mice and were stained for flow cytometry or sorting by standard procedures. Cells were stained with lineage markers  antibodies to B220 (clone RA3-6B2), IgM (clone II/41), IgD (clone 11-26c2a), GL7 (clone GL7), CD38 (clone 90), CD138 (clone 281\u20132) and (NANP)n tetramers and antibodies to B cell markers as described in the supplementary experimental procedures. Antigen specific cells were sorted on a FACS ARIA I or II instrument prior to RNA extraction with the Arturus Picopure RNA isolation kit (Invitrogen) and cDNA preparation using the iScript cDNA synthesis kit (BioRad). BCR sequences were amplified using previously described heavy and kappa chain primers including adaptor sequences allowing subsequent indexing using the Nextera indexing kit (Illumina). Analysis was performed in house using R-scripts and the program MiXCR as described in supplementary experimental procedures.Single cell suspensions from the spleens of immunized mice were stained with (NANP)Variants of the 2A10 antibody were expressed in HEK293 T cells  as described in the supplemental experimental procedures. Binding to the CSP repeat was tested by ELISA and ITC using standard techniques as described in the supplementary experimental procedures.p-value was < 0.05 for a tested difference. .Statistical analysis was performed using Prism6 (GraphPad) for simple T tests and one-way ANOVAs from single experiments. Where data were pooled from multiple experiments, analysis was performed using linear mixed models in R version 3.3.3 . Linear mixed models are a regression analysis model containing both fixed and random effects: fixed effects being the variable/treatment under examination, whilst random effects are unintended factors that may influence the variable being measured. If significance was found from running a linear mixed model, pair-wise comparisons of the least significant differences of means (LSD) was undertaken to determine at which level interactions were occurring. Statistical significance was assumed if the Sequence data generated in this paper is deposited at the NCBI sequence read archive (SRA) with accession number SRP092808 as part of BioProject database accession number PRJNA352758. Atomic coordinates and related experimental data for structural analyses are deposited in the Protein Data Bank (PDB) with PDB codes 5SZF and 5T0Y.S1 FigThe computational prediction of the spectra (A) was performed using DichroCalc , the exp(TIF)Click here for additional data file.S2 FigConformations were clustered by concatenating the trajectory and performing a Jarvis-Patrick analysis. The clusters are sorted by their RMSD from the first cluster (starting geometry). As shown, Run 2 is stable in the starting geometry for several ns, while Run 3 diverged, then reconverged to the starting geometry, where it was stable for several ns. These data suggest the quasi-helical structure observed from the ab initio calculations is stable, and can be spontaneously sampled, on a timescale of several ns.(TIF)Click here for additional data file.S3 Fig6:FAB complex. Root mean square deviation (RMSD) of the (NANP)6:FAB complex as a function of time. Independent simulations are shown in green, black and red.Molecular dynamics simulation of the (NANP)(TIF)Click here for additional data file.S4 FigP. berghei CSPf RAS, live P. berghei CSPf under CQ cover or rCSP. (A) 4 days later the IgM and IgG response to the (NANP)n repeat was analyzed by ELISA (B) At the same time the number of IgD- Tetramer+ B cells was quantified in the spleen. Data are from a single experiment, analyzed using linear models with immunization/treatment as the experimental factor.Mice either treated with an anti-CD4 depleting antibody or an isotype control were immunizaed with either (TIF)Click here for additional data file.S5 FigResidues that are mutated away from the predicted germline sequence in more one or more other antibody heavy chain (2E7 or 3D6) are highlighted in red, mutations that are predicted to be involved in binding to CSP are highlighted in blue.(TIF)Click here for additional data file.S6 FigResidues that are mutated away from the predicted germline sequence in both 2A10 and the related 2E7 antibody are highlighted in red, mutations that are predicted to be involved in binding to CSP are highlighted in blue.(TIF)Click here for additional data file.S1 Movie6 run 3. The trajectory was fitted to minimize alpha-carbon RMSD and then passed through a low-pass filter with a filter length of 8 frames to reduce temporal aliasing.Excerpt from (NANP)(MP4)Click here for additional data file.S2 Movie6 run 3. The trajectory was fitted to minimize alpha-carbon RMSD and then passed through a low-pass filter with a filter length 8 frames to reduce temporal aliasing.Excerpt from 2A10:(NANP)(MP4)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file."}
{"text": "Asthma is the first cause of children hospitalization and need for emergency and impose high economic burden on the families and governments. We aimed to investigate the economic burden of pediatric asthma and its contribution to family health budget in Iran.Overall, 283 pediatric asthmatic patients, who referred to two tertiary pediatric referral centers in Tehran capital of Iran, included from 2010\u20132012. Direct and indirect asthma-related costs were recorded during one-year period. Data were statistically analyzed for finding association between the costs and factors that affect this cost .P=0.011), and 7\u201311 yr old age group (P=0.018). In addition, we found significant association between total asthma costs and asthma control status (P=0.011).Ninety-two (32.5%) females and 191(67.5%) males with the age range of 1\u201316 yr old were included. We found the annual total pediatrics asthma related costs were 367.97\u00b123.06 USD. The highest cost belonged to the medications (69%) and the lowest one to the emergency (2%). We noticed a significant increasing in boys\u2019 total costs (The presence of an asthmatic child can consume nearly half of the health budget of a family. Our results emphasis on improving asthma management programs, which leads to successful control status of the disease and reduction in economic burden of pediatric asthma. Asthma is a chronic obstructive respiratory disease in all age groups and for both sexes. It happens after an allergen stimulation that leads to severe symptomatic attacks of bronchial constriction and recurrent episodes of coughing, chest tightness, wheezing and breathlessness .There is sharp rise in the prevalence of asthma in many parts of the world with adopted western lifestyle . OverallThe world health organization in a study on global burden of diseases estimated 13.8 million disability-adjusted life years (DALYs) per year because of asthma that encompasses 1.8% of total burden of all diseases . Asthma Investigation of burden of diseases published by WHO revealed a strong effect of economic aspect of asthma on quality of life of patients and their families . SeveralAsthma is responsible for death of many children in developed countries . A sharpThe global prevalence of asthma is different by reason of the lack of internationally accepted definition for asthma . AccordiEconomic aspect of asthma is important for both asthmatic patients and health administrators of countries for precise allocating the health budget. In studies on asthma economic burden, attention should be paid for both direct  and indirect costs (i.e. loss of productivity and premature death) , 17.In our previous study  on the eOverall, 283 asthmatic children entered this prospective study. The patients referred to Children Medical Center and Mofid Hospital, Tehran, Iran as the most important tertiary referral centers of pediatrics in Iran. The inclusion criteria for patients was the age \u226416 yr, diagnosis of asthma by pediatric allergist and immunologist based on GINA 2009 guideline , insurance type), the disease history ) and asthma control status . The costs of asthma recorded regularly during one-year follow-up period in second part of questionnaire. The recorded direct costs consisted of doctor visit, asthma medication, radiology, laboratory, skin prick test, spirometry, emergency, hospital admission. The only indirect assessed cost of asthma was transportation. The entire recorded price in questionnaire was as Rial for convenience. All the expenditure were converted to US Dollar based on the exchange data of the Iran Central Bank .t-test and one way ANOVA and post hoc test, Tukey were used to compare means of expenditures. The P-values less than 0.05 considered statistically significant.The data of this study were analyzed statistically by SPSS software version 19 . We used standard statistical descriptions such as mean, minimum, maximum, and standard error for each cost. Association of asthma economic costs according to age, sex, asthma control status, comorbidities and smoking status were investigated. Independent samples Among included asthmatic pediatric patients, 283 patients had successful one-year period follow-up; consisting of 92 (32.5%) females and 191 (67.5%) males, with the age range of 1\u201316 yr old (mean\u00b1SE=6.91\u00b10.18). Only 33(11.7%) patients did not have any health insurance whereas 250 (88.3%) had health insurance. Assessment of parental education level showed that 118 (41.7%) of fathers had a university degree and 124 (43.8%) had high school diploma and others did not complete the high school. Whereas 132 (46.6%) mothers were graduated from university, 109 (38.5) mothers had high school diploma and the rest of mothers were educated less than high school. Patients had 1 to 34 d absent from school due to asthma problems during one year (mean\u00b1SE=1.7\u00b10.21). Patients during one-year period had 1\u201318 times doctor visit (mean\u00b1SE= 4.44\u00b10.18), they needed to emergency bed 0\u20133 times (mean\u00b1SE= 0.07\u00b10.02), hospital bed day 0\u201311 times (mean\u00b1SE= 0.17\u00b10.06). Patients performed X-ray radiography, skin prick test, laboratory test and spirometry, 0.31\u00b10.03, 0.25\u00b10.49, 0.32\u00b10.03, 0.41\u00b10.04 times respectively during this period.We estimated total pediatrics asthma related costs for one-year period as 367.97\u00b123.06 USD. The details of expenditures demonstrated in .P=0.011). In addition, we found significant association between total asthma cost in different age groups (P=0.008), Tukey HSD revealed that 7\u201311 yr old asthmatic children have higher asthma related costs than <7 yr old group (P=0.018).P=0.14). Almost half of patients, 150(53.0%) had not any comorbid diseases of asthma but 55(19.4%) had sinusitis, 25(8.8%) nasal allergy, 24(8.5%) GERD and the rest of patients had other concomitant disease or combination of comorbidities, statistical analysis did not show any significant difference between total cost of these categories (P=0.16). Asthma control assessment showed that 22(11.0%) of patients had un-controlled asthma, 73(33.3%) had partly controlled and 122(55.7%) had well-controlled asthma. Comparing the means of different control groups showed significant association between control status of asthma and total asthma related costs (P=0.011), and Post Hoc test showed that this association is related to significant differences between partly-controlled and un-controlled groups (P=0.047).Sixty (20.2%) of asthmatic children had smoker parents and 223(78.8%) had non-smoker parents , which was the highest asthma related cost. The drug cost was the highest one for adult asthmatic patients  . MoreoveP=0.011).However, medication is the greatest part of asthma treatment regimens, but our results show that medication costs are a bigger proportion (69%) of asthma total costs, which reveals increasing usage of drugs in the therapeutic regimens in Iran. Increased usage of anti-asthmatic drugs maybe relate to poor control situation of asthma in our patients, therefore we investigate the association between total cost of asthma and control situation. We found a significant relationship between total cost of asthma and control situation of asthma , P=0.011Multiple comparisons indicate significant difference between total costs of partly controlled patients (388.69\u00b138.39 USD) and well-controlled patients (329.53\u00b138.79 USD). Our finding is in accordance with other studies that show higher economic burden for patients with severe asthma . In addiP=0.16). According to the relation of the literacy and economic status of families as well as access to medical facilities, we investigated the parents\u2019 education level and economic burden of pediatric asthma but our data did not support any significant relation.Several concomitant diseases are frequently presented with asthma disease especially in cases with poor responses to treatment or severe asthma . ConcomiP=0.14).Although prenatally or after birth exposure to cigarette smoke is connected to increased risk of asthma-like symptoms , the resP=0.011). Before age of 14, male sex is a risk factor for asthma (P=0.08); Tukey test showed that this difference is related to significant differences of total costs between patients less than 7 yr old and those with 7\u201311 yr old. Non-specific and variable clinical symptoms of asthma in young children encounter the asthma management with some problems including difficulties in diagnosis and drug efficacy  have been completely observed by the authors."}
{"text": "Soil N2O emissions from simulated cattle urine patches were quantified with closed static chambers and gas chromatography. At the regional level, rainy season cumulative N2O emissions (3.31 versus 1.91\u2009kg N2O-N ha\u22121) and emission factors (0.42 versus 0.18%) were higher for low vegetative cover compared to adequate vegetative cover pastures. Findings indicate that under rainy season conditions, adequate vegetative cover through proper pasture management could help reduce urine-induced N2O emissions from grazed pastures.A decline in pasture productivity is often associated with a reduction in vegetative cover. We hypothesize that nitrogen (N) in urine deposited by grazing cattle on degraded pastures, with low vegetative cover, is highly susceptible to losses. Here, we quantified the magnitude of urine-based nitrous oxide (N Expanding livestock production is driven by a rapid increase in demand for cattle meat2. This increased demand for animal products together with the development of improved forage options to sustain higher levels of cattle productivity increases pressure on grasslands, the dominant cattle production systems of LAC, resulting in overgrazing and degradation of pastures3. According to Kwon3, an estimated 157 million ha  of the grazing area in LAC is degraded. In Brazil half of the 80 million ha of introduced tropical pastures are estimated to be in some state of degradation as they have, among other symptoms, low soil cover4.The livestock sector accounts for 46% of the agricultural gross domestic product of the Latin America and the Caribbean (LAC) region and grows at 3.7% annually2O) emissions, a powerful greenhouse gas (GHG)5. About 75\u201395% of cattle ingested N is excreted in either urine or dung, which provides N-rich substrate for nitrification and denitrification7. Cattle urine patches can contain very high amounts of soluble N , more than 2\u20133 times of the N uptake capacity of pastures8. Annually, about 1.5 Tg of total global anthropogenic N2O emissions (6.7 Tg N2O-N yr\u22121) are emitted from excreta produced by grazing cattle10 through both direct and indirect (from leached and volatilized excreta nitrogen) emissions. About 2% (0.7\u20136% uncertainty)11 of the nitrogen (N) in deposited urine is lost as N2O. Lower emission factors (EFs) (<0.7%), reported in other studies have been attributed to differences in climatic conditions, texture, soil moisture, and the N concentration in animal excreta12.Cattle excreta deposited on grazed pastures is estimated to represent 16% of global anthropogenic nitrous oxide 17.Pasture degradation may stimulate or constrain N losses. For example low vegetative cover, may reduce N sinks for deposited excreta and thus increase the vulnerability of N to loss through soil microbial processes and leaching. However, the low vegetative cover may also be associated with fewer plant root exudates and thus suppress microbial activity and N2O emissions from urine deposition can influence emission through multiple, often interacting, mechanisms and thus has produced contradictory results in the literature. Previous studies suggest that variations in soil N2O emissions from deposited urine patches in grazed pastures are driven by differences in several factors including ambient temperature18, urine volume and urine-N content19, soil drainage21, and soil moisture23. No previous studies have systematically explored the variation in urine-based soil N2O emissions associated with low vegetative cover in pastures.Clearly, the effect of pasture degradation on N2O emissions from cattle urine deposited on grazed pastures with adequate vegetative cover are less intense than those from pastures with lower vegetative cover by measuring soil N2O fluxes from urine patches deposited on different pastures located at seven contrasting sites, spread across five countries in the LAC region during rainy season.Here we tested the hypothesis that N\u22123 and was generally similar between the low and adequate vegetative cover pastures at each study location. The largest differences in bulk density, soil organic carbon and soil organic nitrogen between low and adequate vegetative cover pastures at the Estel\u00ed location in Nicaragua , Estel\u00ed (Nicaragua) and Taluma (Colombia) Table\u00a0. Soil pHua Table\u00a0.Table 1S2O monitoring period, with the exception of the Trinidad and Tobago site which received 31 days of rainfall  were higher in the AVC (59\u2009\u00b1\u200911\u2009mg N2O-N m\u22122 day\u22121) compared to the LVC (45\u2009\u00b1\u200918\u2009mg N2O-N m\u22122 day\u22121) pasture. The level of N2O emissions observed at Balcarce and Manfredi (Argentina) and Taluma (Colombia) sites were lower than 50\u2009mg N2O\u2013N m\u22122 day\u22121.Ntes Fig.\u00a0; Table\u00a02\u22121  and AVC (0.48% of applied urine-N) pasture were at Estel\u00ed (Nicaragua) and Rio Grande do Sul (Brazil), respectively. On the other hand, the lowest N2O emission factors for LVC (0.02% of applied urine-N) and AVC pasture (0.01% of applied urine-N) were both observed at the Taluma site in Colombia  were observed at St. Augustine (Trinidad and Tobago) and Estel\u00ed (Nicaragua) and the lowest were observed at Taluma (Colombia)  in LVC (0.42\u2009\u00b1\u20090.19% SEM) than AVC (0.18\u2009\u00b1\u20090.08% SEM) pastures. Also at the regional level mean cumulative N2O emissions in the LVC (3.31\u2009\u00b1\u20091.09\u2009kg N2O-N ha\u22121 SEM) were higher than those observed in the AVC (1.91\u2009\u00b1\u20090.78\u2009kg N2O-N ha\u22121 SEM) pasture at the 10% level of significance (P\u2009=\u20090.08), based on results presented in Table\u00a02O emissions at each individual site tended to be higher in LVC pastures than in the AVC pastures, with t-test detecting significant differences at the Nicaragua (Estel\u00ed) and Colombia (Patia) sites (Table\u00a02O emission factors in the LVC (66%) pasture compared to the AVC (88%) pasture  compared to the AVC (0.0041) pastures suggests that urine deposited on LVC pastures is more vulnerable to high N2O losses when exposed to high rainfall. This may explain the significant differences between LVC and AVC pastures that were observed at Estel\u00ed (Nicaragua) and Pat\u00eda (Colombia), where rainfall was high and the observed separation between the LVC and AVC pastures at the Rio Grande do Sul (Brazil) site where rainfall was also high.The key finding of our study is that, at the regional level of LAC, N2O emissions observed in LVC pastures compared to AVC pastures were likely due to lower plant N uptake from soil. Moreover, this may in part explain why AVC pastures generally resulted in lower net cumulative N2O emissions compared to degraded pastures. Despite the significant difference (P\u2009<\u20090.10) in net cumulative N2O emissions between LVC and AVC pastures, at a regional level, site-level comparisons showed that cumulative N2O emissions from LVC pastures were only significantly higher than those of AVC pastures in two sites, at Estel\u00ed (Nicaragua) and Pat\u00eda (Colombia). The fact that soils within the LVC pastures at the Estel\u00ed site were more clayey than those under AVC pastures may have also contributed to the high net cumulative N2O emission in the former. Previous studies have reported higher N2O emissions from urine deposited on fine textured soils26. However, at Pat\u00eda, where both the LVC and AVC pastures were on a clay soil observed differences suggest that despite the obvious influence of soil texture, pasture condition, based on vegetative cover is a driver of N2O emissions.High peaks of N2O observed at the two study sites in Argentina could have been due to the lower mean air temperatures. At the Balcarce location, mean air temperatures and the amount of N in applied urine were higher than at the Manfredi location. Low temperatures are known to reduce microbial activity and thus the rate of N transformation processes such as nitrification and denitrification in soil and, consequently, N2O production27. We assume that low temperatures may also be the cause of low net emissions and emission factors at the site in Brazil. It is important to note that the high soil pH (>7) at the Balcarce site, which would be expected to further increase with urine application may have also resulted in the inhibition of nitrification and high ammonia volatilization30. In addition, at the Balcarce site, the high urine-N levels may have resulted in microbial stresses with possible impacts on soil N transformation31 and thus contributed to the low N2O emissions and emission factors.Low emissions of N2O emission peak following urine application may be because the frequency of measurements at this site was insufficient to capture the expected N2O emission spike. Several other studies using manual static chambers have reported having missed the N2O peak, due to the low temporal resolution34. This problem can be resolved by increasing the frequency of monitoring using manual static chambers or switching to automated chambers. Alternatively, the N content in applied urine was also the lowest at Taluma, which implies that N2O fluxes could have been limited by N substrate availability. In addition, the forage grass (Brachiaria humidicola) that was used at Taluma had high nitrification inhibition capacity37, which could have also contributed to the observed low N2O emissions.At the Taluma location in Colombia, the absence of a N2O emissions, between LVC and AVC pastures at the St. Augustine (Trinidad and Tobago), Balcarce (Argentina), and Manfredi (Argentina) sites was possibly due to the fact that the spatial variation of vegetative cover of grass (soil cover) in the LVC (50\u201370%) pasture was high. As a result, local farmers based their classification on animal productivity differences which are influenced by both quantity (biomass) and quality (e.g. digestibility and crude protein content) of the forage on offer to animals. This further suggests that N2O emission differences between LVC pastures and AVC pastures are driven by differences in soil cover. With high plant density and greater plant vigour, we expect greater uptake of the urine-N by plants which could reduce the amount of N available for microbial transformations in soil such as nitrification and denitrification. It is therefore not surprising that when soil cover was high in the LVC pastures, there was no significant difference in N2O emissions with AVC pastures. This was however not the case for LVC pasture at the Pat\u00eda (Colombia) site, which, though having similar soil cover (50\u201370%) showed significant differences between LVC and AVC pastures. This difference may be due to dissimilar vegetation types at the studied sites which would also affect N uptake and thus N availability for N2O emissions38.Absence of significant differences, in N11. During this short-term study, several of the emission factors were below the uncertainty range of the IPCC Tier 1 emission factor. While this may be due to the short gas monitoring period (1 month), several other studies conducted under temperate conditions42, reported a similar range of emission factors (0.02\u20131.63%) as we observed under warm temperate or sub-tropical conditions in Argentina and Brazil (0.02\u20130.7%). Similarly, the range of emission factors reported from this study under the tropical conditions in Colombia, Nicaragua and Trinidad and Tobago (0.01\u20131.2%) are in agreement with the range of values that have been reported from studies conducted under tropical conditions44.The IPCC Tier 1 emission factor for urine deposited on grazed forages is 2% with an uncertainty range of 0.7\u20136%2O emissions from cattle urine patches. When pasture degradation is associated with a reduction in vegetative cover, N2O emissions are expected to increase. Therefore, better regional understanding of the state of pasture degradation is vital for a robust understanding of N2O emissions from cattle urine deposits. More importantly, these findings suggest that improving soil cover/pasture condition through adoption of appropriate grazing and nutrient management practices may contribute towards mitigating excreta-based soil N2O emissions from grazed pastures during the rainy season. We expect findings from this regional study to contribute towards reducing uncertainties in future assessments on the importance of improving grassland management to achieve global commitments, such as the Bonn Challenge, 20\u2009\u00d7\u200920 initiative45 and the Paris Agreement46.We conclude that in addition to the known effects of rainfall, temperature and the amount of urine-N, the pasture condition based on vegetative cover also influences NThe experimental plots were located at seven different sites in five countries in LAC spanning diverse climatic conditions and soil types  and adequate vegetation cover (>70%) pastures where soil cover is simply the proportion of soil covered by vertical projection of a plant canopy and vegetative biomass  or adequate vegetative cover (AVC). Pairs of LVC and AVC pastures were not always available at the very same location, but were always less than 1\u2009km apart. We used a qualitative approach including expert knowledge, farmer perceptions and an arbitrary ranking system based on soil cover to define pasture conditions based on vegetative cover using criteria that combined those used by Hollman52 with five replicates per treatment  were demarcated within each pasture condition for making measurements of soil properties and N2O emissions. To simulate grazing, grass in each plot was cut to approximately 5\u2009cm sward height, seven days prior to the beginning of the gas and soil sampling.On each pasture (low vegetative cover or adequate vegetative cover), experimental plots were organized following a systematic experimental design54, McLean55, and Vogel56, respectively. A total of 20 cylindrical PVC static chamber bases (10 per treatment) were inserted at the center of each subplot to a depth of 5\u2009cm, five days prior to the start of gas and soil sampling. For each treatment, chamber bases with an internal diameter of 25\u2009cm and a height of 10\u2009cm were distributed in five replicate plots. At each site, cattle urine samples (about 7\u2009L) that were collected from at least 10 local dairy cows were pooled and, immediately, following setting aside a subsample for N analysis, 500\u2009ml of the collected urine was applied to soils to simulate a urination event on soil within each static chamber base at a rate of 1.27\u2009L urine/m2. Nitrogen concentration in urine was characterized in each of the study countries using the direct distillation method described by Hoogendoorn et al.57. Unfortunately, we were unable to quantify urine-N in Trinidad and Tobago as there are currently no laboratories that quantify N in animal urine samples.Prior to starting the experiment, ten soil subsamples (0\u201310\u2009cm) were separately collected from LVC and AVC pastures, using augers with 5\u2009cm diameter, and combined to give one composite soil sample for each pasture condition. Collected soil was characterized for texture, pH, total carbon (C) and organic and inorganic N as described by Gee and Bauder58. Yet, Davidson et al.59 reported the possibility of artefacts with both vented and non-vented chambers, making it difficult to know which chamber yielded the \u2018true\u2019 flux. Since similar non-vented chambers were used at all sites, the chambers did not affect observed differences between AVC and LVC pastures. However, the calculated N2O emissions factors may differ from those measured in other published studies that used vented chambers.Gas measurements were conducted from a total of 20 non \u2013 vented PVC static chambers (10\u2009cm height and 25\u2009cm diameter) fitted with two rubber septa (one for gas sampling and another for inserting a thermometer). On each sampling day, PVC chambers were fitted to the chamber bases and sealed with an air-tight rubber belt. Syringes (15\u2009ml) fitted with hypodermic needles were used to collect four gas samples from each of static chambers following chamber closure and at 15, 30 and 45\u2009minutes after chamber closure. Collected gas samples were transferred to pre\u2013evacuated 10\u2009ml headspace glass vials fitted with rubber butyl septa crimp caps. At each site, at least eight gas samples were collected between the months of November to December in 2015 for the localities of Pat\u00eda (Colombia) and Rio Grande do Sul (Brazil), Estel\u00ed (Nicaragua) and St. Augustine (Trinidad and Tobago). For Taluma (Colombia) and Balcarce (Argentina) measurements were conducted between February to March (2016) and at Manfredi, Argentina in the month of May (2016). Sampling months were chosen to coincide with the wet seasons at each of the study sites. The use of non-vented chambers has been reported to cause bias in the flux estimates2O concentration was analyzed by gas chromatography (GC-2014 Shimadzu), within a month upon arrival. The daily N2O fluxes were calculated by regressing N2O emissions from each chamber on each sampling date against time in order to calculate the hourly flux which was then multiplied by 24 to determine the daily flux. Each calculated flux was corrected for temperature and barometric pressure according to Ideal Gas Law. Subsequently, cumulative fluxes were calculated from daily N2O fluxes by interpolation between measurement days60.Gas sampling frequency was as follows: once before the application of urine, 1\u2009hour after urine application, daily for the first three days following urine application, twice a week during the second and third week and once during last week of the experiment. Due to logistical challenges, less frequent measuring campaigns were done at the Taluma site in Colombia. Immediately after the gas sampling campaign, vials were sent to the Greenhouse Gas Laboratory at the International Center for Tropical Agriculture (CIAT) in Colombia, where N2O\u2013N emission factor for urine patches was calculated according to Sordi et al.61:2O\u2013Nemitted and N2O\u2013NControl are the cumulative N2O emissions from urine or control patches over the 18 to 24 days monitoring period. Napplied represents the amount of N in the applied urine.The N62. Cumulative N2O fluxes and emission factors were, correspondingly, log and square-root transformed to achieve normality and obtain homogenous variances. To determine effects at the region level, cumulative N2O fluxes were analyzed using a split-plot ANOVA where the main plot was the pasture condition  and split-plot was the nitrogen levels (with and without urine application) and the blocking factor was the location and the main plot error term was pasture condition nested within location. In addition, at the regional level the emission factor variable was analyzed using a one-way ANOVA where the treatment effect was the pasture condition and the blocking factor was the location (site). At the individual sites we used the t-test analysis for testing differences in emission factors as influenced by pasture condition.Statistical analyses were conducted using the PROC MIXED procedure of SASSupplementary information"}
{"text": "Excess fluid balance in acute kidney injury (AKI) may be harmful, and conversely, some patients may respond to fluid challenges. This study aimed to develop a prediction model that can be used to differentiate between volume-responsive (VR) and volume-unresponsive (VU) AKI.AKI patients with urine output <\u20090.5\u2009ml/kg/h for the first 6\u2009h after ICU admission and fluid intake >\u20095\u2009l in the following 6\u2009h in the US-based critical care database ) were considered. Patients who received diuretics and renal replacement on day 1 were excluded. Two predictive models, using either machine learning extreme gradient boosting (XGBoost) or logistic regression, were developed to predict urine output >\u20090.65\u2009ml/kg/h during 18\u2009h succeeding the initial 6\u2009h for assessing oliguria. Established models were assessed by using out-of-sample validation. The whole sample was split into training and testing samples by the ratio of 3:1.Of the 6682 patients included in the analysis, 2456 (36.8%) patients were volume responsive with an increase in urine output after receiving >\u20095\u2009l fluid. Urinary creatinine, blood urea nitrogen (BUN), age, and albumin were the important predictors of VR. The machine learning XGBoost model outperformed the traditional logistic regression model in differentiating between the VR and VU groups .The XGBoost model was able to differentiate between patients who would and would not respond to fluid intake in urine output better than a traditional logistic regression model. This result suggests that machine learning techniques have the potential to improve the development and validation of predictive modeling in critical care research. Acute kidney injury (AKI) is common in the intensive care unit (ICU), and there is evidence that even a small increase in serum creatinine may be associated with increased risk of mortality \u20133. AKI cIntravenous fluid challenges are often used in critical care to restore blood pressure to improve urine output in patients with hypotension and oliguria, respectively , 6. ReceIn animal models, fractional excretion of electrolytes was found to perform well in early differentiation between VR- and VU-AKI . HoweverA large US-based critical care database named Medical Information Mart for Intensive Care (MIMIC-III) was analyzed . The MIMPatient eligibility was considered when urine output was less than 0.5\u2009ml/kg/h for the first 6\u2009h after ICU admission. This definition was consistent with the urine output component of the KDIGO criteria . To examThe urine output within an 18-h period following the initial 6\u2009h for defining oliguria was used as the outcome. Patients were considered as VR-AKI if he/she had urine output greater than 0.65\u2009ml/kg/h, corresponding to a 30% increase as compared with the baseline value. Otherwise, they were defined as VU-AKI.Routinely collected clinical and laboratory variables obtained within the first 6\u2009h of ICU admission were assessed for their ability to predict volume responsiveness. For some variables with multiple measurements, both the maximum and minimum values were assessed. Age, gender, ethnicity, admission type, elective surgery, type of ICU and presence of infection, and vital signs including respiratory rate, blood pressure, heart rate, and temperature were analyzed. In addition, laboratory data including glucose, white blood cell count (WBC), hematocrit, chloride, potassium, sodium, lactate, creatinine, blood urea nitrogen (BUN), coagulation profile, PaO2, PaCO2, and pH were included.Because this was a hypothesis-generating epidemiological study, no attempt was made to estimate the sample size of the study; instead, all eligible patients in the database were included to maximize the statistical power of the predictive model. Because missing data may create bias, variables with >\u200970% missing values were excluded from further analysis. Other variables with a lesser degree of missing values were analyzed using multiple imputation method .t test or rank-sum test as appropriate. Chi-square test or Fisher\u2019s exact test was employed to compare the differences of the categorical variables [Clinical characteristics between VR-AKI and VU-AKI groups were compared using either Student ariables . A stepwariables .Extreme gradient boosting (XGBoost) combined with decision trees was employed to predict VR versus VU. A classification tree was used as the weak learner, and the learning objective function was binary logistic. The boosting method works by iteratively refitting a weak classifier (decision tree) to residuals of previous models. Each successive classifier focused more on misclassified observations during the previous round of fitting . In thisOf the 10,795 patients with urine output <\u20090.5\u2009ml/kg/h for the first 6\u2009h after ICU admission, 7491 patients (69.4%) received fluid intake >\u20095\u2009l within the following 6\u2009h. A number of 809 patients were excluded because they received diuretics and/or RRT on the first day. A total of 6682 patients were included in our analysis; 2456 patients had VR-AKI, and 4226 patients had VU-AKI on day 1 in ICU . The maximum serum creatinine concentration  was higher, and the minimum bicarbonate concentration  and maximum hematocrit  were lower in the VU group. The VR group had higher urinary pH , lower urinary creatinine , higher systolic blood pressure , higher albumin , lower rate of mechanical ventilation , vasopressor use , and infection  than the VU group : learning rate = 0.04, minimum loss reduction = 10, maximum tree depth = 9, subsample = 0.6, and number of trees = 300. With these hyperparameters, bootstrap validation (BV) training log-loss decreases as the number of trees in an ensemble increases, and the BV testing log-loss was less than 0.693 and only slightly more than BV training log-loss as the tree grows . The XGBoost had a significantly greater AU-ROC than the logistic regression model , the latter is also associated with increased risk of VU-AKI. The utility of urinary biochemistry to predict AKI outcome has been controversial in the literature. Although urinary biomarkers such as creatinine and fractional excretion of electrolytes were significantly different between VR and VU groups in some animal and human studies , there aThird, we found that patients with AKI and oliguria after elective surgery were more likely to respond to fluid challenges in univariate analysis. Patients who underwent elective surgery are generally in better clinical condition than patients requiring urgent surgery or with a medical emergency. Postoperative oliguria can be explained by hypovolemia due to intraoperative and postoperative insensible fluid loss. As such, they will be more likely to benefit from a larger amount of fluid after major surgery. However, the association of elective surgery with volume responsiveness disappeared after adjusting for other physiological variables, indicating that hypovolemia can be represented by these variables such as systolic blood pressure, heart rate, and hematocrit. We found that an increased hematocrit within the first 24\u2009h of ICU admission was also an independent predictor of VR (in both the logistic and XGBoost models). This result could be explained by the fact that hematocrit has a direct relationship with the intravascular plasma volume and a higher hematocrit may suggest a relative hypovolemic state . ConversThis study has some strengths and weaknesses. The XGBoost modeling is a novel technique that has not been widely adopted in critical care research. The XGBoost algorithm has been successfully used in some complex scenarios such as the prediction of the failure of the treatment for parapneumonic empyema , in whicIn conclusion, this hypothesis-generating study showed that some clinical factors were more likely to be associated with VR-AKI than VU-AKI. The XGBoost modeling technique could identify the predictors of VR-AKI that were not apparent using logistic regression, resulting in a better-performing predictive model to identify patients with VR-AKI. Further epidemiological studies using advanced machine learning techniques to validate our results will help us to identify the most suitable patients to be included in clinical trials assessing the benefits of fluid therapy in AKI."}
{"text": "Cedrela montana and Handroanthus chrysanthus showed the biggest stem shrinkage of up to 2 mm after 10 consecutive dry days. A comparison of daily circumference changes over 600 consecutive days revealed different drought responses between the families concerning the percentage of days with stem shrinkage/increment, ranging from 27.5 to 72.5% for Graffenrieda emarginata to 45\u201355% for Podocarpus oleifolius under same climate conditions. Moreover, we found great difference of recovery times after longer-lasting  VdN drought events between the two evergreen broadleaved species Vismia cavanillesiana and Tapirira guianensis. While Vismia replenished to pre-VdN stem circumference after only 5 days, Tapirira needed 52 days on average to restore its circumference. Hence, a higher frequency of droughts might increase inter-species competition and species-specific mortality and might finally alter the species composition of the ecosystem.Under drought conditions, even tropical rainforests might turn from carbon sinks to sources, and tree species composition might be altered by increased mortality. We monitored stem diameter variations of 40 tree individuals with stem diameters above 10 cm belonging to eleven different tree genera and three tree life forms with high-resolution dendrometers from July 2007 to November 2010 and additionally March 2015 to December 2017 in a tropical mountain rainforest in South Ecuador, a biodiversity hotspot with more than 300 different tree species belonging to different functional types. Although our study area receives around 2200 mm of annual rainfall, dry spells occur regularly during so-called \u201cVeranillo del Ni\u00f1o\u201d (VdN) periods in October-November. In climate change scenarios, droughts are expected with higher frequency and intensity as today. We selected dry intervals with a minimum of four consecutive days to examine how different tree species respond to drought stress, raising the question if some species are better adapted to a possible higher frequency and increasing duration of dry periods. We analyzed the averaged species-specific stem shrinkage rates and recovery times during and after dry periods. The two deciduous broadleaved species  Global change, especially climate change, affects forests worldwide, with adverse effects on biodiversity and ecological services like carbon sequestration. Hence, understanding forest responses to climate variability is key to conservation and protection of forest ecosystems . EspeciaDisentangling the various factors leading to stem circumference changes, such as swelling and shrinkage of phloem, xylem and bark due to water potential and elastic properties of tissues e.g.,  and radiAnalyzing the impact of climatic extremes on tree circumference provides important insights into tree functioning and carbon budget. Hence, analyzing responses of stem girth to extreme meteorological events is an important variable in climate-growth-modeling and helps to improve existing models . Stem ciTapirira and Vismia and draw inferences about possible consequences for forest composition in view of expected climate change. All add up to the main research question, how different humid mountain rainforest tree species respond to drought stress and if some species are better adapted to a possible higher frequency and increasing duration of dry periods.In this paper we analyze one of the largest dendrometer datasets existing in the tropics. Our study is separated into three parts. After examining the general response of stem size variations to climate parameters, we first analyzed how the different tree species in a moist tropical mountain forest in South Ecuador respond to different durations of rare dry spells. Second, we analyzed species-specific differences in growth dynamics and drought response over a 600-day period. Third, we compared recovery from drought in two contrasting tree species Melastomataceae, Lauraceae, Rubiaceae, and Euphorbiaceae  almost exclusively occur during VdN events, except one drought event during 30.06.2010\u201306.07.2010 . In oral-1 for the type DR and 0.0054 \u03bcm \u00b0C-1 for the type DRL26, respectively . Since the maximum temperature differences between consecutive nights in our study area do not exceed 3\u00b0C, no temperature correction was needed for the values derived from band dendrometers. Due to the higher thermal sensor coefficient of 3.29 \u03bcm\u00b0C-1 of the point dendrometers, their maximum error is the range of up to 62 \u03bcm in circumference. An error in this range does not question our main results. Data recorded by dendrometers of DR-type have only been used in the first part of the study. In all cases, dendrometers were installed at breast height (approx. 1.30 m). In case of thick-barked species, parts of the outer bark were removed without injuring the cambial zone to minimize the influence of bark swelling and shrinking due to water uptake or loss. Since active growth by wood formation and stem swelling due to water uptake cannot be differentiated by dendrometer data we will use the term \u201cincrement\u201d in the following for any kind of stem diameter increase. To investigate the impact of drought on different species we selected dry intervals with a minimum of four consecutive days without rainfall during the periods July 2007 to November 2010 and March 2015 to December 2017. We analyzed the averaged stem shrinkage rates during periods from 4 to 9 days. To calculate stem shrinkage, only the daily maximum stem diameter values . From March 2015 to December 2017, logging band dendrometers (LBDs) with a built-in thermometer  were used. For comparability of the two dendrometer types, we converted the data of the point dendrometers into circumference values using the formula: circumference = 2 \u00d7 \u03c0 \u00d7 radius, assuming symmetrical stem geometries of the studied individuals. Due to latest calibration tests, the used dendrometer types show different thermal sensor coefficients of 3.29092 \u03bcm\u00b0Cr values  were comPersea, Nectandra, and Ocotea. Hereafter, we use the genus names for the groups of trees comprising more than one species and the species names, if the genus is represented by only one species. For Prumnopitys, our data regarding dry spells is limited to 6 days due to data logger problems.In total, 40 tree individuals with stem diameters above 10 cm belonging to eleven different tree genera and three tree life forms were studied . Stem diThe dendrometer data were scanned for outliers and measurement artifacts (exceeding 0.5 mm stem circumference change within 30 min). For stem shrinkage and increment calculation we took the maximum daily circumference/diameter values and subtracted the values from the subsequent day. The distinguished groups for the second part of the study  differedH. chrysanthus and C. montana, with average circumference losses of 1.876 and 2.190 mm after nine dry days, respectively  in which no missing dendrometer measurement values, which might compromise the results, occurred. The species In G. emarginata showed the highest percentage of days with stem increment (68.1%) and also the lowest percentage of days with shrinkage (28.5%). These values are followed by V.cavanillesiana (65/29.8%), T. guianensis (62.3/35.8%), Ocotea (58.3/38.7%) and P. oleifolius (55.5/40.3%). The percentages missing to 100% is stagnation of circumference  until the pre-drought stem diameter was regained, but already at the beginning of December the species showed a flattening of the stem increment curve (\u2605).Both species showed a very consistent response pattern among different individuals . Their rG.emarginata appeared very well adapted, showing the smallest absolute stem diameter shrinkage but standardized, it turned more to average. P. oleifolius showed the second smallest stem diameter change in response to ongoing drought, very similar to Ocotea and Inga. V.cavanillesiana responded very alike, but the daily changes of the last three dry days indicated differences in response, probably due to change in drought-coping adaptations like different degrees of leaf-water potential regulation , root and sapwood area (increase), and cavitation resistance (increase), which should protect trees against future droughts . ConsideThe datasets generated for this study are available on request to the corresponding author.AB and VR developed the concept of the manuscript. VR and KP collected the data in the field. VR and SS contributed statistics and graphics. All authors contributed to the text writing.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."}
{"text": "The morbidity of knee arthritis is increasing among aged people and total knee arthroplasty has been its mainstream treatment to date. Postoperative rehabilitation is an important part of the procedure. However, the intense pain during the functional exercise involved has always been a challenge for both patients and health care professionals. The aim of this study is to test the analgesic effect of a mixture of nitrous oxide/oxygeb (1:1) inhalation for patients who are doing functional exercise 1 month after total knee arthroplasty.This double-blind, randomized, placebo-controlled study will be implemented in the Rehabilitation Department in the General Hospital of Ningxia Medical University. Patients aged between 50 and 75\u2009years who underwent a primary unilateral total knee arthroplasty are eligible for inclusion. The key exclusion criteria include: epilepsy, pulmonary embolism, intestinal obstruction, aerothorax. The treatment group (A) will receive a pre-prepared nitrous oxide/oxygen mixture plus conventional treatment , and the control group (B) will receive oxygen plus conventional treatment . Patients, physicians, therapists, and data collectors are all blind to the experiment. Assessments will be taken immediately after functional exercise begins (T0), 5\u2009min (T1) after functional exercise begins, and 5\u2009min after functional exercise has finished (T2). Patients will be randomly allocated between a treatment group (A) and a control group (B) in a ratio of 1:1. Primary outcome, including pain severity in the procedure, will be taken for each group. Secondary outcomes include blood pressure, heart rate, oxygen saturation, side effects, knee joint range of motion, Knee Society Score (KSS), rescue analgesia need, and satisfaction from both therapists and patients.This study will focus on exploring a fast and efficient analgesic for patients who are doing functional exercise after total knee arthroplasty. Our previous studies suggested that the prefixed nitrous oxide/oxygen mixture was an efficacious analgesic for the management of burn-dressing pain and breakthrough cancer pain. The results of this study should provide a more in-depth insight into the effects of this analgesic method. If this treatment proves successful, it could be implemented widely for patients doing functional exercise in the rehabilitation department.ChiCTR-INR-17012891. Registered on 6 October 2017.The online version of this article (10.1186/s13063-019-3472-7) contains supplementary material, which is available to authorized users. Knee arthritis is a chronic joint disease that leads to considerable morbidity in terms of pain, difficulties in performing daily life activities, sleep disorder, functional limitations, mental illness, and decreased quality of life , 2. KneeRehabilitation after TKA generally begins immediately after surgery and, therefore, sufficient analgesia is important. An ideal analgesia for post-TKA rehabilitation will permit adequate knee flexion with minimal pain and without motor impairment, leading to successful mobilization . In ChinOrally administered analgesics are often the main treatment in the immediate- to short-term postoperative period. Despite the effectiveness of opioids, they often lead to some undesirable side effects such as vomiting, constipation, and respiratory depression . It is rPopulation: patients who undergo total knee replacement and are do functional exerciseIntervention: nitrous oxide/oxygen inhalationComparison: oxygen inhalationOutcome: pain severity, adverse events, satisfaction from both patients and therapists, Knee Society Score (KSS), the time for ROM \u2265\u200990\u00b0 and rescue analgesiaStudy design: a randomized controlled trial (RCT)  approach:Potential participants for the study will be sourced from the General Hospital of Ningxia Medical University, which is the biggest general hospital in Ningxia. Recruitment sources will be focused on patients who undergo TKA and are doing functional exercise. A member of the research team will contact the principals, directors, case managers and\u2009other relevant staff for information about the study. Should they wish their organization to cooperate with the study, staff members will be asked to provide written information to individual members whom they deem to be an appropriate candidate for the study. If that member or client is interested in participating, based on the information provided, they will then voluntarily contact the research team. Alternatively, should the potential participant wish to be contacted directly by a member of the research team, they may choose to complete a \u201cconsent to contact\u201d form. At this point, it is made clear that patients are only consenting to the research team contacting them to discuss the possibility of participating in the study as opposed to actually consenting to participate in the study itself. Those who agree to contact the research team will be invited to attend a screening interview to see whether they meet the inclusion criteria . The study will be conducted in the rehabilitation department of the General Hospital of Ningxia Medical University. Patients in the intervention group will receive conventional analgesic regimens  with the premixed nitrous oxide/oxygen mixture inhalation while patients in the control group will receive conventional analgesic regimens  and oxygen inhalation when doing functional exercise  [The study will be implemented in the General Hospital of NingXia Medical University, which is a specialized tertiary hospital. A written informed consent form will be collected from all the participants after a thorough explanation of the procedure by the therapists. The researchers state that voluntary participation and all their demographic data and survey answers will be kept confidential. Patients are eligible if they are undergoing primary unilateral TKA (see Table actures) . The patAccording to the computer-generated random number table in a 1:1 ratio, all the participants will be randomized to the intervention group  or the control group . The allocation sequence of each patient will be decided by a computer-generated schedule, which will be numbered by a statistician. The randomization schedule will be kept and sealed in an independent research room. Apart from the project manager, who is responsible for gas distribution, no other nurses or data collectors will have access to the data allocation. Prior to, and during, the treatment period, the participants, therapists, and the investigators will all be blind to the allocation. Only the project manager will know the allocation. The two gases will be stored in identical-looking canisters labeled A and B  . During The primary endpoint measures will be pain severity at T0, T1, and T2. The anticipated pain score  during functional exercise will be a reduction from to 3 to 5 in the intervention group. Repeated-measures analysis of variance will be used for data analysis.Secondary outcomes will include physiological parameters: blood pressure, heart rate, oxygen saturation at T0, T1, and T2, adverse effects, and satisfaction investigated by a 5-point satisfaction scale  from both patients and therapists, KSS, and the time for ROM \u2265\u200990\u00b0(ROM will be measured by a goniometer). Rescue analgesia should also be an indicator and recorded.Respiratory adverse effects, such as desaturation (pulse oximetric saturation\u2266\u200994%), arterial hypotension or bradycardia\u2014as well as hallucination and gastrointestinal adverse effects  or any other discomfort\u2014will be evaluated and recorded during and after the procedure. If any of these happen, patients will be given oxygen inhalation, and the discomfort should vanish within 5 min.P value of 0.05 , these calculations show that 14 patients are needed (seven per group). We finally decided on a sample size of 140 to meet the Chinese Food and Drug Administration standard for feasibility and the safety of staffs in implementing this analgesic [Pain relief was defined as a 30% decrease in the pain level in T1-T0\u00a0 . Based onalgesic , 28.A DSMB will be established shortly after the project launch and will meet several times during the data collection period. Members will included two pain management specialists; four professional therapists and nursing officers; and a senior academic statistician who served as the Board\u2019s Chair.2 test or Fisher\u2019s exact test for categorical variables and the Mann-Whitney U test for non-parametric data. Statistical significance will be defined as P\u00a0<\u20090.05.Before the study, all data collectors will be given unified training. The data will be analyzed using SPSS Statistics for Windows, Version 22.0. Chicago, IL, USA. Descriptive statistics will be analyzed by medians (inter-quartile ranges), means (standard deviations), and proportions . The baseline parameters will be compared between the two groups  using the chiKnee arthritis is associated with recurrent sharp pain, gait disturbance, joint deformity, and functional degeneration. TKA is the main surgery to solve the problem, but pain along with the need for rehabilitation after TKA has always been a significant clinical challenge for physicians, especially therapists in the rehabilitation department. Endurance has long been strongly represented in the Chinese consciousness. Patients are often told to just get over it, but it's reported that\u00a0for women patients it is pretty hard \u201331. PoorThe use of adjunct, non-opioid agents is integral for pain control following TKA. The premixed nitrous oxide/oxygen displays its superiority. Its effective use in controlling pain and its rare side effects accelerates its wide use in various areas such as venipuncture, lumbar puncture, bone marrow aspiration, and laceration repairs , 32, 33.The limitations of this study must be acknowledged. The study will be conducted in a remote area of China. As a result, our findings may not reflect the national average. Due to the limited resources, the research will be carried out in a hospital only. The results may not represent an ideal research situation and further multi-center studies will be necessary in the future.Additional file 1: Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 Checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 126 kb)Additional file 2: Equipment. (TIF 379 kb)"}
{"text": "The ability of the quinoxaline derivatives to induce apoptosis in A549 cells was assessed using the Acridine Orange/Ethidium Bromide (AO/EB) and Annexin V-FITC/Dead Cell Assay. Of the four quinoxaline derivatives tested, 3- prop-2-ynyl methanosulphate (LA-39B) and 3- prop-2-yn-1-ol (LA-55) displayed a dose-dependent reducing power, free-radical scavenging activity, inhibition of cell viability, and stimulation of ROS production which was accompanied by induction of apoptosis in A549 lung cancer cells. None of the quinoxaline derivatives induced cell death or ROS production in non-cancerous Raw 267.4 macrophage cells. Cytotoxicity was observed in A549 lung cancer, HeLa cervical cancer, and MCF-7 breast cancer cells albeit inhibition was more pronounced in A549 cells. The results of the study suggest that 3- prop-2-ynyl methanosulphate and 3- prop-2-yn-1-ol induce apoptotic cell death in A549 lung cancer cells.Despite major advancements in the development of various chemotherapeutic agents, treatment for lung cancer remains costly, ineffective, toxic to normal non-cancerous cells, and still hampered by a high level of remissions. A novel cohort of quinoxaline derivatives designed to possess a wide spectrum of biological activities was synthesized with promising targeted and selective anticancer drug activity. Hence, this study was aimed at determining in vitro anticancer activity effects of a newly synthesized class of 3- prop-2-ynyl quinoxaline derivatives on A549 lung cancer cells. An assessment of the quinoxaline derivatives ferric reducing power, free radical scavenging activity, cytotoxic activity, and ability to induce reactive oxygen species (ROS) production was performed using the Ferric Reducing Antioxidant Power (FRAP), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 3--2,5-diphenyltetrazolium bromide (MTT) and 2\u2019,7\u2019-dichlorodihydrofluorescein diacetate (H Lung cancer remains among the leading causes of cancer-related death in the world affecting both males and females . Non-smaQuinoxalines are referred to as a benzopyrazines because their structure consists of a benzene ring and a pyrazine ring . They caIn this study, we document the antioxidant properties of 3- prop-2-ynyl methanosulphate (LA-39B), and 3- prop-2-yn-1-ol (LA-55) quinoxaline derivatives and their ability to induce cell death in A549 lung cancer cells. 1 , PdCl2(PPh3)2 , and CuI  were dissolved in 15 mL dry THF followed by the addition of Et3N  and propargyl alcohol . The reaction mixture was stirred at 50 \u00b0C for 18 h under nitrogen atmosphere, thereafter partitioned between EtOAc/water in (20 mL 3:1). The layers were separated and the aqueous layer was washed with EtOAc (3 \u00d7 20 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by recrystallization from acetone and gave 3-prop-2-yn-1-ol as a brown powder , mp = 139.2 to 140.8 \u00b0C (Lit 140 to 141 \u00b0C) 2; \u03b4H  4.610 , 7.789 , 8.077  and 8.887 ; \u03b4C  51.39, 83.03, 91.90, 128.44, 129.16, 130.72, 132.01, 138.72, 141.06, 141.96, and 146.90; Vmax (FTIR) 758, 946, 1014, 1127, 1229, 1429, 1694, 2228, 2922, 3275 cm\u22121; m/z MS (ESI): MH+ 185.1.Quinoxaline derivative LA-55  was synt1 , PdCl2(PPh3)2 , and CuI  were dissolved in 25 mL dry THF followed by the addition of Et3N  and 2-methylbut-3-yn-2-ol . The reaction mixture was stirred at 50 \u00b0C for 18 h under nitrogen atmosphere, thereafter partitioned between EtOAc/water in (20 mL 3:1). The layers were separated and the aqueous layer was washed with EtOAc (3 \u00d7 20 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by recrystallization from acetone and gave 2-methyl-4-but-3-yn-2-ol as a brown powder , mp = 155.3 to 158.4 \u00b0C; \u03b4H  1.695 , 7.778 , 8.076  and 8.871 ; \u03b4C  31.07, 65.51, 79.84, 98.21, 129.14, 129.18, 130.56, 130.75, 139.01, 140.93, 141.96, and 147.09; Vmax (FTIR) 762, 960, 1050, 1229, 1301, 1488, 1538, 2230, 2982, 3290 cm\u22121; m/z HRMS (ESI): MH+ 213.1020.Quinoxaline derivative LA-65C3  was synt, were mixed in 50 mL round bottom flask containing 10 mL dichloromethane and stirred for 30 min at room temperature. Following this, aqueous 10% Na2S2O3.5H2O (10 mL) and aqueous saturated solution NaHCO3 (6 mL) were added into the reaction mixture and stirred for further 5 min. The layers were separated and the aqueous layer was extracted with dichloromethane (10 mL \u00d7 3). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified on prep TLC 3:7 ethyl acetate/n-hexane and gave 3-propionaldehyde as brown powder , mp = 122.2 to 124.8 \u00b0C; \u03b4H  7.873 , 8.139 , 9.021  and 9.532 ; \u03b4C  88.18, 89.25, 129.46, 129.68, 131.37, 132.16, 136.41, 141.85, 124.31, 147.09, and 175.89; Vmax (FTIR) 752, 955, 1024, 1090, 1122, 1295, 1367, 1485, 1662, 2200 cm\u22121; m/z HRMS (ES): MH+ 183.0552.Quinoxaline derivative LA-16A  was synt3N  in 10 mL dry THF, cooled to 0 \u00b0C, followed by drop-wise addition of MeSO2Cl . The reaction was maintained at 0 \u00b0C for 2.5 h under nitrogen atmosphere. The reaction was quenched by the addition of aqueous saturated solution of NaHCO3 (10 mL), the layers were separated and the aqueous layer was extracted with Et2O (10 mL \u00d7 3). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified on prep TLC using 3:7 ethyl acetate/n-hexane and gave 3- prop-2-ynyl methanesulfonate as a brown solid , mp = 93.8 to 96.7 \u00b0C; \u03b4H  3.193 , 5.159 , 7.795 , 8.067  and 8.885 ; \u03b4C  38.95, 57.12, 84.54, 86.12, 129.25, 131.00, 131.20, 137.56, 141.33, 141,97, and 146.60; Vmax (FTIR) 523, 649, 765, 801, 938, 1008, 1172, 1355, 1490, 2953 cm\u22121; m/z HRMS (ES): MH+ 263.0481.Quinoxaline derivative LA-39B  was syntThe FRAP assay was carried out on the four quinoxaline derivatives to evaluate their reducing power potential. The DPPH assay was carried out to evaluate the free-radical scavenging abilities of the quinoxaline derivatives. The ability of quinoxaline derivatives to induce cancer cell death was assessed using the MTT assay after challenging various cancer cell types with the four selected quinoxaline derivatives. The two quinoxaline derivatives, LA-39B and LA-55, with high free-radical scavenging activities were tested on Lipopolysaccharide (LPS)-activated Raw 264.7 mammalian macrophages. Reactive oxygen species (ROS) profiles are used as an indicative measure of oxidative stress levels in mammalian cells ,15,16,17Free-radical levels in cancer cells play a crucial role in DNA stability and the ability to proliferate ,15,16,17Induction of apoptosis was analyzed through staining of A549 cells with Acridine Orange and Ethidium Bromide wherein double-staining with the two dyes display an orange fluorescence indicative of apoptotic cell death whereas green nuclear fluorescence designates viable cells. Apoptosis profiles are displayed in a quadratic format showing various stages of apoptosis after treatment of A549 lung cancer cells with LA-39B and LA-55. Despite major advancements in the management of cancer through chemotherapy, the progressive pledge in research of discovering new treatment strategies with improved efficacy is of crucial importance ,15. The Antioxidant properties have been identified for their preventive action against cancer through a number of cellular processes which include apoptosis . In thisApoptosis is a programmed and regulated process in induction of cell death which is characterized by numerous cellular and molecular changes . An apopThe FRAP assay is principled on the ability of antioxidant-containing compound to react with potassium ferricyanide to form ferrocyanide. This will then react with ferric chloride to form ferrous complex that has absorption maximum at 700 nm. Antioxidant activity of quinoxalines was assessed following the ferric reducing power assay protocol as previously described . BrieflyThe principle of the assay is based on the ability of antioxidant containing compounds to scavenge the DPPH stable free radical by reducing the purple colored DPPH to yellow color. The assay was carried out as described previously . Briefly2 environment in cell culture flasks. Raw 264.7 cells and A549 cells were maintained in RPMI-1640 culture medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine and 1x penicillin, streptomycin, and neomycin mixture (PSN). Cervical cancer HeLa and breast cancer MCF-7 cells were maintained under the same conditions but maintained in Dulbecco\u2019s modification of Eagle medium (DMEM) medium supplemented with 10% FBS, 2 mM L-glutamine, and 1\u00d7 PSN. Cell density was determined by diluting cells with 10\u00d7 trypan blue dye and counted using a Life technologies countess II FL automated cell counter .Raw 264.7 macrophage cells, lung cancer A549 cells, breast cancer cells MCF-7, and cervical cancer HeLa cells were maintained at 37 \u00b0C, in a humidified 95% air and 5% CO4 pre-well in a 96 well-plate and incubated overnight. Cell were then treated with various concentrations of quinoxaline derivatives , 0.5% DMSO in culture medium and 20 \u03bcg/mL Actinomycin-D  for 24 hrs. Prior to the addition of the MTT reagent, cell imaging was conducted. MTT of 5 mg/mL  was added and after 4 h of incubation the aqueous medium was replaced with 100 \u00b5L DMSO. The blue formazan crystals were allowed to dissolve in DMSO by incubating in the dark for 30 min. Absorbance was then measured at 570 nm using GloMax-Multi microplate reader . Cell viability was determined using MTT  assay which is principled on the ability of mitochondrial succinyl dehydrogenase in viable cells to convert soluble yellow tetrazolium salt into an insoluble formazan product. Briefly, cells  were seeded at a density of 6 \u00d7 102DCFDA dye to permeate the plasma membrane and fluoresce to 2\u2019,7\u2019-dichlorofluorescein (DCF) upon encounter with ROS after removal of the diacetate moiety by cellular esterases. Briefly, cells were seeded in 12 well plates at a density of 6 \u00d7 105 cells/mL for 24 h. Oxidative stress was stimulated in Raw 264.7 cells using Lipopolysaccharides (10 \u00b5g/mL) for 24 h and in A549 cells using hydrogen peroxide (1 mM) for 1 h before staining. Cells were then treated with quinoxaline derivatives at 25 \u00b5M and Actinomycin D 20 \u00b5g/mL for 24 h. Cells were detached, centrifuged, and re-suspended in culture medium with H2DCFDA for 30 min at 37 \u00b0C in the dark. Analysis was then carried out using the Muse\u2122 Cell Analyzer.This fluorescence-based assay is principled on the ability of the H5 cells/mL on coverslips in 6 well-plates overnight. Cells were then treated for 24 h with quinoxaline derivatives at 25 \u00b5M and Actinomycin D at 20 \u00b5g/mL Plates were centrifuged at 5000 rpm for 5 min then washed with PBS. Cells were then fixed with 2.7% paraformaldehyde for 30 min and centrifuged for 5 min at 5000 rpm. Cells were stained with Acridine Orange 100 \u00b5g/mL for 5 min, centrifuged and washed then stained with Ethidium Bromide 100 \u00b5g/mL for 5 min and washed again after centrifuging. Coverslips were then mounted onto slides and viewed under the microscope using the Nikon Ti-E inverted microscope.The extent of apoptosis in cells was assayed using the Acridine Orange/Ethidium Bromide assay. This assay is based on staining cells with Acridine Orange (AO) which stains the DNA and RNA of live cells and Ethidium Bromide (EB) which stains exposed DNA and RNA of dead/necrotic cells . The com\u00ae Annexin V and Dead Cell Assay kit was used according to manufacturer\u2019s instruction. Briefly, cells were seeded in 12 well-plates at a density of 6 \u00d7 105 cells/mL for 24 h. They were then treated with quinoxaline derivatives at 25 \u00b5M and Actinomycin D 20 \u00b5g/mL for 24 h. Cell were detached, centrifuged, and resuspended in 1% FBS. Muse\u2122 Annexin V and Dead Cell Reagent was then added to the suspension and incubated for 20 min in the dark. Analysis was then carried out using the Muse\u2122 Cell Analyzer.This assay is principled on Annexin V and PI stain which is based on the staining of cells undergoing early apoptosis with Annexin V which binds to phosphatidylserine which is being expressed on an impermeable membrane, while with late apoptosis, PI will be able to bind to the DNA due to permeability of the membrane. MuseIn this study, the four quinoxaline derivatives were designed to possess the signature benzopyrazine core molecule made of a benzene ring and a pyrazine ring capable of triggering antimicrobial activities and induce anticancer properties due to their ability to bind DNA ,32. FourTM 3 software with variants test (ANOVA), Tukey\u2013Kramer to compare samples. Each value represents the mean \u00b1 SD of three experiments performed in triplicate wherein p-value represents the level of significant changes and p < 0.05 is considered statistically significant. Levels of significance are denoted using an associated asterix as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.All experiments were carried out in duplicate and repeated three times to confirm consistency and determine level of variation. Data was reported as mean \u00b1 SD and statistical analysis was performed using Graph Pad InstatThe results obtained demonstrate that quinoxaline derivatives, 3- prop-2-ynyl methaonosulphate (LA-39B) and 3- prop-2-yn-1-ol (LA-55), can potently induce significant anticancer properties against A549 lung cancer cells while displaying no cytotoxicity against the non-cancerous Raw 264.7 macrophage cells. These newly developed series of quinoxaline derivatives may present new alternatives with potential abilities for use in lung cancer treatment."}
{"text": "Background: In persons with Parkinson\u2019s disease (pwPD) any additional somatosensory or distractor interference can influence the posture. When deprivation of vision and dual-task are associated, the effect on biomechanical performance is less consistent. The aim of this study was to evaluate the role of the visual deprivation and a cognitive task on the static balance in earlier stage PD subjects. Methods: Fifteen off-medication state pwPD (9 women and 6 men), 67.7 \u00b1 7.3 years old, diagnosed PD since 5.4 \u00b1 3.4 years, only Hoehn and Yahr state 2 and fifteen young control adults (7 women and 8 men) aged 24.9 \u00b1 4.9 years, performed semi-tandem task under four randomized experimental conditions: eyes opened single-task, eyes closed single-task, eyes opened dual-task and eyes closed dual-task. The center of pressure (COP) was measured using a force plate and electromyography signals (EMG) of the ankle/hip muscles were recorded. Traditional parameters, including COP pathway length, ellipse area, mediolateral/anteroposterior root-mean-square and non-linear measurements were computed. The effect of vision privation, cognitive task, and vision X cognitive was investigated by a 2 (eyes opened/eyes closed) \u00d7 2  repeated-measures ANOVA after application of a Bonferroni pairwise correction for multiple comparisons. Significant interactions were further analyzed using post-hoc tests. Results: In pwPD, both COP pathway length (p < 0.01), ellipse area (p < 0.01) and mediolateral/anteroposterior root-mean-square (p < 0.01) were increased with the eyes closed, while the dual-task had no significant effect when compared to the single-task condition. Comparable results were observed in the control group for who COP pathway was longer in all conditions compared to eyes opened single-task (p < 0.01) and longer in conditions with eyes closed compared to eyes opened dual-task (p < 0.01). Similarly, all differences in EMG activity of pwPD were exclusively observed between eyes opened vs. eyes closed conditions, and especially for the forward leg\u2019s soleus (p < 0.01) and backward tibialis anterior (p < 0.01). Conclusions: These results in pwPD without noticeable impairment of static balance encourage the assessment of both visual occlusion and dual-task conditions when the appearance of significant alteration during the dual-task could reveal the subtle worsening onset of the balance control. Parkinson\u2019s disease (PD) is a neurodegenerative disease leading to plastic changes in the primary motor cortex, with a multifaced physiopathology, whose cardinal motor symptoms are bradykinesia, rigidity, rest tremor and postural instability . PosturaThe semi-tandem stance, a position where the toes of one foot are in line with the inside arch of the other foot, has been found to be the most challenging quiet stance task that can be sustained by healthy young and older subjects for at least 30 s  of PD coThis study was set to test how visual deprivation and dual-task affect body sway in OFF-state pwPD who hold a semi-tandem stance. A control group of healthy subjects underwent the same protocol. We expected an effect of both visual deprivation and dual-task on the semi-tandem performances, especially in the patients. We hypothesized a significant increase in neuromuscular activity of the leg muscles during the more challenging condition, in relation with an increase of ankle contribution to maintain body sways .Fifteen pwPD and fifteen healthy young adults  participAll subjects provided informed written consent as required by the Declaration of Helsinki. The experiment was approved by the local ethics committee of the University Paris-Saclay (EA4532) and by the \u201cComit\u00e9 de Protection des Personnes Ile-de-France XI\u201d under identification number 19028-60429.Clinical assessments of pwPD were conducted at least 30 min before the study procedure. These tests included the Unified Parkinson\u2019s Disease Rating Scale motor examination , Berg BaAll subjects  had to stand for 60 s on a force platform with their feet in a semi-tandem position  with theThe subjects completed three trials for each of the four randomly-assigned conditions: semi-tandem only with eyes open (EO-ST) and eyes closed (EC-ST), semi-tandem with additional cognitive task with eyes open (EO-DT) and eyes closed (EC-DT). In the eyes open conditions (EO-ST and EO-DT), they were instructed to fix the gaze on a 10 cm diameter target placed at eye level at 6 m distance.The subjects sat for 3 min between the trials to prevent them from extensive fatigue. The feet outline was drawn on the platform to ensure identical foot positions between all measurements. During the mentally idle condition, instructions such as \u201cconcentrate on the task\u201d ,26, or \u201cThe ground reaction forces and moments were acquired using a floor-embedded force plate, sampling at 1000 Hz. Due to the inter-centeral study design the measurements were conducted with different systems but identical data collection settings: AMTI BP9001800  for the pwPD and a Kistler 9260AA  for the young adults.The positions of the center of pressure (COP) in the mediolateral (ML) and anteroposterior (AP) directions were computed offline using a custom-made MATLAB  routine and were bandpass filtered between 0.1 and 5 Hz  with a n2] . The PL was defined as the total length of the COP trajectory on the platform [mm]. MeanFreq came from the power spectrum of the COP sway in both AP and ML directions. The EA represents the area of the smallest ellipse which covered 95% of the COP samples [mm2] .Three non-linear methods\u2014Sample Entropy (SampEn), Multi-Scale Entropy (MSE), and Multivariate Multi-Scale Entropy (MMSE)-were used to measure the regularity of the COP signals from the unfiltered signal and to compare their sensitivity between the incremented time series and the original time series. SampEN is essentially a negative logarithm of the conditional probability of the sequences of a data vector. If a vector of length N has repeated itself in tolerance \u03b3 for m points, it will also do so for m + 1 point. The conditional probability means the ratio of counts of repeated time of m + 1 point to that of m points. Thereby, high SampEN arises from a low probability of repeated sequences in the data. Higher SampEN means lower regularity and more complexity in the data. Based on SampEN, MSE is a method evaluating the complexity of signals over different time scales while MMSE generalizes the analysis to the multivariate case . In shorEMG activities were analysed for the M. tibialis anterior (TA), M. soleus (SOL), and M. Tensor of Fascia Lata (TFL). According to the respective leg positions, Muscle were determined as frontal and backward TA, SOL and TFL. Signals were recorded using a wireless EMG system in the pwPD  and in the young adults . The standard guidelines recommended by the SENIAM  were appp < 0.01 was selected to consider significant results.Descriptive statistics were computed for all variables under the four conditions. The normality of distribution (Shapiro-Wilk test) and homoscedasticity of data (Levene\u2019s test) were verified before the application of the parametric tests. Since the criteria were not satisfied for all parameters, sway metrics were log-transformed to meet the normality request for further analysis. In pwPD and controls, the effect of vision privation, cognitive task, and vision X cognitive were judged by a 2 (eyes opened/eyes closed) \u00d7 2 (without/with cognitive task) repeated-measures ANOVA. Significant interactions were further analyzed using Bonferroni post-hoc tests. Also, a Bonferroni pairwise correction was applied to account for multiple comparisons  and a threshold of The characteristics of the pwPD and participants in the control group are displayed in p < 0.001, longer with eyes closed: EO-ST 1606 \u00b1 1060 mm vs. EC-ST 2546 \u00b1 930 mm, EO-DT 2105 \u00b1 1749 mm vs. EC-DT 2874 \u00b1 1776 mm), ellipse area , RMS of AP and ML amplitude . No differences were found between other conditions for the other parameters, including non-linear metrics. Regarding muscle activities, forward SOL and backward TA resulted in increased RMS with eyes closed than with eyes open (p < 0.01), while the RMS of backward SOL and forward TFL were higher during EC-DT than during EO-ST (p < 0.01). In addition, multiple regressions have been conducted with age, sex, UPDRS and Berg Balance Scale as potential confounders. No effects were observed.In pwPD , differep < 0.01, EO-ST 1654 \u00b1 220 mm, EC-ST 1985 \u00b1 223 mm, EO-DT 1793 \u00b1 272 mm, EC-DT 2041 \u00b1 332 mm) and longer with eyes closed compared to EO-DT (p < 0.0001). Ellipse area was larger with eyes closed compared to EO-ST (p \u2264 0.01). Differences were found in MSE for AP and ML amplitudes (p \u2264 0.01), demonstrating a lower regularity for the eyes closed conditions than during EO-ST. EMG activities did not differ across the four conditions.The analyses of the control group lead to differences in the COP pathway length and ellipse area. The COP pathway was longer in all conditions compared to EO-ST (No differences between pwPD and controls were found for the length of the COP pathway, MeanFreq AP and few conditions for MeanFreq ML and MedianFreq ML. The other parameters were significantly different between groups, with better outcomes for the controls .In this study we investigated challenging postural tasks in pwPD with no noticeable balance impairments, to determine whether, and to which extend, subtle balance disorders can be detected. The current protocol, where visual deprivation and dual-task paradigms were evaluated during semi-tandem stance, provided new insights. Overall trends in the results were similar in pwPD and young controls, although COP path length was significantly longer for controls when performing dual-task than single-task with the eyes opened. Maintaining postural stability involves sensorimotor transformations that continuously integrate several sensory inputs and coordinate motor outputs to muscles throughout the body . In thisThe lack of visual inputs implies a greater reliance on the integration of the other sensory outputs, with a suitable informational processing speed. A healthy person relies on visual, somatosensory, and vestibular information for balance control, but all or some of the components of this system may be dysfunctional in Parkinsonian patients . TherefoVuillerme and Nafati  observedOn the other hand, all difference in EMG activity have been exclusively observed between eyes opened vs. eyes closed conditions, especially for the forward leg\u2019s M. soleus and backward M. tibialis anterior, which demonstrated increased RMS when eyes were closed. This result was expected based on the M. soleus\u2019 fundamental support role during quiet standing , with a More surprising were the results under the dual-task condition. The pwPD included in the study demonstrated no alteration to biomechanical outcomes or EMG RMS when a cognitive task was added to the postural primary task, independently of the vision condition. The pwPD have issues in automating movements, which increases the attentional demand during daily activities and generates difficulties associating a simultaneous cognitive task to a motor task . NieuwhoIn the most complex task, under dual-task with eyes closed, the increase in both M. soleus and M. tibialis anterior activity of the backward leg might have reflected the necessity for the pwPD to use an ankle strategy with co-activation of the muscles and could underly the postural instability during quiet standing , since tIn addition to the most standard COP metrics, we analyzed non-linear parameters, such as entropy, to leverage information that traditional measurements do not reveal. While the use of non-linear methods has a long tradition to distinguish between visual conditions in elderly patients , former Some limitations must be discussed. The findings around dual-task are hard to interpret. First, the cognitive performance during the dual-task was not assessed and the trade-off between cognitive and balance tasks cannot be determined. Then, the findings may be due to the mild disease of the participants, or perhaps cortical control is not as important in early disease or compared to other motor tasks such as gait. One recent review suggests the importance of cerebellum for postural control . RegardiFuture studies should first overcome these limitations. The use of a conceptual model would be helpful to characterize patterns of cognitive-motor dual-task interference , especiaIn pwPD without noticeable impairment of static balance, visual deprivation seems to alter more standing performance during semi-tandem stance than the addition of a secondary cognitive task. However, for clinical assessment, it can be valuable to combine both visual occlusion and dual-task conditions, when studies in pwPD with higher functional impairments usually demonstrated significant poorer balance on dual-task than single-task . Appeara"}
{"text": "After exclusion of values obtained from four patients with ECMO or Cytosorb\u00ae adsorber the median concentration was 0.91 \u00b5g/mL  in the RRT group. In addition to previous recommendations we propose to monitor ISA trough plasma concentrations in certain circumstances including RRT, other extracorporeal treatments and obesity.Isavuconazole (ISA) is a triazole antifungal agent recommended for treatment of invasive aspergillosis or mucormycosis. The objective of this study was to evaluate ISA levels in a real world setting in a mixed patient cohort including patients with non-malignant diseases and extracorporeal treatments, and to correlate findings with efficacy and safety outcomes. We investigated 33 ISA treatment courses in 32 adult patients with hematological and other underlying diseases and assessed the clinical response, side effects and ISA trough plasma concentrations. ISA treatment led to complete and partial response in 87% of patients and was well tolerated. The median ISA plasma concentration was 3.05 \u00b5g/mL  in patients without renal replacement therapy (RRT) or extracorporeal membrane oxygenation (ECMO) and significantly lower in patients with RRT including cases with additional ECMO or Cytosorb Isavuconazole (ISA) is a triazole antifungal agent recommended for treatment of invasive aspergillosis or mucormycosis ,2,3. In One larger study investigated 283 plasma concentrations from real world ISA use, and found ISA plasma concentrations of >1 \u00b5g/mL in >90% of patients, resembling findings from the SECURE trial . HoweverThe objective of this study was to evaluate ISA levels in a real world setting in a mixed patient cohort including patients with non-malignant diseases and extracorporeal treatments, and to correlate findings with efficacy and safety outcomes.This observational national multicenter cohort study prospectively included patients aged 18 years or older undergoing treatment with ISA at four centers in the Southern and Eastern part of Austria, the Medical University of Graz, the Landeskrankenhaus (LKH) Graz 2, LKH Hartberg, and the Klinikum Baden from November 2016 to October 2019.Based on recommendations of the local drug advisory committees ISA was allowed to be administered in patients with possible/probable/proven invasive aspergillosis using revised EORTC MSG criteria and/or Blot criteria in case of invasive aspergillosis in critical ill patients ,14. PatiPatients\u2019 medical records were reviewed individually by using a standardized data collection template in order to collect demographic information and clinical data, mycological laboratory test results, ISA trough plasma concentrations, as well as ISA formulation, dosing information, termination of ISA treatment as well as other antifungal therapy, clinical response, serious adverse events, and breakthrough infections. Treatment success, failure and death were classified according to EORTC/MSG criteria . BreakthThe day of the first administration of ISA was considered as day 0. Samples for determination of ISA trough plasma concentrations were obtained in the morning immediately prior to scheduled ISA infusion or intake as ordered by the treating physician. As no routine TDM algorithm for ISA was available by the current literature ,6,7, plap-value of <0.05 was considered significant.The study was approved by the local ethics committee, Medical University Graz, Austria (protocol number 29\u2013444 ex 16/17). All statistical analyses were performed using R version 3.5.1. Continuous data  are presented as medians (inter-quartile ranges [IQR]). The data at patient level were summarized by calculating the median  of Isavuconazole levels. More specifically, in patients with more than one single measurement, the median and maximum levels of Isavuconazole were calculated. By doing so, each patient is represented by one single summary measurement (median or maximum) and standard statistical tests can be applied. We used the median as the main variable for comparison between groups and the maximum value was used for sensitivity analysis. Measurements of patients assigned to specified groups  were considered independent. Group comparisons were performed by Wilcoxon-rank-sum test. A 2 . A total of 14 patients (44%) had hematological diseases , whereas the 18 other patients (56%) had mixed underlying diseases  male, nine (28%) female; median age 60 years, range 24 to 85 years, IQR 46\u201369). Study patients had a median body mass index of 24.6 kg/mdiseases .\u00ae adsorber therapy .Fourteen of 32 patients (44%) had proven invasive fungal disease, nine (28%) had probable and nine (28%) had possible invasive fungal disease. One patient with two courses of ISA was classified as suffering from probable invasive fungal disease on both occasions. The median duration of ISA administration was 45 days . In 12/33 (36%) ISA courses the patients received only the intravenous and in 6/33 (18%) courses only the oral formulation of ISA. In 15/33 (45%) ISA courses patients received the intravenous and oral formulation. The intravenous ISA formulation was used for a median of 13 days  whereas the tablet was used for a median of 70 days . In a total of 20/33 courses (61%) the patients received antifungal therapy or prophylaxis prior to ISA, in six of these 20 courses (30%) ISA was used due to failure and in 13/20 courses (65%) ISA was used due to side effects of preceding antifungals. A total of 11/32 patients (34%) were on renal replacement therapy , while three of these patients additionally had extracorporeal membrane oxygenation (ECMO) and one additionally had CytosorbFusarium solani blood stream isolate and died six weeks later . ISA plasma concentrations were 1.51 \u00b5g/mL on day 4, 1.32 \u00b5g/mL on day 6 of ISA treatment, and 1.36 \u00b5g/mL two days after termination of ISA treatment. In 3/33 (9%) ISA courses no classification was possible . Overall, a total of 11/32 patients (34%) had a fatal outcome during hospitalization including the three IFI related deaths. No breakthrough infection during ISA administration was observed.Complete response occurred in 18/30 (60%) ISA courses, partial response in 8/30 (27%), and stable disease in 1/30 (3%) ISA courses. Assessment was not possible in three cases. Three out of 32 patients (9%) had a fatal outcome during hospitalization attributable to fungal disease. One patient with pulmonal and cerebral aspergillosis died on day 4 and had no ISA plasma concentrations measured. One patient with possible invasive mould disease died on day 10 of ISA treatment and had 1.36 \u00b5g/mL ISA plasma concentration on day 3 (without RRT) and 1.69 \u00b5g/mL on day 6 while on continuous hemodialysis. The remaining patient received six days of ISA, was switched to lipAmpB plus voriconazole after receiving the antifungal susceptibility testing of a Six of 33 (18%) ISA treatments led to adverse events including one case with an anaphylaxis , one with leucopenia (1.52G/L), two with elevated liver enzymes, one with paraesthesia and one with erythema and elevated liver enzymes. All of these patients had concomitant medication, thus the role of ISA in the reported adverse events remains unclear. All patients recovered fully from adverse events.\u00ae adsorber therapy) the median concentration was 0.91 \u00b5g/mL  in the RRT group. Patients without extracorporal treatments also had higher maximum Isavuconazole trough levels compared to the groups with extracorporal treatments . Two patients (body mass index 23 and 26 kg/m2) with influenza and ARDS received ECMO  for pulmonary support and continuous RRT. One ECMO patient was treated with a standard dose of ISA for invasive aspergillosis since preceding and dose escalated intravenous voriconazole did not reach target levels of >1 \u00b5g/mL. On day 12 after initiation of intravenous ISA the plasma concentration was measured and was 1.79 \u00b5g/mL. The patient died 2 days later due to a gangrenous bowel. The other ECMO patient had a standard dose of ISA for treatment of probable intraabdominal Candida parapsilosis infection after failure of caspofungin. On day 1 and 4 after initiation of intravenous ISA the plasma concentration was 0.74 \u00b5g/mL (during loading dose) and 0.57 \u00b5g/mL, respectively. ECMO was terminated on day 6. Subsequent ISA plasma concentration during the second ISA treatment course of this particular patient was 2.44 \u00b5g/mL while receiving RRT. The patient died 5 weeks later due to secondary sclerosing cholangitis. The third patient was treated with a Cytosorb\u00ae cytokine adsorber within a continuous RRT circuit for 4 days and had an ISA plasma concentration of 1.3 \u00b5g/mL immediately prior and 0.82 \u00b5g/mL on the last day of adsorber treatment. Subsequent ISA plasma concentrations determined 14 and 32 days after the adsorber treatment but during ongoing RRT were 0.62 \u00b5g/mL and 0.91 \u00b5g/mL. This patient suffered from pulmonary invasive aspergillosis and had a favorable response after 56 days of ISA treatment. The fourth patient developed ARDS after major cardiac surgery and was treated for pulmonary invasive aspergillosis. ISA concentration was measured daily during a period of 18 days. During ECMO the median ISA concentration was 1.7 \u00b5g/mL. On day 12 additional RRT was initiated and ISA concentration decreased to 0.8 \u00b5g/mL. ECMO was discontinued on day 15 but RRT was still ongoing and ISA concentration remained below 0.9 \u00b5g/mL. Boxplots of ISA plasma concentrations with and without extracorporeal treatments are shown in A total of 145 ISA plasma concentrations were measured. Five ISA plasma concentrations were excluded due to erroneous sampling time points . Thus, 140 ISA plasma concentrations were analyzed from 29 courses of ISA treatment with a median of three measurements per patient (range 1\u201318). There was no change in the dosage due to the measured plasma levels. In three out of four patients with missing ISA plasma concentrations ISA was administered for less than 5 days  while it was administered for one month in one patient. Median time of first measurement after initiation was 2.28 days . The median plasma concentration was 2.35 \u00b5g/mL (range 0.66 to 9.1 IQR 1.49\u20133.71). After exclusion of values during RRT or ECMO the median ISA concentration was 3.05 \u00b5g/mL  . The medAmong the six of 33 (18%) ISA treated patients with adverse events, four had ISA plasma concentrations measured, which were always below 5.5 \u00b5g/mL .In our cohort ISA was used for treatment of invasive fungal diseases in patients with malignant and non-malignant underlying diseases. Overall, ISA was well tolerated compared to previous studies ,10. EighAs influenza might result in ARDS requiring extracorporeal membrane oxygenation (ECMO) and was reported to be an independent risk factor for invasive pulmonary aspergillosis  the selep < 0.001; 42% reduction rate of ISA concentration) [These findings are of course limited by low numbers but may highlight the need of ISA TDM in patients with ongoing ECMO, a fact that was also previously reported for other antifungals like voriconazole or caspofungin. Based on ISA concentrations measured in our ECMO patients one might assume that ISA is sequestrated within the ECMO circuit during the first days of treatment leading to low plasma concentrations according to an assumption previously described for voriconazole . As bothtration) . Our critration) .\u00ae cytokine adsorber four times for 16 h each within a RRT circuit. Whereas no data of this cytokine adsorber are available determination of ISA plasma concentrations in our patient suggest that this particular device might lower ISA plasma concentrations. However, as the patient subsequently showed ISA plasma concentrations of 0.68 \u00b5g/mL and 0.91 \u00b5g/mL 14 and 32 days after cytokine adsorption the exact effect of the Cytosorb\u00ae cytokine adsorber remains unclear.Cytokine reduction by hemoadsorption aims to attenuate the overwhelming systemic levels of pro-inflammatory and anti-inflammatory mediators released in the early phase of sepsis. One of our ISA treated patient had a Cytosorb2 (140 kg body weight) which was outside the BMI and weight standard deviations of published ISA data [2 the patient received the standard dose of ISA for treatment of probable invasive aspergillosis and was closely monitored by ISA plasma concentrations. The obtained ISA plasma concentrations were 1, 2.42, 3.68 and 3.42 \u00b5g/mL, respectively. The patient had a complete response.One of our patients had a body mass index (BMI) of 39.6 kg/mISA data ,24. PrevISA data . DespiteA post hoc analysis of ISA exposure-response relationship for measures of efficacy and safety in patients with invasive aspergillosis and infections by other filamentous fungi from the SECURE clinical trial showed that no statistically significant relationship between ISA exposure and either efficacy or safety endpoints. The authors concluded that lack of association between exposure and efficacy indicates that the ISA exposures achieved by clinical dosing were appropriate for treating the infecting organisms in the SECURE study and that side effects were not related to increase in exposures . ComparaIn summary, ISA was used in patients with and without hematological malignancies for treatment of invasive fungal diseases. ISA was efficacious, well tolerated and showed ISA plasma concentrations comparable to previously successfully treated patients even in cases with high body weight (BMI). Plasma concentrations in patients receiving RRT were significantly lower compared to patients without. However, due to low numbers of patients with high body weight and RRT as well as inconsistent or low levels obtained in patients with ECMO or cytokine adsorber we propose to monitor ISA plasma concentrations in such patients to attain previously suggested but still uncertain target levels of ISA >1 \u00b5g/mL ,9,22."}
{"text": "Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, \u03b1-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from  Ion ce signal ,2, whilee signal . At the e signal ,7, malfue signal ,9, and de signal .The artificial lipid bilayer recording technique measures the ion transport activities of channel proteins along with other ion transporting proteins and peptides . This teThe physical processes that occur during lipid bilayer formation have been extensively studied and are described in standard textbooks . PrincipMembrane proteins, including channel proteins, are generally not inserted into bilayers directly from the aqueous solution, with certain exceptions such as voltage-dependent anion channels (VDACs). The most efficient method used to incorporate channel proteins into bilayers is vesicle fusion. However, because vesicles stochastically fuse with the bilayer, the process cannot be controlled, and thus sometimes takes a long time.Streptomyces lividans (KcsA) or from Methanobacterium thermoautotrophicum (MthK) channels by binding them to cobalt affinity gel beads with a histidine tag and then forming a lipid bilayer membrane with the channels on the bead by pushing the lipid layer onto the bead [In order to improve the measurement efficiency, several approaches have been proposed. For example, bilayer membranes can be promptly made in a lipid solution at the droplet\u2013droplet interface ,14,15, dthe bead . In anotthe bead . By contthe bead ,20,21. In general, we have developed techniques for channel current recordings using various types of substrate-supported bilayers to improve the measurement efficiency of conventional artificial bilayer methods. Here, we report further enhanced efficiency by simplifying the bilayer formation technique and shortening the required time for the measurement preparation. This component technology could potentially lead to the miniaturization of a measurement apparatus. 2+ affinity gel beads  from Clontech , and Sepharose 4B beads were from Cytiva Japan . All other chemicals were commercial products of analytical grade.Asolectin, alamethicin, and gramicidin were purchased from Sigma , \u03b1-hemolysin was from Abcam plc , CoStreptomyces lividans (KcsA channel) gene cloned into pQE-30 vectors, including an N-terminal hexahistidine tag, was a gift from Dr. Kubo of the National Institute of Advanced Industrial Science and Technology. We used a KcsA mutant (E71A) made by using the QuickChangeTM site-directed mutagenesis kit (Stratagene), because the E71A mutation is known to inhibit inactivation.The cDNA of mouse voltage-dependent anion channel 1 (VDAC1) (ENSMUST00000102758.7) was subcloned into a multi-cloning site (NdeI-XhoI) of the pET21b vector. The potassium channels from Escherichia coli BL21(DE3) and then overexpressed in the presence of 1 mM isopropyl-d-thiogalactopyranoside (IPTG) for 4 h. The BL21(DE3) pellets were incubated in BugBuster Protein Extraction Reagent  containing protease inhibitors  and benzonase (Merck) for 5 min at room temperature, and then incubated in BugBuster Protein Extraction Reagent containing 250 \u03bcg/mL lysozyme (Merck) for 5 min at room temperature. After they were centrifuged at 16,000\u00d7 g for 20 min, the precipitates were sonicated in binding buffer ) and then centrifuged at 16,000\u00d7 g for 10 min. The supernatant was mixed with nickel resin  and rotated at 4 \u00b0C for 60 min. Nonspecific bound proteins were removed with 5-fold volume of binding buffer, 5-fold volume of wash buffer ), and 5-fold volume of refolding buffer , 50 mM Tris-HCl (pH 8.0)). Then, VDAC1 was eluted with elution buffer ) and dialyzed with a dialysis membrane . VDAC1 protein was further purified by cation exchange using \u00c4KTA explorer 10S  and RESOURCE\u2122 S column .The pET21b plasmids containing the VDAC1 sequence were transformed into Escherichia coli XL1-Blue and overexpressed by the addition of IPTG to a final concentration of 0.5 mM for 2 h. The expressed channels were extracted from membrane fractions by 10 mM n-dodecyl-d-maltoside . Co2+ affinity gel beads  equilibrated with normal buffer , 100 mM KCl) were added to the extracted channel protein solution and incubated for 30 min at 4 \u00b0C in order to bind histidine tags to the gel beads. Nonspecific bound proteins were removed with washing buffer . The pQE-30 plasmids containing the KcsA sequence were transformed into g. The supernatant was centrifuged for 40 min at 14,000\u00d7 g and then for 20 min at 9500\u00d7 g. The pellet was resuspended in solution  with a Teflon pestle homogenizer. Samples were incubated on ice for 1 h and centrifuged for 1 h at 42,000\u00d7 g. The pellets were resuspended in 400 mM KCl, 10% sucrose (w/w), and 5 mM Na-PIPES, pH 6.8. The microsomes were placed on top of a discontinuous sucrose gradient (w/w): 20:25:30:35:40%. The gradient was centrifuged at 67,500\u00d7 g in a swinging rotor for 18 h. Membranes obtained from the 20:25% sucrose interface were used in this study.Myometrium plasma membrane vesicles containing BK channels  were prepared according to the method of Toro et al. . Membran2+ affinity gel bead, on which KcsA proteins were immobilized, was used in the case of KcsA channel recordings and a Sepharose 4B bead was used for other types of channels. A glass capillary  was pulled to make a pipette with a very fine tip (<1 \u03bcm) using a pipette puller , then the tip was cut to make a large aperture (>10 \u03bcm). The tip was fabricated using a microforge  in order to smooth the surface. 2+ affinity gel bead fixed at the tip of a glass pipette by suction. The glass pipette was set with a pipette holder and a bead was fixed at the tip of the pipette by suctioning it through a tube from the holder with a syringe. In A hydrogel bead was immobilized to the aperture at the glass pipette tip by suction. A Con-decane or hexadecane) was layered over the recording solution layer. Bilayers were made by moving the bead fixed at the pipette tip through this lipid solution layer into the recording solution  by an Ag\u2013AgCl electrode defined the membrane potential. Unless otherwise noted, the signal was filtered at 1 kHz, sampled at 10 kHz, digitized, then stored on a PC. One of two channel forming peptides , the hemolytic protein \u03b1-hemolysin, or detergent-solubilized VDAC1 was added to the recording solution at appropriate concentrations before the bilayer formation. These molecules were spontaneously incorporated into the bilayer formed on the Sepharose 4B bead to make ionic channels. KcsA channel proteins were spontaneously incorporated into the bilayers by forming bilayers with a Con-decane). The aqueous recording solution was extruded out from a polyethylene tube (0.1\u20130.5 mm inner diameter) placed near the bead by manually exerting positive pressure with a syringe, such that it contacted the bead in the lipid solution. The contact was observed with a microscope. A bilayer membrane was promptly formed at the water\u2013gel interface. The solution in the tube was held at virtual ground level so that the voltage at the solution level in the glass pipette connected to a patch clamp amplifier by an Ag\u2013AgCl electrode defined the membrane potential , which agreed well with the value obtained by conventional methods [ methods .n = 5). The current fluctuation showed voltage dependency, as reported in previous papers [s papers . The opeStaphylococcus aureus, which is known to form a heptamer in bilayers, forming a large ion channel pore [n = 5), which was identical to the values obtained by conventional bilayer methods [nel pore ,28. The  methods , again vn = 5), and the channel showed voltage dependent gating as reported previously [eviously . The above confirmed that our method can be used to measure the currents of several types of channels. Importantly, the channel current properties observed were in good agreement with the results obtained from other techniques, such as the conventional planar bilayer technique and patch clamp analysis. Thus, our method can be used to study a wide range of channels. Moreover, the channel current measurements were relatively easy, as we only needed to form bilayers with recording solutions containing the channel peptides or proteins. All of the channels shown in Streptomyces lividans. It is one of the best-characterized channel proteins because its structure is very simple. As described in the Materials and Methods section, purified KcsA channels were solubilized with detergent and immobilized on a gel bead via histidine tags. The bead was moved to contact the aqueous solution through the lipid solution layer, and immobilized KcsA channels were reconstituted immediately after the bilayer formation.Next, we immobilized physiological channel proteins (which cannot be directly inserted into bilayers from an aqueous solution) on the surface of a hydrogel bead and reconstituted them into bilayers to measure their currents. As we previously reported, some types of physiological channel proteins that have been solubilized with detergent and immobilized on a gel  or solid+ channel purified from pig uterus. Prior to the bilayer formation, a hydrogel bead immobilized at the glass pipette tip was dipped into a membrane vesicle suspension that contained BK channels in the vesicular membrane. Immediately after contact between the gel and lipid\u2013water interface, the current began to fluctuate, showing that BK channels were inserted into the bilayer by vesicle fusion. It is likely that the incorporation rate was high compared with conventional bilayer methods, because the space between the gel and the lipid was narrow, meaning that the vesicle concentration was kept high. In Physiological channel proteins were also measured by incorporating them into bilayers through vesicle fusion. Bilayers were promptly made by pushing out the aqueous solution from a fine tube, such that the solution contacted a gel bead. The effect of this strategy is to miniaturize the apparatus. Above, we showed that we could measure the properties of various types of channels by using bilayers formed on a gel bead. Although our technique makes the recordings more efficient than the conventional bilayer technique, we should develop a simultaneous parallel recording technique to further increase the measurement efficiency. However, because our apparatus contains a bulky manipulator, it is not suitable for the parallel recordings of many channels. Therefore, we modified the bilayer formation technique to miniaturize the apparatus.Next, we used this modified bilayer formation technology to perform simultaneous recordings of different types of channels. The current recordings shown in By improving our previously developed channel recording techniques, here we developed an efficient method for measuring ion channel activities. We measured the natural properties of various types of channels incorporated into gel-supported bilayers. We were able to simultaneously measure different types of channels in different environments. This method allows miniaturization of a recording system. In the future, by making this system multi-channel, we may obtain a high-throughput device for channel current measurement."}
{"text": "Systemic CD8+ T cell depletion abolished NLGP-mediated metastasis inhibition and reappeared upon adoptive transfer of NLGP-activated CD8+ T cells. Interferon \u03b3-secreting from CD8+ T cells inhibit the expression of angiogenesis regulatory vascular endothelial growth factor and transforming growth factor \u03b2 and have an impact on the prevention of colonization. Neem leaf glycoprotein modulates dendritic cells (DCs) for proper antigen presentation by its DC surface binding and upregulation of MHC-I/II, CD86, and CCR7. Neem leaf glycoprotein\u2013treated DCs specifically imprint CXCR3 and CCR4 homing receptors on activated CD8+ T cells, which helps to infiltrate into metastatic sites to restrain colonization. Such NLGP's effect on DCs is translation dependent and transcription independent. Studies using ovalbumin, OVA257\u2212264, and crude B16F10 antigen indicate MHC-I upregulation depends on the quantity of proteasome degradable peptide and only stimulates CD8+ T cells in the presence of antigen. Overall data suggest NLGP inhibits metastasis, in conjunction with tumor growth restriction, and thus might appear as a promising next-generation cancer immunotherapeutic.Neem leaf glycoprotein (NLGP), a natural immunomodulator, attenuates murine carcinoma and melanoma metastasis, independent of primary tumor growth and alterations in basic cellular properties . Colonization event of invasion\u2013metastasis cascade was primarily inhibited by NLGP, with no effect on metastasis-related invasion, migration, and extravasation. High infiltration of interferon \u03b3 (IFN-\u03b3)\u2013secreting cytotoxic CD8 Metastasis, an eminent hallmark of cancer , is an i+ T cells via influencing maturation of dendritic cells (DCs)] primarily intervenes two hallmarks of cancer, that is, \u201cavoiding immune destruction\u201d and \u201cangiogenesis\u201d (Neem leaf glycoprotein (NLGP) therapeutically restricts melanoma, carcinoma, and sarcoma solid tumor growth \u201315 by imgenesis\u201d , 7 of di+ T cell depleting antibody (clone 2.43) was purchased from Taconic .RPMI-1640, Dulbecco modified Eagle medium (DMEM)-high-glucose, heat-inactivated fetal bovine serum (FBS), tri-reagent, and carboxy-fluorescein-diacetate succinimidyl ester (CFSE) were purchased from Invitrogen . CD4\u2013fluorescein isothiocyanate (FITC)/Cy-chrome, Foxp3-phycoerythrin (PE) monoclonal antibodies (mAbs), and Cytofix/Cytoperm kit were procured from BD Pharmingen . Anti-CD25 (PE), anti\u2013MHC-I , anti-MHC-II (PE), anti-CD80 (PE), anti-CD86 (PE), anti-CCR7 (PE), anti-CD44 (PE), and anti-CD69 (FITC) were purchased from Biolegends . Purified anti\u2013mouse transforming growth factor \u03b2 (TGF-\u03b2), vascular endothelial growth factor (VEGF), and interferon \u03b3 (IFN-\u03b3) mAbs, secondary antibodies either fluorescence or peroxidase labeled, were obtained from e-Biosciences . Cytotoxicity detection kit [based on lactate dehydrogenase (LDH) release] Cyber-Green were procured from Roche Diagnostics . Aminoethylcarbazol (AEC) chromogen solution and aqueous mounting media were purchased from VECTOR Laboratories Inc. . Reverse transcription\u2013polymerase chain reaction (RT-PCR) primers and cDNA synthesis kit were procured from MWG-Biotech AG  and from Fermentaus . DAPI-shield, peroxidase secondary antibodies were purchased from Sigma . Different inhibitors were procured from Calbiochem-Merck-Millipore and Sigma. CD8Azadirachta indica) leaves of same size and deep green color (indicative of same age), plucked from neem trees , were shed-dried and pulverized. Permission was taken to use such natural product from the National Biodiversity Authority (NBA), an autonomous and statutory body of the Ministry of Environment and Forests, Government of India (ref no. NBA/Tech Appl/9/518/12/14-15/4587). Leaf powder was soaked overnight in phosphate-buffered saline (PBS), pH 7.4; supernatant was collected by centrifugation at 1,500 revolutions/min (rpm). Crude neem leaf preparation was then extensively (four to five times) dialyzed against PBS, pH 7.4, and concentrated by Centricon membrane filter  with 10-kD molecular-weight cutoff. The purity of NLGP was checked by size exclusion\u2013high-performance liquid chromatography (SE-HPLC) in a protein PAK 300 SW column of 7.5 mm (ID) \u00d730 cm. The protein peaks were determined by absorption at 280 nm in a UV recorder. Batch consistency of the NLGP is checked by HPLC and tumor growth restriction assay as per standard protocol.Mature neem  were obtained from the animal facility of CNCI, Kolkata. Autoclaved dry pellet diet  and water were given l-glutamine and penicillin (50 U/mL), and streptomycin (50 \u03bcg/mL) at 37\u00b0C with 5% CO2 and 95% humidity. As the cell lines were obtained from cell bank, cell authentication was not done by us.B16F10 (melanoma) cell line was obtained from the National Center for Cell Sciences, Pune, India, and LLC (carcinoma) cell line was obtained from ATCC, Manassas VA, USA. Cells were cultured in complete DMEM high-glucose media supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM 5) cells subcutaneously on the right flank of mice. After palpable tumor formation, one group of mice was subcutaneously injected with NLGP (1 \u03bcg/g of body weight/injection); the other group was treated with PBS weekly for 4 weeks in total or once only as per the experimental requirement. Solid tumor growth (in mm2) was measured using the formula: (width \u00d7 length). Tumor-bearing mice were euthanized by overdose of ketamine HCL (160 mg/kg) + xylazine (20 mg/kg) intraperitoneally (i.p.) as per Committee for the Purpose of Control and Supervision of Experiments on Animals guideline when tumor size reached 20 mm in either direction or the animal looks sick or any necrosis of tumor was occurred. The animal's health was monitored every day.Solid tumors were developed in C57BL/6J mice by inoculation of B16F10 or LLC (5 \u00d7 105 cells) were injected through tail-vein (t.v.). Following tumor inoculation, mice were subcutaneously injected with NLGP (1 \u03bcg/g of body weight/injection) weekly for 4 weeks in total, keeping a PBS-treated group as control.For the development of lung metastasis, B16F10 or LLC cells . Fixed cells were stained with primary fluorescence antibody, mounted with fluoroshield-DAPI , and observed under fluorescence-microscope [LeicaDM4000B  or Olympus BX53 ] or confocal microscope .Cells were harvested by cytospin  on poly-Western blot of IFN-\u03b3, granzyme B, TGF-\u03b2, VEGF, and cleaved caspase 3 was performed as described . Lungs wFlow-cytometric analysis was done as described . The datCells were stained according to the protocol given by the manufacturer, BD Pharmingen. Cells were washed twice with cold PBS and then resuspended in 1\u00d7 binding buffer. Annexin V\u2013FITC and propidium iodide (PI) (5 \u03bcL of each) were added and mixed gently with vortexing. Cells were incubated for 15 min at room temperature (25\u00b0C) in the dark followed by resuspending the cells with 400 \u03bcL of 1\u00d7 binding buffer. Cells were analyzed immediately by flow cytometry.Cells were harvested, fixed in chilled 70% ethanol with vortexing, followed by incubation at \u221220\u00b0C overnight. Cells were washed twice with PBS containing 0.1% sodium azide, followed by 50 \u03bcL (100 ng/mL) RNase treatment. Cells were stained with 200 \u03bcL (50 \u03bcg/mL) PI to analyze flow-cytometrically immediately.Ki67 staining was done as described . In brieDifferent protumorous cytokines from tumor conditioning media  and cell supernatant from DC:T cell coculture  were measured  by enzyme-linked immunosorbent assay (ELISA) using microplate reader .Cellular total RNA was isolated using Trizol . Random hexamers were used to generate corresponding cDNA according to the manufacturer's protocol . Amplification was performed using 2\u00d7 green mix . Amplification of the target genes was done by gene-specific primers as described . Primer A scratch was made with a scratcher on confluent B16F10 cells, followed by NLGP treatment (1.5 \u03bcg/mL). Wells were photographed at different time points to check the healing of wound (scratch).4 and 2 \u00d7 104 cells for migration and invasion, respectively) in serum-free media in presence or absence of NLGP. Migration or invasion was measured against the 10% FBS containing media for 12 h. Following incubation, cells were fixed with 2% paraformaldehyde and stained with 0.01% crystal violet. Cells in the upper chamber were removed by wiping with cotton swabs. Serum-free gradient was used as a negative control.Overnight serum-starved B16F10 or LLC cells were seeded in the upper chamber of either Transwell or BD invasion chamber (4 \u00d7 105) cells were adoptively transferred through t.v. injection. Lungs were harvested at desired time points and digested with collagenase (1.5 mg/mL) and DNase I (0.1 mg/mL) for 30 min at 37\u00b0C for single-cell preparation, and CFSE+ cells were analyzed by flow cytometry. In a separate set, harvested lungs were prepared for cryosectioning by standard method as described (B16F10 or LLC cells were stained with CFSE (5 mM) according to the manufacturer's protocol. Tumor  were either cocultured with DCs or transferred adoptively in mice.CD8tic lung  with the+ T cell depletion status in peripheral blood was monitored by flow cytometry.Tumor-bearing mice were peritoneally injected with CD8-depleting antibody (100 \u03bcg/50 \u03bcL) 24 h prior to NLGP administration on each time point. CD8+ T cells were isolated by magnetic bead\u2013based positive selection (+ T (2 \u00d7 105) cells were adoptively transferred through t.v. injection.Metastatic lungs were harvested from PBS- and NLGP-treated mice at desired time points  and digeelection . Isolate+ T cells were isolated from PBS- and NLGP-treated lungs. Cellular cytotoxicity of those CD8+ T cells was checked by measuring LDH release assay according to the manufacturer's protocol (Roche Diagnostics). For antigen restimulation assay, CD8+ T cells were restimulated, and secreted IFN-\u03b3 was measured by ELISA. Assay was performed by the method as described (CD8escribed .Evans blue solution (0.1% in PBS) was injected through t.v. After 30 min of incubation, mice were sacrificed, and macroscopic observation was made.6 cells/mL) were cultured with complete RPMI-1640 medium containing 10% (vol/vol) heat-inactivated FBS, 2 mM l-glutamine, and Pen-Strep , with recombinant mouse Granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (10 ng/mL) and recombinant mouse Interleukin 4 (rmIL-4) (5 ng/mL) at 37\u00b0C in 5% CO2 for 6 days. Media including the supplements was refreshed every third day. On day 6 of culture, non-adherent cells were harvested and considered as immature bone marrow\u2013derived DCs (BmDCs).A single-cell suspension was obtained after flushing bone marrow from tibia and femurs. Erythrocyte lysed (by ACK lysis buffer) cells  after filtration with 0.22-\u03bcm membrane filter .B16F10 or LLC cells were cultured in complete DMEM at 37\u00b0C in 5% COAcid stripping of MHC-I was done according to the method described . Then MHImmature DCs were stained with MHC-I and then incubated at 37\u00b0C in presence or absence of NLGP, keeping controls at 4\u00b0C. After 12 h, surface MHC-I was removed by acid stripping, and internalized MHC-I was measured flow-cytometrically.Different inhibitors MG132 (0.5 \u03bcm), chloroquine (50 \u03bcm), ammonium chloride (10 mM), brefeldin A (10 nM), cytochalasin D (10 \u03bcm), cytochalasin B (20 \u03bcm), bortezomib (1 ng/mL), leupeptine (100 \u03bcm), dimethyl amilorite (200 \u03bcm), and cycloheximide (5 \u03bcm) were used to pretreat BmDCs. B16F10, LLC lysates, ovalbumin, and SIINFEKL were also used for different assays.7 cells/mL) in serum-free DMEM (measured by trypan blue dye exclusion assay) were lysed by 6 or 3 freeze (in liquid nitrogen)\u2013thaw (at room temperature) cycles for lysate or necrotic cell preparation, respectively. The lysate was centrifuged at 15,000 rpm for 30 min at 4\u00b0C, and supernatant was collected. Total protein concentration was measured using Folin's phenol reagent. Necrotic cells were centrifuged at 2,500 rpm for 5 min, and collected cells were used in different experiments.Viable B16F10 or LLC cells (1 \u00d7 105 cells/mL), after treatment with NLGP and cell lysate (for 24 h), were fixed with 0.008% glutaraldehyde, washed with PBS, and then cocultured with autologous purified CD8+ T cells (1 \u00d7 106 cells/mL) in a total volume of 200 \u03bcL in complete RPMI-1640 media for 7 days in a 96-well plate. Cells and supernatants were collected for further analysis.Dendritic cells  or three to six (for in vitro assays) independent experiments. Statistical significance was established by unpaired Student t-test (for two groups) or one-way analysis of variance followed by Tukey post-hoc test (for more than two groups) using GraphPad Prism 5 software . For survival study, Kaplan-Meier survival analysis followed by data analysis with log-rank (Mantel-Cox) test was used. Differences between groups attaining a p-value of 0.05 are considered as significant.ImageJ  was used for image analysis, brightness\u2013contrast adjustment, quantification of agarose gels, manual cell counting for invasion/migration assay, and measurement of wound healing. Quantity one (Bio-Rad) was used for Western blot gel scanning and Image Studiolite  was used for Western blot analysis. All reported results represent the mean \u00b1 SEM of data obtained in either one  in C57BL/6J mice keeping PBS-treated mice as control . In addiConsidering the linear relationship between primary tumor volume and metastasis, we checked the impact of NLGP-mediated primary tumor reduction on metastasis development. Accordingly, we used the experimental metastasis model (EMM) where tumor cells were inoculated intravenously without developing any primary tumor. On treatment completion , we founin vitro NLGP treatment has no direct effect on tumor cells or their secretomes  were injected t.v.  tumor cells were inoculated t.v., and significantly fewer number of tumor cells were detected in the lungs compared to the PBS-treated group  assay revealed higher IFN-\u03b3 release from the same T cells of the NLGP-treated mice group only  into a separate mice cohort, already inoculated with B16F10 cells  cells were collected after 48 h of NLGP treatment and found higher per-cell MHC-I expression along with increased CD80 and CCR7 in comparison to control  on purified CD8+ T cells after DC:T (1:10) cell coculture  enhanced NLGP-induced MHC-I expression. As phagosome and lysosome fusion gets inhibited by chloroquine and NH4Cl, it creates opportunity for antigen within phagosome to get export into cytosol for proteasome-mediated degradation. Hence, this leads to increase in total pool of proteasome-derived peptide, which in turn augments in the NLGP-mediated MHC-I increment. Cysteine protease inhibitor, leupeptin, which inhibits the cathepsin family protein within the phagolysosome, probably does not aid in antigenic protein export within cytosol, and hence does not augment NLGP-mediated MHC-I upregulation to a significant level. Phagocytosis inhibitors, cytochalasin D and cytochalasin B, and macropinocytosis inhibitor, dimethyl amiloride, did not show any effect (4Cl) augments this upregulation and indicates the possible involvement of NLGP in cross-presentation as cross-presentation is very relevant in tumor biology, where extracellular proteins are presented in the context of MHC-I.As NLGP upregulates MHC-I, next we wanted to know how alterations in antigen processing and presentation affect this upregulation. We found that in the presence of proteasome inhibitors, MG132 and bortezomib, NLGP-mediated MHC-I upregulation was inhibited. This observation clearly suggested that, in the absence of proteosome-generated peptide, NLGP treatment does not increase the surface expression of MHC-I and hence does not alter the basic principle, which states that only antigen-peptide\u2013bound MHC-I gets exported to the cell surface. Protein transport inhibitor, brefeldin A, also inhibits NLGP-mediated MHC-I upregulation, which is also in line with the basic principle of cellular export, whereas phagosome and lysosome fusion inhibitor  as a foreign antigen. Two different antibodies were used; one specifically detects SIINFEKL-bound H2Kb, and the other detects antigen-bound H2Kb/H2Db. More MHC-I expression was detected by H2Kb/H2Db-specific antibody in the NLGP-treated group, whereas slight increment was detected by SIINFEKL-specific H2Kb . Apart from CD8+ T cell\u2013dependent killing, we macroscopically detected reduced angiogenesis in the tumor and its adjacent areas; those are which were in line with the previous observation (+ and VEGFR2+ cells), along with the decreased level of angiogenesis regulatory factors such as VEGF and TGF-\u03b2, was detected in metastatic lungs after NLGP treatment. This may have an indirect role in metastasis attenuation. However, this study does not demonstrate the particular mechanism of reduced angiogenesis, but immune-mediated (mainly by IFN-\u03b3) angiogenesis inhibition is already documented by us , and further elucidation of the signaling pathway is ongoing. T cell activation by dectin-1\u2013stimulated DCs has been reported by others in context to fungal infection; however, the literature on antitumor role of dectin-1\u2013activated DCs is very limited \u201348. Leibry tumor , 45. The antigen . Overallic drugs , 50. On + T cell dependence of NLGP action. Preclinical exploration of human tumor model specifically demands the use of nude or SCID mice, and those are immune compromised and lack CD8+ T cells, in particular.Validation of NLGP's effect in preclinical human tumor models is not possible because of CD8+ T cells, which skews the ongoing dialogue between the immune system and cancer cells to prevent the primary tumor growth and metastatic colonization. Based on our accumulated data from present or previous studies, we are in good faith that adjuvant and/or neoadjuvant treatment with NLGP in combination with other immunotherapy and/or chemotherapy will exhibit synergistic benefits in clinical outcome.A conglomeration of all these results proves that NLGP, via altering maturation of DCs, generates antigen-specific activated CD8Additional Files). Raw data related to this publication are available from the corresponding author on reasonable request.The data and material related to the findings of this study are presented in main figures within the article or as supplementary information files .ABh, ABo, and RB designed the study, analyzed the data, and wrote the manuscript. ABh, IG, and NG performed the research. AS and SD performed PCR and WB. AD performed confocal microscopy. PN, SG, TG, EH, and SB provide resources. ABo and RB supervise the project. RB acquired the fund. All authors read and approved the final manuscript.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."}
{"text": "Loss of smell and taste are commonly reported symptoms associated with coronavirus disease 2019 (COVID-19); however, the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in people with acute loss of smell and/or taste is unknown. The study aimed to determine the seroprevalence of SARS-CoV-2 antibodies in a community-based population with acute loss of smell and/or taste and to compare the frequency of COVID-19 associated symptoms in participants with and without SARS-CoV-2 antibodies. It also evaluated whether smell or taste loss are indicative of COVID-19 infection.n = 392) of participants were female. A total of 567 (96.1%) had a telemedicine consultation during which their COVID-19\u2013related symptoms were verified and a lateral flow immunoassay test that detected SARS-CoV-2 immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies was undertaken under medical supervision. A total of 77.6% of 567 participants with acute smell and/or taste loss had SARS-CoV-2 antibodies; of these, 39.8% (n = 175) had neither cough nor fever. New loss of smell was more prevalent in participants with SARS-CoV-2 antibodies, compared with those without antibodies , whereas taste loss was equally prevalent . Seropositivity for SARS-CoV-2 was 3 times more likely in participants with smell loss  compared with those with taste loss. The limitations of this study are the lack of a general population control group, the self-reported nature of the smell and taste changes, and the fact our methodology does not take into account the possibility that a population subset may not seroconvert to develop SARS-CoV-2 antibodies post-COVID-19.Text messages, sent via primary care centers in London, United Kingdom, invited people with loss of smell and/or taste in the preceding month, to participate. Recruitment took place between 23 April 2020 and 14 May 2020. A total of 590 participants enrolled via a web-based platform and responded to questions about loss of smell and taste and other COVID-19\u2013related symptoms. Mean age was 39.4 years (SD \u00b1 12.0) and 69.1% , an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, was declared a pandemic in March 2020.COVID-19 can cause loss or reduced ability to smell (anosmia) or taste, without cough or fever, but few countries recommend self-isolation and testing on the basis of smell or taste changes alone.This study aimed to find out the proportion of people who have developed SARS-CoV-2 antibodies in a community-based population with a newly developed loss in their sense of smell and/or taste in London, UK.Text messages were sent out to people registered with a number of primary care centers in London inviting people with a new loss in their sense of smell and/or taste to participate.Recruited participants completed online questionnaires regarding demographics, their loss of smell and/or taste, and other COVID-19 symptoms, before they had a telemedicine consultation with a healthcare professional who confirmed the history of their symptoms and supervised a test to find out if they had SARS-CoV-2 antibodies.A total of 78% of 567 people with smell and/or taste loss had SARS-CoV-2 antibodies; of these, 40% had neither cough nor fever, and participants with loss of smell were 3 times more to have SARS-CoV-2 antibodies, compared with those with loss of taste.Loss of smell is a highly specific symptom of COVID-19.COVID-19 can present with loss of smell and/or taste without cough or fever.Loss of smell should be take into consideration in case isolation, testing, and treatment strategies for COVID-19. Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has spread exponentially, with 27,417,497 cases and 894,241 deaths reported from 216 countries by 9 September 2020 \u201d. Participants were then directed to an online platform  with the study information and an eligibility check. Inclusion criteria were age \u226518 years, proficiency in written and spoken English, and access to video-calling. Exclusion criteria were any preexisting loss of the sense of smell or taste of longer than a month\u2019s duration. Participation was voluntary, and written informed consent was obtained electronically. Enrolled participants completed an online questionnaire see , capturiParticipants were subsequently sent a point-of-care testing kit detecting the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to SARS-CoV-2. A healthcare professional arranged a telemedicine video consultation with each participant. At the beginning of the consultation, they asked the participant to describe the changes that they had experienced in their sense of smell and/or taste. Participants were then supervised in performing the antibody test using a finger-prick sample of whole blood, and their results were discussed with them. Photographs of the test cassette were obtained and reviewed independently by a second healthcare professional to confirm the result. Participants with a prior COVID-19 PCR test result were offered antibody testing irrespective of their result. Testing was carried out between 24 April 2020 and 22 May 2020.The antibody test used detects the presence of IgM and IgG antibodies to SARS-CoV-2 via lateral flow immunoassay  . As partThe study received ethical approval from the National Health Service Queen\u2019s Square Research Ethics Committee  and was conducted in line with the declaration of Helsinki and Good Clinical Practice.https://www.graphpad.com/scientific-software/prism/) and STATA version 15 (https://www.stata-uk.com/). Data analysis was planned on completion of SARS-CoV-2 antibody testing. There was no prospective study protocol or analysis plan, and no data-driven changes to analyses took place. Descriptive analyses included the calculation of means (plus standard deviation [SD]) for continuous variables and numbers  for categorical variables. Chi-squared tests were performed on categorical data as part of the secondary analysis. The significance level was adjusted for multiple comparisons by applying a Bonferroni correction when comparing symptoms other than loss of smell or taste. Logistic regression analysis was performed to estimate the association between loss of smell and/or taste and the presence of SARS-CoV-2 antibodies.A sample size calculation was undertaken in order to determine the study\u2019s recruitment target, using the information on reported symptoms from the web-based COVID symptom study app developed by King\u2019s College London and symptom reporting between the 24\u201329 March 2020 . To calcn = 392) were female, 30.5% (n = 173) male, and 0.4% (n = 2) of other sex. A total of (n = 311) 79.3% of female participants had a positive test result, compared with 73.4% (n = 127) of male participants (p = 0.120).A total of 33,650 text messages were sent out to people registered with 4 participating primary care centers. A total of 650 participants completed the registration process; 60 participants were ineligible and excluded. The participant flowchart  is illusn = 531) reported loss of their sense of smell. This was described as complete smell loss by 69.9% (n = 371) and as a partial smell loss by 30.1% (n = 160). No smell loss was reported in 10.0% (n = 59). A total of 89.8% (n = 530) of participants reported loss of their sense of taste. This was described as complete by 47.4% (n = 251) and as partial by 52.6% (n = 279). No taste loss was reported in 10.1% (n = 60) of participants. Combined loss of smell and taste was reported by 80.0% (n = 472).Among the 590 participants who completed questionnaires, 90.0% (n = 439) had a positive SARS-CoV-2 result . One further participant was included with a positive PCR result (77.6% [n = 440] positive). Participants with and without SARS-CoV-2 antibodies were comparable in terms of age, sex, ethnicity, smoking status, and frequency of other reported symptoms (n = 229) of the participants with SARS-CoV-2 antibodies had no history of cough, and 39.8% (n = 175) had neither a fever nor a cough.Of 590 eligible participants, 567 participants (96.1%) underwent SARS-CoV-2 testing. A total of 77.4% (symptoms . Importan = 567), 89.9% (n = 510) reported loss of their sense of smell and 89.7% (n = 509) taste loss. The frequency of reported loss of smell and taste in participants with and without SARS-CoV-2 antibodies are presented in n = 307) of those with complete smell loss had a positive test result. A total of 77.8% of participants with taste loss and 86.0% of those with complete taste loss (n = 209) had a positive test result. Extracts from questionnaire responses from participants with positive SARS-CoV-2 antibodies describing their loss of smell and taste can be seen in Among the participants who underwent SARS-CoV-2 antibody testing , and participants with combined loss of smell and taste were 4 times more likely to have SARS-CoV-2 antibodies . These findings remained unchanged after adjusting for sex, age, ethnicity, and smoking status.Participants with loss of smell alone were nearly 3 times more likely than participants with isolated taste loss to have SARS-CoV-2 antibodies  of the study participants reported neither cough nor fever. In our study cohort, loss of smell was more prevalent in participants with SARS-CoV-2 antibodies compared with those without antibodies , whereas taste loss was equally prevalent . Furthermore, participants with acute smell loss were 3 times more to be seropositive for SARS-CoV-2  compared with those with taste loss.In this community-based cohort study, undertaken during the peak of the COVID-19 outbreak in London, the seroprevalence of SARS-CoV-2 antibodies in participants with new onset loss of sense of smell and/or taste, was 77.6%. A total of 39.8% ; thus, it is plausible that the loss of taste reported by participants who also report loss of smell reflects impaired retronasal olfaction and hence, represents loss of flavor perception, as a consequence of smell loss. Interestingly, 6.6Globally, as populations are released from lockdown, early identification by the public of COVID-19 symptoms and rapid self-isolation and testing will be of vital importance to limit disease spread. Currently, as highlighted in SARS-CoV-2 specific antibody testing performed via telemedicine consultation enabled us to confirm participants\u2019 reported symptomatology and verify their identity and test results, which adds to the quality of our data. Although viral nucleic acid detection using real-time PCR remains the gold standard for diagnosis, there are several limitations to this method, including a high processing time, labor intensity, and a high false negative rate \u201322. FurtThe main limitation of our study is the lack of a general population control group without loss of smell and/or taste. The study only recruited participants who reported acute smell and/or taste loss. Although this enabled us to study this presentation and its relevance to COVID-19, this also presents a degree of selection bias; hence, our findings refer to a population subset with new acute loss of smell and/or taste. In addition, although we used a questionnaire to assess COVID-19 symptoms and subsequently validated participants\u2019 responses during the telemedicine interview, we did not undertake any objective assessments of smell or taste. However, from a COVID-19 disease containment perspective, the general public are unlikely to have rapid access to formal objective smell/taste testing. Moreover, our data show a high seroprevalence of SARS-CoV-2 antibodies in people who recognized a loss of their sense of smell. Furthermore, as our study relied on antibody testing via telemedicine, we were unable to account for COVID-19 patients who may not have seroconverted to develop IgG or IgM antibodies, which may lead to our findings underestimating the prevalence of COVID-19 in this population. However, our findings suggest that a key public health message is that people who notice a loss in their ability to smell everyday house-hold odors such as garlic, onions, coffee, and perfumes should self-isolate and seek PCR testing.The majority of our participants were female; this finding may reflect previous findings that females have a higher frequency of loss of smell and/or taste with COVID-19 than males ,9,28. WeIn a community-based population, the vast majority of participants with new onset loss of smell were seropositive for SARS-CoV-2 antibodies. Acute loss of sense of smell needs to be considered globally as a criterion for self-isolation, testing, and contact tracing in order to contain the spread of COVID-19.S1 STROBE checklist(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file."}
{"text": "Healthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals. In this study we sought to determine if tracking smell sensitivity and loss using an at-home assessment could identify SARS-CoV-2 infection in HCW.P < .01). 6/9 (67%) of SARS-CoV-2-positive HCW reporting smell loss reported smell loss prior to having a positive SARS-CoV-2 test, and smell loss was reported a median of two days before testing positive. Neurological symptoms were reported more frequently among SARS-CoV-2-positive HCW who reported smell loss compared to those without smell loss .We performed a prospective cohort study tracking 473 HCW across three months to determine if smell loss could predict SARS-CoV-2 infection in this high-risk group. HCW subjects completed a longitudinal, behavioral at-home assessment of olfaction with household items, as well as detailed symptom surveys that included a parosmia screening questionnaire, and real-time quantitative polymerase chain reaction testing to identify SARS-CoV-2 infection. Our main measures were the prevalence of smell loss in SARS-CoV-2-positive HCW versus SARS-CoV-2-negative HCW, and timing of smell loss relative to SARS-CoV-2 test positivity. SARS-CoV-2 was identified in 17 (3.6%) of 473 HCW. HCW with SARS-CoV-2 infection were more likely to report smell loss than SARS-CoV-2-negative HCW on both the at-home assessment and the screening questionnaire (9/17, 53% vs 105/456, 23%, In this prospective study of HCW, self-reported changes in smell using two different measures were predictive of SARS-CoV-2 infection. Smell loss frequently preceded a positive test and was associated with neurological symptoms. It is increasingly clear that asymptomatic and pre-symptomatic infection play an important role in the ongoing spread of COVID-19 , 3. HealOlfactory impairment is a common manifestation of COVID-19, with meta-analyses indicating a prevalence of 77\u201386% in studies using standardized olfactory testing . Among pS1 Table), to track ratings of odor intensity perception using two household olfactory stimuli. High risk HCW undergoing routine (every 3 days) viral screening for SARS-CoV-2 infection were assessed for self-reported smell loss, along with symptoms commonly used to screen for COVID-19.The aim of this study was to determine if tracking smell sensitivity and loss using an at-home assessment could identify HCW who are infected with SARS-CoV-2. Since testing for objective smell loss using standard laboratory or clinical techniques is not feasible for widespread testing, we employed a brief at-home smell sensitivity screen, the Yale Jiffy  (IMPACT) study at Yale University . The goal of the parent study was to prospectively follow SARS-CoV-2-negative HCW at high risk of acquiring infection due to occupational exposures. The study recruited HCW working in the medical ICU or dedicated COVID-19 units at Yale New Haven Hospital (YNHH), a 1,541-bed tertiary care hospital located in New Haven, CT, USA. All participants provided written and/or verbal informed consent. Inclusion criteria for the IMPACT study included: a) aged 18 or older; b) English-speaking; c) working in a health care facility ; d) possible moderate to high risk exposure to COVID-19, or work in a COVID-19 unit, and e) SARS-CoV-2 negative at study entry. For this analysis of smell alterations and COVID-19, we excluded subjects without at least one SARS-CoV-2 PCR result and those who had not completed at least one daily symptom questionnaire or Yale Jiffy. All reported data were collected between March 31 and July 7, 2020.SARS-CoV-2 real-time quantitative polymerase chain reaction (RT-qPCR) testing was performed on self-collected nasopharyngeal and saliva specimens every three days. Specimens were processed and tested on the same day as collection following previously described protocols , 17. TwoThe Yale Jiffy is an online survey developed to screen for smell loss that can be conducted in under two minutes using readily available household items. The questionnaire includes two sections using self-ratings of: 1) ability to smell, and 2) strength of smell in response to olfactory and trigeminal stimuli. Peanut butter (or jam/jelly) was used as the olfactory stimulus as it has minimal or no trigeminal component, allowing for isolation of effects on the olfactory system. We also included a stimulus with a trigeminal component as a control stimulus .First, participants were asked to rate their ability to smell on a categorical scale (Poor/Average/Good/Very Good), to report any reduction in smell in the past week on a categorical scale (None/Slight/Moderate/Severe), and to rate the degree of reduction on a 10cm (0.0\u201310.0) visual analog scale (VAS). Next, participants were asked to hold the olfactory stimulus one inch from their nose and to provide ratings of both the strength of smell (0.0\u201310.0) and how different it smells from normal (0.0\u201310.0). HCW then held the trigeminal stimulus one inch from their nose and rated strength of sensation of irritation and difference from normal, as above. Since olfactory sensitivity fluctuates across the day , HCW werS2 Table), indicating how often they were bothered by common complaints caused by smell distortions, with responses ranging from \u201calways\u201d (1 point) to \u201cnever\u201d (4 points). Parosmia was defined as a cumulative score less than or equal to 14 (out of 16). HCW responded to four hypogeusia screening questions for saltiness, sourness, sweetness, and bitterness, by indicating that they could detect these tastes \u201cEasily\u201d (3 points), \u201cSomewhat\u201d (2 points), or \u201cNot at all\u201d (1 point). Hypogeusia was defined as any total score less than the maximum of 12 points.As part of the IMPACT study, HCW completed an online daily symptom questionnaire. HCW were given a list of symptoms and prompted to indicate whether they had developed such symptoms in the past 24 hours. Listed symptoms included objective fever (>100.4\u00b0F), subjective fever, cough, shortness of breath, stuffy nose, sore throat, chills, sweating, malaise, fatigue, muscle pain, anorexia, nausea, vomiting, diarrhea, abdominal pain, and dizziness. In addition, we included screening questions for parosmia\u2013changes in odor quality perception \u2013and for Descriptive statistics were used to characterize the study population. The Fisher\u2019s exact and Wilcoxon rank-sum tests were used to compare SARS-CoV-2-positive and negative HCW. Multivariable logistic regression models were developed to calculate adjusted odds ratios for the associations between smell symptoms and SARS-CoV-2 infection. Each model included one symptom as the predictor and additionally adjusted for age, sex, body mass index (BMI), ethnicity, and number of symptom surveys completed.To further examine the strength of association between symptoms of smell loss and SARS-CoV-2 infection, we compared HCW ever reporting smell loss to a subset of HCW never reporting smell loss who were well-matched on age, sex, ethnicity, and the number of daily symptom surveys completed. We used the R package \u2018MatchIt\u2019 for this analysis , specifyFinally, we summarized responses to the Yale Jiffy and daily symptom questionnaires. We also examined longitudinal responses among HCW who completed the questionnaires multiple times.P < .05 using R statistical software (version 3.4.2).All analyses were conducted with a two-sided statistical significance level of Fig 1). Within the sub-study population, 373 (79%) participants were female; the mean (SD) age was 37.5 (11.2) years; 375 (79%) were white non-Hispanic/Latino; 261 (55%) were registered nurses (RNs) and 98 (21%) were medical doctors (MDs) (Table 1). Compared to HCW included in this analysis, IMPACT HCW ineligible for the sub-study were more likely to be Black, Asian, or other ethnicity (P < .001) and have higher BMI (P = 0.004) (S3 Table).588 HCW were recruited and consented in the IMPACT study between March 31 and July 7, 2020, of whom 473 (80%) were eligible for the smell sub-study, while 115 (20%) were excluded because they did not undergo SARS-CoV-2 testing or they did not submit either a Yale Jiffy or symptom survey . Among the 313 (66%) HCW who completed the Yale Jiffy at least once, HCW completed a median of 10  Jiffy questionnaires.5771 SARS-CoV-2 RT-qPCR tests were performed on 473 HCW in the sub-study (median = 11 tests per HCW) between March 31 and July 7. Of these, 17 (3.6%) HCW tested positive for SARS-CoV-2.Table 1). Nine of the 17 (53%) SARS-CoV-2-positive HCW completed the Yale Jiffy at least once. SARS-CoV-2-positive HCW were more likely to report categorical smell loss on the Jiffy, with 5/9 (56%) SARS-CoV-2-positive HCW versus 43/304 (14%) SARS-CoV-2-negative HCW reporting smell loss  (Table 1). The five SARS-CoV-2-positive HCW who reported categorical smell loss via the Jiffy had a mean (SD) reduction in smell of 5.8 (4.0)cm on a 10cm (0.0 to 10.0) scale; those indicating severe loss had a mean 8.3 (3.0)cm decrease compared to 2.2 (0.5)cm in those with slight smell loss. For individuals with any smell loss reported via Jiffy, SARS-CoV-2-positive HCW reported more severe smell loss than SARS-CoV-2-negative HCW (P < .001) . SARS-CoV-2-positive HCW reported severe  or slight 2/5, 40%) smell loss on a categorical scale, while SARS-CoV-2- negative HCW reported slight  or moderate  smell loss.The demographic characteristics of SARS-CoV-2-positive HCW were similar to those of SARS-CoV-2-negative HCW (Table 1). Six of those seven also reported smell loss.All 17 SARS-CoV-2-positive HCW eligible for the smell sub-study completed at least one daily symptom questionnaire. Eight (47%) reported parosmia versus 83/456 (18%) SARS-CoV-2-negative HCW . Hypogeusia was reported in seven (41%) SARS-CoV-2-positive HCW and 104/456 (23%) SARS-CoV-2-negative HCW  . Of those who reported smell loss, 3/9 (33%) reported changes in smell as their first symptom; all 9 reported symptoms of COVID-19 in addition to changes in smell.Relative to Day 0 (defined as the day of test positivity), the median timing of reported smell loss was Day -2  among SARS-CoV-2-positive HCW reporting smell loss, with 6/9 (67%) reporting smell loss before test positivity .Overall, in either the daily symptom questionnaire or Yale Jiffy, nine (53%) SARS-CoV-2-positive HCW reported smell loss or parosmia, compared to 105/456 (23%) SARS-CoV-2-negative HCW . After adjusting for age, sex, BMI, and number of symptom questionnaires, smell loss remained a significant predictor of SARS-CoV-2 infection . Among other symptoms in the questionnaire, only dyspnea  and headache  were more strongly predictive of SARS-CoV-2 infection. In the matched analysis, unadjusted conditional logistic regression estimated a significant association between smell loss and SARS-CoV-2 infection  Table 2 summarizes findings among SARS-CoV-2-positive HCW stratified by smell loss as reported by either measure. Neurological symptoms as assessed by the daily symptom questionnaire, including headache, fatigue, and dizziness, were reported in all nine SARS-CoV-2-positive HCW reporting smell loss, and three (38%) who did not report smell loss. Prolonged neurological symptoms (> seven days after positive test) were reported in four (44%) SARS-CoV-2-positive HCW with smell loss versus one (13%) without smell loss. Three SARS-CoV-2-positive HCW, all of whom experienced smell loss, had significantly prolonged neurological symptoms (> 20 days after positive test).In this prospective study of a high-risk HCW population, we assessed smell loss alongside routine viral testing, finding that HCW who acquired SARS-CoV-2 infection over the course of the study had significantly increased odds of reporting smell loss compared to SARS-CoV-2-negative HCW. This finding was consistent across bivariate (OR = 3.7), regression-adjusted (OR = 4.5), and matched (OR = 6.0) analyses. Likewise, SARS-CoV-2-positive HCW reported significantly more severe smell loss compared to SARS-CoV-2-negative HCW. Overall, our findings add to the accumulating evidence demonstrating the efficacy and feasibility of using prospective, self-administered, at-home assessments of smell sensitivity to track changes over time in a group of individuals at high risk for SARS-CoV-2 infection.Our findings are consistent with recent research demonstrating robust associations between smell perturbations and SARS-CoV-2 infection. Specifically, smell and taste impairments are reported prior to (20%) or during 13%) hospitalization , typical% hospitaInterestingly, we found an association between reduction in smell ability and neurological symptoms in HCW with SARS-CoV-2 infection. While the exact pathophysiology underlying COVID-19 related smell loss remains incompletely understood, the association between smell loss and other neurological symptoms suggests a potential common mechanism underlying these symptoms. A growing body of evidence has demonstrated the potential for SARS-CoV-2 to infect neurons , 27, andOur study has several limitations. Despite performing almost 6,000 SARS-CoV-2 tests on nearly 500 HCW during the first peak of the COVID-19 pandemic in Connecticut, only 17 (3.6%) HCW tested positive, limiting the statistical power of our analysis. Nevertheless, we found large, significant effects. Smell sensitivity and loss using the Yale Jiffy were evaluated using both categorical responses and a VAS . Indeed,In our study, self-reported changes in smell perception were predictive of SARS-CoV-2 infection in a healthcare worker population. At-home smell assessments should be considered for non-invasive screening of groups that are at high risk for COVID-19.S1 FigResults of matching on age, sex, profession, ethnicity, and number of symptom questionnaires completed for HCW reporting smell on either survey , the daily symptom survey only , and the Yale Jiffy only . A, C, and E show the change an absolute standardized difference in means between the unmatched (\u201cAll Data\u201d) and matched datasets. B, D, and F compare the distributions, as histograms, of the propensity scores between HCW with smell loss (\u201cTreated\u201d) and those without (\u201cControl\u201d) both before (\u201cRaw\u201d) and after (\u201cMatched\u201d) matching. In all cases, matching resulted in a decrease in the absolute standardized difference for the overall distance measure and a more similar distribution (as shown by histograms) of propensity scores.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableLaryngoscope. 2010;120(8):1708.Adapted from Landis BN, Frasnelli J, Croy I, Hummel T. Evaluating the clinical usefulness of structured questions in parosmia assessment. (DOCX)Click here for additional data file.S3 Tablea Unadjusted P values are Wilcoxon rank sum test (continuous variables) or Fisher\u2019s exact test .Data are presented as median (IQR) for continuous variables and no. (%) for categorical variables. Abbreviations: BMI, body mass index; HCW, healthcare workers; IMPACT, Implementing Medical and Public Health Action against Coronavirus (CT); MD, medical doctor; RN, registered nurse. (DOCX)Click here for additional data file.S4 Tablea Odds ratios are presented for the association between smell loss and SARS-CoV-2 infection as calculated from unadjusted conditional logistic regression after matching on demographics and symptom questionnaire participation.Abbreviations: CI, confidence interval. OR, odds ratio. (DOCX)Click here for additional data file."}
{"text": "Some nonlinear radiations such as superfluorescence can be understood as cooperative effects between atoms. We regard cooperative radiations as a manifested effect secondary to the intrinsic synchronization among the two-level atoms and propose the entanglement measure, concurrence, as a time-resolved measure of synchronization. Modeled on two cavity-coupled qubits, the evolved concurrence monotonically increases to a saturated level. The finite duration required for the rising to saturation coincides with the time delay characteristic to the initiation of superfluorescence, showing the role of synchronization in establishing the cooperation among the qubits. We verify concurrence to be a good measure of synchronization by comparing it with asynchronicity computed from the difference between the density matrices of the qubits. We find that the feature of time delay agrees in both measures and is determined by the coupling regimes of the cavity-qubit interaction. Specifically, synchronization is impossible in the weak coupling regime. How synchronizations arise in different situations has since remained a problem of interest2. In recent years, the study of the classical phenomenon has been revived under the quantum regime. Synchronization is observed between a pair of nanomechanical oscillators3 and on the motions of the Cooper pairs among Josephson-insulated superconducting islands4. It is also ubiquitously predicted between a qubit and an oscillator5, between a cavity field and an oscillator6, between two oscillators7, among a trapped group of cold atoms8, and even between a quantum Van der Pol oscillator and an external drive9.Centuries ago, Huygens studied the correlation among the motions of pendulums and discovered the synchronization pattern of these individual oscillators under the influence of a common oscillator they are coupled to11. This nonlinear fluorescent effect embodies Dicke\u2019s formulation of superradiance12, where the nonlinearity exemplifies in the N atoms. Furthermore, the emitted photonic pulse peaks after a positive time delay14 that bears no direct dependence on the atom-field coupling strength, showing the collective interatomic motion to be a nonlinear process. Experimentally recorded on hydrofluoric gas15, cesium16, and most recently rubidium vapor17, this delay shows the necessity of a finite time during which the atoms establish cooperation before the radiation is initiated18.Here we study the synchronization of two qubits coupled indirectly to each other through a cavity field, in specific relevance to the quantum optical phenomenon of superfluorescence21 of two cavity-coupled qubits, which is the minimum-dimensional system that emulates the structure of an atomic ensemble undergoing cooperated radiation.Such a delay is also characteristic of synchronization: it takes finite time for the classical Huygens pendulums to reach in-phase oscillations. If one regards the correlated atoms as quantized Huygens oscillators concentrated on the lowest two levels at the weak-energy limit, it is not hard to find the resemblance between cooperated radiation and synchronization. In this paper, we formalize the study of this resemblance by analyzing the entanglement dynamics22, quantum dots23, and resonator modes24. We note that synchronization as a quantum correlation can be studied using quantum discord25, as it was for two oscillators26, and non-locality. The advantage of entanglement approach is avoiding the unnecessary constraint to two-party correlation. We use specifically, in contrast to quantum discord, concurrence27 as the entanglement measure to quantify quantum synchronization in order to extract the temporal features of atomic cooperation. Concurrence is broadly applicable, especially for multipartite system. After it was introduced for two-qubit systems27, it was soon extended to the cases of three qubits28 and N-qubits29. Concurrence has then been generalized to two multi-level systems31 and finally to multipartite multi-level systems32. Given its generality, concurrence permits synchronization to be exemplified not only between two qubits, but among three parties that include the cavity field. In addition, synchronization is present in a Hilbert space of finite dimensions33, so non-locality is not an essential factor to initiate synchronization.Many different metrics have been proposed over the years to measure synchronization of different quantum systems, such as a set of two-level systems in a shared bath35. Therefore, we extend the employment of multipartite concurrence to the dynamical analysis of the finite-dimensional cavity-qubit system. It is found that the entanglement among the components universally begins at a zero level and rises to a saturated value after a finite delay. The length of the delay and the saturated level depend nonlinearly on the cavity-qubit coupling strength. We use numerical simulations to show that the nonlinear dependence is divisible into weak, strong, and ultrastrong coupling regimes, verifying the role of operation regimes36 in the dynamics of superconducting circuits. Under all coupling strengths, the saturated entangled state is sustained thereafter, where oscillations in concurrence only exists locally about the saturation offset with an amplitude much smaller than the offset. These distinctive features of a synchronized state stand in constrast to the sudden death or death and revival37 one usually sees in entanglement evolution.Further, concurrence is proved to be a good measure for static analysis of collective phenomena such as radiationx,\u00a0p)-quadratures of quantum oscillators24. The transition point in time produced from the synchronization measures matches exactly the delay time found in the concurrence evolution, proving that the qubit-to-qubit synchronization is well registered in the entanglement. Moreover, since two-qubit systems on a superconducting circuit can produce superfluorescent pulses38, the synchronization delay is associated with the initiation of superradiance, thereby establishing the dynamic correlation between entanglement and the atomic cooperation for collective phenomena.To verify the coincidence between the maximization of concurrence and the dynamic process of synchronization, we introduce a synchronization measure computed from the density matrix, which is modified from the synchronization measure introduced on the  system where each qubit is coupled to the cavity through dipole-field interaction under the rotating wave approximation. The parameters adopted for the numerical analysis are sourced from the superconducting circuit implementationThe total system Hamiltonian A and B when writing the product states and let the first letter denote the state of the left qubit, the middle letter that of the cavity mode, and the last letter that of the right qubit , B (the right qubit), or C (the cavity), i.e. The permitted dressed level transitions induced by the external driving can be found by substituting Eq.\u00a0 into Eq.A with j and k being the row and the column indices, respectively. We denote A is integrable, then solving the linear system for In the rotating frame s in Eq.\u00a0, one canTo see that the evolution of the state vector can initiate the synchronization of the two delocalized qubits, we assume the cavity mode is initially driven by the external field to reach a partial population inversion while setting the qubits initially at the ground. In other words, the expansion coefficients at the initial moment are: 40: 42.We plot out the evolutions of these coefficients in Fig.\u00a0entclass1pt{minimaentclass1pt{minimaWe observe that for both the lower set and the upper set of states, there exists a transition point of the oscillations of the coefficients, which is located at about To fully capture the evolution characteristics of the two cavity-coupled qubits from a holistic point of view, we apply two entanglement measures\u2014bipartite concurrence and tripartite concurrence\u2014to the state vector of the total system.The bipartite concurrence quantifies the inseparability of the joint pure state of two coupled systems of arbitrary dimensions by inverting the density matrix. For our case here, the joint state is the product state Applying \u27e9 in Eq.\u00a0 to the fentclass1pt{minima28. Extending the original formulation on three-qubit systems, we generalize the inversion operations for two arbitrary-dimensional systems given above to three arbitrary-dimensional system. That is, we introduce the superoperatorI denotes the identity matrix while The cavity mode plays an active part in initiating the entanglement between the two qubits. From the entanglement-theoretic point of view, the concurrence is distributed among the qubits as well as the cavity. Taking away the pairwise entanglements between any two parties in the tripartite system, one obtains the residual concurrence that remains as an equally distributed entanglement among all three partiessing Eq.\u00a0, we plotComparing Figs.\u00a0The concurrences plotted in Fig.\u00a044 and even up to hundreds of \u00b5s when an error-correcting feedback mechanism is implemented45.We note that the dynamics originated from the equations of motion \u201318) doe doe18) dThe synchronization between the qubits is also affected by how strong they are driven by the cavity mode, i.e. the coupling strengths Multi-partite concurrence as a measure of synchronization reveals a gradual increase between two qubits in the time domain, explaining the existence of a delay in the superfluorescent pulse of cooperated radiation from a system-intrinsic point of view. This synchronization is affected by many factors, among which the symmetry of the system parameters plays an important part. Tuning the system from a symmetric setting to an asymmetric setting is accompanied by tuning the transition rates of the qubits from a synchronous setting to an asynchronous setting. For the latter, we refer to the scenario where the population of the left qubit oscillates at a Rabi frequency not synchronous to that of the right qubit.24 for continuous variable systems to discrete systems. We consider instead the measure of asynchronicityHowever, since two oscillators sharing a common oscillating platform are able to synchronize after certain time duration according to classical mechanics, we expect the qubits sharing the cavity resonator would behave similarly. To precisely describe the transition process from asynchronous regime to synchronous regime, we extend the quantum synchronization measure introduced in Ref.46. For example, a 43. To consider the scenario where the sychronization is initiated from the cavity field as the medium, we let the initial state be only populated in the first and second excited state of the cavity mode, i.e. No matter the coupling strength, there exists a transition point after which the asynchronicity remains at a stable value. This transition point is identical to the transition point shown in Fig.\u00a0Before reaching the stable minimal value, the asynchronicity increases from a non-zero value for a certain duration, which are spent on the cooperation by the qubits. When the coupling is sufficiently weak  for for\\docu"}
{"text": "What is the representation of racial and ethnic minority populations in studies incorporating precision oncology objectives in the US?This cross-sectional analysis evaluates breast, prostate, lung, and colorectal cancer studies in the Clinicaltrials.gov registry with precision medicine objectives and reporting race and ethnicity\u2014a total of 93 studies with 5867 total enrollees. An underrepresentation of minority racial groups and an overrepresentation of non-Hispanic White participants relative to their incidence in the US cancer population was found in precision oncology studies.These findings demonstrate an urgent need to increase enrollment of participants from diverse racial and ethnic backgrounds onto precision oncology studies, so that meaningful precision data can be collected and stratified to traditionally underrepresented participants. This cross-sectional study evaluates the representation of racial and ethnic minority groups in breast, prostate, lung, and colorectal cancer studies with a focus on precision medicine objectives. Precision oncology is revolutionizing cancer care, allowing for personalized treatments to improve outcomes. Cancer research has benefitted from well-designed studies incorporating precision medicine objectives, but it is unclear if these studies are representative of the diverse cancer population.To evaluate racial and ethnic representation in breast, prostate, lung, and colorectal cancer studies incorporating precision oncology objectives in the Clinicaltrials.gov registry and compare with the incidence of these cancer types in racial and ethnic minority groups in the US population.This cross-sectional study identified US-based breast, prostate, lung, and colorectal cancer studies incorporating precision oncology objectives for reporting of race and ethnicity. The Surveillance, Epidemiology, and End Results and US Census databases were used to determine cancer incidence by race and ethnicity, linked with cancer type and median year of enrollment for each trial. Data were collected and analyzed between December 2020 and April 2021.The expected number of participants per study by each racial and ethnic group was calculated based on the corresponding US-based proportion. Under- and overrepresentation was defined as the ratio of the actual number of enrolled cases to the expected number of cases for each trial by cancer type. Ratios above 1 indicated overrepresentation while a ratio below 1 indicated underrepresentation. Random-effects meta-analysis of representation ratios of individual trials was performed to weigh each individual study.Of 93 studies encompassing 5867 enrollees with race and ethnicity data; 4826 participants (82.3%) were non-Hispanic White, 587 (10.0%) were Black, and 238 (4.1%) were Asian. Per observed-to-expected ratios, White participants were overrepresented in all studies, with a ratio of 1.35 , as well as Asian participants, with a ratio of 1.46 , while Black participants , Hispanic participants , and American Indian and Alaskan Native participants  were underrepresented. By individual cancer site, White participants were consistently overrepresented in all studies, while Black and Hispanic participants were underrepresented.This analysis found that precision oncology studies for breast, lung, prostate, and colorectal cancers vastly underrepresent racial and ethnic minority populations relative to their cancer incidence in the US population. It is imperative to increase diversity among enrollees so that all individuals may benefit from cancer research breakthroughs and personalized treatments. Additionally, knowledge learned from studies that are designed to collect translational data can be used as part of discovery for predictive and prognostic biomarkers, which then can be applied to personalized therapeutic regimens.Precision medicine has revolutionized oncology in the past 2 decades and is expected to continue to transform cancer management. The principle of personalized cancer therapy incorporates a multi-omic  approach along with other tumor- and patient-specific factors. The identification of biomarkers facilitates stratifying patients and guiding treatment individualization, leading to improved outcomes.2 as well as Native American ancestry.3 Separately, growing evidence suggests underlying tumor genomic differences between African American vs White men with prostate cancer.5 This information can lead to additional study and discovery as well as novel treatment options for those who harbor the specific tumor alteration or biomarker. However, the use of race and ethnicity in such analyses has multiple limitations. Race and ethnicity must be understood as social constructs. We also recognize that the traditionally broad racial and ethnic categories do not capture the heterogeneity that exists within each group.6 Consequently, even the aforementioned findings should be further explained and contextualized in terms of other social determinants of health, such as health insurance status, zip code, socioeconomic status, environment, and other characteristics. The intersection of social, environmental, and genomic and/or biologic factors and the resultant influence on disease is poorly understood, with calls to action to capture these data points more robustly in all future precision medicine studies to evaluate and understand these interactions.8Several targetable biomarkers have been found to have racial and ethnic differences in incidence for different cancer types such as epidermal growth factor receptor variants in lung cancer associating with East Asian ethnicity10 However, genetic ancestry data are not yet readily available for clinical practice or for use in clinical studies, nor have that data been historically captured.11 The historical static groupings of individuals by race and ethnicity with regards to reported health data over time have led to the acceptance and use of these categories in biomedical research.7 Stratification by various racial and ethnic groupings has identified several important differences in disease risk as well as response to certain treatments.11 In addition, the historical use of race and ethnicity as currently defined to differentiate patients in the study of disease has led to the emergence and recognition of medically underserved and underrepresented minority populations.12 Thus, although imperfect, understanding disparities by racial and ethnic groupings is of value, particularly pertaining to the study of precision medicine, as it is important to understand from which populations we are collecting our biologic and biomarker data for use in the general cancer population.The use of genetic ancestry has been lauded as a tool that can better differentiate populations in the study of biology and genomics.14 enrollment of a diverse patient population into cancer clinical trials lags behind, including for racial and ethnic minority groups.17 As more clinical studies specifically incorporate precision oncology principles into their design, it is unknown whether ethnic and racial minority populations are adequately represented, an important consideration when one critical question is whether precision oncology discoveries in clinical trials are broadly applicable to the general cancer population. Is precision oncology research missing crucial findings that could be benefiting traditionally marginalized groups, thus further widening inequality and contributing to disparities in cancer care? Moreover, any identifiable differences between groups in a single or across a collection of studies must be further evaluated, particularly if personalization of a therapeutic is identified for 1 group vs another, as sample sizes would be too small to gather appropriate safety data. Both prespecified analyses in postapproval settings as well as the use of real-world study would be necessary to interrogate safety and efficacy data.18 However, the opportunity to look for differences between various racial and ethnic groupings requires a concerted effort towards recruitment of underserved populations to these studies in their initial stages. Therefore, we sought to understand how well precision oncology studies are representative of the diverse US cancer population. Focusing on the top causes of new cancer cases in the US ,19 we evaluated clinical studies with precision medicine objectives for their reporting of race and ethnicity, and asked whether these study demographics were representative of the diverse US cancer population.Despite rapid diversification of the US cancer population,immunohistochemistry, histopathologic, DNA, RNA, DNA sequencing, RNA sequencing, sequencing, proteomics, tumor mutational burden, tumor biomarkers, biomarkers, tumor analysis, mutation testing, mutational analysis, microarray, whole exome, molecular analysis, genomics, genetics, gene expression, expression, signatures, genetic testing, and prognostic testing. Studies were excluded if they included site locations outside the US, did not include diagnosis of cancer , or did not incorporate precision oncology measures. Eligible studies were evaluated for reporting of racial and ethnic demographic data. Additional data gathered included type of clinical study  and type of funding . Participant demographics were obtained from Clinicaltrials.gov if present and corroborated with primary report journal articles or abstract and/or presentation if unpublished (per availability). Racial and ethnic groupings were mutually exclusive as follows: American Indian/Alaskan Native, Asian, Black, Hispanic, and Non-Hispanic White participants. Participants with race and ethnicity not reported or unknown were removed from the analysis. Overall, these individuals made up less than 3% of the total enrollees per each cancer type . Clinicaltrials.gov was queried in December 2020 with an update of the query and analysis in April 2021.In this cross-sectional study, the Clinicaltrials.gov registry was queried for completed US-based clinical studies by cancer type  with results incorporating precision medicine objectives based on a set of precision oncology search terms: 20 Age-standardized incidence rates adjusted to the 2000 standard US population by race and ethnicity were used to calculate cancer cases as a measure of proportion of cancer burden by race and ethnicity using US Census Bureau tables.22 This study was determined to be exempt from human participant research guidelines because it was a secondary analysis of publicly available published reports and data by the Mass General Brigham institutional review board. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional studies and the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline.Cancer incidence by race and ethnicity within the US cancer population, correlated with cancer type and median year of enrollment for each study, was collected from the National Cancer Institute Surveillance, Epidemiology, and End Result (SEER) database.For each trial, the expected number of participants of each of the 5 racial and ethnic groups was calculated by multiplying the total number of participants in a trial with the US-based proportion of each group for a given cancer type and median year of enrollment. We defined underrepresentation and overrepresentation as the ratio of the actual number of enrolled cases and the expected number of cases for each trial and per cancer type, with 95% exact binomial CIs estimated. A ratio greater than 1 indicated overrepresentation, while a ratio smaller than 1 indicated underrepresentation. Since the expected number of participants for groups other than non-Hispanic White is small, especially for smaller trials, we calculated totals of expected to enrolled ratios in all precision studies and per cancer type.2 and 2-sample Wilcoxon rank-sum tests were performed, respectively; P\u2009\u2264\u2009.05 was considered significant in 2-sided tests.Separately, we also performed random-effects meta-analysis of underrepresentation and overrepresentation ratios of individual trials , with the exclusion of trials with no enrolled participants of a given racial or ethnic group. Therefore, the weighted average of underrepresentation and overrepresentation effect sizes from the meta-analysis might be biased for minority populations with small numbers of enrolled participants . Results are presented using forest plots , followed by Black participants (587 [10.0%]), Asian participants (238 [4.1%]), Hispanic participants (200 [3.4%]), with the lowest representation of American Indian/Alaskan Native participants (16 [0.3%]). By cancer type, lung cancer studies had the highest proportion of White (2054 of 2399 [85.6%]) and Asian (116 [4.8%]) participants, and the lowest proportion of Black (199 [8.3%]), Hispanic (26 [1.1%]), and American Indian/Alaskan Native (4 [0.2%]) participants. Colorectal cancer studies had the highest proportion of Hispanic participants (59 of 999 [5.9%]). Prostate cancer studies had the highest proportion of Black participants (67 of 596 [11.2%]) and American Indian/Alaskan Native participants (5 of 596 [0.8%]), and the lowest proportion of Asian participants (6 of 596 [1.0%]). When evaluating racial and ethnic distribution by funding type, Asian individuals were statistically overrepresented on studies with NIH funding.When evaluating observed-to-expected ratios by race and ethnicity in all precision oncology studies, White and Asian participants were overrepresented with a ratio of 1.35  and 1.46 , respectively . The othIn the meta-analysis, which weighs individual studies, White participants remained overrepresented overall with a ratio of 1.34  and by cancer type . Overrep15 There is an urgent need to increase equitable recruitment of diverse participants to cancer clinical trials, as well as to understand racial and ethnic differences in treatment outcomes. However, the field of oncology has been moving toward personalized treatments based on presence of tumor molecular markers as well as genetic and genomic variation. Despite this, there is limited knowledge of underlying cancer biology in racial and ethnic minority groups. Current personalized treatments that are broadly generalized to all individuals based on the presence or absence of a biomarker without fully studying the implications of such treatment in racial and ethnic minority populations may not be appropriate. Even if biological differences are discovered that are unique to certain racial or ethnic minority subgroups, these must be further validated and evaluated for safety and efficacy. Similarly, large biorepositories from which to study precision omics are disproportionately dominated by individuals of European ancestry.24 This is a well-documented issue with genomewide association studies,10 particularly for cancer research, with European-ancestry groups making up 96% of participants, while 0.11%, 0%, and 0.5% of subjects are from African, African American and Afro-Caribbean, and Hispanic and Latin American groups in studies at the initial discovery phase in 2019,25 prompting the call for more inclusive samples and inclusive research communities. Thus, it is insufficient to take our current understanding of tumor biomarkers and biology and generalize findings to the entire diverse cancer population. Although race and ethnicity have been used historically to stratify populations, to fully understand any biological findings that differ by these social constructs, it is critical that future studies prospectively capture additional data that accounts for social determinants that influence disease, as well as data incorporating the use of genetic ancestry.11The underrepresentation of racial and ethnic minority populations in clinical trials relative to their cancer burden in the US has been previously reported.29 Yet, data now demonstrate differential biology (as it relates to somatic driver mutations) among Hispanic participants with lung adenocarcinoma, underscoring the importance of their increased representation on precision oncology lung trials, as well as capturing of social determinants of disease to analyze these associations further. Separately, Hispanic individuals also had poor representation in prostate cancer studies (by 67%). Overall, our findings data highlight the need to increase enrollment of these groups into future clinical studies. All individuals deserve to benefit from cancer research breakthroughs and deep understanding of the underlying tumor biology not only in the context of race and ethnicity, but in the context of social determinants of health as well.As contemporary clinical studies specifically incorporate precision oncology principles, it is unknown whether racial and ethnic minority populations are adequately represented overall and by cancer type relative to their cancer incidence in the US cancer population. By focusing on the most common cancer types in the US , we found that most precision oncology studies do not report the race and ethnicity of participants. Of those studies with racial and ethnic reporting, we demonstrated an underrepresentation of minority racial groups and an overrepresentation of Non-Hispanic White participants in precision oncology studies. Black and Hispanic participants were the most underrepresented on lung cancer studies by 68% and 69% per meta-analysis, respectively. On the other hand, Asian individuals were overrepresented in lung cancer studies by 196%, consistent with the prominent role of targetable oncogene drivers in this population. There is likely underlying genetic susceptibility for these alterations among the Asian population in addition to the epidemiologic association with nonsmoking status.18 There are multiple factors at play, including medical mistrust.30 Prior work has found that individuals from underrepresented racial and ethnic groups\u2019 willingness to participate in research stems from perceived trustworthiness of the researchers, the institutions conducting the research, and the information provided about the applicable research studies.31 There may be more mistrust surrounding genetic and biological studies\u2014for example, genetic counseling participation is low among Black women for BRCA1/2 testing.32 This finding was exacerbated among women with higher rates of medical mistrust,32 fear of discrimination by insurance companies if considered high-risk,33 and fear of carrying the mutation.34 A targeted study evaluating African American and Hispanic perspectives on the prospect of precision medicine found that, although groups believed that precision medicine can improve health outcomes, both groups were concerned that current barriers to health care would prevent their communities from benefiting from precision medicine.35There is a long history surrounding underrepresentation of medically underserved participants on trials.36 Finally, precision oncology studies should increase the reporting of race and ethnicity of participants so that improvements in diversity and representation can be observed over time, and this analysis can serve as a benchmark with which to compare future progress.Multiple strategies must be implemented to increase diverse enrollment onto precision oncology trials, including patient navigation programs, patient education about multi-omic studies, patient education about genetics and genetic counseling, unique trial designs, education for all researchers or members involved to combat bias, and other educational resources to address barriers to enrollment for trial participation. More research is needed to fully understand all barriers and fears surrounding research recruitment. An increase in racial and ethnic diversity among scientists, biomedical researchers, physicians, and clinical trialists can help to increase trust among patients from minoritized groups.14 This highlights the opportunity for future studies or trials to capture these critical nuances in the future, along with demographic, social, environmental, and other determinants of health, to create fully annotated databases to enhance the study of biology and multi-omic research with a broader lens to understand the interaction of these multiple factors.Limitations to this analysis include those associated with use of Clinicaltrials.gov. We queried studies with reported results, which could miss noncompliant and incompletely accrued studies. Our search criteria could have missed slow-accruing precision oncology studies, or those that remained open after reaching their primary end point. We may be missing crucial precision oncology studies that were not identified with the search terms used, as well as studies that did not prespecify these analyses and were instead performed post hoc. Second, no information about race and ethnicity assessment was provided\u2014it is important to emphasize that the racial and ethnic categories as reported do not capture the cultural heterogeneity within these groups. Third, there is no information on multiracial or multiethnic patients, which fails to capture patients who might identify as more than 1 racial or ethnic group. We recognize that this is imperfect, particularly given that in the latest 2020 US Census, over 33.8 million Americans identified themselves as being multiracial, resulting in an increase of nearly 276% since the 2010 census.Increased emphasis on equitable recruitment and enrollment for precision oncology studies is essential, as resulting discoveries are used to personalize treatments, and it is unclear whether current precision medicine breakthroughs can be broadly applicable to, or safe for, our diverse cancer population. We demonstrate that precision oncology studies for breast, lung, prostate, and colorectal cancers underrepresent racial and ethnic minority populations relative to their cancer incidence in the US population. All relevant stakeholders should implement strategies to increase diverse clinical study enrollment to address this disparity. A continued lack of diversity among trial enrollees may further leave behind undeserved minority populations in the era of precision oncology."}
{"text": "Micronutrient malnutrition is a global health issue and needs immediate attention. Over two billion people across the globe suffer from micronutrient malnutrition. The widespread zinc (Zn) deficiency in soils, poor zinc intake by humans in their diet, low bioavailability, and health consequences has led the research community to think of an economic as well as sustainable strategy for the alleviation of zinc deficiency. Strategies like fortification and diet supplements, though effective, are not economical and most people in low-income countries cannot afford them, and they are the most vulnerable to Zn deficiency. In this regard, the biofortification of staple food crops with Zn has been considered a useful strategy. An agronomic biofortification approach that uses crop fertilization with Zn-based fertilizers at the appropriate time to ensure grain Zn enrichment has been found to be cost-effective, easy to practice, and efficient. Genetic biofortification, though time-consuming, is also highly effective. Moreover, a Zn-rich genotype once developed can also be used for many years without any recurring cost. Hence, both agronomic and genetic biofortification can be a very useful tool in alleviating Zn deficiency. Food and nutritional security are key to human health. Food insecurity, imbalanced diet, consumption of food grains with poor nutritional quality, lack of dietary diversity, etc. negatively affect human health ,2. In faLow-income countries largely trust staple foods while relying less on fruits, vegetables, and foods from animal sources compared to high-income countries. Micronutrient malnutrition, due to a lack of sufficient micronutrients in the diet results in serious but often invisible health consequences, referred to as hidden hunger. In the world, over 2 billion people are affected by micronutrient deficiency ,7. This Amongst different micronutrients, Zn inadequacy is common in both plants and animals ,16. Zn dFrom the discussion above, it is very clear that a healthy diet can not only improve human health and maximize human resource potential. It can also reduce the costs related to health. We need suitable intervention like biofortification to make nutritionally superior food available to every person in the world at an affordable cost. In this direction, biofortification of staple food grains like rice, wheat, and maize with zinc and their inclusion in the human diet can help in alleviating zinc malnutrition.Zn is a very crucial nutrient for maintaining optimal human health. The essentiality of Zn for human was established in the year 1961 . Around Though the mechanism of Zn deficiency-induced impairment of growth and development is not clearly understood, it is one of the most studied effects of Zn deficiency. The effect of Zn deficiency is more significant especially in the time of rapid growth like infancy, puberty, and pregnancy . Intake Considering the importance of Zn in human health , there iZn is an essential plant nutrient and known as a micronutrient because of its low requirement. Zn remains in plants in either free ionic form or as a complex with many low molecular weight compounds . Though Zn is a constituent of many important enzymes of great significance such as carbonic anhydrase, alcohol dehydrogenase, and superoxide dismutase ,49. CarbZn is also an integral part of the Zn finger family of transcription factors that controls cell proliferation and differentiation . Zn playThe possible role of Zn in water uptake and transport in plants and a short-term tolerance to heat and stress tolerance has been reported ,57,58,59Zn deficiency in crops limits crop yield and grain nutritional quality ,65. CereThe biofortification approach aims at enriching the grains with minerals like iron, Zn, selenium, iodine, etc., so that, their intake can be improved among people consuming those grains ,9,14. AsUnderstanding the physiological basis of micronutrient accumulation in the grains is of utmost importance for successful biofortification. Micronutrient concentration in the edible portion of the crop is decided by nutrient availability to plants and the process of absorption, translocation, and redistribution of micronutrients in the plant, which are being regulated by homeostatic mechanisms that allow accumulation of micronutrients in an adequate amount, yet at a non-toxic level ,16. The + from root cells and the release of metal complexing compounds and reductants can also be very useful root characteristics for increasing Zn uptake [The crop also plays a very important role in the absorption of micronutrients. For enhancing Zn uptake, the micronutrient level in the root\u2013soil interface should be increased. Changing root morphology for increasing the absorptive surface area can enhance Zn uptake. Efflux of Hn uptake . Understn uptake . The micn uptake ,11. The When the soil is inherently deficient in Zn content and/or the solubility of Zn in soils is low, then the plant cannot absorb sufficient Zn to meet its physiological or metabolic need. In fact, nearly 50% of cereal growing areas in the world have been found deficient in Zn . This leAgronomic biofortification is cheap and provides the dual advantage of yield enhancement and improvement of grain Zn concentration. Though genetic biofortification also shows a lot of promise, developing a variety takes a lot of time and effort . MoreoveZn is provided to the crops by soil application, foliar application, seed application (priming), or by a combination of these methods ,70,71,723 induced rise in pH, direct sorption of Zn to the precipitated CaCO3, and formation of insoluble calcium Znate [The efficiency of soil-applied Zn fertilizer depends on soil pH. The availability of Zn is relatively higher at acidic soil pH. The solubility of soil decreases hundred-fold with each unit increase in pH . Liming um Znate . Alkalinum Znate ,74. Otheum Znate ,75,76.Arbuscular mycorrhiza in soil also plays an important role in mobilizing Zn to plants [As Zn reaches the plant root predominantly through diffusion , therefoo plants . The effo plants ,18. The The efficiency of foliar Zn application greatly depends on the type of fertilizer, crop characteristics, especially, leaf characteristics and genetic potential of the crop . The folA poor correlation between DTPA-Zn concentration of soil and grain Zn concentration has been reported . This law/v) ZnSO4. 7H2O at the heading and milk stage of wheat resulted in a significant increase in grain Zn concentration across locations in seven different countries over 2 years of an experiment. The result suggested an average increase in Zn concentration by 83.5%, while soil Zn application showed an average increase of 12.3% over no Zn application [The time of foliar application is very critical for the effectiveness of foliar-applied Zn ,92. Highlication . Foliar lication  and suchlication ,95.High seed Zn content has been found to improve seedling vigour and crop stand in the field . When plSeed priming of wheat with Zn significantly increased grain Zn concentration by 12%, while the concentration of chickpea and maize improved by 29% and 19%, respectively . Seed prDifferent combinations of application methods (soil + foliar and seed + foliar) have also been studied to show their effectiveness in improving grain zinc concentration ,70,98. CNutrient uptake from different application methods may vary due to environmental (soil and atmospheric) and crop characteristics . Agronomic management plays an important role in altering the crop environment, thus agronomic management practices other than Zn fertilization are also expected to affect Zn uptake.3\u2212 ion resulting in more release of OH- from rice roots, which ultimately precipitates Zn thus reducing its\u2019 level [The moisture status of soil may vary depending on the irrigation practices followed. Moisture deficiency in crop field reduces the diffusion of Zn, limiting their absorption by the plant. Zn deficiency has been observed in rice under contrasting environments, i.e., flooded conditions as well as aerobic conditions . In aeros\u2019 level .In addition to Zn fertilization, the status of other nutrients in the soil and their application also affects Zn uptake. Nutrient interaction may alter the availability of nutrients either positively or negatively. A sufficient level of nitrogen supply is very critical for increasing the grain Zn concentration of wheat. Nitrogen application improves uptake as well as remobilization of Zn in wheat . The rolIn addition to enhancing grain Zn concentration and sometimes yield, agronomic biofortification gives some additional benefits. The soil application of Zn under a Zn deficient condition has been found to reduce the uptake and accumulation of phosphorus. This Zn\u2013phosphorus antagonism is one of the most discussed nutrient antagonisms. The reduced phosphorus uptake and accumulation may reduce the phytate content in grains . Under ZIn addition to human health benefits, grains enriched with Zn can give additional agronomic benefits. Seeds with low Zn concentration show poor tolerance to environmental stresses . When seThe second approach for enhancing grain Zn concentration in crops is genetic biofortification. Genetic biofortification follows the breeding approach to increase the concentration and bioavailability of grain Zn. Genetic biofortification can serve as a cost-effective strategy to alleviate Zn deficiency. A superior genotype, once developed, can be used for many years without any additional recurring cost.Plant breeding and/or transgenic approaches provide a hopeful and long-term strategy to overcome micronutrient malnutrition by developing genotypes with a high level of Zn in the edible plant parts ,34,67. TThe overall steps involved in breeding include the following minimum steps: finding suitable genetic variation and selection of parents, long term crossing and backcrossing, stabilization of target traits across multiple environments/climatic conditions, and adaptation of the biofortified genotypes to the regional agronomic management practices . The breA superior genotype for Zn biofortification needs to have the following characteristics: high Zn acquisition efficiency, readily translocate Zn to grain/edible part of plant, efficient remobilization of Zn from vegetative tissues to grain or edible part of the plant, and availability of Zn in the plant in a bioavailable form that can be utilized by the person consuming it ,118. SevExisting genetic variability, trait heritability, gene action, the association among traits, available screening techniques, and diagnostic tools are commonly used criteria to estimate the potential genetic gains . A largeIn addition to grain Zn concentration, the bioavailability of the Zn should be given importance in the breeding programs. Only 25% of the Zn in the staple food grains are thought to be bioavailable . The bioArabidopsis thaliana in barley roots increased the Zn concentration in grain [A transgenic approach can be followed to develop crop varieties with a high Zn content. Evidence of ZIP family iron and Zn transporter proteins in improving grain micronutrient concentration is available ,125,126.in grain .In the agronomic biofortification section, we have briefly discussed how various soil characteristics affect a plant\u2019s availability of Zn. Under such a condition, the varieties developed to accumulate more Zn in the edible parts may not be able to show its full potential. To achieve a grain Zn concentration in the edible portions of the plant that can bring a measurable biological impact, the plant must be grown in a soil environment with sufficient plant-available Zn . As the Moreover, crop improvement to develop Zn rich variety is a fairly lengthy process and requires a lot of effort in germplasm selection/screening, crossing, and performance assessment in the multilocation trial. However, as Zn concentration is not subjected to genetic erosion, little maintenance breeding is needed after the incorporation of the desired gene into the gene pool .The success of genetic biofortification may be jeopardized if sufficient Zn is not available in soil . The capThe unavailability and unaffordability of a healthy diet are largely responsible for the prevalence of malnutrition across the globe. As per an estimate, over 3 billion people worldwide are far from easy access to a healthy diet. Thus, both accessibility and affordability of a healthy diet must be ensured. By 2030, diet-related health cost linked to death and non-communicable diseases are expected to exceed USD 1.3 trillion while, the diet-related social cost of GHG (greenhouse gas) emission is projected to be greater than USD 1.7 trillion. Though these costs are often ignored, a dietary shift to a healthy diet can reduce the cost related to health and climate change. In fact, adoption of a healthy diet can reduce the direct and indirect health costs up to 97% and a 41\u201374% reduction in social cost in GHG emission by 2030 [As the application of Zn may not necessarily increase the yield, farmers may be sceptical to apply Zn as it incurs an additional cost. However, the health benefits obtained from Zn application are usually overlooked in such conditions. Zn and other micronutrient deficiency cause huge economic losses in developing countries and have a huge impact on gross domestic product (GDP) and costs related to health care ,130. MicWang et al.  calculatThe suboptimal utilization of the human resource potential is expected to reduce the work productivity and thus will have an undesirable impact on economic output at all levels, starting from the individual to the national level. Intervention is required to reduce micronutrient malnutrition so that human resource potential can be fully utilized to their potential and cost of health can be reduced. Biofortification may be seen as an investment in human health, which will also reduce the cost of health. Unless we evaluate this health benefit of biofortified grains and see grain yield as the only criteria for evaluating the economic output of crop production; then the benefits of biofortification can be hardly realized. Social awareness on the importance of micronutrient nutrition, policies to promote micronutrient application in crops to realize the benefits of agronomic and/or genetic biofortification, and investment in the research and development on biofortification can help in sustainably alleviating micronutrient malnutrition.A comprehensive 4R  approach of Zn application can be developed for different crops at the regional level and the best combination can be found for achieving high grain Zn concentration.Physiological constraints of grain Zn accumulation must be identified for different crops under different conditions and agronomic and genetic approaches for ameliorating these constraints may be found to further improve the grain Zn density.Biofortification options must be studied under stressed environments and their effects must be evaluated under such conditions. As climate change is expected to bring more weather anomalies, a stress-proof biofortification approach must be developed.The bioavailability of Zn obtained through foliar application can be compared with other application methods. Agronomic management that improves grain Zn bioavailability should be studied.The environmental implications of continuous Zn application should be studied. Continuous application of Zn over a long period may cause Zn toxicity and therefore should be regularly monitored.The performance of Zn-efficient genotypes under different soil Zn availability should be evaluated. The beneficial effects of combining the agronomic and genetic biofortification approach should be explored.Though research has advanced in biofortification, some key areas need to be addressed or improved further. Some have been highlighted below:From this comprehensive review, it can be concluded that the biofortification approach has outstanding potential for ameliorating the problem of micronutrient malnutrition. The cost-effectiveness of this approach makes it a suitable option for low-income countries. As biofortification improves the micronutrient concentration of the staple food grains that is predominantly consumed by people it does not involve any dietary change and can be adapted by people quickly. Agronomic biofortification not only improves the grain Zn concentration providing health benefits, but it can also help in reducing the extent of Zn deficiency especially in regions where intensive cropping is practiced and micronutrient application is overlooked. With the advent of genetic engineering and molecular tools, the breeding approach of biofortification can also be fastened and developing a superior Zn-efficient and Zn-rich varieties will be comparatively easier to find. Policy initiative and government support will also help in further research and dissemination of biofortification technologies and practices."}
{"text": "ZmZIP3, ZmZIP4, ZmZIP5, ZmZIP7, and ZmZIP8 in the roots of two inbred lines. These results indicate that extended time length of low-Zn stress will enlarge the difference of multiple physiological traits, especially biomass, between Zn-sensitive and Zn-tolerant inbred lines. There were significant genotypic differences of root morphology in response to heterogeneous Zn supply. Compared with split-supply with +Zn/+Zn, the difference of above-ground biomass between Zn-sensitive and Zn-tolerant inbred lines under split-supply with \u2013Zn/+Zn was higher. Under the condition of heterogeneous Zn supply, several ZmZIP genes may play important roles in tolerance to low Zn stress, which can provide a basis for further functional characterization.All over the world, a common problem in the soil is the low content of available zinc (Zn), which is unevenly distributed and difficult to move. However, information on the foraging strategies of roots in response to heterogeneous Zn supply is still very limited. Few studies have analyzed the adaptability of maize inbred lines with different Zn efficiencies to different low Zn stress time lengths in maize. This study analyzed the effects of different time lengths of low Zn stress on various related traits in different inbred lines. In addition, morphological plasticity of roots and the response of Zn-related important gene iron-regulated transporter-like proteins (ZIPs) were studied via simulating the heterogeneity of Zn nutrition in the soil. In this report, when Zn deficiency stress duration was extended (from 14 to 21 days), under Zn-deficient supply (0.5 \u03bcM), Zn efficiency (ZE) based on shoot dry weight of Wu312 displayed no significant difference, and ZE for Ye478 was increased by 92.9%. Under longer-term Zn deficiency, shoot, and root dry weights of Ye478 were 6.5 and 2.1-fold higher than those of Wu312, respectively. Uneven Zn supply strongly inhibited the development of some root traits in the -Zn region. Difference in shoot dry weights between Wu312 and Ye478 was larger in T1 (1.97 times) than in T2 (1.53 times). Under heterogeneous condition of Zn supply, both the \u2013Zn region and the +Zn region upregulated the expressions of  Zinc (Zn) is an essential micronutrient in plant growth and development. It plays an important role in various enzymatic reactions, metabolic processes, redox reactions, plant hormone metabolism, promoting the development of plant reproductive organs, resistance to infection by certain pathogens, and improving plant resistance to stress , followed by rice (up to 27%) and maize (9%)  transcription factor gene family, bZIP19 and bZIP23, contributes to the upregulation of ZIPs and improves the adaptation to low Zn supply  are highly induced in roots of Zn-deficient plants. Tissue-specific expression in roots supports the roles of these genes in uptake and root-to-shoot translocation of Zn under Zn starvation conditions : 0.5 NH4NO3, 0.5 CaCl2, 1.5 Ca(NO3)2, 0.75 K2SO4, 0.65 MgSO4, 0.1 KCl, 0.25 KH2PO4, 1.0 \u00d7 10\u22123 H3BO3, 0.35 Fe(II)-EDTA, 8.0 \u00d7 10\u22123 CuSO4, 1.2 \u00d7 10\u22122 MnSO4, 4.0 \u00d7 10\u22125 (NH4)Mo7O24, and 4.0 \u00d7 10\u22123 NiCl. Growth chamber condition was set as a 14-h light period from 8:00 to 22:00 with 28\u00b0C and a 10 h dark period with 22\u00b0C. The average light intensity measured at canopy was 350 \u03bcmol m\u22122 s\u22121. The pH of solution was adjusted to 5.5\u20136.0.Maize seeds of inbred lines Wu312 and Ye478 were sterilized for 30 min in a 10% solution of H4\u00b77H2O. However, a study showed that compared with ZnSO4, ethylene diamine tetra acetic acid-Zn (EDTA-Zn) was found to be better for growth and yield of rice  and +Zn (8 \u03bcM EDTA-Zn) in Treatment 1 (T1), and with +Zn (8 \u03bcM EDTA-Zn) and +Zn (8 \u03bcM EDTA-Zn) in Treatment 2 (T2), respectively . After 4In Experiment 1 and 2, shoots and roots for plants were separately collected in an envelope. All samples were heat-treated at 105\u00b0C for 30 min and dried at 75\u00b0C until constant weight. Zn concentrations in shoots and roots were analyzed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). Zn uptake efficiency, Zn transport efficiency, and Zn use efficiency were calculated using the following equations from (1) to (3), respectively.\u22121) = Zn uptake efficiency (\u03bcg root dry weight gZn transport efficiency (%) = \u22121) = Zn use efficiency .Total RNA was isolated from each root sample for Wu312 and Ye478 with TRIzol (Takara). We used 1.5 \u03bcg of total RNA to synthesize cDNA. Quantitative real-time polymerase chain reaction (PCR) was performed using SYBR Green Real-time RT-PCR (Applied Biosystems) and an ABI7500 Fast Real-Time PCR System (Applied Biosystems). The primers used for real-time PCR are shown in t-test. A probability level of p < 0.05 was required for statistical significance.Each experiment was arranged in accordance with a randomized complete design. Means among different treatments were compared using the least significant difference (LSD) test. Means between inbred lines Wu312 and Ye478 were compared using In Experiment 1, when the Zn supply concentration was 0.5 \u03bcM, shoot and root dry weights of Ye478 were 154 and 109% higher than those of Wu312, respectively. Compared with Zn-sufficient supply, shoot dry weights of Wu312 and Ye478 under Zn deficient condition (0.5 \u03bcM) were decreased by 33.6 and 18.8%, respectively , while rIn Experiment 2, at the nil Zn supply (0 \u03bcM), plant of Wu312 was tended to be dead and Ye478 showed serious Zn-deficient symptoms . When thIn Experiment 2, when the Zn supply level was 0.5 \u03bcM, compared with Zn-sufficient treatment (8 \u03bcM), leaf soil plant analysis development (SPAD) of Wu312 was decreased by 38.7% and SPAD of Ye478 was increased by 14.9%. When the Zn supply concentration was increased to 16 and 32 \u03bcM, leaf SPAD values of two inbred lines were significantly reduced . Under dBased on visible phenotypic variation of plants, shoot and root dry weight, and leaf SPAD of Wu312 and Ye478 in Experiments 1 and 2, Zn nutritional status of 8 \u03bcM was determined to be the Zn-sufficient condition (control treatment). When Zn concentration was 0.5 \u03bcM, compared with 21-day Experiment 2, a 14-day Experiment 1 was designed as a short-term zinc deficiency experiment. In addition, shoot dry weights of Ye478 under Zn-deficient (0.5 \u03bcM EDTA-Zn), and Zn-sufficient condition (8 \u03bcM EDTA-Zn) under short-term stress were 1.5- and 1.3-fold higher than that of Wu312 in Experiment 1, respectively. Under longer-term Zn deficiency stress in Experiment 2, these multiple values were reduced to 6.5 and 2.1, respectively.Relative values of shoot dry weight, which were estimated by the ratio of shoot dry weight under 0.5 \u03bcM low Zn stress to the shoot dry weight under Zn-sufficient condition (8 \u03bcM EDTA-Zn), were considered as Zn efficiency (ZE) based on shoot dry weight. ZEs for Zn-sensitive inbred line Wu312 showed no significant difference between Experiment 1 and 2. However, ZE for Zn-tolerant inbred line Ye478 in Experiment 2 was 192.9% higher than that in Experiment 1 . This fiIn Experiments 1 and 2, shoot and root Zn concentration of Wu312 and Ye478 gradually enhanced with the increase of Zn supply , 4C,D. EIn Experiment 1, compared with Zn sufficient condition (8 \u03bcM), Zn uptake and transport efficiency of Wu312 and Ye478 under Zn deficient condition (0.5 \u03bcM) was decreased by 59.5 and 67.8%, and 42.9 and 38.8%, respectively . And Zn Under the condition of heterogeneous Zn supply, total root length, root surface area, and lateral root number of the Wu312 and Ye478 under Zn deficiency were significantly reduced  comparedSpecific root length of two inbred lines differed greatly, but the difference in main root length was relatively small . In the ZmZIP1, ZmZIP2, and ZmZIP6 showed no significant difference . Also, the expression levels of these genes in Zn-efficient and Zn-inefficient genotypes were also different.Under the condition of heterogeneous Zn supply, compared with homogeneous Zn-sufficient supply, the expression levels of fference . Howeverifferent  while Zmfference . Uneven of Wu312 . Heterogn region . CompareZmZIP1-7 . These rIn Experiment 1, shoot dry weight of Ye478 under Zn-deficient condition (0.5 \u03bcM) was 1.5 times higher than that of Wu312 despite there being no significant difference in root dry weight between two inbred lines . HoweverApart from extending stress duration, we also investigated the effects of excess Zn on maize via increasing the Zn supply to 16 and 32 \u03bcM. Previous studies have shown that excess Zn inhibited the production of biomass, chlorophyll, total soluble protein, and strongly increased accumulation of Zn in both root and shoot mainly because Zn-induced oxidative stress can cause loss of plasma membrane integrity, electrolyte leakage, increase in malondialdehyde content, and damage photosynthesis to produce reactive oxygen species  indicated that the root surface of Wu312 have begun to rot and become grayish brown since the 21st day after transfer, which would probably lead to necrosis in the whole plant . But theThe R/S ratio of genotypes increased as Zn application decreased , Ye478 could continue to grow healthily, while the development of Wu312 was significantly inhibited. This may be because genotypes with different tolerance to Zn stress have different minimum Zn requirements  in ZIP genes are crucial in plant adaptation to low and fluctuating Zn availability in soil . The expression levels of each gene in the two lines are basically consistent with the results of this study. According to the results of this study, the expression of ZmZIP1 in two inbred lines under the condition of non-uniform Zn supply did not show a significant difference compared with the control  of the nodes and responsible for the preferential distribution of Zn to developing tissues  mainly accumulate Zn in the roots rather than in shoots. OsZIP4 is a Zn transporter that localizes to the phloem cells of stems, vascular bundles of leaves, and roots. Constitutive expression of it changes the Zn distribution within rice plants . However, low levels are found in the ZmZIP5i (RNAi line) plants. ZmZIP5 may play a key role in Zn uptake and root-to-shoot translocation , ZmZIP7 may display characteristics of low-affinity Zn transport in the plant  demonstrated a much stronger activity than OsZIP5  and other rice ZIP proteins .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "The high-areal-capacity full cells demonstrate hundreds of cycles under high Zn utilization conditions.Rechargeable aqueous zinc-ion batteries (RZIBs) provide a promising complementarity to the existing lithium-ion batteries due to their low cost, non-toxicity and intrinsic safety. However, Zn anodes suffer from zinc dendrite growth and electrolyte corrosion, resulting in poor reversibility. Here, we develop an ultrathin, fluorinated two-dimensional porous covalent organic framework (FCOF) film as a protective layer on the Zn surface. The strong interaction between fluorine (F) in FCOF and Zn reduces the surface energy of the Zn (002) crystal plane, enabling the preferred growth of (002) planes during the electrodeposition process. As a result, Zn deposits show horizontally arranged platelet morphology with (002) orientations preferred. Furthermore, F-containing nanochannels facilitate ion transport and prevent electrolyte penetration for improving corrosion resistance. The FCOF@Zn symmetric cells achieve stability for over 750\u2009h at an ultrahigh current density of 40\u2009mA\u2009cm Rechargeable aqueous zinc-ion batteries are promising but the zinc anode suffers from dendrite growth and electrolyte corrosion. Here, the authors develop a fluorinated covalent organic framework film as a protective layer for aqueous zinc anode battery. Unfortunately, previous Zn anodes showed poor reversibility in aqueous electrolytes5. Issues including Zn dendrite formation, continuous parasitic hydrogen evolution reaction (HER), and irreversible by-products, resulting in low Coulombic efficiency (CE), and shortened battery life9. To stabilize the Zn anode, various strategies including electrolyte optimization , surface coating materials  and Zn bulk structure engineering  have been proposed to achieve higher performance Zn anodes. However, there are still some unsolved issues with these strategies, which restricts them to subdued performance levels in Zn batteries. For instance, manipulating previous electrolyte compositions leads to an increase in overall costs15, sacrifices rate performance of the batteries owing to their slow ionic conductivity and the HER is only lowered, not eliminated2. Interfacial modification layers are effective for suppressing HER16, however, the huge volume change during repeated Zn plating/stripping can damage protective layers, even peeling them completely off the Zn matrix17. Employing conductive 3D hosts could help to realize high-rate Zn deposition18, but adds additional porosity and weight, thereby reducing the volumetric/gravimetric energy density of the batteries. Therefore, developing alternative techniques to achieve dendrite-free Zn anodes while maintaining fast Zn deposition is urgently needed.Zinc (Zn) anode-based aqueous batteries are attracting tremendous interest owing to Zn\u2019s high theoretical capacity  and (103) planes, which mitigates dendrite formation and reduces the later corrosion rates. Substrate such as stainless steel modified with an aligned graphene layer5, shows good lattice matching with Zn, which induces epitaxial deposition of Zn along the (002) planes, achieving ultra-long cycling life. Recently, fields generated by rotating disc electrodes19 are reported to promote the crystallographic reorientation of Zn to be grow parallel to the substrate, and the reversibility of Zn deposition/stripping is greatly increased. Therefore, correlating the crystallography and morphology to deeply understand and regulate the electrodeposition behavior of Zn is of great significance for developing long-life Zn batteries. However, there is an obvious lacks of fundamental elucidation of the mechanism controlling planar Zn deposition. Furthermore, the surface stability of inorganic crystals has long been thought to be dominated by their surface energy33. From the perspective of crystal growth, controlling the surface energy of Zn crystal planes offers exciting opportunities to realize planar zinc deposition.The crystallinity and morphology of Zn electrodeposits dominates the reversibility of Zn plating/stripping\u22122). The assembled full cells paired with manganese dioxide (MnO2) cathodes show a\u00a0stable cycle life for over 250 cycles under practical condition of lean electrolyte, high areal capacity cathode and limited Zn excess.Here, by using two-dimensional (2D) covalent organic frameworks (COF) as a multi-functional platform Fig.\u00a0, we deveThe imine-linked FCOF thin films are prepared through a solvothermal procedure Fig.\u00a0. In a tyq\u2009=\u20090.20, 0.35, 0.40, 0.53\u2009A\u22121 correspond to the planes (100), (110), (200) and (210), respectively, consistent with a previous report34, confirming the high degree of crystallinity of the as-prepared film. Findings from theoretical simulation and Pawley refinement confirm that the 2D stacked structure in FCOF is an AA stacking mode  of the N1s spectra . The high intensity of the peak at 398.96\u2009eV in the N1s spectra  measurement Fig.\u00a0. From thter Fig.\u00a0, which itra Fig.\u00a0 is assigtra Fig.\u00a0 shows thtra Fig.\u00a0 further 2 and polyvinylidene difluoride (PVDF) hybrid matrix (2.67\u2009GPa)36. The good mechanical strength is greatly beneficial to buffer volume expansion and retard dendrite propagation during the dissolution/deposition of Zn anodes.The F atoms are the crucial elements within the FCOF films for achieving high-performance Zn anodes. The element mapping obtained from energy dispersive X-ray spectroscopy (EDX) indicates that F is evenly distributed in the film  results  than bare Zn. In particular, the Rct of Zn anodes coated by the 100\u2009nm thick FCOF film is lowest (90 \u03a9), about half that of bare Zn (180 \u03a9). Apparently, the Zn2+ transport is increased by the fluorinated 1D nanochannels. This is mainly due to the F atoms surrounded within the nanopores that endow the film a strong hydrophobic effect. When hydrated Zn2+ transport through fluorinated nanochannels is driven by electric field force, water molecules coordinated with Zn2+ will be repelled because of the strong hydrophobicity of F element in the FCOF film covering the Zn metal. Consequently, a large portion of water molecules is retarded and not able to penetrate the fluorinated nanochannels. The de-solvation effect of Zn2+ is therefore significantly promoted by F element. To confirm the ability of FCOF film to promote de-solvation, a study of the activation energy (Ea) is made, Ea represents the de-solvation barrier to Zn2+ transport. A computation for Ea based on temperature-dependent EIS is performed  for FCOF@Zn is 14.5\u2009kJ\u2009mol\u22121, whilst that for bare Zn is significantly greater at 32.2\u2009kJ\u2009mol\u22121. The lower activation energy for FCOF@Zn over bare Zn strongly suggests that the FCOF layer ensures fast de-solvation of Zn2+ and facilitates fast ion-transference. Studies have demonstrated that COFs are an excellent ionic conductor39. The inherent ordered structures provide regular ionic coordination sites to facilitate ions to hop along the 1D aligned nanochannels. This highly boosts ionic conductivity. To study Zn2+ transport in FCOF, the transport sites for Zn2+ on the COF skeleton are determined theoretically. The charge density distribution of the chemical structural unit in FCOF is computed. As is shown in Supplementary Fig.\u00a039. Because of electrostatic attraction, the positively charged Zn2+ hops around the F atom sites and transports along the 1D aligned channels during charge/discharge . The impedance of the FCOF@Zn symmetric cells increases from 180 to 600 \u03a9 within 8\u2009h (h) after cell assembly 6\u00b75H2O (JCPDS# 39-0688)11. When plain Zn anodes are immersed in aqueous electrolyte for 48\u2009h, the peak intensity of by-products increases sharply. Much less irreversible by-product is accumulated on the surface of FCOF@Zn anodes during the same time duration. To evaluate corrosion resistance of the FCOF film, potentiodynamic and open-circuit, tests are conducted using a home-made test apparatus  decreased from 2.5 \u00d7 10\u22123\u2009A\u2009cm\u22122 for bare Zn to 4.1 \u00d7 10\u22125\u2009A\u2009cm\u22122 for FCOF@Zn. Despite 8 days of electrolyte immersion, FCOF@Zn continued to exhibit a greater corrosion potential (Ecorr) of \u22121.063\u2009V and a lower corrosion current (Icorr) of 4.8 \u00d7 10\u22124 A cm\u22122, compared with bare Zn of, respectively, \u22121.068\u2009V and 1.2 \u00d7 10\u22123 A cm\u22122. The open circuit potential (OCP) for both electrodes is shown in Supplementary Fig.\u00a04 remained stable at ~ \u22121.042\u2009V vs. SCE, whilst that for FCOF@Zn is ~ \u22121.020\u2009V in the initial stage, and remained greater at \u22121.032\u2009V than that for bare Zn of \u22121.041\u2009V, despite 5000\u2009s. It is reliably concluded therefore that the corrosion resistance for Zn anode is significantly improved by FCOF film surface protection.The corrosion resistance of the FCOF film on the Zn surface is investigated in aqueous electrolyte 6 \u20225H2O (JCPDS NO: 39-0688) by-product. In contrast, these by-product signals are significantly weaker for Zn deposits under the FCOF film, strongly evidencing that the FCOF film provides protection for Zn, and inhibits accumulation of by-product. The orientation of the Zn deposits can also be quantified by calculating the texture coefficient41  of Zn deposits underneath the FCOF film is 19.2, much higher than that of the deposits on bare Zn (11.5), verifying the preferential growth on the (002) plane of Zn modulated by an FCOF film. XRD patterns for Zn deposits following the 30th and 80th cycles are shown in Supplementary Fig.\u00a0Tc for the (002) plane is stable with increase in cycle number , however is higher than that for bare Zn . These evidences validate that the preferred (002) plane orientation is maintained, despite long cycling.In addition to the fast ion conduction and suppression of side reactions features, the morphology and texture of Zn deposits has been proven to have large impact on the cycling life of Zn batteries. Attaining an even planar deposition can ensure the batteries running for a prolonged time without short circuiting. An electronic resistance property is important in Zn deposition underneath the FCOF film. From the polarization I\u2013V curve, the 100\u2009nm FCOF film exhibits an electronic resistance of 3 \u00d7 10ion Fig.\u00a0, disordelms Fig.\u00a0, while tlms Fig.\u00a0. This chlms Fig.\u00a0, and are42 . As shown in Fig.\u00a0d-spacings of 0.230 and 0.133\u2009nm with an interfacial angle of 90\u00b0, corresponding to the (100) and (1\u201320) planes, respectively. The HRTEM result is in accord with the indexed SAED diffraction spots. To further verify the indexing results, an atomic arrangement model of Zn along the (001) direction is simulated, as shown in Fig.\u00a0X-ray diffraction pole figures are used to further identify the texture information of Zn deposits. The (002) pole figure Fig.\u00a0 of Zn un42 Fig.\u00a0. In cont42 Fig.\u00a0 shows al42 Fig.\u00a0, while f42 Fig.\u00a0. This in2+ transport, and corrosion resistance properties enabled by the FCOF film are expected to greatly improve the electrochemical performance of Zn anodes. The reversibility of Zn anodes can be measured by a procedure that wherein a specific amount of Zn is plated on the substrate and then stripped away. Coulombic efficiency (CE) is an important index to evaluate such reversibility. The CE using the half cells in FCOF@Ti/Zn and Ti/Zn configurations is measured. At a moderate current density . Remarkably, as evidenced from the AFM height and phase imaging . The average height difference  is only 170\u2009nm 2 or Zn4SO4(OH)6\u00b7nH2O on Zn surface16. Raman spectroscopy is carried out to reveal the components on Zn deposits surfaces after cycling . Sharp peaks at 1152, 1110, 1011, and 967\u2009cm\u22121 are observed on the Zn deposits on bare Ti 6\u00b75H2O43. In contrast, the peaks of the Zn deposits underneath FCOF are not obvious and their intensity is much lower  is also far superior to most previous reported values  manganese dioxide (MnO2) cathodes. For the FCOF@Zn/MnO2cells, cyclic voltammetry curve (CV) curves demonstrate a larger current density at 0.1\u2009mV\u2009s\u22121 and a smaller voltage gap between typical redox peaks than in Zn/MnO2 cells  is lower than that of Zn/MnO2 cells . Reducing the capacity ratio of the negative electrode to the positive electrode (N/P) during full cell operation is a key parameter to achieve high energy density45. In previous studies, many systems chose to use thick zinc foil (\u2265100 \u00b5m) paired with low mass loading cathodes to assemble full cells, the N/P reported in these studies is typically higher than 50, which is not beneficial for achieving high energy density. In our case, the excellent performance of the FCOF@Zn anodes allows us to further evaluate the cycle performance of full cells under harsh conditions. Using thin FCOF film-protected Zn plates as anodes (the thin Zn plates is rolled to desired thickness to satisfy the required N/P condition), FCOF@Zn/MnO2 cells with N/P = 10:1 and N/P = 5:1 show stable specific capacity at current density of 4\u2009mA\u2009cm\u22122 for over 300 and 200 cycles, respectively  and controlled electrolyte-to-capacity ratio  is assembled. When the mass loading of MnO2 increases from 8 to 16\u2009mg\u2009cm\u22122, the charge and discharge curves for FCOF@Zn-MnO2 full cell exhibit low polarization as shown in Supplementary Fig.\u00a02 full cell  is cycled at a current density of 3\u2009mA\u2009cm\u22122. The charge/discharge curves determined from different cycles almost overlap , which is significantly increased (by approx. 6.5 times) compared with many reported Zn/MnO2 cells using low mass loading cathodes and thick Zn foils30  indicator  plane with lowest the surface energy tends to stay exposed instead of its intrinsic thermodynamically stable plane. In our case, the uniformly distributed F atoms within FCOF film have a strong binding interaction with the Zn atoms, thereby regulating the relative surface energy value of the (101) and (002) planes and influencing the deposited morphology. To verify this, the surface energy and adsorption energy of the two crystal planes terminated by F atoms are studied by first-principles calculations  and lower conduction band (CB). With the formation of Zn\u2013F interaction, however, localized states of Zn 4s are observed, indicating that F atoms are prone to stabilize Zn atoms. To qualitatively determine the strength of the interaction between F atoms and Zn atoms and its impact on different crystal planes, the electron states of the Zn 3d and F 2p are analyzed. Due to the strong electron-withdrawing property of F atoms, the F 2p could accept electrons from the Zn 4s. As a result, the Zn\u00a04s planes would partially lose electrons, forming quasi-stable bonds with the F species. Thereafter, the electrons from the Zn 3d may be excited toward the Fermi level. The displacement of Zn 3d electrons from the FT-(002) planes is more energetically-favorable than from FT-(101) planes. On the other hand, compared with the F\u00a02p electron states of the in FT-(101) planes, the F 2p electron distribution in a FT-(002) plane is more localized. These results clearly suggest that F atoms interact more strongly with the (002) planes than with the (101) plane. The XPS data for the F 1s and Zn 2p at the FCOF-Zn interface also indicate the existence of strong F\u2013Zn interaction50  crystal planes of Zn. The growth of (002) planes is most stable during the deposition process, and anisotropic growth of Zn along other planes is done in such a way that a (002) plane is formed, resulting in platelet-like Zn deposits. Given that the F atoms bonded within the FCOF film are arranged in parallel along the surfaces of the current collectors, which induces each Zn platelet to also be arranged in parallel. Furthermore, the F-containing porous nano channels endow good hydrophobic effects on the film, which is beneficial for de-solvation and facilitates fast transport of hydrated Zn2 bath) and degassed by three freeze-pump-thaw cycles and finally sealed under vacuum conditions. The sealed tube is heated at 120\u2009\u00b0C for 3 days. For the postprocessing, the tube is opened, washed with acetone several times and then the tube is charged with deionized water overnight. The free-standing FCOF films easily detach from the tube wall by gently shaking the tube. The obtained FCOF films is immersed in acetone for 2 days to wash out any impurities. Finally, the as-prepared FCOF films is transferred to various substrates via a pulling method in acetone solvent. The thickness of the membrane/film can be also easily tuned by controlling the concentration of monomers. The MnO2 cathodes are synthesized using an aging method according to a previous report51. First, 5.07\u2009g manganese sulfate monohydrate (MnSO4\u00b7H2O) is put in 100\u2009mL deionized water, followed by ultrasound and stir to ensure dissolution (named Solution 1). Then 4.32\u2009g sodium hydroxide (NaOH) and 20\u2009mL hydrogen peroxide  are dissolved in 180\u2009mL deionized water (named Solution 2). Solution 2 is then dropwise added into Solution 1 under vigorous stirring conditions. The mixed solution is stirred for 1\u2009h and aged at 25\u2009\u00b0C for 24\u2009h. The precipitated product is centrifuged for three times, washed with deionized water, and dried to obtain MnO2.For the typical synthesis of FCOF film, a pyrex tube is charged with 3.75\u2009mg (0.0182\u2009mmol) 2, 3, 5, 6-tetrafluoroterephthalaldehyde (TFA), 4.40\u2009mg (0.0125\u2009mmol) 1,3,5-Tris(4-aminophenyl) benzene (TAPB), 1.8\u2009mL dioxane (Dio) and 0.2\u2009mL 1, 3, 5-trimethylbenzene (TMB). After sonication for 10\u2009min, 0.1\u2009mL HOAc (1.5\u2009M) solution is added. After this, the tube is frozen at 77\u2009K (liquid N\u03bb = 0.154056\u2009nm), using an operating voltage of 40\u2009kV and a 40\u2009mA current. Fourier transform infrared (FTIR) spectra are collected on a ThermoFisher Nicolet 6700 spectrometer. N2 adsorption\u2013desorption isotherms are measured at 77\u2009K on a Micromeritics TriStar II 3020 volumetric adsorption analyzer after degassing in a vacuum at 120\u2009\u00b0C overnight. Atomic force microscope (AFM) is performed on a NT-MDT NTEGRA Spectra II microscope. Field-emission scanning electron microscopy (FESEM) images are acquired from a Zeiss Gemini SEM500, equipped with an Aztec X-Max Extreme energy dispersive spectrometer (EDS). High-resolution transmission electron microscope (HRTEM) images are collected from a JEM-2010F transmission electron microscope. XPS measurements are carried out with a Thermo Scientific K-Alpha+ spectrometer under vacuum. Nano-indentation surface hardness measurements are conducted on a TI-950, NHT.Wide-angle X-ray scattering (WAXS) measurement is conducted on a XenocsXeuss2.0 with 8\u2009KeV Cu K\u03b1 radiation. X-ray diffraction (XRD) data is measured by a Bruker D8 Advance with Cu-K\u03b1 X-ray radiation  in a mass ratio of 7:2:1. The mixture is compressed onto a Ti grid. The electrodes are dried at 80\u2009\u00b0C under vacuum for 12\u2009h and then punched into disks. All the electrochemical properties are tested by assembling 2016-coin cells with glass fiber separators. All Galvanostatic charge-discharge measurements are carried out on a battery testing instrument  at different current densities. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) are performed by a CHI 660E electrochemical workstation. CV curves of full cells are recorded over the voltage range of 1\u20131.85\u2009V. EIS is measured in a frequency range of 100\u2009kHz to 0.1\u2009Hz at open circuit potential and an amplitude of 5\u2009mV. The fitting parameters of the equivalent circuit are analyzed by ZSimpWin software.Cycling tests for symmetric cells and Zn/Ti half cells of bare Zn or FCOF@Zn are conducted with 2\u2009M ZnSOThe DFT calculations are carried out using the Vienna Ab-initio Simulation Package (VASP) with the frozen-core all-electron projector-augment-wave (PAW) method. The Perdew\u2013Burke\u2013Ernzerhof (PBE) form of the generalized gradient approximation (GGA) is adopted to describe the exchange and correlation potential. The cutoff energy for the plane-wave basis set is set to 500\u2009eV. The Monkhorst-Pack k-point6 sampling is set to 3\u2009\u00d7\u20093\u2009\u00d7\u20091. The geometry optimizations are performed until the forces on each ion is reduced below 0.05\u2009eV/\u00c5. The vacuum slab models are used to calculate the adsorption of F atom on Zn (002) and (101) surfaces. These Zn surface slabs comprise four layers of Zn atoms, and a vacuum region of 20\u2009\u00c5 above them is used to ensure the decoupling between neighboring systems. For the geometry optimization, the atoms in the 2-bottom layers of slab are fixed to their bulk positions.The adsorption energy, 31:2.The surface free energy (Supplementary Information"}
{"text": "This review addresses the physiological and biochemical connections between steroidal implants and Zn and their interaction to influence growth in beef cattle. Steroidal implants have been widely accepted as a growth-promoting technology that provides an unmatched economic return to the producer and improves beef production\u2019s environmental sustainability. Likewise, decades of research have indicated Zn is vital for skeletal muscle growth. Considering Zn is an essential trace mineral, strategic Zn supplementation to implanted cattle may optimize beef production. Although many interrelationships between steroidal implants and Zn are new and forthcoming, the literature reviewed hereafter indicates roles for Zn in a multitude of growth processes pertinent to steroidal implant-induced growth and uncover changes in Zn metabolism due to steroidal implant use.Growth-promoting technologies such as steroidal implants have been utilized in the beef industry for over 60 years and remain an indispensable tool for improving economic returns through consistently improved average daily gain via increased skeletal muscle hypertrophy. Zinc has been implicated in skeletal muscle growth through protein synthesis, satellite cell function, and many other growth processes. Therefore, the objective of this review was to present the available literature linking Zn to steroidal implant-induced protein synthesis and other metabolic processes. Herein, steroidal implants and their mode of action, the biological importance of Zn, and several connections between steroidal implants and Zn related to growth processes are discussed. These include the influence of Zn on hormone receptor signaling, circulating insulin-like growth factor-1 concentrations, glucose metabolism, protein synthesis via mTOR, and satellite cell proliferation and differentiation. Supplemental Zn has also been implicated in improved growth rates of cattle utilizing growth-promoting technologies, and steroidal implants appear to alter liver and circulating Zn concentrations. Therefore, this review provides evidence of the role of Zn in steroidal implant-induced growth yet reveals gaps in the current knowledge base related to optimizing Zn supplementation strategies to best capture growth performance improvements offered through steroidal implants. Increasing supplementation of trace minerals (TM)  and moreSteroidal implants have been used since the 1950s in the U.S. beef industry although they are no longer an accepted growth-promoting technology in several countries. This technology routinely improves cattle gains by 16\u201320%  and is a2) or the synthetic prodrug of E2, estradiol benzoate (EB). The estrogenic component can be included alone, but most often, it is included in combination with either progesterone, testosterone propionate, or trenbolone acetate (TBA) thymidine, which functions as a labeled DNA precursor, linearly decreased in the liver, kidney, and spleen, reflective of the importance of Zn in DNA synthesis. Additionally, protein synthesis was markedly decreased in the kidney and spleen of rats, measured by [14C]lysine incorporation in these tissues. These studies were instrumental in demonstrating a vital role for Zn in protein utilization in the body.Zinc functions in mediating whole body growth and protein synthesis. It was demonstrated that in piglets fed Zn deficient diets, growth retardation was evident as early as 1 week following dietary treatment administration . FurtherOfficial beef cattle TM requirements are established to prevent deficiency and allow for growth . The curAlthough Zn supplementation is greater across the beef industry  than NASYet, Zn absorption may be influenced by additional factors that alter whole body use of Zn, such as growth. Skeletal muscle and bone comprise a large percentage of Zn storage. However, classified as slow Zn metabolizers, release of Zn from muscle and bone is less likely . Seeing 2 receptor\u2019s signaling in some manner.Zinc has been implicated in many pathways downstream of hormone receptors often with a role in affecting the phosphorylation state of the cell, these responses typically begin at the receptor. Similar to the non-genomic pathways by which hormone signaling occurs, Zn treatment can also directly influence GPER signaling. Interestingly, Pisano et al.  found th2+ for 2 h resulted in increased release of hbEGF from EGFR, resulting in EGFR phosphorylation [Treatment of normal human bronchial epithelial cells with 100 \u00b5M Znrylation . Phosphorylation . Zinc surylation . Furtherrylation  observedrylation ,68. As arylation ,70, MMP rylation ,72. Furtrylation . Althougrylation ,26, thes2 and androgen hormone receptor signaling.Although Zn has not been directly related to androgen receptor function like the previously discussed estrogen receptor, a role for Zn in androgen signaling is emerging. Interestingly, the Zn transporter ZIP9 was discovered as a membrane androgen receptor that facilitates non-genomic signaling in fish  and huma2 implant [Steroidal implants have consistently been shown to increase both hepatic and/or local skeletal muscle IGF-I production in steers implanted with a combination TBA + E implant ,77,78. I implant . Additio implant . The imp implant , who repAlthough Zn supplementation has been shown to improve growth rates in rats , links bThe majority of circulating IGF-1 is bound by a binding protein that acts to prolong the half-life of IGF-1 . Due to Circulating concentrations of IGF-1 and/or IGFBP-3 have been observed to increase with Zn supplementation ,8,87,88.As previously suggested, the literature strongly indicates a role for Zn concerning glucose metabolism. While ruminants derive a substantial amount of energy from volatile fatty acids, gluconeogenesis plays an important role in ruminant energy metabolism . GlucoseThe behavior of proinsulin and insulin in the presence of Zn suggests Zn is important in \u03b2-cell production of insulin for most animal species. Zinc is an essential TM that plays a vital role in the formation of insulin, stimulation of phosphorylation of the \u03b2-subunit of this hormone receptor, activation of PI3K, and the translocation of glucose transporter 4 (GLUT4) ,93. The Wu et al.  showed tAdditionally, Ohashi et al.  reported2 resulted in a dose-dependent increase in protein synthesis rate coupled with a decrease in protein degradation rate [Downstream of GH and IGF-1 signaling, a considerable body of the literature reveals additional interactions for Zn and steroidal implants in protein synthesis exist . Steroidion rate .Zinc appears to be relevant to many of these same protein synthesis pathways. Nimmanon et al.  reportedAs it pertains to cattle, BSC provide the additional nuclei source necessary to support postnatal skeletal muscle fiber hypertrophy and are critical in determining the extent of muscle growth . Satelli2 influence satellite cell function. While it is clear Zn plays a role in satellite cell function, past evidence provides controversy about the exact relationship of Zn to proliferation and differentiation of myoblasts. Differential expression of Zn transporters were analyzed by Paskavitz et al. [2+ indicates cellular Zn concentration is dynamic during the myogenesis process. Petrie et al. [In addition to playing a structural role, Zn appears to have an important regulatory function related to cell proliferation and differentiation in satellite cells that likely interacts or overlaps with the mechanisms by which TBA and Ez et al.  to deterz et al.  demonstre et al.  found in2 on myogenic proliferation and differentiation and found that exogenous Zn treatment increased both myoblast proliferation and differentiation. Mnatsakanyan et al. [\u22121\u00b7d\u22121 of Zn as Zn-methionine. Skeletal muscle SC, extracted and cultured from these steers, possessed increased Pax7 gene expression. This indicates animals treated with Zn-methionine had greater capacity to increase total nuclei present in skeletal muscle through proliferation and differentiation of the Pax7 SC. Although some of the previous literature is in disagreement with regard to how Zn specifically impacts different aspects of SC lineage, these data do indicate that Zn does in fact play a role in SC proliferation and differentiation and require further investigation.Additionally, Petrie et al.  reportedn et al.  also repn et al.  found Znn et al.  reportedn et al.  and Ohasn et al. , contrasn et al.  conducteAlthough TM recommendations for beef cattle were recently updated , few cha4 was fed to heifers and steers, steroidal implant administration improved average daily gain (ADG) by more than 17% [4 (100 mg Zn/kg DM) resulted in a 24% improvement in ADG over non-implanted steers fed the same Zn treatment [4 resulted in a linear improvement in performance of steers administered a high potency implant during the first 18 d post-implant administration. In contrast, no differences in performance were observed due to Zn supplementation within non-implanted steers [Indeed, Zn supplementation influences steroidal implant-induced performance of cattle. When 200 mg Zn/kg DM from ZnSOthan 17% . Howeverthan 17% . Interesreatment . Both ofd steers . These d4 (30 vs. 100 mg Zn/kg DM) [Although supplementation of Zn to cattle utilizing growth-promoting technologies has been proven advantageous, timing of Zn supplementation may be vital to capturing this growth response. Peak payout of hormone from steroidal implants occurs within 40 d of implant administration , resultin/kg DM) . These dAlthough effects of Zn supplementation on cattle performance clearly indicate the importance of Zn in growth, changes in liver and plasma Zn concentrations provide compelling evidence of modified physiological Zn requirements due to steroidal implants. In lambs implanted with zeranol (12 mg), Zn retention was increased by 60% . FurtherRecent studies have found increasing evidence of implants altering Zn requirements by assessing plasma and liver Zn concentrations . It is i2 cannot be quantified and accounted for in a controlled study. Therefore, the effects of steroidal implants on Zn metabolism in heifers may not be as reliable as in steers or spayed heifers.In contrast, Huerta et al.  observed4 or Zn-methionine [In addition to circulating Zn concentrations, steroidal implants have been found to influence concentrations of Zn in the liver. As previously mentioned, liver Zn concentrations are more difficult to change than plasma due to the less labile nature of Zn stored in the liver. Interestingly, Messersmith et al.  observedthionine . In compthionine . This rethionine  observedthionine  suggeststhionine  observedthionine  Zn recomThese data indicate changes in plasma Zn concentrations may be more consistent than liver Zn concentrations due to steroidal implant administration. Although the detected differences in Zn concentrations remained within the wide reference ranges  for plasStrategic supplementation of TM, including Zn, to cattle based upon expected performance and management practices such as steroidal implant use is critical to a future that features precision cattle feeding. This review details the proposed multi-faceted interactions between Zn and steroidal implants on growth ranging from effects on hormone receptors to protein synthesis. The literature reveals supporting roles for additional dietary Zn in implanted cattle and many avenues for future research. With more than 20 million cattle on feed in the U.S. each year and Zn being a low-cost input, strategic supplementation of Zn could potentially better capture the growth performance advantages offered by steroidal implants while improving producer return on investment and the efficiencies of beef production. However, the optimal concentration of supplemental Zn to capitalize on the genetic potential and implant-induced growth rates of cattle is unclear. Similar to the energy and protein demands of implanted cattle, Zn requirements of the beef animal are likely increased to accommodate these higher rates of growth. An association between Zn metabolism and steroidal implant-induced growth, as outlined in this review, provide evidence of Zn as a potential limiting micronutrient in beef cattle growth.Furthermore, optimal timing of Zn supplementation is unknown. Pinpointing a Zn supplementation window may prove useful in best accommodating the micronutrient demands of fed beef cattle. Phase feeding of greater concentrations of Zn during peak hormonal payout or other periods of high growth rates may optimize performance and best utilize TM resources. Supplementation of Zn at high concentrations in cattle poses minimal risk for toxicity, and supplemented concentrations are well below pharmacological concentrations fed in other species . Therefo"}
{"text": "This allowed us to calculate the fraction of Zn in the shoots that was derived from fertilizer, soil, and seed over 4 successive cuts. In addition, we measured the 67Zn:66Zn isotope ratio with the diffusive gradients in thin films technique (DGT) on soils labeled with 67Zn and incubated with the same fertilizers. After 48 days of growth, the largest fraction of Zn in the ryegrass shoots was derived from the soil (79\u201388%), followed by the Zn-containing fertilizer (11\u201320%); the least (<2.3%) came from the seed. Only a minor fraction of the Zn applied with the fertilizer was transferred to the shoots (4.7\u201312%), which indicates that most of the freshly added Zn remained in the soil after one crop cycle and may thereby contribute to a residual Zn pool in the soil. The 67Zn:66Zn isotope ratios in the DGT extracts and the shoots measured at cut 4 were identical, suggesting that the DGT and plant took up Zn from the same pool. The proportion of Zn derived from the fertilizers in the DGT extracts was also identical to that measured in ryegrass shoots at cut 4. In conclusion, this work shows that stable Zn isotope labeling of the soil available Zn can be used to precisely quantify the impact of complex organic fertilizers on the Zn nutrition of crops. It also demonstrates that DGT extractions on labeled soils could be used to estimate the contribution of Zn fertilizers to plant nutrition.Manure and sewage sludge are known to add significant amounts of zinc (Zn) and other metals to soils. However, there is a paucity of information on the fate of Zn that derives from complex organic fertilizers in soil\u2013plant systems and the contribution of these fertilizers to the Zn nutrition of crops. To answer these questions, we grew Italian ryegrass in the presence of ZnSO Tella et al. (65Zn strongly enhanced the uptake of Zn derived from the soil (non-labeled) by wheat. An increased solubility of soil Zn may be caused by nitrification of ammonium added with the organic fertilizer, which leads to acidification, and also by the decomposition of organic compounds leading to the release of water-soluble organic compounds that can form complexes with soil Zn  in agroecosystems . AUE is the difference between the amount of Zn taken up by a plant that has been fertilized with Zn and the amount of Zn taken by the same plant grown in the absence of Zn fertilizer, divided by the amount of Zn added as fertilizer. Using this mass balance approach, Berenguer et al.  estimate67Zn can be used to assess the plant uptake of Zn derived from wheat straw compost ICP-MS. Hence, labeling the pool of soil exchangeable Zn with 67Zn could be used to quantify the plant uptake of Zn derived from the soil and from sewage sludge or animal manure. Moreover, the isotope label can be used to calculate the recovery of the Zn applied with the fertilizer in the plant. Such recovery calculations can provide valuable insights about the potential fate of Zn in soil\u2013plant systems after harvest  is a well-established tool to predict the plant available pool of Zn in the soil. A strong correlation between the amount of Zn extracted by the DGT sampler and the concentration of Zn in plant tissues has been shown in several studies  was grown as a model plant that takes up most of its Zn from the soil isotopically exchangeable pool  precisely quantify the amount of Zn in plants derived from fertilizer, soil, and seeds in soils amended with distinct complex organic fertilizers, (2) determine the fate of Zn input with complex organic fertilizer in soil\u2013plant systems, and (3) test whether DGT can be employed to predict the relative contribution of soil and fertilizer sources to the plant uptake of Zn. To this end, two cropped soils, an acidic soil from Heitenried, Switzerland, and an alkaline soil from Strickhof, Switzerland, were labeled with 67Zn-enriched isotope (67Zn abundance = 32%) used in a wheat growth trial . Poultry manure and cattle manure in the form of dried pellets were bought from the Landi farmers' shop (https://www.landi.ch). These organic fertilizers were finely ground before their application. Mineral Zn fertilizer was supplied as a ZnSO4 solution. These Zn-containing fertilizers were thoroughly mixed with each labeled soil. The application rate was based on the Zn concentration of the fertilizer, leading to similar amounts of Zn added per kilogram of soil  but different N and C inputs among these treatments  was sown in each pot. As observed in our previous isotope labeling studies on phosphorus and Zn, the root system of ryegrass can colonize well in a pot with a relatively small volume of soil over a short growth time, which is a prerequisite so that the plant shoots can provide a proper estimation of the isotopic signature of the available P and Zn , 55 mg P kg\u22121 soil (phosphate), 138 mg K kg\u22121 soil (K+), 31 mg S kg\u22121 soil (sulfate), 23 mg Mg kg\u22121 soil (Mg2+), 5 mg Fe kg\u22121 soil (Fe-EDTA), 232 \u03bcg B kg\u22121 soil (boric acid), 127 \u03bcg Mn kg\u22121 soil (Mn2+), 31 \u03bcg Cu kg\u22121 soil (Cu2+), and 54 \u03bcg Mo kg\u22121 soil (molybdic acid). Distilled water was supplied regularly to the pot by weighing to maintain soil moisture between 40 and 80% water-holding capacity. No drought symptoms were observed on the ryegrass plants throughout the experiment. Each treatment had 4 replicates with 1 pot per replicate. These pots were randomly rearranged after each watering. The shoots were harvested 4 times by cutting at 21, 30, 38, and 48 days after sowing, to minimize the influence of seed Zn on the tracing of fertilizer Zn. After each cut, the nutrient solution was resupplied at a rate of 20% of the first load.The plant growing conditions were similar to those described in D\u00fcrr-Auster et al. . BrieflyA soil incubation experiment was carried out in parallel with the growth trial for deploying the DGT sampler to extract Zn from the soil available Zn pool. To this end, one pot per treatment was prepared for each type of soil, but no ryegrass was grown. The pot received only the first load of the abovementioned nutrient solution and was then placed in the same growth chamber as the growth trial. Distilled water was supplied weekly to adjust the soil moisture at 80% water-holding capacity to keep the soil moisture in the same range as for the plant trial. The soil incubation lasted until the fourth cut of the ryegrass .3 for 24 h .After the incubation, 4 beakers, each containing about 50 g of incubated soils , were prepared for each incubated pot, to make 4 replicates. Following the protocol described in Hooda et al. , the soi3 in a high-pressure single-reaction microwave chamber . The same digestion was applied to the dried fertilizers. An aliquot of the digest was purified to extract Zn from the sample matrix. Sample purification was shown to be necessary for Zn isotope analysis with quadrupole single-collector (Q-)ICP-MS in stable Zn isotope soil labeling experiments ICP-MS  for the purified samples and corrected for mass bias using standard sample bracketing, as explained in D\u00fcrr-Auster et al. , with i varying from 1 to 4] was obtained from the Zn concentration [CZni (\u03bcg g\u22121 DW)] and the dry weight [DWi (g kg\u22121 soil)] of the corresponding cut:For each cut of each pot, the Zn uptake in the ryegrass shoots at cut QZni was normalized to 1 kg of soil to describe the transfer of Zn from the soil. The values of DWi, CZni, and QZni in the ryegrass shoot at each cut are shown in QZni was composed of the uptake of Zn derived from the fertilizer [QZndfferti (\u03bcg kg\u22121 soil)], soil [QZndfsoili (\u03bcg kg\u22121 soil)], and seeds [QZndfseedi (\u03bcg kg\u22121 soil)]:Zndfferti (%)], soil [Zndfsoili (%)], and seed [Zndfseedi (%)]add up to 100%:In the ryegrass shoot, the relative proportion of Zn derived from the fertilizer 2\u22124:Zndfferti and Zndfsoili in the ryegrass shoot at each cut and Zndfseed at cut 1 and cuts 2\u20134 are shown in The values of QZndfferti and QZndfsoili in the ryegrass shoots at cut i were then calculated:QZndfseed, QZndfferti, and QZndfsoili in the ryegrass shoot at each cut are shown in The values of ZnReci (%)] in the ryegrass shoot at cut i was calculated using:The recovery of Zn from the fertilizer [QZninput is the input of Zn into the soil with the Zn-containing fertilizers ], total Zn uptake [QZnsum (\u03bcg kg\u22121 soil)], and averaged Zn concentration [CZnavg (\u03bcg g\u22121 DW)] in the ryegrass shoots of the 4 cuts are discussed. They were calculated for each pot, as follows:In this paper, the total dry weight  and the soil  of the 4 cuts were calculated:Then, the total uptake and averaged proportion of Zn derived from the fertilizer [ZnRecsum (%)] in the ryegrass shoot at cuts 1\u20134 was calculated:The total recovery of Zn from the fertilizer [AUE (%)] was also calculated to compare the results to ZnRecsum, which relies only on the relative increase of QZnsum resulting from the addition of Zn with the fertilizer:The apparent fertilizer use efficiency  was calculated following the same principles as for the plant (Equation 5). In the equation, 67Zn:66Zn ratios measured in the DGT extracts were used instead of those in the plant, whereas the soil Zn isotope abundances of 67Zn and 66Zn were measured in the DGT extracts in the no Zn treatment instead of the abundances measured in the ryegrass shoots. In ZndffertDGT values were compared with Zndffert4 values measured in the ryegrass shoots at cut 4 . Meanwhile, ZndffertDGT and Zndffert4 were highly positively correlated in each of the soils (p < 0.05) and in both soils , compared with the no Zn treatment . For the higher shoot dry weight in the Strickhof soil, one explanation would be that the growth of Italian ryegrass was favored under neutral to alkaline soil conditions  may have caused the overall higher shoot dry weight, lower Zn concentration and uptake, and lower hof soil . These rhof soil . The highof soil . The lowZndffertavg and QZndffertsum were significantly higher with the application of the animal manures than with ZnSO4 , followed by the Zn-containing fertilizer (11\u201320%), and the seed (<2.3%) after 48 days of growth. The plant recovery of Zn derived from freshly applied complex organic fertilizers was low (4.7\u201312%), whereas the major fraction of fertilizer Zn remained in the soil (>88%). These findings illustrate that complex organic fertilizer may contribute to the buildup of a Zn residual pool in the soil. In both the acidic Heitenried soil and the alkaline Strickhof, animal manures increased the plant uptake of Zn more effectively than the water-soluble ZnSOThe original contributions presented in the study are included in the article/B-FY wrote original draft, formal analyses, data curation, and visualization. TD-A involved in methodology, resources, validation, experiments, and data collection. EF conducted conceptualization, methodology, validation, data curation, writing review and editing, and funding acquisition. MW contributed to review and editing the manuscript and to investigations. All authors contributed to the article and approved the submitted version.This study was funded by the Swiss State Secretariat for Education and Research within the framework of the COST (European Cooperation for Science and Technology) action Mineral-Improved Crop Production for Healthy Food and Feed (FA0905) with the Project Number C10.0085 and by the Group of Plant Nutrition.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "Foliar Zn application significantly increased the Zn concentration and bioavailability in wheat grain and flour. If rural households consumed Zn-biofortified flour instead of self-cultivated flour or flour purchased from supermarkets, 257\u2013769 or 280\u2013838, 0.46\u20131.36 million or 0.50\u20131.49 million disability-adjusted life years (DALYs) lost, respectively, could be saved in Quzhou County and China. Amounts of 2.3\u201312.0 million and 5.5\u201322.6 billion RMB could be obtained via Zn-biofortified flour in Quzhou County and China, respectively. The current study indicates that Zn-biofortified flour via foliar Zn application is a win-win strategy to maintain the yield and combat human Zn deficiency in rural households in China. More health and economic benefits could be obtained in rural household dependent on wheat flour purchased from supermarkets than in those dependent on self-cultivated wheat flour.Zinc (Zn) malnutrition is a common health problem, especially in developing countries. The human health and economic benefits of the replacement of conventional flour with Zn-biofortified wheat flour in rural household diets were assessed. One hundred forty-five wheat flour samples were collected from rural households in Quzhou County. Then, field experiments were conducted on wheat at two Zn levels (0 and 0.4% ZnSO As an essential micronutrient, zinc (Zn) plays a vital role in crop production and human nutrition. At present, Zn deficiency, also called hidden hunger, is a common public issue worldwide, contributing to many health problems . Zn defiThe widespread occurrence of Zn malnutrition in humans mainly arises from a low dietary intake of Zn . Current\u22121 in China, indicating a wide gap to the target value of 40 mg kg\u22121 , consisting of 10 townships and 342 administrative villages. However, a previous survey has revealed that 39% of the children in Handan city suffers from Zn malnutrition  to analyze the current Zn intake based on samples of the wheat flour consumed daily in rural households in Quzhou County, (2) to study the effects of foliar Zn application on the Zn concentration and bioavailability in wheat grain and flour under diverse agricultural practices in Quzhou County, and (3) to comprehensively assess the health and economic impacts of the replacement of conventional wheat flour with Zn-biofortified flour in rural household diets in Quzhou County and China via scenario simulation.Field experiments were conducted at 16 locations in Quzhou County  in the North China Plain . Quzhou 4 \u00b7 7H2O, w/v). Foliar Zn was applied twice as follows: the first spraying was conducted at the early milk stage, and the second spraying occurred a week later. A Tween  solution was applied at 800 L ha\u22121 in the foliar Zn application treatment. Spraying was conducted on cloudy days or after sunset under windless conditions across all 16 locations. The area of each plot was 100 m2 at all 16 locations. In addition to foliar Zn application, routine cropping practices were implemented in the present study, including various seeding rates, fertilizer application, herbicide sprays, and irrigation.Each location included two treatments: the control treatment  and foliar Zn application , and the rate of flour extraction was ~75%, which is similar to the general flour extraction rate in China market . (2) Flour purchased from the supermarket by the rural households (n = 21). These two wheat flour types were separately consumed by the rural households in Quzhou County as part of their daily diets.A total of 145 wheat flour samples was also randomly collected from 21 villages (including the 16 test locations) to estimate the current Zn intake via wheat flour consumption in the rural households in Quzhou County. We divided the sources of wheat flour into two groups: (1) flour milled from wheat grain that was self-cultivated by the rural households . The micronutrient concentrations in the digested solutions were determined via inductively coupled plasma optical emission spectroscopy . Standard wheat grain material (IPE182) was acquired from the Wageningen Evaluation Programs for Analytical Laboratories  and used to ensure consistency and quality. The PA concentrations in the wheat grain and flour samples were analyzed according to a previous study (\u22121 diethylene triamine pentaacetic acid (DTPA)  . The soid (DTPA) .A trivariate mathematical model of Zn absorption was adopted to predict the Zn bioavailability :\u22121), TDZ and TDP are the total daily dietary Zn (mmol Zn day\u22121) and PA (mmol PA day\u22121), respectively, AMAX is the maximum Zn absorption (0.091), KR is the equilibrium dissociation constant of the Zn-receptor binding reaction (0.680), and KP is the equilibrium dissociation constant of the Zn-PA binding reaction (0.033) (\u22121) as the sole source of Zn and phytate for adults  , while tr adults , which iex ante assessment tool to estimate the burden of micronutrient malnutrition and the health impact of micronutrient-biofortified wheat flour  is an at flour . The curat flour . Infantsat flour . The totat flour . The staectively . Based oeach day . The daieach day .The following equation was adopted to calculate the economic benefit of Zn-biofortified wheat flour:Economic benefit = total DALYs saved \u00d7 PCNI \u2013 Zn fertilizer cost.\u22121) according to Wang et al. . Similarly, the average of the above three parameters (the Zn and PA concentrations and the estimated Zn bioavailability) for the two sources of wheat flour collected from rural households were also compared via independent t-tests (P < 0.05).Excel 2010  and SPSS software (version 26.0) were used for the calculations and statistical analysis. The effects of foliar Zn application on the Zn and PA concentrations and the estimated Zn bioavailability in wheat grain and flour were assessed via one-way analysis of variance (ANOVA) followed by independent \u22121 under the control and foliar Zn treatments, respectively.Foliar Zn application imposed no significant effects on the wheat grain yield at any location . The aveThere was a large variation in the Zn and PA concentrations and estimated Zn bioavailability in the wheat flour samples irrespective of the source. The Zn concentration in the self-cultivated wheat flour was significantly higher than that in the wheat flour purchased from supermarkets. However, no significant differences occurred in the PA concentration and estimated Zn bioavailability between the two sources of wheat flour .\u22121, respectively (\u22121) was obtained at 12 field locations due to foliar Zn application. Foliar Zn application imposed no significant effects on the PA concentration in wheat grain and flour, and the PA concentration in wheat grain was much higher than that in flour across the 16 locations  (\u22121) (\u22121 (n = 188), which was lower than our result of 7.1 mg kg\u22121. Different manufacturers and production and processing methods may explain this difference. In addition, the current study demonstrated that the Zn concentration in the flour purchased from supermarkets was lower than that in the flour self-cultivated by the rural households. The reason for this may be that the extraction rate of the flour purchased from supermarkets is lower than that of the flour self-cultivated by rural households, and more Zn is lost during milling   and high\u22121) (\u22121) , which l\u22121) (\u22121) , 34. Wei\u22121) (\u22121)  reported milling .Considering Zn homeostasis in human intestines, the Zn bioavailability in wheat flour is more important than the Zn concentration in wheat flour. The observed difference in wheat flour Zn bioavailability between the self-cultivated flour and the flour purchased by rural households suggests a relatively high vulnerability to Zn malnutrition of rural households dependent on wheat flour purchased from supermarkets . Rosado \u22121 due to foliar Zn application, which matches the biofortification target of Zn in wheat grain.In the present study, foliar Zn application imposed no significant effect on the wheat grain yield, which is consistent with previous studies , 38. In As a store of P and energy, PA plays an important role in plant growth and development and functions as an antioxidant and anticarcinogen in the human body . UnfortuThe current study suggested that the estimated Zn bioavailability in wheat grain and flour was significantly enhanced by foliar Zn application, which is consistent with the findings of Li et al. . Based oConsidering the role of wheat flour as a staple food in Quzhou County and the estimated Zn bioavailability in Zn-biofortified flour being significantly higher than that in the self-cultivated flour or the flour purchased by rural households, substitution of conventional wheat flour with Zn-biofortified flour in rural household diets could highly alleviate Zn malnutrition.\u22121) (\u22121)  in Quzhou County was greater if Zn-biofortified flour replaced the flour purchased from supermarkets than that if the flour produced on rural household farmlands was replaced. This occurs because the Zn concentration and bioavailability in the flour produced on rural household farmlands were higher than those in the flour purchased from supermarkets. Our results also revealed that the reduction in the burden of Zn deficiency in China (12.33\u201313.43% under the pessimistic scenario and 36.87\u201340.18% under the optimistic scenario) due to foliar Zn application was larger than that due to biofortification in India (2\u201312%) and Pakistan (5\u201333%) , 50. In \u22121)  and Chin\u22121) (\u22121) , a relat\u22121) (\u22121) . In summOur farm field experiment demonstrated that foliar Zn application effectively increased the Zn concentration and bioavailability in wheat grain and flour irrespective of the agricultural management practices in Quzhou County. Zn-biofortified wheat flour provided ~57% of the daily Zn requirement. Based on the defined scenarios, more health and economic benefits could be obtained by the replacement of self-cultivated flour or flour purchased from supermarkets with Zn-biofortified wheat flour in Quzhou County and China. Therefore, foliar Zn application is a win-win agronomic strategy to maintain the yield and combat human Zn deficiency in rural households in China.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.B-GY: resources, data curation, and writing\u2014original draft. Y-ML, X-XC, W-QC, and T-BD: investigation. C-QZ: conceptualization, writing\u2014review, and editing. All authors contributed to the article and approved the submitted version.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."}
{"text": "Over the past few years, rechargeable aqueous Zn-ion batteries have garnered significant interest as potential alternatives for lithium-ion batteries because of their low cost, high theoretical capacity, low redox potential, and environmentally friendliness. However, several constraints associated with Zn metal anodes, such as the growth of Zn dendrites, occurrence of side reactions, and hydrogen evolution during repeated stripping/plating processes result in poor cycling life and low Coulombic efficiency, which severely impede further advancements in this technology. Despite recent efforts and impressive breakthroughs, the origin of these fundamental obstacles remains unclear and no successful strategy that can address these issues has been developed yet to realize the practical applications of rechargeable aqueous Zn-ion batteries. In this review, we have discussed various issues associated with the use of Zn metal anodes in mildly acidic aqueous electrolytes. Various strategies, including the shielding of the Zn surface, regulating the Zn deposition behavior, creating a uniform electric field, and controlling the surface energy of Zn metal anodes to repress the growth of Zn dendrites and the occurrence of side reactions, proposed to overcome the limitations of Zn metal anodes have also been discussed. Finally, the future perspectives of Zn anodes and possible design strategies for developing highly stable Zn anodes in mildly acidic aqueous environments have been discussed. Renewable energy supplies have drawn worldwide attention because of the scarcity of fossil fuels and the increasing global warming ,2,3. Nev\u22121) than most organic solvents 2@Zn anodlectrode c. In add3 coating to control the Zn deposition  . On the other hand, the battery with the pristine Zn anode had a lower initial capacity of 188 mAh g\u22121, which remained only 124 mAh g\u22121 after 1000 cycles. Later, Zeng et al. proposed a similar strategy using conductive CNT scaffolds . T. T3 coatctively) b. The ba 1 A g\u22121 c. Its cacaffolds . The hig2+ ion flux. As a pioneer in this field, Zhao et al. proposed a solid-state interphase comprising of polyamide (PA) and zinc trifluoromethane sulfonate (Zn(TfO)2) . . 123]. Uer 200 h b. This sWith regard to PVDF, Liu et al. synthesized a MOF-PVDF composite coating layer consisting of PVDF and hydrophilic microporous metal-organic framework (MOF) particles . The met3 (BTO) can effectively restrain the Zn dendrite growth (2O)62+) were easily attracted by the O atom in BTO via hydrogen bonding, which facilitated the diffusion of Zn ions and restricted the Zn dendrite formation [\u22122, 1 mAh cm\u22122  cormation . In contmAh cm\u22122 c and formAh cm\u22122 d. After 2+ was preferentially absorbed by CuO and dispersed evenly on the Zn surface, which was advantageous for Zn nucleation. Furthermore, as illustrated in 2+ distribution on the surface, small Zn nanosheets were consistently developed on the CuO nanowires, as shown in the SEM image  was produced using magnetron sputtering as a scaffold combining a 3D architecture and an interfacial control to efficiently promote the uniform nucleation of Zn metal and reduce the growth of Zn dendrites [2+ transfer and deposition could be accelerated by the desolvation process of Zn2+ with surface chemistry control. The 3D hierarchical conductive carbon scaffold uniformly distributed the electric fields and lowered the local current density. The N-containing functional groups in NC served as nucleation sites to distribute the Zn nuclei uniformly across the electrode surface. As Zn was constantly deposited, an electric charge was generated in the uneven nucleation sites. The carbon scaffold doped with N atoms encouraged the homogenous nucleation and growth of Zn, thus constraining the formation of Zn dendrites. The N atoms generated by the addition of pyrrole lowered the migration energy barrier and promoted the redistribution of Zn ions. Furthermore, the zincophilicity of the NC scaffold was enhanced by the strong electrostatic interaction between the negatively charged pyrrole N sites in the carbon lattice and the Zn atoms, resulting in the homogeneous nucleation and growth of Zn. The negative state of the pyrrole N site can be explained by the formation of \u03c3-bonds between the doped N atoms and the adjacent carbons from which electrons moved toward pyrrole N through the inductive effect. As a result, the assembled half-cell showed a high zinc stripping/plating CE of 98.8%. The NC-Zn symmetrical cell showed stable operation at a high current density of 5 mA cm\u22122 with an overpotential as low as 11 mV after 210 cycles.Zhang et al. investigated CuO nanowires grown on a Cu mesh (CM@CuO) by anodic oxidation, followed by thermal annealing in air . BecauseEM image b. Zn remmage  for AZIBs in mildly acidic electrolytes are summarized in 2+, and controlling the electric field. Corrosion and HER can be resolved by reducing the chemical activity of H2O and introducing the protective layer on the Zn metal anode. As can be seen from According to the strategies discussed above for modifying the Zn metal surface, the drawbacks must be addressed concurrently to achieve a practical improvement of Zn metal anodes. As for the dendrites, they can be effectively suppressed by increasing the Zn nucleation sites, redistributing the ionic flux of Zn2 evolution. The presence of uneven nucleation sites on Zn metal facilitates uncontrollable charge transfer, which accelerates the generation of an uneven electric field, causing the growth of dendrites. The corrosion of Zn metal anodes can be categorized as self-corrosion or corrosion by an electrochemical reaction. The latter refers to the irreversible Zn consumption due to the removal of Zn from the electrode as well as the side reactions between the electrode and electrolyte. H2 evolution is a complicated process that is affected by the reduction potential, overpotential, surface area, electrolyte pH, and operating temperature. To address these issues, researchers have proposed various modification strategies, which can largely be categorized as (i) shielding the Zn metal from side reactions, (ii) regulating the Zn deposition behavior (controlling the nucleation sites and redistributing the Zn2+ ion flux), and (iii) establishing a uniform electric field.In summary, the major challenges associated with the application of Zn metal anodes in mild aqueous electrolytes and the modification strategies employed to overcome these challenges are discussed to provide an insight into the development of high-performance AZIBs. AZIBs are regarded as promising alternatives to LIBs  owing to their safety, cost-effectiveness, environmental benignity, and high energy density. The main challenges associated with the use of Zn metal anodes in neutral or mildly acidic media are dendrite growth, corrosion, and HDespite the considerable progress made in this field, there is still a long way to go to address the complex challenges discussed thus far. An efficient approach to produce a dendrite-free and highly reversible Zn anode is to strategically tailor the 3D structure of the electrode. The construction of 3D nanostructured Zn metal anodes has not been fully explored yet. Fabricating Zn anodes with hierarchical 3D architectures provides new opportunities for increasing the rate capability and service life of AZIBs because it allows a more uniform distribution of the electric current and provides a large number of reaction sites. The fabrication of 3D Zn architecture can be realized by the coating of Zn metal  on a well-defined conductive 3D framework  or by the selective etching of the sacrificial components in Zn composites. The caveat here is that while the increase in the surface area of the electrode can improve the energy density and suppress the dendrite development, it can accelerate the corrosion of Zn metal. This can be avoided by coating 3D Zn metal with corrosion inhibitors or by introducing organic/inorganic additives in the plating electrolyte. In addition, although the behavior of Zn electrodes in mild aqueous electrolytes differs from that in alkaline electrolytes, the knowledge of the operation of Zn batteries in alkaline aqueous media can provide time-saving guidance for the study of AZIBs. Furthermore, more progress can be made by investigating the previously reported strategies for modifying metal anodes .4 and Zn(CF3SO3)2 are mainly used within a certain concentration range. In most cases, ZnSO4 is preferable because of its relatively low price as compared to that of Zn(CF3SO3)2. However, ZnSO4 suffers from the formation of hydroxide sulfate by-products (Zn4(OH)6SO4\u00b7nH2O). Exploring other Zn salts for application under mild aqueous conditions and some appropriate ZnSO4-based additives is another potential approach. Mn-based materials are the most widely used cathode materials. As revealed by many studies on AZIBs cathodes, Mn-based oxides can be operated at high working voltages, but it is still difficult to fully understand their reaction mechanism because of the diverse phase transitions occurring during the reactions. Apart from Mn-based cathode materials, V-based materials and Prussian blue analogs can be potential candidates for application in conjunction with Zn metal anodes for AZIBs. However, in this case, the limitations of these materials  should be considered.In addition to the Zn metal anode, the other components (cathode and electrolyte) of the cell should also be taken into consideration to improve the performance of the anode. The type of electrolyte, electrolyte concentration, and additives are important factors that strongly influence the electrochemical reactions on the Zn metal anode. In particular, in mild aqueous electrolytes, ZnSOFinally, most studies on Zn metal anodes have mainly focused on coin cells or pouch cells. To meet the market demands , these studies need to be expanded to larger cells . In practical applications, unknown challenges that have not been encountered in coin cells can be encountered. The know-how of various cell configurations can narrow the gap between the lab-scale results and the commercialization of AZIBs. Finally, the development of flexible and wearable AZIBs can be an intriguing research topic because of their intrinsic safety, low cost, and facile fabrication (without the need for a glovebox). To realize this, not only should a sufficiently thin Zn metal electrode  be developed, but appropriate components  compatible with Zn anodes should also be carefully designed."}
{"text": "In this study, we evaluated the leaf antioxidative responses of three wheat varieties  treated with two different forms of zinc (Zn), Zn-sulfate and Zn-EDTA, in concentrations commonly used in agronomic biofortification. Zn concentration was significantly higher in the flag leaves of all three wheat varieties treated with Zn-EDTA compared to control and leaves treated with Zn-sulfate. Both forms of Zn increased malondialdehyde level and total phenolics content in varieties Srpanjka and Divana. Total glutathione content was not affected after the Zn treatment. Zn-sulfate increased the activities of glutathione reductase (GR) and guaiacol peroxidase (GPOD) in both Srpanjka and Divana, while glutathione S-transferase (GST) was only induced in var. Srpanjka. Chelate form of Zn increased the activities of GST and GPOD in both Simonida and Divana. Catalase activity was shown to be less sensitive to Zn treatment and was only induced in var. Srpanjka treated with Zn-EDTA where GPOD activity was not induced. Concentrations of Zn used for agronomic biofortification can induce oxidative stress in wheat leaves. The antioxidative status of wheat leaves could be a good indicator of Zn tolerance, whereas wheat genotype and chemical form of Zn are the most critical factors influencing Zn toxicity. Zinc (Zn) is an essential metal for plants, and adequate availability of this micronutrient is vital at all stages of plant development. This micronutrient acts as an enzyme cofactor and thus plays an important role in regulating metabolic processes like the synthesis of nucleic acids and proteins, pollen formation, carbohydrate metabolism, and auxin synthesis ,2. HowevZn fertilizers are widely used in agronomic biofortification to elevate Zn concentrations in grains of wheat and different cereals ,15. AddiThis work aimed to investigate the effects of two different forms of foliar-applied Zn on the leaf antioxidative status of three wheat varieties, var. Divana, a Croatian standard of high quality, var. Srpanjka, a standard for high yield and the most common variety in Croatia, and var. Simonida, a standard for high yield and the most common variety in agricultural production in Serbia. We hypothesized that the antioxidative response would be variety specific, and would be dependent on the applied Zn-form. The results given in this paper provide a better understanding of the possible toxicity of Zn used in agronomic biofortification and antioxidative leaf status as a potential biomarker of its toxicity. Additionally, this study emphasizes the importance of genetic factors of different wheat varieties that determines the variety-specific antioxidative response to Zn treatment.Foliar application of Zn-sulfate did not cause changes in the Zn content in the flag leaves, while the treatment with Zn-EDTA significantly increased the concentrations of Zn in the flag leaves of all three wheat varieties compared to the control plants . VarietyThe lipid peroxidation products, such as MDA, are usually measured as an indicator of oxidative stress. A significant increase in lipid peroxidation levels was showed in flag leaves of varieties Srpanjka and Divana treated with both forms of Zn . A slighIn leaves of all three varieties tested, treatment with Zn in both forms had no impact on the total glutathione (tGSH) content a. The acThe antioxidant enzymes (guaiacol peroxidase (GPOD) and catalase (CAT)) activities were affected due to Zn treatment, although the effects depended on the wheat variety and the Zn form . The actTreatments with both forms of Zn caused an increase in the total soluble phenolics content but only in leaves of var. Srpanjka and Divana. In the leaves of var. Simonida, Zn treatment did not cause significant changes in the content of total phenolics .Agronomic biofortification is a promising strategy for increasing Zn concentration in grains of different cereals . AlthougLablab purpureus), and okra (Hibiscus esculentus cv. Hassawi) treated with Zn [Corchorus capsilaris L. where the addition of EDTA diminished the copper toxic effects and, at the same time, improved copper accumulation Zn [Phytotoxic concentrations of Zn stimulate lipoxygenase activity leading to the induction of lipid peroxidation, one of the most reliable indicators of oxidative stress . In this with Zn ,31. Alth with Zn ,33. Althation Zn . Diaz etation Zn  found siThe results of studies conducted in different transgenic and wild-type plants, Zn accumulators, hyperaccumulators, and Zn-resistant plant genotypes, showed that higher levels of GSH and increased activity of enzymes involved in the metabolism of GSH result in intrinsic resistance to excess HMs ,37,38. GIn this research, Zn treatment did not cause significant changes in the GSH content compared to the control leaves  a. On theCajanus cajan), rice (Oryza sativa), golden bean (Vigna radiata), and corn (Zea mays) treated with different HMs [In our study, foliar application of Zn-sulfate increased GR activity in the flag leaves of var. Srpanjka, while in var. Divana, both sources of Zn resulted in a significant increase in GR activity with a more pronounced effect of EDTA form b. On therent HMs ,46,47,48rent HMs . The autrent HMs . The incrent HMs , as confrent HMs .P. vulgaris) exposed to elevated concentrations of Zn [In our experiment, there was no correlation between the concentration of tGSH and GR activity, which was also found in hydroponic-grown seedlings of beans (ns of Zn .Vigna radiata) [Hordeum vulgare) can be induced by a variety of metals, including Zn. As a stable and efficient response, GST activity is induced under conditions of increased oxidative stress when the basal antioxidant enzymes are exhaust [In addition to the ascorbate-glutathione cycle, high status of GSH is essential for the detoxification effect of GST. It was found that exposure to cadmium causes an increase GST activity in leaves of golden beans (radiata) , while Hradiata)  showed i exhaust .Our results showed that increased activity of GST in the leaves might be the result of treatment with Zn c. In the2O2 [2O2; thus, it is activated only when high levels of ROS are produced [2O2. An increase of total phenolics content was observed in different plant species exposed to HMs [GPOD and CAT are the most important antioxidative enzymes in plants that detoxify H2O2 . Their aproduced . In addiproduced ,54,55. Id to HMs ,56. Ma ed to HMs  found thd to HMs , while td to HMs . Such and to HMs ,60. In tThis research provides a better understanding of the wheat antioxidative defense mechanisms in response to Zn, and could contribute to the Zn-biofortification strategies and the sustainability of wheat production. In conclusion, we showed that concentrations of Zn used in agronomic biofortification can induce oxidative stress in wheat leaves. Leaf antioxidative response depends on the genotype and the chemical form of Zn used in the experiment. This research suggests that the antioxidative status of wheat leaves could be a reliable indicator of Zn tolerance, with var. Simonida being the most tolerant genotype. To understand the regulation of the processes of absorption, remobilization, compartmentalization, and transport of Zn to the grains, gene expression analysis of Zn-transporters should be performed.Triticum aestivum L.) varieties  were grown under field conditions in seasons 2012/13 on Mollic Gleysol in the Danube region . Sowing depth was 4\u20135 cm with seeding rate 220 kg/ha (Divana) or 260 kg/ha (Srpanjka and Simonida) seeds for 550 spikes/ha (Divana) or 700 spikes/ha . The Mollic Gleysol was calcareous (4.95% CaCO3), moderately alkaline (pHH2O = 8.13), with moderate SOM (3.00%) total Zn content (53.5 mg kg\u22121) extracted by aqua regia , and plant available Zn content (1.11 mg kg\u22121) extracted by EDTA [Three winter wheat ( by EDTA .2). The Zn foliar application treatments were as follows:Control without Zn application;\u22121 Zn in the form of ZnSO4 \u00d7 7 H2O (6.6 kg ha\u22121);Zn-sulfate: application of 1.5 kg ha\u22121 of Zn in the form of Zn-EDTA (10 kg ha\u22121).Zn-EDTA: application of 1.5 kg haAll treatments were distributed in complete randomized blocks with three replications of basic experimental plots (size 20 m\u22121 of solutions (0.25% Zn w/v) or water for control treatment, plus 0.1% (v/v) surfactant, early in the morning between heading (Feekes 10.3) and beginning of flowering (Feekes 10.51). The leaves for analyses were collected by sampling across the whole plot for the average sample.All treatments were applied using 600 L ha3 and H2O2 mixture  in a microwave oven (CEM Mars 6) at 180 \u00b0C for 60 min. The concentrations of Zn were determined by ICP-OES (PerkinElmer Optima 2100 DV) using an internal pooled plasma control and the reference material  prepared in the same way as were the plant samples. All samples were analyzed in duplicate, and the results were expressed in mg kg\u22121 of dry weight.The dried leaf samples were ground into a fine powder using a heavy metal-free ultra-centrifugal mill (Retsch ZM 200). All samples were digested with 10 mL of HNOw/v) solution of trichloroacetic acid (TCA). After centrifugation of the homogenates, the resulting supernatant was mixed with the thiobarbituric acid  prepared in a 20% (w/v) TCA solution. The result of this reaction was the formation of red coloration, whose intensity was determined spectrophotometrically by measuring the absorbance at 532 nm and 600 nm. The absorbance at 600 nm was subtracted from the absorbance at 532 nm due to the correction for non-specific reactions. The amount of MDA was calculated based on the extinction coefficient (\u03b5 = 155 mM\u22121 cm\u22121) and expressed in nmol per g fresh weight (nmol MDA g\u22121 FW).The products of lipid peroxidation in wheat leaves treated with Zn were determined with the method of Verma and Dubey  based onw/v) in 5% 5-sulfosalicylic acid solution and centrifuged at 10,000\u00d7 g for 10 min at 4 \u00b0C. The resulting supernatant was transferred to the reaction mixture that contained 100 mM phosphate buffer with 1 mM EDTA (pH 7.0), 0.031 mg mL\u22121 DTNB, and 0.115 units mL\u22121 of GR in a final volume of 1.05 mL. The reaction was initiated by adding NADPH at a final concentration of 48 \u03bcM. The total amount of GSH was determined by a standard curve of GSH, and the results were expressed as nmol g\u22121 of FW.Content of tGSH in wheat leaves treated with Zn was determined using a kinetic method based on a continuous reduction of 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) by catalytic amounts of GSH, where GR and NADPH recycle the GSSG . The forg and +4 \u00b0C. The resulting supernatants were stored at \u221280 \u00b0C and used for the spectrophotometric determination of the activity of the enzymes CAT (EC 1.11.1.6), GPOD (EC 1.11.1.7), GR (EC 1.8.1.7), and GST (EC 2.5.1.13).Frozen leaf powder (about 200 mg) macerated with liquid nitrogen with the addition polyvinylpyrrolidone (PVP) was extracted for 15 min on ice with the addition of 1 mL of cold 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA. Obtained homogenates were centrifuged for 15 min at 20,000\u00d7 2O2 was catabolized [2O2. CAT activity was expressed in enzyme units (U) representing the amount of enzyme that catalyzes the oxidation of 1 \u00b5mol H2O2 per min at 25 \u00b0C and pH 7.0 per g of fresh weight.The total CAT activity was measured by following the decrease in absorption at 240 nm for 2 min as Habolized . The enz2O2 in 0.2 M phosphate buffer (pH = 5.8) [The total GPOD activity was determined spectrophotometrically by measuring the absorbance increase at 470 nm. The reaction mixture contained 5 mM guaiacol and 5 mM HH = 5.8) . The enzThe activity of GR was determined according to the method described by Halliwell and Foyer . The reaThe GST activity was conducted spectrophotometrically by monitoring the formation of the reaction product of conjugation between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm ,67. The 2O, 100 \u00b5L of Folin-Ciocalteu reagent, and 300 \u00b5L of sodium carbonate saturated solution was incubated in a water bath at 37 \u00b0C for 1 h. The absorbance of the prepared samples was determined at 765 nm, and total phenol content in ethanol extract was calculated from the calibration curve with the gallic acid used as a standard. Soluble phenolic content in fresh tissue was expressed as mg of GA equivalents (GAE) per g of fresh weight.The ethanol extracts of wheat flag leaves were obtained by extracting 100 mg of frozen leaf powder with 1 mL of 80% ethanol in a bath at 80 \u00b0C for 30 min. The total phenol content was determined spectrophotometrically by the method of Folin-Ciocalteu . The rea"}
{"text": "This study enriches the fundamental comprehension of Ohmic contact interfaces on the Zn deposition, which may shed light on the development of other metal battery anodes.Zn metal holds grand promise as the anodes of aqueous batteries for grid\u2010scale energy storage. However, the rampant zinc dendrite growth and severe surface side reactions significantly impede the commercial implementation. Herein, a universal Zn\u2010metal oxide Ohmic contact interface model is demonstrated for effectively improving Zn plating/stripping reversibility. The high work function difference between Zn and metal oxides enables the building of an interfacial anti\u2010blocking layer for dendrite\u2010free Zn deposition. Moreover, the metal oxide layer can function as a physical barrier to suppress the pernicious side reactions. Consequently, the proof\u2010of\u2010concept CeO 2+ diffusion kinetics and a reduced Zn nucleation barrier, thus achieving a dendrite\u2010free and side\u2010reaction\u2010free Zn deposition chemistry for significant improvement of the electrochemical performance in both the symmetric cells and full cells.A universal Zn\u2010metal oxide Ohmic contact interface model is demonstrated to effectively enable improved Zn The dendrite formation may pierce the separator and eventually renders battery failure or even safety hazards. In addition, the continuous accumulation of irreversible by\u2010products 6\u00b7xH2O and H2) not only reduces the Coulombic efficiency (CE) of the cell, but also increases the inner pressure that may inflate the cell to failure. Therefore, solving the dendrite and side reaction issues are of great significance for the commercialization of Zn anodes.Rechargeable aqueous Zn\u2010ion batteries have garnered an extensive research interest due to their attractive merits of low price, non\u2010pollution, and rich abundance in the earth's crust.1 Howe electrolyte modulation, and a three\u2010dimensional (3D) design, have been developed to deal with the above\u2010mentioned critical problems. In particular, surface modification attracts tremendous attention due to its remarkable protective effect and easy manipulation. The recent endeavors are primarily concentrated on metals, carbon matrix, polymers, metal\u2010organic frameworks, and metal oxide coatings. Among these candidates, metal oxides show a great potential to effectively suppress the dendrite growth on the Zn anode, which will propel the commercialization of aqueous Zn\u2010ion batteries. For instance, ZrO2 featuring a high dielectric constant and good chemical stability was demonstrated as an excellent artificial coating layer that provides more nucleation sites and enhanced Zn2+ transport kinetics through \u201cspace charge polarization\u201d, which guarantees the uniform Zn stripping/plating and long cycling stability. An ultrathin Al2O3 layer was coated on a Zn anode to enable enhanced corrosion resistance and suppressed dendrite. TiO2 coating was also revealed to be capable of suppressing the corrosion and HER of Zn anode. More recently, Wang et\u00a0al. revealed that TiO2 with exposed (001) facet exhibits relatively low Zn affinity, thereafter preventing the vertical growth of Zn dendrites and stabilizing the Zn\u2010electrolyte interface. Despite these great endeavors, however, the underlying mechanism on why and how these metal oxides affect the Zn deposition still remains unclear.To date, a variety of design strategies, such as surface modification,4 elec than many metal oxide semiconducting materials, such as TiO2 (4.4\u20135.0\u00a0eV), WO3 (4.3\u20134.8\u00a0eV), MoO3 (6.2\u20136.7\u00a0eV), and CeO2 (4.3\u20134.7\u00a0eV). The high work function difference could drive electrons flowing from metallic Zn to the oxide semiconductors, thus building an Ohmic contact interface  electrode exhibits a long lifespan up to 1300 h with high CE, and moreover, a full cell based on CeO2@Zn//MoS2 also delivers a brilliant rate capability and long cycling stability.It is important to notice that metallic Zn owns lower work function (3.6\u20133.8\u00a0eV)16 tha Figures\u00a0. Inspire2 forming a typical Ohmic contact interface from a physics viewpoint  distinctly exhibit linear features, which reveals the non\u2010rectifying Ohmic contact characteristics. It should be noted that the negative charge accumulation will lead to the establishment of an electron enrichment region at the interfaced metal oxides, which is called an \u201canti\u2010blocking layer\u201d herein. When immersed into the electrolyte, the negative charges in the anti\u2010blocking layer will have crucial effects in attracting the cations but repelling the anions through electrostatic interaction, thus regulating the cation flux for homogeneous Zn deposition . Scanning electron microscopy (SEM) image reveals the nanoparticle morphology of CeO2  technique. As shown in Figure\u00a02 (PDF: 03\u2010065\u20105923), respectively.To demonstrate the practicality of this unique Zn\u2010metal oxide interface model, as a proof\u2010of\u2010concept, mesoporous CeO2 Figure\u00a02a. The lectively.242@Zn anodes. Notably, the pristine Zn exhibits a metallic luster character, while the surface of CeO2@Zn is slight yellow stemming from the intrinsic color of nano\u2010CeO2. Contact angle measurements were performed to compare the hydrophilia of the two samples. Impressively, the CeO2@Zn electrode delivers dramatically improved electrolyte wettability (63\u00b0) compared to the pristine Zn (87\u00b0), . The synergistic combinations of good wettability, sufficient sub\u2010nano transportation channels, and high potential gradient are of significant benefit for improving the Zn2+ diffusion capability.Figure\u00a0, Figure\u00a0. The enhng layer.25 In 2+ transference number (tZn2+) was then applied to quantitatively estimate the Zn2+ diffusion capability of CeO2 layer. Here, the Bruce\u2013Vincent method was executed to evaluate the tZn2+ value in the system ; I0 and R0 are the initial current and interface resistance, respectively; Is and Rs represent the steady\u2010state current and interface resistance, respectively. As displayed in Figure\u00a0tZn2+ value in the bare Zn system is as low as 0.33, which can be ascribed to the unrestricted migration of SO42\u2212 anions compared to hydrated Zn2+. In sharp contrast, the value for CeO2@Zn symmetric cell dramatically increases to 0.77 due to the improved Zn2+ diffusion kinetics and restrained anions transfer, which is highly desirable for high performance battery operation.Where 2 coating layer on suppressing the side reactions, both CeO2@Zn and bare Zn electrodes were soaked in 2 m ZnSO4 for 10 days. As shown in Figure 4SO4(OH)6\u00b7xH2O by\u2010product 6\u00b7xH2O species are fundamentally absent  measurements. Remarkably, the CeO2@Zn electrode shows a higher HER overpotential of 0.90\u00a0V at 5\u00a0mA cm\u22122 and a higher Tafel slope of 336\u00a0mV dec\u22121, compared to that of the bare Zn (0.78\u00a0V and 199\u00a0mV dec\u22121) . The high and stable CE and lower polarization potential electrochemically elucidates the importance of the CeO2 layer on promoting the Zn platting/stripping reversibility and kinetics.The electrochemical plating/stripping behavior of Zn was investigated using asymmetric Zn\u2014Cu and CeO2@Zn and bare Zn electrodes. Figure\u00a0\u22122. Impressively, the CeO2@Zn cell delivers a long life\u2010time up to 1300 h, which is far better than that of the bare Zn (<100 h). In addition, the CeO2@Zn anode shows a small voltage hysteresis of 42\u00a0mV with ultrastable voltage\u2013time curves even after 1000 h (inset). In strong contrast, a high voltage hysteresis of 67\u00a0mV and a quick short circuit are observed for bare Zn. The similar phenomenon is further identified at high current densities of 1.0  than that of bare Zn (47\u00a0mV) at 0.5\u00a0mA cm\u22122, suggesting that the Zn2+ nucleation barrier is significantly reduced. Even at high current densities of 1 and 5\u00a0mA cm\u22122, a similar trend can also be concluded  compared to the bare Zn cells, indicating rapid Zn2+ transference stemming from the mesoporous CeO2 layer. In order to clarify the effect of polyvinylidene fluoride (PVDF) binder on the Zn anode, we also evaluated the electrochemical behaviors of PVDF\u2010coated Zn anode. As shown in Figure 2+ deposition kinetics. Therefore, the enhanced Zn2+ deposition kinetics of CeO2@Zn originates from CeO2 layer instead of PVDF.Symmetric cells were assembled to evaluate the cycling stability of CeO2 Figure\u00a0. The com2+ on the anode surface. As shown in Figure\u00a02+. This nucleation mode will cause the subsequent Zn nucleus to aggregate and grow into Zn dendrites, which is typical of the noted \u201ctip effect\u201d. In sharp contrast, the CeO2@Zn electrode manifests a typical 3D diffusion and nucleation process as the planar nucleation in a CeO2@Zn symmetric cell accounts for only 5 s, suggesting the regulated Zn2+ diffusion and nucleation upon deposition.Chronoamperometry (CA) measurement was carried out to fundamentally analyze the deposition behavior of Znigure\u00a02+.9 This2+ nucleation and growth on bare Zn and CeO2@Zn. Figure\u00a0\u22122 at current densities of 0.5, 1, and 5\u00a0mA cm\u22122, respectively. After Zn deposition, large protrusions are unevenly distributed on the surface of Zn at 0.5 and 1\u00a0mA cm\u22122, which may be due to the rampant 2D diffusion at low current densities that leads to the accumulation of Zn at the initial nucleus, corresponding well with the electrochemical analysis in Figure\u00a0\u22122. These large protrusions can reach a size of 100\u00a0\u00b5m, which can easily pierce the separator and cause the battery short circuit . More importantly, the specific capacity of CeO2@Zn//MoS2 battery stabilizes at 167 mAh g\u22121 when the current density goes back to 0.1 A g\u22121, while the bare Zn//MoS2 cell undergoes dramatic capacity fading. This excellent rate capability can be attributed to the improved Zn2+ diffusion kinetics in the CeO2@Zn/MoS2 battery, as further elucidated by the decreased Rct value . When assembled with the MoS2 cathodes, the CeO2@Zn enables the full cells to manifest a superior rate (96 mAh g\u22121 at 4 A g\u22121) and cycling performance. It is believed that our fundamental findings herein will provide a valuable guideline for developing ultrastable metal anodes beyond Zn\u2010ion batteries.In summary, a universal Zn\u2010metal oxide Ohmic contact interface model has been established to regulate the homogeneous Zn deposition behavior. The as\u2010demonstrated CeOThe authors declare no conflict of interest.Supporting InformationClick here for additional data file."}
{"text": "Contrary to other head and neck subsites, oropharyngeal squamous cell carcinoma (OPSCC) has shown a considerable increase in incidence over the past 20 years. This growing incidence is largely due to the increasing place of human papillomavirus (HPV)-related tumors. HPV-positive and HPV-negative OPSCC are two distinct entities with considerable differences in terms of treatment response and prognosis. However, there are no specific recommendations yet in the therapeutic management of OPSCC patients according to their tumor HPV-status. The aim of this review is therefore to discuss the therapeutic management of patients with OPSCC and the impact of HPV status on treatment selection.Since there is no published randomized study comparing surgical and non-surgical therapeutic strategies in patients with oropharyngeal squamous cell carcinoma (OPSCC), the therapeutic management of these patients remains highly controversial. While human papillomavirus (HPV)-positive and HPV-negative OPSCC are now recognized as two distinct diseases with different epidemiological, biological, and clinical characteristics, the impact of HPV status on the management of OPSCC patients is still unclear. In this review, we analyze the current therapeutic options in patients with OPSCC, highlighting the most recent advances in surgical and non-surgical therapies, and we discuss the impact of HPV status on the therapeutic strategy. Head and neck squamous cell carcinoma (HNSCC) accounts for more than 600,000 new cases each year worldwide and represents the 6th cause of cancer deaths ,2,3. In There has been considerable debate over the last three decades regarding the initial therapeutic management of OPSCC ,11. To dIn the light of the results of larynx preservation programs, the 1990s saw an increased use of non-surgical treatments, for OPSCC, combining radiotherapy (RT) with chemotherapy (CT), commonly referred to as an organ-sparing therapy protocol ,12. ThisThe aim of this review article is therefore to discuss current therapeutic strategies in patients with OPSCC and the potential impact of tumor HPV status.HPV-positive OPSCC is a unique entity both clinically and demographically ,7. HPV-pAt the molecular level, HPV-induced carcinogenesis leads to functional abrogation of p53 and pRb pathways, mediated by the expression of the viral oncoproteins E6 and E7 . E6 bindIn a comprehensive genomic characterization of HNSCC, the Cancer Genome Atlas Network showed that HPV-associated tumors are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1 . By contRegarding clinical presentation, HPV-positive OPSCC are characterized by a frequent discordance between small primary tumor size and significant lymph node involvement . This exMultiple studies demonstrated that HPV tumor status was the main prognostic factor in OPSCC patients ,7,29. ThSecondly, HPV-positive OPSCC are characterized by an improved chemo- and radiosensitivity compared to HPV-negative OPSCC ,30. CompThirdly, HPV-positive OPSCC patients display a lower risk of second primary neoplasia than HPV negative OPSCC patients ,20. The All these differences and particularly the considerable discrepancy in terms of prognosis have led to the creation of two distinct TNM classifications, in the 8th UICC/AJCC staging system, according to the p16 tumor status of OPSCC patients ,37. The Interestingly, several studies demonstrated that, besides p16 tumor status, tobacco consumption was an important prognostic factor in OPSCC patients ,40. IndeAmerican and European guidelines on the management of OPSCC patients do not differ according to the HPV status of the tumor ,43,44. I2 laser and especially robotic surgery  [OPSCC oncologic surgery has undergone an intense transformation over the past 30 years. Although complete tumor removal with free surgical margins remains the cornerstone of surgical treatment, surgical techniques have evolved from radical non-conservative open surgery to functional organ preservation surgery ,46,47. S System) ,50. Tran System) . Some au System) .Similar to primary tumor resection, neck surgical treatment has evolved into more conservative procedures, from radical neck dissection to modified and selective neck dissection . With thAlong with the development of more conservative surgical procedures, considerable advances have been made in head and neck reconstructive surgery. This progress is mainly linked to the development and refinements of microvascular surgical techniques ,53,54. TEarly-stage OPSCC  can be managed by either surgery or RT alone ,44. WhenIn patients with locally advanced  OPSCC treated by upfront surgery, postoperative RT is almost always indicated. A low proportion of patients with a pT3N0 tumor or with a small unique metastatic lymph node in the upper neck without any other adverse pathological feature may avoid postoperative RT ,44. PostNon-surgical treatment of patients with OPSCC is mainly based on definitive RT. RT alone on the primary tumor and neck is sufficient for early-stage OPSCC, whereas it should be potentiated by a systemic therapy for locally advanced OPSCC ,44.Similar to head and neck surgery, RT techniques have largely evolved over the past 20 years. The use of intensity-modulated RT (IMRT) has become the gold standard in patients with OPSCC and has demonstrated an improved preservation of salivary function compared with conventional RT, without compromising locoregional control or survival . IMRT alThree-weekly (100 mg/m\u00b2) cisplatin-based concurrent CRT is the gold standard non-surgical treatment of locally advanced OPSCC ,63. In pIn contrast to de-escalation studies conducted in HPV-positive OPSCC, other studies have examined the opportunity to enhance treatment intensity by combining additional therapy with the conventional cisplatin-based CRT ,68. ThesEarly-stage  OPSCC can be treated by either surgery or definitive RT alone. There is no demonstrated survival advantage with primary surgery compared to definitive RT for early-stage HPV-positive OPSCC ,73. IndeWhatever the therapeutic strategy, the overall prognosis of these patients is excellent and the main objective should be therefore to preserve functions and QoL. Consequently, primary surgery should not be the first therapeutic option if the surgeon is not confident that it will provide optimal functional results. This depends mainly on the anatomical subsite and extension of the tumor. If surgery is selected, a transoral approach should be preferred. Indeed, minimally invasive surgery (TORS and elective neck dissection or SNB) has demonstrated promising results in terms of swallowing function and could be an interesting option to avoid late side-effects of RT ,75. SeveAt the opposite, several retrospective studies showed that, for HPV-negative OPSCC, upfront surgery was associated with improved oncologic outcomes, including for early-stage tumors ,15. IndeIn locally advanced  resectable OPSCC, primary surgery is associated with significant functional impairments depending on the anatomical subsite and tumor extensions ,76. Althp = 0.10) [p = 0.002). However, the high rate of successful surgical salvage for locoregional recurrence in the non-surgical group  explained the absence of significant impact on OS [In HPV-positive locally advanced OPSCC, there is no clear benefit in terms of survival of primary surgery followed by adjuvant (C)RT compared with definitive CRT ,72,78. Ip = 0.001 for the whole cohort; HR = 0.74, 95% CI: 0.60\u20130.91, p = 0.005 in the HPV-negative group; and HR = 0.85, 95% CI: 0.70\u20131.03, p = 0.098 for the HPV-positive group) [p = 0.01), DSS (p = 0.02), and RFS (p = 0.02), compared with non-surgical treatment  [Conversely, although there is no randomized controlled study comparing surgery followed by adjuvant (C)RT and definitive CRT in patients with HPV-negative locally advanced OPSCC, most recent cohort studies suggested that primary surgery provided a clear benefit in terms of oncologic outcomes in this population ,15,80. D. 42.2%) . Altoget. 42.2%) .HPV-positive OPSCC occurring in smokers (>10 pack-years) exhibit an intermediate prognosis between HPV-positive tumors in non-smokers, which are associated with the best prognosis, and HPV-negative tumors, which are associated with the worst prognosis ,83,84. IIn addition to oropharyngeal tumors invading the pterygoid process, the skull base, the nasopharynx, or the carotid artery, those with a large tongue base involvement crossing the midline have to be classified in this category because primary surgery would lead to unacceptable functional impairment  ,85.Definitive cisplatin-based concurrent CRT is the gold standard treatment for locally advanced unresectable OPSCC, whatever the HPV-status ,86.Patients with HPV-negative unresectable OPSCC bear a poor prognosis with reported 5-year OS rates inferior to 35% ,86,87. AAt the opposite, even with a T4 unresectable tumor, HPV-positive OPSCC patients display satisfactory survival rates, particularly if they are non-smokers ,89. HoweTreatment of recurrent and/or metastatic OPSCC is similar to that of other recurrent and/or metastatic HNSCC. There is no specific recommendation for oropharyngeal tumors and no particularities according to the HPV status ,44,90. HBriefly, salvage surgery, when feasible, is the gold standard therapy for loco-regional recurrence ,92. WherLocal therapy  is a valid therapeutic option for patients with a single metastatic or oligometastatic disease . In otheThe three main perspectives in the treatment of OPSCC can be summarized as follows: 1\u2014to determine the optimal therapeutic strategy between primary surgery and RT alone in patients with early-stage OPSCC; 2\u2014to improve oncologic outcomes in patients with HPV-negative locally advanced OPSCC with an intensified therapeutic regimen that does not raise acute and chronic treatment-related toxicities; 3\u2014to reduce long-term functional impairment and improve QoL in HPV-positive locally advanced OPSCC patients through de-escalation therapeutic strategies without compromising survival.As mentioned previously, primary surgery and definitive RT lead to satisfactory and comparable oncologic outcomes in patients with early-stage OPSCC ,72,73,96Approximately half of patients with HPV-negative locally advanced OPSCC will present tumor recurrence and will die from their cancer . There iGiven the favorable prognosis of HPV-positive OPSCC patients, several studies have investigated the possibility to reduce treatment intensity without compromising oncologic outcomes  ,99,100. The role of tumor HPV-status on therapeutic decision-making in OPSCC patients is not yet well defined. However, the tumor HPV status will have, in the near future, a major impact on the therapeutic management of OPSCC patients . There is no published randomized phase III clinical trial comparing surgical vs. non-surgical therapeutic strategies in OPSCC patients. Nevertheless, there are convergent data supporting the use of primary surgery in patients with HPV-negative OPSCC since it is associated with improved oncologic outcomes, if an acceptable functional result can be reasonably expected. In patients with HPV-negative unresectable OPSCC, cisplatin-based CRT remains the gold standard treatment since recent studies aiming at intensifying therapeutic strategy have failed to improve both oncologic and functional outcomes. In patients with early-stage HPV-positive OPSCC, surgery and RT lead to comparable survival outcomes and treatment selection should be mainly based on treatment-related morbidity and preservation of swallowing function and QoL. In patients with HPV-positive locally advanced OPSCC, upfront surgery plus adjuvant (C)RT is associated with increased morbidity and functional impairment and no substantial gain in terms of survival compared with definitive CRT. In these patients, cisplatin-based concurrent CRT remains the cornerstone of the treatment but research is being undertaken to assess new therapeutic regimens  in order to minimize treatment-related toxicities."}
{"text": "Background: Perceptions of cannabis-related risk are changing, and many are viewing cannabis as harmless despite the biopsychosocial risks. Perceptions of risk have an impact on behavior as individuals who are less likely to view cannabis as risky are more likely to use it problematically. Purpose: This study examined how mental health professionals who use cannabis perceive the risks related to use. Methods: Interpretative phenomenological analysis was utilized to understand how participants made sense of the harm related to personal and client use. Interviews were conducted with a sample of social workers, nurses, and psychotherapists who work with cannabis-consuming clients. Results: Participants reported cannabis use is related to anxiety, relational challenges, impaired driving, psychosis, cognitive impairment, educational/employment dysfunction, and addiction in some users. Conclusion: Assessing risk perceptions among cannabis users can reveal subtle psychosocial problems the user may be experiencing. Mental health workers may benefit from further education regarding cannabis-related physical health harm. Perceptions of cannabis-related risk have been changing across Canada since cannabis legalization in 2018. Many view this drug to be relatively harmless despite the known psychological, physical, and social risks . PerceptAcute cannabis consumption can cause anxiety, panic, cognitive impairment, and psychotic symptoms in some users, as well as motor vehicle accidents among impaired drivers . FrequenPrevious studies have applied rational choice theory to understand cannabis use among adult Canadians and related risk perceptions . This leCannabis use has long been considered a means to relieve stress , and somOnly 2% of lifetime cannabis consumers in Canada have sought professional support for related problems . While mThis study followed a qualitative design informed by interpretative phenomenological analysis (IPA). This approach involves exploring perspectives, meanings, and lived experiences of a specific phenomenon, with emphasis on understanding how participants make sense of their personal and social worlds . Interprn = 7) of Canadian mental health professionals. Convenience and purposive sampling were utilized. Eligible participants (a) were regulated mental health professionals, (b) had direct service experience with cannabis-using clients, and (c) had recreationally consumed cannabis at least once in the past month. The sample consisted of three registered social workers, two nurses (one nurse practitioner), and two registered psychotherapists. The four female and three male professionals reported employment in community mental health, hospital, and private practice settings. They described serving clients including children, youth, families, and adults with a broad range of psychosocial challenges. The study\u2019s methods were approved by a Canadian University Research Ethics Board (REB), and participants provided written informed consent to participate in the study. Guidelines proposed by the REB limited questioning participants around negative personal experiences with cannabis to prevent undue distress. Additional consideration was given to privacy and confidentiality due to the sensitive nature of the subject matter. Participants were de-identified in all possible documentation and given an opportunity to review the findings to ensure quotes could not reveal their identities  disclosed cannabis personally increased their anxiety at times, with Participant 6 stating: \u201cI don't really smoke in public nowadays \u2018cause it kind of gives me social anxiety.\u201d Anxiety is clinically related to avoidance, and the potential for cannabis to be used to \u201cavoid painful emotions\u201d and \u201cnot allow [clients] to really examine what they\u2019re feeling\u201d was also discussed by Participants 1 and 7.A lot of parents have rules in their home of not using, or kids aren\u2019t allowed in if they\u2019re clearly high \u2026 it makes the other parts of just living more challenging. So if they\u2019re not allowed home on Sunday night because they\u2019re clearly high, then it\u2019s harder to have somewhere to sleep at night, and a meal that evening, and get \u2026 to school the next day. [Participant 3]Six participants described how cannabis could negatively affect interpersonal relationships. Four reported it could impair one\u2019s \u201cability to function [and] socially cope\u201d by \u201climit[ing] your ability to interact with others, \u2018cause you can get panicky\u201d [Participant 2]. Participant 7 explained: \u201cI had young clients\u2026 getting high with their friends because that\u2019s what everyone\u2019s doing. But they\u2019re having a horrendous, horrific experience while everyone else is enjoying it. But they don\u2019t want to say \u2018no\u2019 because of peer pressure.\u201d It was noted by Participant 2 that some clients are reluctant to accept recommendations to reduce or discontinue use because they fear \u201cthey\u2019re going to lose connection[s]\u201d with peers. Three informants expressed concerns about the potential for cannabis to \u201ccause some tension in a relationship\u2026 if a parent or a spouse really feels strongly that it is not a good thing to be using\u201d [Participant 2]. According to one professional who works with youth:A component of relational challenges identified by participants 4 and 5 was \u201csocial stigmatization.\u201d Negative assumptions about the effects of cannabis can result in judgments toward users being \u201ccriminal[s]\u201d or \u201clazy\u201d [Participant 4]. These social perceptions were reported to contribute to self-stigma: \u201cSome people say, like, they\u2019re just getting known as \u2018the stoner.\u2019 So, they do have a label of \u2018this is who I am.\u2019 \u2026 I do wonder [how] that impacts them and how they view themselves\u201d [Participant 2].1 Six participants described \u201cdriving while high\u201d as risky, with Participant 6 stating: \u201cBeing stoned and driving is obviously a huge no-no.\u201d This behavior was also described in relation to \u201cchronic\u201d use and employment problems as some workplaces require operation of a vehicle and the harm associated with impaired driving can increase when it occurs while at work.Another common risk identified by the professionals was the potential for cannabis-impaired driving.Five participants reported cannabis use \u201ccould lead to psychosis\u201d or exacerbate symptoms of schizophrenia. According to Participant 5, \u201cThere are certain people that \u2026 I\u2019ve recommended probably should stop [using cannabis] because \u2026 they were experiencing symptoms that \u2026fell along the schizophrenia [spectrum].\u201d Participant 4 stated: \u201cI\u2019ve had clients where doctors identified marijuana as being the reason for [developing] schizophrenia.\u201d The potential for cannabis to make young people more vulnerable to psychosis or long-term users more resistant to antipsychotic treatment was also discussed.Five participants described how cannabis could affect various dimensions of cognitive functioning. Four expressed concerns about the potential for disrupted adolescent brain development: According to Participant 2: \u201cIf they\u2019re under the age of 25, there\u2019s some pretty good literature on brain development in teenagers. So right away, if you\u2019re under 25, I\u2019m going to be giving some education on what happens [to] the brain [while using cannabis].\u201d Three informants described how it could negatively affect attention and concentration, with Participants 5 and 6 personally disclosing: \u201cit decreases my concentration\u201d and \u201cif I smoke too much, my mind starts to wander.\u201d Participants 4 and 7 discussed how cannabis could contribute to \u201cdecreased motivation\u201d and reduced \u201cability to function on a day-to-day basis.\u201d Concerns about impaired memory were noted by Participants 2 and 1, particularly in relation to \u201cretaining information\u201d and \u201cremember[ing] key facts when they\u2019re starting a new role\u201d at work or school. One professional [Participant 6] shared the story of a client with an acquired brain injury who struggled with \u201cmanaging his confusion \u2026 just remembering things was an issue. But when he stopped smoking [cannabis], you know, those issues kind of, like, went away.\u201dThe potentially deleterious effects on academic and employment functioning were noted by five participants. Concerns were expressed regarding cannabis impacting clients\u2019 ability to secure, prepare for, and maintain employment. According to Participant 1: \u201cI have had clients that are using daily that I\u2019ve encouraged to reduce their use, especially surrounding when they\u2019re going to be starting a new job.\u201d Others also shared concerns about clients and members of the public \u201csmoking at work,\u201d with Participant 6 explaining, \u201cif they\u2019re chronic [users] then it can affect their ability to work \u2026 operate a vehicle [and] interact with others\u2026 you forget things, obviously, when you\u2019re at work and stoned.\u201d Several professionals described how cannabis could affect academic functioning, with Participant 3 stating: \u201cSome [students] are skipping school to go and smoke\u2026 [then] they have a harder time focusing or, like, being an active participant in school.\u201dI\u2019m really pissed off at myself that I couldn\u2019t [cope] myself and make myself feel better\u2026 I\u2019m not using cannabis to get blasted and completely detach. I\u2019m using it \u2026 to calm [myself] when I can\u2019t use the available skills in that moment.The potential for cannabis consumption to become a \u201cchronic\u201d habit or addiction was noted by four participants. According to Participant 6: \u201cthe risk for addiction is very real\u2026 it can be really, really hard to stop.\u201d Participant 7 expressed concerns about clients developing \u201cnegative, unhealthy pattern[s]\u201d of consumption or needing to \u201cwake and bake\u201d (smoke after waking in the morning) as signs of dependence. Two participants were personally concerned about developing a \u201cpsychological addiction.\u201d Participant 6 shared: \u201cThere are times when I tell myself, oh, I shouldn\u2019t do it, but I do it anyway, you know? Um, so it was prominently habitual\u2026 sometimes I regret smoking or smoking so much.\u201d Participant 2 shared that sometimes after using it to manage physical or psychological discomfort:The risks identified by the participants in this study are interconnected and reflect how the harms associated with cannabis use impact one another. The potential for this drug to cause or worsen anxiety and psychotic symptoms in some users was noted by most participants, with these effects reportedly contributing to relational difficulties. On occasions when social anxiety or panic is felt after taking cannabis, the consumer may find it difficult to communicate effectively or navigate complex social situations. Difficulties managing stressful interactions can intensify interpersonal anxiety, at times prompting increased cannabis use in an attempt to reduce or avoid that feeling. A cycle of cannabis causing anxiety and anxiety leading to more cannabis use may ensue, with the drug being consumed to manage a condition it may be aggravating. As noted by one participant, the experience of cannabis-induced panic in a social situation where others appear to be enjoying themselves can be isolating, although peer pressure creates barriers to limiting consumption. For those who belong to social circles where cannabis is common, recommendations from professionals to reduce or stop may be met with ambivalence, reluctance, or resistance. This reality produces barriers to treatment for individuals whose anxiety escalates to psychotic symptoms such as paranoia or perceptual disturbances. Cannabis-induced psychosis, panic, and/or \u201cbad trips\u201d can result in hospitalization while wreaking havoc on one\u2019s social and family life.Several participants linked the themes of cognitive impairment and addiction to problems with school, employment, and driving. When cannabis intake becomes a habit or dependence, changes to memory, motivation, attention, and concentration can become entrenched . These dA personal risk/reward analysis was described by almost all the professionals in this study. While cannabis was reported to increase anxiety and hinder cognition in some users and two participants noted concerns about dependence, the majority indicated that the benefits of consumption eclipsed the costs. This group weighed the harms they knew could be incurred against the benefits they perceived and determined that consumption was a reasonable personal choice. Particularly for those managing pain or insomnia, cannabis was reported to be the best option for relief. In studies by Two participants in this study indicated that they sometimes regretted using this drug. One described ambivalence, at times espousing the benefits and at other times wishing they had alternative skills to cope. Another alluded that the negative effects of social anxiety, sleep disturbance, and wandering thoughts overshadowed the benefits despite ongoing use. While The professionals in this study demonstrated an intricate understanding of the psychological, social, familial, and functional risks associated with cannabis use. Their cumulative, off-hand knowledge reflected many of the concerns outlined in a World Health Organization report on nonmedical cannabis use . Their pInterestingly, only one participant mentioned the potential for smoking cannabis to cause physiological damage. While many Canadians acknowledge the risk for pulmonary injury and medical professionals link long-term use to cardiovascular disease, none of the informants noted these harms . This abThis study shed light on how Canadian mental health professionals who use cannabis can reduce stigma and barriers to mental health care for cannabis users. The participants\u2019 responses challenge public perceptions that cannabis use is harmless and emphasize the value of providing fact-based education for clients and families tailored to address their specific concerns. The findings also challenge the notion that cannabis use is inherently irrational and underscore the importance assessing the individualized psychosocial effects of the drug. Through dispelling myths about cannabis and creating an inclusive therapeutic environment, clients can feel comfortable enough to share their problems, motivations, goals, and appraisals of risk. As these professionals shift clinical and public discourse toward the facts and away from unfounded assumptions, barriers to care for cannabis users who are seeking support  can be diminished.This study\u2019s findings suggest assessing perceptions of cannabis-related risk among consumers in clinical settings may help reveal latent psychosocial problems. Examining how cannabis affects anxiety, relationships, driving, psychotic symptoms, cognition, and educational and employment functioning can provide a deeper understanding of lived experiences of harm. Helping clients weigh the perceived risks and benefits can set the foundation for therapeutic approaches that address ambivalence and promote recovery. The findings suggest mental health workers may benefit from education regarding the physical health harms associated with this drug. As mental health professionals and services evolve to adapt to the changing health climate, a need remains for improved access to evidence-informed, destigmatized care for people who use cannabis.This study was limited by time constraints, small sample size, and convenience sampling. Future research using larger samples of professionals and mixed methods approaches may offer more comprehensive understandings of the associations between cannabis use and specific health or clinical outcomes. An analysis of perspectives on stigma, legalization, and barriers to accessing services would have enhanced this study. The findings of this study may not be generalizable to professionals in regions where cannabis is not legal as concerns related to criminal sanctions are likely to be perceived as a risk in jurisdictions where cannabis possession remains a criminal activity. Interviews were conducted prior to widespread awareness of the coronavirus pandemic in Canada and it is unclear how this global crisis may have impacted participant views. This study did not examine choice of cannabis products due to ethical concerns as only legal market products provide validated product information and inquiry into potentially illicit activities was intentionally avoided. There are significant variations in psychoactive effects among various cannabinoids, plant strains, and formulations. Future studies should investigate the perceptions of risk related to specific cannabis products."}
{"text": "TP53, APC, KRAS, and PIK3CA. In total, 16 somatic mutations were detected in the CRC group and in either the IBD or CP group. In addition, our data showed that 51% of total somatic variants were CRC-specific variants. The average number of CRC-specific variants per sample is 2.4. The top genes carrying CRC-specific mutations are APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4. It seems obvious that TP53 and APC genes were the most affected genes with somatic mutations in all groups. Of interest, 85% and 28% of the APC and TP53 deleterious somatic mutations were located in Exon 14 and Exon 3, respectively. Besides, 37% and 28% of the total somatic mutations identified in APC and TP53 were CRC-specific variants, respectively. Moreover, we identified that, in 29 somatic mutations in 21 genes, their association with CRC patients was unprecedented. Ten detected variants were likely to be novel: six in PIK3CA and four variants in FBXW7. The detected P53, Wnt/\u03b2catenin, Angiogenesis, EGFR, TGF-\u03b2 and Interleukin signaling pathways were the most altered pathways in 22%, 16%, 12%, 10%, 9% and 9% of the CRC patients, respectively. These results would contribute to a better understanding of the colorectal cancer and in introducing personalized therapies for Egyptian CRC patients.This study aims at identifying common pathogenic somatic mutations at different stages of colorectal carcinogenesis in Egyptian patients. Our cohort included colonoscopic biopsies collected from 120 patients: 20 biopsies from patients with inflammatory bowel disease, 38 from colonic polyp patients, and 62 from patients with colorectal cancer. On top of this, the cohort included 20 biopsies from patients with non-specific mild to moderated colitis. Targeted DNA sequencing using a customized gene panel of 96 colorectal related genes running on the Ion Torrent NGS technology was used to process the samples. Our results revealed that 69% of all cases harbored at least one somatic mutation. Fifty-seven genes were found to carry 232 somatic non-synonymous variants. The most frequently pathogenic somatic mutations were localized in  Its rank jumped in females, however, to the fifth place  [Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity worldwide. It is the third most common neoplasm and the second leading cause of cancer-related death worldwide . In Egypin 2015) . It is eNext generation sequencing (NGS) technology is an efficient means to characterize mutations associated with the disease in the patients\u2019 genomes . It is fA number of studies have been published to characterize the somatic mutations and related genes associated with CRC worldwide ,5,6,7. In = 120) were collected from the patients classified into (1) inflammatory bowel disease , (2) colonic polyp  and (3) colorectal carcinoma patients  as well as extra participants with chronic non-specific mild to moderate colitis without any colonoscopic abnormalities . The collected colonoscopic biopsies were stored in MACS Tissue Storage Solution at \u221280\u00b0 freezer until DNA extraction. Before the study began, each patient who was enrolled signed a written informed consent form. The Institutional Review Board of the National Cancer Institute (NCI), Cairo University, Egypt has authorized all human subject protocols and procedures .Fresh colonoscopic biopsy samples , and they are summarized in Our laboratory uses the Ion Torrent  sequencing technologies. The first version of the ready-to-use colorectal cancer gene panel kit based on this technology was the Ion AmpliSeq Colon/Lung panel. This panel appeared a few years ago and was composed of 22 genes. The recent version of this panel from Thermo Fischer is the Ion Torrent Oncomine Colorectal and Pancreatic Panel, composed of 24 genes . For our study, we wanted to have a disease focused panel, but with more genes specific to colorectal cancer. To this end, we studied the gene lists available in the commercial cancer gene panels, such as the Comprehensive Cancer Panel  composed of 409 genes, the different versions of the Oncomine Comprehensive panels , and the Qiagen\u2019s GeneRead colorectal panel, composed of 38 genes. This is in addition to studying gene lists from literature, TCGA, and different commercial tests registered in the Gene Test Registry. Our final list included 96 genes, and it is given in \u00ae DNA mini kit  according to the manufacturer\u2019s instructions. The purified DNA was measured using Qubit\u00ae 3.0 Fluorometer  with Qubit\u2122 dsDNA HS assay kit .The DNA was isolated from the collected biopsies using the QIAampThe QIAseq Targeted DNA technology from Qiagen was used to develop kits for our customized gene panel composed of the selected 96 genes . The NGS libraries were constructed according to the manufacturer\u2019s instructions. After library preparation, the QIAxcel  was used to check the fragment size and concentration with the QIAxcel DNA high resolution kit . The prepared libraries were quantified using QIAseq Library Quant Assay Kit . Then, the libraries with different sample indexes were combined in equimolar amounts to achieve a similar sequencing depth for each combined library. The fragment size distribution in our libraries ranged between 200 and 1000 bp. The Ion PI Hi-Q Chef Kit , running on the Ion Chef, was used to load the combined libraries on the Ion PI Chip . The Ion Proton Platform was used for next generation sequencing using the Ion Proton Sequencing 200 Kit v2 .http://www.webgestalt.org (accessed on 3 November 2021)), Ingenuity Variant/Pathway Analysis , and web Reactome (https:/reactome.org (accessed on 15 November 2021)). For the alignment step, we used the human reference genome version hg19. The sequencing of a sample was accepted if the depth was larger than 100\u00d7 and the coverage was more than 95% of the target regions.The Ion Torrent Suite was used for base calling, alignment, and variant analysis. First, the low-quality reads were excluded and the bases with base quality less than Q30 were trimmed. The alignment and variant calling proceeded in two parallel directions to identify the somatic variants: In the first direction, we used the somatic workflow in the Torrent Suite  to call the somatic variants. In the second, we used the QIAGEN GeneGlobe Data Analysis Center. Then, the variant lists from the two platforms were combined together and submitted to the annotation workflow. For annotation, we used QIAGEN GeneGlobe Data Analysis Center, Ingenuity Variant Analysis  , and theSomatic mutations in the cancer patients were identified based on the following multi-step filtering process introduced in . First, 2 test or Fisher\u2019s exact test, when appropriate for the categorical variables, and by Mann\u2013Whitney test or Student\u2019s t-test when appropriate for the continuous variables. p-value was considered significant when p-value \u2264 0.05.The clinic-pathological features of the assessed patients were analyzed using SPSS software package (version 22). Continuous variables were expressed as mean \u00b1 SD and range, while categorical variables were expressed as percentages. Comparisons between groups were analyzed by \u03c7p-value < 0.001). Nearly half of the CP group (47%) had atypical lesions. The most predominate grade was grade II, found in 64% of the CRC group. Most of the CRC patients presented with non-metastatic and non-recurrent status   .In total, there were 232 somatic non-synonymous variants (73% SNPs and 27% Indels). The mean depth of coverage of the non-synonymous variants ranged from 500 to 1000\u00d7 in all studied groups . Most ofTP53 , APC , KRAS , PIK3CA , POLE , MSH6 , FBXW7 , SMAD4 , ATM , and FGFR3 . In the CRC group, TP53 , APC , KRAS , PIK3CA , MSH6 , FBXW7 , SMAD4 , ATM , FGFR3 , BRAF , and POLE  were the top mutated genes. In the CP group, APC , TP53 , BRAF , FGFR3 , KRAS , and POLE  were the most mutated genes, while TP53 , APC , and POLE  were the most mutated ones in the IBD group.Fifty-seven genes out of the studied genes were found to carry somatic mutations . The topTP53, APC, and KRAS genes harbored the most frequently detected somatic mutations  in the total cohort. In the CRC group, TP53, APC, PIK3CA, KRAS, and ATM genes harbored the most frequently detected somatic mutations . In the CP group, APC, TP53, BRAF, FGFR3, and KRAS genes had the most frequently detected variants . Regarding the IBD group, TP53 and APC genes harbored the most frequent somatic mutations (five and four). The somatic mutational burden per sample in the CRC group was the highest, and it was about three variants per sample on average; this is followed by the CP (2.7) and the IBD groups (2.4). As for the somatic mutations that were detected in the top genes in each group, TP53 and APC genes were found to be the most affected genes, with somatic mutations in all groups, a schematic representation of their somatic mutations at protein level was made, as shown in TP53 harbored 36 mutations from 31 samples  and APC harbored 30 mutations from 26 samples . We found that exons 3 and 4 of the TP53 gene (NM_001126115) possessed a high number of mutations (28% and 25% respectively). In APC (NM_001127511), exon 14 harbors most of the mutations (85%). The \u03b2-catenin binding and down-regulation site was the most affected region at the APC protein, whereas the transactivation and the proline rich sites were the most affected regions at the TP53 protein.As the TP53 and APC are the genes with the greatest number of mutations . This is followed by the KRAS gene with two mutations, as shown in There are, in total, 16 somatic mutations detected in the CRC group and in either the IBD or the CP group: 10 SNPs and 6 Indels. APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4, and they were housing 12, 11, 7, 6, 5, and 5 mutations, respectively. CRC-specific variants harbored by the top genes were reported in 23 out of total 35 CRC patients carrying CRC-specific mutations. Average CRC-specific variants per sample is 2.4, as shown in Eighty-four somatic variants harbored by 36 genes were found only in the CRC group, representing 51% of total somatic variants. The top genes carrying CRC-specific mutations are PIK3CA (NM_006218) and four in FBXW7 (NM_001013415), as shown in PIK3CA and FBXW7 mutations were reported in seven and four cases, respectively. Out of these 10 novel mutations, 8 mutations were found in the CRC group only, while a mutation in PIK3CA (c.1013T>A) is found in CP group only and a single mutation in FBXW7 (c.248C>T) is found in IBD group. The ten novel mutations have not been previously reported in any of the public databases.Moreover, ten novel heterozygous mutations were identified, including six in p-value = 1.28 \u00d7 10\u221208), Wnt signaling pathway (p = 0.0028), Angiogenesis , EGF-receptor , TGF-beta signaling , and Interleukin signaling . The most altered pathways in CRC patients and the distribution of the mutations of cancer driver genes are in Pathway analysis revealed that the following pathways were strongly suggested to be altered in the CRC group: P53-signaling pathway  were exon 3 and 4. In accordance with a recent study by Kassem et al. [TP53 somatic mutations c.628C>T, c.448C>T, c.347G>A, and c.128G>A in our cohort are specific to the CRC group, which suggests that they play a key role in the CRC in the Egyptian population. Additionally, we detected two TP53 variants (c.164C>G and c.137delC) that were previously reported in the esophageal and lung cancers. Interestingly, and according to the COSMIC database, this is the first study to report the presence of such variants in CRC [Moreover, eleven m et al.  on the Es in CRC ,24,25,26ATM gene, as a DNA repair gene, occur in many tumor types including colorectal cancer. In colorectal cancer, the loss of ATM protein expression is associated with worse prognosis [ATM gene was mutated in 12% of the CRC cohort; five out of the seven detected somatic mutations were found only in the CRC group. All of the observed ATM mutations had previously been linked to CRC [Somatic mutations of the rognosis . Therefod to CRC , with thd to CRC ,16,17,18ATM mutational status could be used to help in the clinical decision-making for those patients along with the development of specific targeted strategies [Nowadays, novel therapies have been developed to selectively target patients with ATM-deficient cancers. Those therapies induce synthetic lethality due to lacking an efficient repair mechanism such as platinum drugs . Thus, trategies . Thus, iAPC gene, a multi-functional tumor-suppressor gene, is an early event in the development of CRC and result in activation of Wnt/\u03b2-catenin signaling pathway, which is a key event for epithelial development [APC, Axin2, and AMER1 (APC-recruitment protein) disrupt the formation of the \u03b2-catenin destruction complex leading to stabilization and accumulation of \u03b2-catenin protein, which in turn induces overactivation of Wnt/\u03b2-catenin signaling and promotes the proliferation, invasion, and metastasis of cancerous cells [APC gene (NM_001127511) was the second ranked mutated gene (23% of the CRC cohort). There were 12 APC somatic mutations with identified loss of function detected only in the CRC group. Interestingly, exon 14 was the most affected exon and it was found to harbor 11 out of 12 detected mutations in the CRC group only. Thus, sequencing this exon could be used as a genetic test assay for CRC diagnosis. Of interest, most of our identified APC somatic mutations were located in the \u03b2-catenin binding and down-regulation site, which may result in an altered Wnt/\u03b2catenin pathway. Meanwhile, the somatic mutations detected in AXIN2 (two mutations) and FZD3 (one mutation) were reported only in CRC patients, and they were participating in Wnt/\u03b2catenin pathway as well. The two AXIN2 mutations (c.2347G>T and c.1102G>A) were previously addressed as being associated with small cell lung and prostate cancers, respectively [FZD3 mutation (c.674dupT) was previously reported in oral squamous cell carcinoma (OSCC) [Mutation of the elopment . Mutant us cells ,60. We hectively ,30, whila (OSCC) . TherefoAPC bi-allelic mutations, which in turn decrease the \u03b2-catenin degradation [CTNNB1 mutations are considered an alternative method for activation of Wnt signaling pathway [APC and CTNNB1 mutations is infrequent [CTNNB1 gene. It is another protein that acts as a gene expression regulator for Wnt downstream genes which is responsible for cell proliferation and differentiation [CTNNB1 gene are rare. Here in our study, we identified one CTNNB1 (NM_001098209) pathogenic variant in CRC patients and it was located on exon 10 (c.1561C>T). This was in agreement with Giannakis et al., who had detected the same mutation in one CRC patient, as well [The Wnt signaling cascade is mostly activated by radation . However pathway . Furtherfrequent . Beta-cantiation ,65. Besintiation ,67,68,69 as well .KRAS, NRAS, BRAF and PIK3CA) can determine the response to this target therapy. Hence, they can be used as predictive biomarkers. The following mutations were previously known to contribute to the acquired resistance to anti-EGFR target therapy, mutations in KRAS  [NRAS , BRAF (exon 15), and PIK3CA (exon 20) [PIK3CA and KRAS are often co-mutated and therefore can predict the lack of response to anti-EGFR therapy [Epidermal growth factor receptor (EGFR) is a trans-membrane protein. Bad prognosis and drug resistance in CRC is usually associated with EGFR overexpression ,72. The , and 4) ,74,75, Nexon 20) . PIK3CA  therapy ,78.KRAS and NRAS are members of RAS gene family. KRAS is one of the most frequently mutated genes, with alterations observed in 30\u201340% of CRC patients. On activation of KRAS, a signal transduction cascade will be initiated that will eventually promote many cell processes  through the subsequent activation of several target effectors  [NRAS mutations are identified in ~4% of CRCs [KRAS and NRAS mutations have less favorable prognoses, shorter survival, and increased tumor aggressiveness [KRAS and NRAS mutations in CRC predict lack of response to anti-EGFR MoAbs therapy [KRAS and NRAS mutations among our CRC patients were detected. In CRC patients, three were found to carry KRAS mutations in codon 13 (c.38G>A) while five CRCs carried KRAS mutations in codon 12  and both codons are located on exon 2. As for the NRAS mutations, one mutation in codon 12 on exon 2 (c.35G>T) was identified in one CRC patient only.In light of this, the genes implicated in the EGFR-signaling pathway will be discussed in the following lines.  PIK3CA) ,80. On t of CRCs . Additiosiveness . Moreove therapy ,83,84. IBRAF) which encompasses 18 exons. This protein is associated with the MAPK pathway, which is involved in carcinogenesis of many cancers [BRAF mutation leads to a constitutive activation of the MAPK signaling pathway, eliciting cellular proliferation, angiogenesis, and differentiation [BRAF alterations have been identified [BRAF mutations were observed in less than 10% of CRC. BRAF mutations may indicate an initial event in tumorigenesis [BRAF mutation is the V600E subtype  [BRAF 10 times relative to the BRAF wild type, which in turn promotes cell survival via the ERK or MEK signaling cascade [BRAFV600E inhibitors in CRC [BRAFV600 mutations in CRC patients  and these findings were also in agreement with Yanus et al. and Won et al. [Serine or threonine protein kinase is encoded by a proto-oncogene  . This mu cascade . Our dat cascade . To dates in CRC . Most ofs in CRC ,97. Heren et al. ,99.PIK3CA mutations are frequently associated with other gene mutations which are involved in significant cancer-related pathways, such as the Wnt/beta-catenin pathway and tyrosine kinase receptors K-Ras/BRAF/MAPK. The PIK3CA gene encodes the alpha catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), which is mutated in several malignancies  [PIK3CA mutations have been detected in 10 to 32% of colorectal tumors [PIK3CA somatic variants; out of these, five of them were likely to be novel , and they were located on exons 2, 2, 2, 21, and 20, respectively. In addition, our results are in agreement with Samuels et al., who has reported an association between PIK3CA mutation  and CRC [PIK3CA variant  detected in our CRC patients and colorectal cancer. This variant was previously addressed with lung cancer [KRAS, BRAF, and PIK3CA mutations, which was consistent with an Egyptian study by Farghal et al. [and CRC) . PIK3CA l tumors ,102. In  and CRC . Our stug cancer . FurtherKRAS located on exon 2, one mutation in NRAS located on exon 2, one BRAFV600, two non-BRAFV600 mutations located on exon 15, and one PIK3CA mutation located on exon 20. Thus, these data may provide beneficial information that helps in the clinical management regarding anti-EGFR therapy using a personalized medicine approach for the colorectal cancer patients in Egypt. In addition, we detected other mutations in CRC patients that are known to participate in the EGFR-signaling pathway, such as ERBB2 , and EGFR (c.2116C>T and c.630G>A).Matching with what we have mentioned before, we have detected somatic mutations in eleven CRC patients (18%) that are linked to the resistance to anti-EGFR target therapy, three variants in TP53 and ATM genes. Up-regulation of the Wnt/\u03b2catenin pathway in CRC patients due to mutations in APC, AXIN2, and FZD3 reveal that the Wnt/\u03b2catenin pathway plays a major role in sporadic colorectal carcinogenesis. Therefore, the dysregulation of both pathways in our CRC group may arise as an attractive therapeutic target [RAS gene can lead to PI3K/AKT/mTOR pathway activation that will promote the expression of other angiogenesis-associated factors  [AKT mutations [APC, CTNNB1, NRAS, PIK3CA, AKT2, and BRAF genes in our CRC patients, the dysregulation of the Angiogenesis pathway could be attributed to the interaction between Wnt/\u03b2-catenin, PI3K/AKT/mTOR, and MAPK signaling pathways, and this is in agreement with Lee et al. and Jeong et al. [When we performed pathway analysis, it revealed dysregulations of six pathways. Inactivation of P53 signaling pathway was detected in CRC patients due to the presence of deleterious mutations in c target ,106. Meac target . On actic target . In addioietins) . Angiogeoietins) . Moreoveutations . In the g et al. ,112,113.g et al. .SMAD4 mutations with the CRC, we detected five somatic mutations only in the CRC group [SMAD4 gene acts as an intracellular mediator of TGF-\u03b2 superfamily signals. TGF-\u03b2/SMAD4 signaling maintains DNA damage response (DDR) and DNA damage repair [SMAD4 promotes malignant progression via acquiring resistance to TGF-\u03b2 superfamily growth inhibition [SMAD4 occurs in the CRC in frequencies ranging from 9 to 67% [SMAD4 loss was also associated with worse clinical outcome and resistance to fluoropyrimidine-based chemotherapy [SMAD4 mutations may not benefit from fluoropyrimidine-based treatment.Matching with two studies that reported the association of RC group . The SMAe repair . Additioe repair . In thishibition . Moreovehibition . Isaksso9 to 67% . Moreoveotherapy , implicaotherapy . Thus, wEP300 gene has been previously observed in gastric, breast, pancreatic, and colorectal cancers. In addition, Gayther et al. reported a great relevance of the EP300 loss in colorectal carcinogenesis [EP300 gene harbored two missense mutations in CRC patients (c.1058G>A and c.3671+1G>A). They were previously detected in breast and gastric cancers, respectively [EP300 overexpression was an indicator of good prognosis in the CRC patients [EP300 gene might serve as a predictor of bad prognosis in Egyptian CRC patients. One of the most frequently detected somatic mutations in the CRC is in the tumor suppressor FBXW7 gene. Loss of the FBXW7 was reported to promote epithelial\u2013mesenchymal transition (EMT) and metastasis in the CRC cells [FBXW7 gene that were found associated with CRC: two were previously reported with CRC patients , a single mutation was previously reported with breast cancer but was not addressed with CRC before (c.167A>G), and another three likely novel mutations  [The ogenesis . Our stuectively ,35. Morepatients . TherefoRC cells . The pre1030T>C) .ARIDA1 gene has been previously reported as a frequent event in the colorectal carcinogenesis [ARIDA1 mutations in CRC patients (only a single case in the CRC group). ACVR2A (activin A receptor type 2A) has a mutation rate of about 60%, making it the most frequently mutated gene in hypermutated colon cancer [The functional loss of the tumor suppressor ogenesis ,126. Hown cancer . It medin cancer . Here, wTP53), (c.38G>A in KRAS), and (c.4337_4338del in POLE). We found no significant increase in frequency of those mutations from IBD to, finally, CRC. Thus, we recommend increasing sample size to validate the association of these variants with disease progression in further studies.Regarding the common somatic mutations detected in all the studied groups, we have found that four mutations were the most frequent (c.137delC and c.422G>A in APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4 were the top genes carrying CRC-specific mutations. APC and TP53 genes were the most affected genes in all groups; most of their deleterious somatic mutations were located in exon 14 and exon 3, respectively. Twenty-nine somatic mutations in 21 genes were found to be associated with CRC patients exclusively. Additionally, ten likely novel variants in PIK3CA and FBXW7 were identified in Egyptian CRC patients. P53, Wnt/\u03b2catenin, Angiogenesis, EGFR, TGF-\u03b2, and Interleukin signaling pathways were found to be the most altered pathways in CRC patients. Furthermore, our findings revealed that 18% of CRC patients had somatic mutations linked to resistance to anti-EGFR target therapy, implying that 82% of patients could benefit from this treatment. This study may provide a better understanding of colorectal cancer and identification of cancer driver genes with cancer-specific variants in our patients, and these findings may assist in the development of diagnosis and novel personalized treatment regimens suited to Egyptian colorectal cancer patients. It is worth noting that our findings are confined to grade II patients because they account for 64% of all CRC cases.To the best of our knowledge, this is the first study on the sequencing of a multiple-gene panel for disease progression of Egyptian CRC patients. Our results revealed that"}
{"text": "Anemia, which is a condition with reduced healthy red blood cells, is reported to be closely related to the development of infectious diseases. We aimed to investigate the association between history of anemia and 12-year mortality rate due to infections, and compare it with that among non-anemic individuals.Data from the National Health Insurance Service Health Screening Cohort were used in this population-based cohort study. Adults who underwent standardized medical examination between and 2002\u20132003 were included, and the mortality rate due to infection between 2004 and 2015 was analyzed. Individuals were considered to have a history of anemia if the serum hemoglobin\u2009level in 2002\u20132003 was <\u200912\u2009g/dL for women and\u2009<\u200913\u2009g/dL for men. The severity of anemia at that time was categorized as mild (12\u2009g/dL\u2009>\u2009hemoglobin \u226511\u2009g/dL in women and 13\u2009g/dL\u2009>\u2009hemoglobin \u226511\u2009g/dL in men), moderate (hemoglobin 8\u201310.9\u2009g/dL), or severe (hemoglobin\u2009<\u20098\u2009g/dL). Propensity score (PS) matching and Cox regression analysis were used as statistical methods.P\u00a0<\u20090.001) than that in the non-anemic group. In the subgroup analysis, the mild and moderate anemia groups had 1.38-fold  and 2.02-fold  risk of mortality due to infection compared to that of the non-anemic group, respectively. The severe anemia group did not have a significantly different risk of mortality due to infection (P\u00a0=\u20090.448).Overall, 512,905 individuals were included in this study. The mean age of the participants was 54.5\u2009years old (range: 40\u201398), and 49,042 (9.6%) individuals were classified in the anemic group, which comprised of 36,383 (7.1%), 11,787 (2.3%), and 872 (0.2%) participants in the mild, moderate, and severe sub-groups, respectively. After PS matching, 49,039 individuals in each group were included in the analysis. The risk of mortality due to infection in the anemic group was 1.77-fold higher (hazard ratio [HR]: 1.77, 95% confidence interval [CI]: 1.52\u20132.60; History of anemia was associated with increased mortality rate due to infection at 12-year follow-up.The online version contains supplementary material available at 10.1186/s12879-021-06377-0. Infection is defined as the entrance and development of an infectious agent in a human, regardless of whether it develops into an infectious disease . As a maAnemia is a chronic disease in which a person\u2019s hemoglobin levels are lower than required to meet their physiological needs, and affects roughly one-third of the global population .In South Korea, the total prevalence of anemia was 6.0%, and the prevalence of severe anemia was 0.92% . Since iTherefore, this study aimed to investigate the hypothesis that history of anemia might be an independent risk factor for higher mortality rate due to infection.This study involved human participants, and all procedures were conducted in accordance with the guidance provided by the relevant ethics boards. The Institutional Review Board (IRB) of Seoul National University Bundang Hospital  and the Health Insurance Review and Assessment Service (NHIS-2020-2-067) approved the study protocol. Informed consent was waived by IRB of Seoul National University Bundang Hospital, because data analyses were performed retrospectively using anonymized health records derived from the South Korean NHIS database. Data were extracted by an independent medical record technician at the NHIS center who was unaffiliated with this study.The NHIS-National Health Screening Cohort (NHIS-HEALS) was used in this study . As the All individuals were divided into two groups: the anemic (who had a history of anemia) and non-anemic groups. Individuals who had hemoglobin\u2009levels <\u200912\u2009g/dL for women and\u2009<\u200913\u2009g/dL for men, during 2003\u20132003, were considered to have anemia based on the World Health Organization (WHO) criteria. The severity of anemia at that time was categorized as mild (12\u2009g/dL\u2009>\u2009hemoglobin \u226511\u2009g/dL in women and 13\u2009g/dL\u2009>\u2009hemoglobin \u226511\u2009g/dL in men), moderate (hemoglobin 8\u201310.9\u2009g/dL), or severe (hemoglobin\u2009<\u20098\u2009g/dL), using the WHO criteria . Serum hIn this study, mortality due to a primary infection was considered as the study endpoint. The NHIS database provided data on the death date and main cause of death for all individuals. Mortality rate due to infection was evaluated for a period of 12-years, from January 1, 2004 to December 31, 2015. The specific diagnoses for mortality due to infection are presented as ICD-10 codes in 2). The national income level was registered in the NHIS database to determine the insurance premium of all individuals. Annual income level was divided into five groups using quintile ratio , and underlying disability was divided into two groups . In South Korea, all physical disabilities should be registered in the NHIS to receive various benefits, and are divided into six levels considering their severity. Thus, in this study, disabilities in the 1st (most severe) to 3rd levels were classified in the severe disability group, while those in the 4th to 6th (most mild) levels were classified in the mild to moderate disability group. Smoking status was divided into four groups , and alcohol consumption was divided into six groups . Exercise frequency was divided into six groups . The Charlson comorbidity index was calculated using registered ICD-10 codes from to 2002\u20132003, as shown in The following variables were collected as covariates for this study: demographic information , socioeconomic status related information , comorbidity related information (underlying disability and Charlson comorbidity index), and lifestyle information . Residence was divided into three groups , and BMI was categorized into four groups  matching between the anemic group (those with a history of anemia) and non-anemic group to reduce confounders . For thiP\u00a0<\u20090.05 was considered statistically significant.As a second sensitivity analysis, we constructed a multivariable Cox regression model for mortality due to infection using the entire cohort to determine: (1) whether the results obtained from the PS-matched cohort were generalizable to the entire cohort, and (2) the risk of mortality due to infection in the anemic group with other important covariates in context, and not in isolation. All covariates were included in the multivariate Cox model for adjustment. Using multivariable Cox regression modeling, we performed subgroup analyses to investigate whether mild, moderate, and severe anemia in the past, were associated with mortality due to infection compared to the non-anemic group. In addition, considering sex is associated with development of anemia , we perfIn the NHIS-HEALS data, a total of 514,795 individuals received standardized medical examination from 2002 to 2003. Among them, 1320 individuals were excluded due to death during 2002\u20132003, and 570 individuals were excluded due to missing data regarding hemoglobin level. Thus, a total of 512,905 individuals were included in this study. Among them, 49,042 individuals were classified as the anemic group (9.6%) using serum hemoglobin level, and 463,863 individuals (90.4%) were classified as the non-anemic group. After PS-matching, a total of 98,078 individuals  were included in the analysis  than that in the non-anemic group. The incidence of mortality due to infection in the two groups had a similar trend, as shown in Fig.P\u2009<\u20090.001) than that in the non-anemic group.Table\u00a0P\u00a0<\u20090.001; Model 1). In the subgroup analysis, the mild and moderate historical anemia groups had 1.38-fold  and 2.02-fold  higher mortality due to infection than the non-anemic group, respectively. However, the severe anemic group did not show a significant difference in mortality due to infection compared to that of the non-anemic group (P\u00a0=\u20090.448). The C-index of the multivariable model was 0.85 (95% CI: 0.84 to 0.86). The Table\u00a0n\u00a0=\u2009277,846) had a 1.57-fold higher mortality due to infection than the male non-anemic group . The female anemic group  had a 1.36-fold higher mortality due to infection than the female non-anemic group .Table\u00a0up P\u00a0=\u20090.48. The CThis population-based cohort study showed that history of anemia was independently associated with increased infectious mortality in South Korea. Interestingly, in the subgroup analyses, it was more evident in the mild and moderate anemic groups than the severe anemic group. We hypothesized that patients with a history of anemia have a higher risk of death due to infection during the long-term follow-up period. Considering that the prevalence of both anemia and infection was high in the low-income countries , our resAnemia, iron deficiency, and infections have been reported as three major causes of mortality and morbidity during childhood worldwide . These tHowever, a previous study reported that >\u200950% of anemia was developed due to other reasons such as deficiency of micronutrients  rather than IDA alone . The malHistory of anemia might also affect the prognosis of individuals after infection. Anemia has been reported to be common among hospitalized patients with acquired pneumonia, and it was associated independently with higher 90-day mortality . MoreoveA recent study has reported that the comorbid status of anemia is associated with an enhanced risk of severe COVID-19 infection, with an odds ratio of 2.44 (95% CI: 1.75 to 3.40) . In the The results of the subgroup analysis are notable: the severe anemic group (hemoglobin <\u20098\u2009g/dL) was not associated with mortality due to infection; whereas, the mild and moderate anemic groups were associated with a higher risk of mortality due to infection. Anemia has been reported to increase hospitalization and mortality in older adults , and it Our study has some limitations. First, we did not distinguish the type of anemia in detail because we only used the measured hemoglobin level in the standardized medical examination. Furthermore, information on treatment of the anemia was not evaluated during follow-up period (2004\u20132015) in this study, and it might affect the result of this study. Second, PS-matching, and multivariable adjustment only adjusted for known confounders, and there might be unmeasured confounders that may have affected the results in this study. Third, we used ICD-10 codes to define comorbid status and calculate the Charlson comorbidity index, but actual underlying diseases might differ with the registered ICD-10 codes in this study. For example, if individuals with diabetes mellitus did not visit outpatient clinic due to mild symptom or poor accessibility to healthcare utilization, they were not registered as diabetes mellitus in the NHIS database in this study. Fourth, we did not exclude women who were pregnant during 2002\u20132003 in this study. However, since our inclusion criteria were adults \u226540\u2009years old, we believe that the impact of pregnancy might be limited in this study. Fifth, lifestyle information was collected using a questionnaire, and there might be a selection bias due to non-response to the surveys in the standardized medical examination . Lastly,We showed that a history of anemia was associated with increased mortality due to infection among the adult South Korean population, and this was more evident in patients with mild to moderate anemia than those with severe anemia. However, considering the limitations of this study, further studies are warranted to confirm our findings.Additional file 1.Additional file 2.Additional file 3."}
{"text": "The different structures of the new materialslead to compounds with a range of band gaps with MXDBiI5 having the lowest at 2.15 eV. We have explored the tunabilty ofMXDBiI5 through halide substitution by preparing a seriesof samples with composition MXDBiIx5\u2013Brx and determined the halide contentusing energy dispersive X-ray spectroscopy. A large range of solidsolution is obtained in MXDBiIx5\u2013Brx, resulting in the formation of single-phasematerials for bromine contents from x = 0 to 3.71(iodine contents from 1.29 to 5). This highlights the fact that zero-dimensionalorganic\u2013inorganic halides are highly tunable, in a similarmanner to the higher-dimensional perovskite counterparts. Such newmaterials open up the opportunity for further studies of the physicsand optoelectronic properties of these materials.Over the past decade,the efficiency of photovoltaic devices basedon CH 2PbI6, MXDBiI5 and MXD3Bi2Br12\u00b72H2O have been determined. Halide substitution has been exploredin MXDBiIx5\u2212Brx, and a large region of solid solution has been found. MXDBiI5 has short intercluster I\u2212I distances and \u03c0\u2212\u03c0stacking between the MXD cations while also possessing the lowestband gap of the materials studied here.The structures of MXD The structure consists of corner-sharing PbI6 octahedra, with the organic cation being disordered on theperovskite A-site at room temperature.6 As the size of the organic ammonium cation is increased, differentperovskite-related structure types can form.7 In particular, layered perovskites based on the Ruddlesden\u2013Popperand Dion\u2013Jacobson structure types have received considerableinterest. A notable breakthrough came when a power conversion efficiencyof 12.52% was achieved for (C4H9NH3)2(CH3NH3)3Pb4I13, which adopts the Ruddlesden\u2013Popper structure.8 Very recently, the \u201cMemory Seed Effect\u201dhas been reported, which involves dissolving presynthesized crystalsin solvents such as DMF, which enables the preparation of high-qualitythin films for a variety of layered perovskites.9 The solutions have been shown to retain \u201cmemoryseed\u201d crystallites, which facilitates the production of phase-purelayered perovskites.9 The use of this \u201cMemorySeed Effect\u201d has led to solar cells with power conversion efficienciesof 17.1% for (C4H9NH3)2(CH3NH3)3Pb4I13.9CH10 The A, B, and X sites can bedoped in order to fine-tune the electronic structure and band gapfor a range of optoelectronic applications.11 In particular, as the electronic structureof organic\u2013inorganic metal halides depends on the B-site cationand halide, halide substitution is an excellent way of tuning theband gap of these materials.13 For example, halidesubstitution in FAPbI3-xBrx (FA = formamidinium) resulted in the band gap being tunedfrom 1.48 to 2.23 eV.3 Unfortunately, photoinducedhalide segregation has been reported in CH3NH3PbIx3\u2013Brx, yielding iodine- and bromine-rich domains in the thin films.14 Nevertheless, such tunability of the materialsis of interest, particularly in the fields of indoor photovoltaicsor tandem solar cells, which can utilize two perovskites with differentcompositions and band gaps.4 One methodof preventing halide segregation is to use multidentate ligands whichcreate interfaces with a low defect content.15Organic\u2013inorganic halideperovskites exhibit great compositionalflexibility.meta-phenylenediammonium (mPDA).18 Several of these perovskitesadopt the Dion\u2013Jacobson structure.17 Interestingly, the position of the aminomethyl group on the pyridiniumring in 4AMPY and 3AMPY influences the stacking of the inorganic layercontaining lead iodide octahedra and results in a decrease in bandgap when going from (4AMPY)(CH3NH3)n-1PbnIn+13 to (3AMPY)(CH3NH3)n-1PbnIn+13.16 (3AMPY)(CH3NH3)3Pb4I13 exhibited a power conversion efficiencyof 9.20%. In the (mPDA)(CH3NH3)n-1PbnIn+13 system, fabrication of phase pure thin films hasbeen challenging for some values of n.17 (PEA)2(CH3NH3)2Pb3I10 has been shown to haveenhanced moisture stability with respect to CH3NH3PbI3.18Recently, several new families of layered perovskites havebeenprepared which incorporate methylammonium in addition to cations whichcontain aromatic rings such as phenylethylammonium (PEA), 4-aminomethylpyridinium(4AMPY), 3-aminomethylpyridinium (3AMPY), or 6 or edge-sharing octahedra to make M2X10 or face-sharingoctahedra to make M2X9. Zero-dimensional materialsare already showing interesting properties; for example, 2Bi2I10\u00b72H2O has beentested as a photodetector, and reproducible photocurrents could bedrawn from this material.19 (CH3NH3)3Bi2I9 shows highlyanisotropic photoluminescence and has shown evidence of quantum cutting.20 In addition, isovalent doping in the zero-dimensional(CH3NH3)3Bix2\u2013SbxI9 hasshown a band-bowing phenomena.21 Zero-dimensionalall-inorganic halides such as A4PbX6 and Cs4SnX6  are alsoof interest, particularly for white light emission, and work by Mohammedet al. has recently found that the local octahedral distortions enabledthe formation of self-trapped states.22The term \u201czero-dimensionalperovskite\u201d has been coinedfor organic\u2013inorganic metal halides which consist of isolatedclusters of metal halide octahedra which are separated by organiccations, although strictly speaking these materials do not possessthe perovskite structure. Depending on the nature of the metal andhalide, different connectivities of octahedra may exist in the cluster,such as isolated MX18 alongwith the huge compositional and structural diversity of organic\u2013inorganichalides, we have explored the synthesis and tunability of new organic\u2013inorganichalides which use the meta-xylylenediammonium  cation.Motivated by reports of enhanced moisture stability when aromaticorganic ammonium cations are used,3NH3PbI3.23 The appropriate metal oxide was dissolved in 5 mL of HX and heatedto 80 \u00b0C. Separately, 0.5 mL of HX was added to 0.4 mL m-xylylenediamine, resulting in the formation of crystals.These crystals were heated until dissolved. The two solutions weremixed and stirred for 30 min. The solution was cooled to room temperature,and the resulting crystals were filtered. The crystals were driedin a vacuum oven at 80 \u00b0C for 2 h.The synthesis of all materials was based on modificationsof themethod reported by Poglitsch and Weber for the preparation of CHx5\u2013Brx, 0.47 g Bi2O3 was added to a mixtureof HX acid . The volumetric ratioof HI:HBr used in the reactions are given in m-xylylenediamine(0.14 mL) was added to the solution, and the mixture was stirred for30 min. After cooling to room temperature, the crystals were filteredoff and left to dry in the fume hood.For the synthesis ofMXDBiI\u03b11 radiation in Bragg\u2013Brentanogeometry. Data were collected from 5\u00b0 to 70\u00b0 with a stepsize of 0.017\u00b0 and a time per step of 0.94 s. PXRD data wereanalyzed using Topas Academic ver. 6.24Powder X-ray diffractiondata were collected on a Panalytical EmpyreanDiffractometer using CuK25 Thedata was processed using CrysAlisPro software.26 Structure solution was carried out using SHELXT,27 and structure refinement by full matrix least-squaresagainst F2 was carried out with SHELXL (2018/3).28 Non-hydrogen atoms were refined anisotropically,and carbon-bound hydrogens were refined using a riding model. Ammoniumhydrogens were located from the difference Fourier map and refinedisotropically subject to distance restraints. Selected crystallographicdata are presented in 2151572\u20132151574 contains the supplementary crystallographic datafor this paper. These data are provided free of charge by the jointCambridge Crystallographic Data Centre and FachinformationszentrumKarlsruhe Access Structures service www.ccdc.cam.ac.uk/structures.Single crystal diffraction data were recorded at either 173or293 K using a Rigaku FR-X Ultrahigh brilliance Microfocus RA generator/confocaloptics and Rigaku XtaLAB P200 diffractometer [Mo K\u03b1 radiation(\u03bb = 0.71073 \u00c5)]. Intensity data was collected using \u03c9steps accumulating area detector images spanning at least a hemisphereof reciprocal space .2 Jeol DrySD EDS detector anda Jeol JSM 5600. UV\u2013vis diffuse reflectance spectra were collectedon a Jasco V650 spectrophotometer equipped with an integrating sphere,in the wavelength range 190\u2013900 nm. BaSO4 was usedas a reference.Scanning electron microscopystudies were carried out using a JSMIT200 equipped with a 25 mm2O3 and HI or HBr, along with PbO and HI,produced the crystalline products MXDBiI5, MXD3Bi2Br12\u00b72H2O, and MXD2PbI6. Their structures were determined from single-crystalX-ray diffraction data, and selected parameters are tabulated in The reactions of MXD with eitherBi2PbI6 consists of isolated PbI6 octahedra separated by MXD cations  to 3.2443(2) \u00c5 PbI4 recentlyreported.17 The aromatic core of the MXDcation is not directly above adjacent MXD cations but is offset. Theorientations of the adjacent MXD cations result in NH3 groupspointing in three different directions to give a Y-shaped arrangementof the NH3 groups, with one pair of NH3 groupsbeing almost eclipsed (3 hydrogens and iodinefrom the PbI6 octahedra range from 3.546(2) to 3.718(2)\u00c5. Figure S1 shows the PXRD data ofthe bulk sample. Pawley fits were carried out using the unit cellparameters and space group obtained from single-crystal diffraction.Twelve Chebyshev background parameters, sample displacement, and profileparameters were also refined. The resulting fit is shown in Figure S1, and this confirms the phase purityof the sample.The structureof MXD cations 1. Althou443(2) \u00c5 3. The I\u2013eclipsed 1c. Hydro2PbI6,which contains isolated PbI6 octahedra, the structure ofMXDBiI5 consists of BiI6 octahedra, which shareedges to form Bi2I10 dimers (31 The Bi2I10 units are separated by the MXDcations. The Bi\u2013I bond lengths range from 2.9710(5) to 3.2459(6)\u00c5 (trans positions and from 85.756(15)\u00b0to 94.581(17)\u00b0 in the cis position. The Bi octahedrashow some distortion, with a bond length distortion of 10.39 and bondangle variance of 13.13. The orientation of the MXD cation plays animportant role in holding the structure together. The aromatic ringsof adjacent MXD cations are strictly parallel and have a centroid\u2013centroiddistance of 3.509(5) \u00c5. The close register of the rings forcesthe ammonium groups to point to the same side of the ring for eachcation, so when considered for each pair of cations, all four NH3 groups point in opposite directions. The I1\u2013I4 interclusterdistance is 4.1357(6) \u00c5, and this is of particular note, as thisis comparable to the short interlayer I\u2013I distances of 4.00and 4.04 \u00c5, which were reported in (H3NC6H4NH3)(CH3NH3)Pb2I7 and (H3NC6H4NH3)(CH3NH3)2Pb3I10, respectively.17 Althoughthese are thought to be the shortest interlayer iodine\u2013iodinedistances in Dion\u2013Jacobson perovskites, we note that in zero-dimensionalorganic\u2212inorganic halides, short intercluster distances of3.7253(16) \u00c5 have been reported in (H3NC6H4NH3)2Bi2I10\u00b74H2O, while short interchain distances of 3.871(10)\u00c5 have been reported in (H3NC6H4NH3)Bi2I8\u00b7I2.32 Hydrogen bonds between NH3 hydrogens and iodine in the Bi2I10 unitsranged from 3.712(5) to 3.812(6) \u00c5. Figure S2 shows the PXRD data of the bulk MXDBiI5 sample.Pawley fits were carried out using the cell parameters and space groupobtained from single crystal diffraction MXDBiI5 and thesame nonstructural parameters that were described for MXD2PbI6. The resulting fit is shown in Figure S2, and this confirms the phase purity of the sample.In contrast to the structure of MXD0 dimers 2. This k459(6)\u00c5 3. TheI\u20133Bi2Br12\u00b72H2O consists of isolated BiBr6 octahedra, with theasymmetric unit containing two inequivalent BiBr6 octahedra(cis bond angles and 170.57(2)\u00b0 to 176.29(3)\u00b0 for trans angles, while for Bi(2), the bond angles range from 174.21(2)\u00b0to 177.20(3)\u00b0 for trans bond angles and 85.46(2)\u00b0to 95.13(2)\u00b0 for cis bond angles. Calculationof the bond length distortion and bond angle variance show that theBiBr6 octahedra exhibit different levels of distortion,with Bi(1) showing the greater angular variance than the Bi(2), whileBi(1) shows less distortion of bond lengths than Bi(2)  in the structure. They are also significantly smaller thanthe bond angle variance reported for (mPDA)PbI4, but arecomparable to those reported for some quinoline and isoquinoline leadhalides.33 The shortest hydrogen bonds are formed betweenthe NH3 and H2O, with N\u00b7\u00b7\u00b7O distancesof 2.757(12) \u00c5 and 2.822(10) \u00c5. Figure S3 shows the PXRD data of the bulk MXD3Bi2Br12\u00b72H2O sample. Pawley fits were carriedout using the cell parameters and space group obtained from singlecrystal diffraction and parameters described for MXD2PbI6. The resulting fit is shown in Figure S3, and this confirms the phase purity of the sample. SEM imagesof MXD2PbI6, MXDBiI5, and MXD3Bi2Br12\u00b72H2O are shownin The structure of MXDctahedra3. For thctahedra3. In addan Bi(2) 3. The bo2PbI6, MXDBiI5, and MXD3Bi2Br12\u00b72H2O are shown in 2PbI6 displays aggregates of crystals,with a needle-like morphology, of dimensions of approximately 600\u03bcm by 40 \u03bcm. Crystallites of MXDBiI5 also showa needle-like morphology, although the crystals are much shorter thanthose reported for the MXD2PbI6, with dimensionsof approximately 50\u201375 \u03bcm by 20 \u03bcm. The MXD3Bi2Br12\u00b72H2O sampleconsists of much thinner crystallites with a narrow, plate-like morphology.A range of crystallite sizes can be observed, with typical dimensionsbeing around 50 \u03bcm by 100\u2013200 \u03bcm. Although thecontrol of morphology was not considered in this study, this willbe of interest in the future, when manufacturing these materials intothin films, as any pinholes in thin films as a result of surface morphologycan influence factors such as device performance in photovoltaicsor LEDs.SEM images of MXD34 Therefore, isovalent doping on the anion site,e.g., replacing I\u2013 with Br\u2013, isa particularly useful technique to tune the band gap of the CH3NH3PbI3 perovskites.35 This is often termed halide substitution and results in the creationof materials with compositions such as CH3NH3PbIx3\u2013Brx. Zero-dimensional inorganic\u2013organic halides commonlyexhibit a similar electronic structure to the 3D analogues, but withgreater orbital decoupling;36 therefore,we decided to probe halide substitution in MXDBiIx5\u2013Brx, in order to determinethe doping limit of Br\u2013 in the MXDBiI5 structure type. A photograph of the MXDBiIx5\u2013Brx samples is shownin Figure S5).36 We note that similar phenomena havebeen reported when two different cations are used in the synthesisof Ruddlesden\u2013Popper phases, as nonstoichiometric ratios ofreagents must be used to isolate the phase pure product.37 In addition, in the synthesis of Cs2SnX6  mixed halides, the products werefound to be richer in the Cl or Br than would be expected given theratios in the precursors, and this has been attributed to the differencein solubility of halides in solution.38It is well-known that the valenceband and conduction bands in organic\u2013inorganic halides compriseorbital contributions from both the halide and the inorganic cation.x5\u2013Brx samples are shown in 4.11Br0.89 is shown in Figure S5. As can be seen, the 100% Br sample exhibits a completelydifferent PXRD pattern to the other samples prepared in this series,which indicates that it adopts a completely different structure type,MXD3Bi2Br12\u00b72H2O(vide supra). However, with only a small amount of HI in the precursorsolution , the MXDBiI5 structure type is adopted, and all peaks can be indexed to a monoclinicunit cell in space group I2/m. Thisstructure type is adopted for all MXDBiIx5\u2013Brx compositions which have a brominecontent, x, between 0 and 3.71 . In order to determine the extent ofthe solid solution in MXDBiIx5\u2013Brx, Pawley refinements were carriedout. During the refinements, 12 Chebyshev polynomial terms were usedto fit the background, and in addition, unit cell parameters, profileparameters, and specimen displacement were all refined. The resultingvariation of unit cell volume with iodine content is shown in Supporting Information . As can be seen from x) and unit cell volume,in agreement with Vegard\u2019s law, with the exception of a slightleveling off at the lowest bromine contents. This shows that thereis a large region of solid solution in the MXDBiIx5\u2013Brx system. This regionof solid solution ranges from a bromine content, x, of 0 to 3.71, and it is likely that a full solid solution couldbe obtained with further optimization of synthetic conditions. Asthe bromine content increases, the \u03b2 angle increases, but thereis a leveling off at the highest iodine contents (which correspondsto the lowest bromine contents). The a, b, and c unit cell parameters all show a linear relationshipwith bromine content and exhibit a smaller leveling off at the lowestbromine contents. The decrease in unit cell parameters with increasingbromine content is expected due to the smaller size of Br\u2013 (1.96 \u00c5) with respect to I\u2013 (2.20 \u00c5).39The PXRD data of MXDBiIx5\u2013Brx samples and MXD3Bi2Br12\u00b72H2O are shown in 2PbI6 spectrum is shown in Figure S7. Two absorption features are observed in the spectra forMXDBiIx5\u2013Brx. For MXDBiIx5\u2013Brx, the first feature is in the 2.36\u20132.06 eVrange (corresponding to 525\u2013600 nm) and the second featureis centered around 1.9 eV (\u223c650 nm), while for MXD2PbI6, the peak occurs at \u223c500 nm (2.5 eV) We notethat dual features have been observed in both UV\u2013vis spectroscopyand photoluminescence measurements for both 2D, layered materials,and zero-dimensional materials.42 For example, Nag et al. noticeddual-emission in photoluminescence studies of single crystals of (PEA)2SnI4 (PEA = phenylethylammonium), which was alsoaccompanied by two absorption features in UV\u2013vis absorptionspectra of the same material.41 A similarbehavior was also observed for (4-AMP)SnI4 (4-AMP = (4-aminomethyl)piperidinium).41 Dual PL emission has also been observed forthe zero-dimensional TPA2SbCl5 (TPA = tetrapropylammonium).42 To date, the presence of dual features in thephotoluminescence or absorption spectra has been attributed to differencesin the edge or bulk of the crystals or self-trapped excitons.42 As the bromide content is increased in MXDBiIx5\u2013Brx, the absorption edgeshifts to larger energies, which is in agreement with an increasein band gap with increasing Br content. The resulting band gaps wereestimated using Tauc plots and are listed in 5 exhibiting thelowest band gap in the series (2.15 eV) and MXD3Bi2Br12\u00b72H2O the largest (2.86 eV).In contrast, the band gap of MXD2PbI6 was determinedto be 2.33 eV. The variation of band gap with bromine content is shownin x5\u2013Brx canbe tuned over a similar range to that observed for the FAPbIx3\u2013Brx perovskites, whichallowed tuning of the band gap from 1.48 to 2.25 eV.3 The high crystallinity of these samples warrants furtherinvestigation into the photostability of MXDBiI5-xBrx, as in the mixed cation, mixed halideperovskites, CsyFAy1\u2013PbIx3\u2013Brx, a high level of crystallinity was found to suppresshalide segregation.43The Kubelka\u2013Munk transformationsof the UV\u2013vis diffusereflectance spectra of the MXDBiImeta-xylylenediammonium (MXD) cation: MXDBiI5, MXD3Bi2Br12\u00b72H2O, and MXD2PbI6. MXDBiI5 has short intercluster I\u2013Idistances and \u03c0\u2013\u03c0 stacking between the MXD cations,while also possessing the lowest band gap of the materials studiedhere. We explored the tunability of MXDBiI5 through halidesubstitution and found that a large region of solid solution existsin the MXDBiIx5\u2013Brx system (where x = 0 to 3.71). Thiswork highlights the fact that zero-dimensional organic\u2013inorganichalides are also highly tunable semiconductors and opens up the wayfor further studies of the physics and the long-term stability ofthese materials.Here, we have reported the synthesis and characterization of threenew organic\u2013inorganic metal halides, using the"}
{"text": "His bookLes d\u00e9moniaques dans l'artis a representation of hysterical symptoms in religion and religious art. This paper aims to discuss Charcot's descriptions of hysteria in religion and his \u201chysterical saints\u201d.Professor Jean-Martin Charcot was the founder of clinical neurology and one of the prominent researchers in the field of hysteria in the 19 Hysterik\u00f3s, meaning \u201crelative to the womb\u201d. This correlation was established in ancient Greece because most cases of hysteria occurred in women, but only in the 16thcentury the term hysteria was regularly applied to designate such functional disorders.The term hysteria is derived from the Greek wordIn the middle age, hysteria gained a religious connotation, and the hysterical phenomenology was attributed to demonic possession or witchcraft. A similar situation to the one experienced by patients with other neurological conditions, such as stroke and epilepsy.thcentury, Jean-Martin Charcot (1825\u20131893) (In the 195\u20131893)  developLes d\u00e9moniaques dans l'art. In this work, illustrated by Richer, they discussed how hysteria was represented in religious art, presenting works of art featuring some of the saints of the catholic church that were possibly presenting a hysterical event.Les d\u00e9moniaques dans l'art, emphasizing Charcot's \u201chysterical saints.\u201dThis interest motivated Charcot to publish a book in 1887, alongside his pupil Paul Richer (1849\u20131933) :Les d\u00e9It is curious that Charcot took the burden of untangling the cloudy subject of hysteria since he had no prior interest in mental illnesses. His contact with the works of Pierre Janet and Briquet, besides the influence of Desir\u00e9e Bourneville, one of the few of his pupils with experience with alienism, were determinants for his interest in hysteria.Charcot had strong political and religious standpoints. An anti-clerical and fierce defendant of laicism in all scientific investigations, Charcot opposed religious intervention in scientifical affairs.Permanent deformations of the hand from the point of view of medical semiotics, by Henri Meillet with drawings of Richer. Impressed by the quality of his art, Charcot invited Richer to join his service at theSalp\u00eatri\u00e8re. Richer's artistical prowess also contributed to his own thesis,\u00c9tudes cliniques sur l'hyst\u00e9ro-\u00e9pilepsie ou grande hist\u00e9rie.Charcot was very fond of art, with a predilection for the classics. A gifted artist himself, he produced numerous self-portraits, drawings, and sketches.Les d\u00e9moniaques dans l'artwas preceded by the start of Bourneville's work, theBiblioth\u00e8que diabolique, nine books, published between 1882 and 1902.A brief discourse of a disease called the suffocation of the mother, in 1603. This book is considered a turning point in the understanding of hysteria, presenting it as a disease instead of a religious event.Another worthy contribution was Edward Jorden's publicationLes d\u00e9moniaques dans l'art, Charcot presents his classical description of the grand hysterical attack, divided in four periods .Iconographie photographique de La Salp\u00eatri\u00e8re.Instigma. Hallucinations are common, often with a religious nature. This phenomenon is commonly described as an epiphanic, religious event, sometimes possessing an erotic connotation.Charcot describes a variant of the third period of the grand hysterical attack, with a predominance of the feeling and constant ecstatic facial expression. The patient is typically quiet, presenting a delusional speech, and might present negative sensorial symptoms, such as achromatopsia, blindness, and anesthesia. These sensorial symptoms are referred to asSainte Catherine de Sienne, providing a copied fragment of the fresco that decorates Saint-Dominique Church, in Sienna (Sainte Catherinein an attitude of ecstatic contemplation, with a facial expression of joy. Other representations of the \u201cHysterical saints\u201d are mentioned by Charcot, such as \u201cSaint Fran\u00e7ois recevant les stigmates\u201d (Sainte Marguerite de Cordoue en extase\u201d (\u201cSaint Fran\u00e7ois en extase\u201d (Charcot illustrates the ecstatic crisis with the case of Sienna . The pimates\u201d  \u201cSaintxtase\u201d , and\u201cSxtase\u201d , but thL'hyst\u00e9rie de Sainte-Th\u00e9r\u00e8se, in hisBiblioth\u00e8que diabolique, reinforcing the relevance of Bernini's work in depicting ecstatic phenomenology.A lacking honorable mention is Gian Lorenzo Bernini's sculpture \u201cThe Ecstasy of Saint Teresa\u201d , the prThis sexual and orgasmic description is commonplace among the ecstatic religious events, as seen in the illustrations of Saint Catherine and Saint Francis. Charcot pointed this out, defining this uniformity as almost scientific, praising such artistic rigor.In modern times, these ecstatic events were also related to epileptic activity, typically secondary to non-dominant temporal lobe abnormalities, sometimes with a sexual and orgasmic phenomenology, giving rise to the term \u201cOrgasmic epilepsy\u201dLa transfiguration, the opisthotonos of the clownism period in an image of Jesus performing an exorcism fromLa Bible de Picart, and the bizarre dystonic postures of the final delirium in a tableau from Saint-Ambroise church, at Genoa.Charcot and Richer also present examples of religious art illustrating the other periods of the grand hysterical attack, such as the limb circumduction movements of the epileptoid period in a fragment of Deodat Delmont'sth-century Parisian society, despite prior contributions in the literature. Charcot's publication ofLes d\u00e9moniaques dans l'artis a tribute to laicism in science, demonstrating his unswerving respect for neurology.In conclusion, the depiction of religious events as a possible manifestation of functional disorders was unusual to the conservative 19"}
{"text": "A 48-year-old woman was referred for a bone scan as an assessment of bone metastasis from breast cancer. Surprisingly, two hot spots of lung uptake were present in the left lung without any abnormality on CT slices. No history of pulmonary disease was observed. An optimized CT scan with fine slices performed the same day was strictly normal (without any micronodule). A lung ventilation/perfusion scintigraphy showed no significant perfusion defect. A follow-up bone scan performed eight months later was normal and without any lung uptake. After exclusion of the main etiologies described in the literature, such as amylosis, sarcoidosis, abscess, or hypercalcemia, radiotracer microembolism seems to be the most likely hypothesis in this patient."}
{"text": "Background: The infectious disease society of America (IDSA) recommends routine laboratory tests for all patients receiving outpatient parenteral antimicrobial therapy (OPAT) to monitor for adverse events. There are no data to support how often patients should take monitoring laboratory tests. In addition, the relevance of different laboratory tests commonly used for OPAT follow up is not clearly known. Methods: We conducted a retrospective observational cohort study over a 7-year study interval (1 January 2014 to 31 December 2021). Clinical data were obtained to identify the risk factors associated with abnormal laboratory tests and determine if abnormal laboratory tests led to antibiotic change or hospital readmission. Results: Two hundred and forty-six patients met the inclusion criteria for this study. In our multivariate analysis, the Charlson comorbidity index (CCI) of 0\u20134 , the use of ceftriaxone without vancomycin  and an OPAT duration of 2\u20134 weeks  were associated with a lower risk of OPAT complications. A CCI of 5 or more ) and an OPAT duration of 5 or more weeks  were associated with a higher risk of OPAT complications. An abnormal complete metabolic panel or vancomycin levels, but not an abnormal complete blood count, were associated with antibiotic change or readmission. Conclusion: Patients with fewer comorbidities, ceftriaxone and short OPAT durations are at lower risk for OPAT complications. These patients could be followed with less frequent laboratory monitoring. Outpatient parenteral antimicrobial therapy (OPAT) is a preferred method to administer long-term intravenous (IV) antibiotic therapy for various infections. OPAT reduces the length of hospital stays and costs while improving patient satisfaction ,2. The uThe IDSA OPAT guidelines on monitoring laboratory results for individual antibiotics are based on known adverse events associated with that antibiotic . The guiAfter reviewing the medical records of 326 patients, 246 eligible patients were included. A total of 116 patients had OPAT-related complications and 130 patients had no complications. Demographic and clinical data characteristics of the two groups of patients are shown in Infections with gram-positive bacteria other than MRSA and polymicrobial infections were the most common infections identified in 91/26 (37%) and 53/246 (21.5) patients, respectively. Beta-lactams with or without vancomycin were the most common antibiotics prescribed. Ceftriaxone and ertapenem were the most frequently used beta-lactams. Two-hundred and nineteen (89%) patients received one or two antibiotics for OPAT and 175/246 (71.1%) were receiving OPAT for 5 weeks or more. The majority of patients  received OPAT at home. Seventy-eight (67.3%) patients with complications and 102 (78.5%) without complications followed up in the outpatient clinic within the first 3 weeks of discharge. Anemia was the common baseline laboratory abnormality, seen in 85 (73.3) patients with complications and 86 (66.2%) patients without complications.There was no patient who had abnormal urinalysis results while receiving OPAT. We assessed the risk of readmission or antibiotic change based on laboratory abnormalities in our patient population. Fifty-two patients required readmission or antibiotic change . In a muOPAT is a health care cost-saving strategy for patients who require long-term parenteral antibiotics ,14. PatiIn our study, only 14 (5.7%) patients received OPAT at a skilled nursing facility (SNF). The risk of rehospitalization did not differ between the group who received OPAT at home and those receiving OPAT at SNF. This could be because of the small number of patients who had OPAT at SNF. A prior study showed that patients who received OPAT at SNF had a 46% increased risk of rehospitalization compared to patients who received OPAT at home ,19.Our results showed that patients with few comorbidities (CCL < 5), patients who used ceftriaxone alone and patients with an OPAT duration of 4 weeks or less could have laboratory monitoring less frequently than once a week. This study suggests that patients on ceftriaxone are less likely to experience OPAT complications and could require less frequent monitoring. Ertapenem was associated with more complications, but this could be incidental since it was more commonly used with vancomycin. Vancomycin use has a strong association with adverse events, as has been shown in multiple studies, so patients should have at least weekly vancomycin troughs and serum creatinine levels ,12.In our study, penicillin and semisynthetic penicillin were the most common causes of abnormal laboratory results. It has been previously reported that semisynthetic penicillins are associated with higher rates of adverse events compared to cephalosporins and ertapenem ,6,7,20. Metronidazole and rifampin were common oral antibiotics used with either vancomycin or beta-lactams for broader coverage and for a better activity in biofilm, respectively. Neither were associated with OPAT complications that warranted hospitalization, but two patients developed leukopenia while on rifampin. Metronidazole is generally safe for use during OPAT, and to our knowledge, there is only one report that showed an unexpected increase in AKI in patients receiving metronidazole, but these patients were also receiving vancomycin which was likely the main cause for AKI . RifampiIn addition to weekly laboratory follow ups, other different strategies have been employed to decrease rehospitalization rates. It has been reported that regular telephone calls after hospital discharge to ensure adherence is associated with lower readmission rates . BecauseIn our study, early clinic follow ups  were associated with fewer OPAT complications, whereas late clinic follow ups were associated with more OPAT complications. The time from discharge to clinic follow up is known to have an association with an increased risk of OPAT complications . PatientSettings: This study was conducted at Saint Louis University Hospital, a 350-bed tertiary academic medical center, over a 7-year interval (1 January 2014 to 31 December 2021). Patients that were discharged home and receiving OPAT received antibiotics from a home infusion company for self-administration. A home health care nurse visited weekly to maintain the peripherally inserted central catheter (PICC) line and obtain samples for weekly laboratory tests. In our center, we routinely order weekly CBCs with differential, CMPs and urine analyses (UA) with microscopy for all OPAT patients. In addition, we order specific serum drug levels for patients on vancomycin or aminoglycoside and creatinine kinase (CK) levels for patients on daptomycin weekly. Infectious disease (ID) fellows review laboratory results regularly and follow patients in the clinic under the supervision of ID attendings.Data collection: After approval from the Institutional Review Board, we collected clinical information from our institution\u2019s electronic medical records. The data collected included age, gender, comorbidities, indications for OPAT, microbiology, prescribed antibiotics, OPAT duration, discharge location, time from discharge to outpatient clinic follow up, baseline laboratory abnormalities and the type and number of abnormal laboratory results while receiving OPAT. If patients were readmitted while receiving OPAT, we documented the date and reason for hospitalization. If the physician changed antibiotics, we documented the date of the change and reasons for the change.Inclusion and exclusion criteria: The patients included in this study were 18 years old or older, required two weeks or more of OPAT and was evaluated by an infectious diseases consult team while in the hospital. We excluded those that entered hospice care, had missing data and were readmitted for more than one week for causes not directly related to antibiotic treatment or abnormal OPAT follow-up results.Definitions and Variables: We separated patients into a cohort of OPAT patients with complications and patients with no complications. We defined an OPAT complication as a clinically significant laboratory abnormality, a need to change antibiotic class and hospital readmission for any reason. We defined immunosuppression as chronic glucocorticoid use, acquired immunodeficiency syndrome with CD4 < 200/mL, use of disease-modifying antirheumatic drugs (DMARD), solid organ transplant or bone marrow transplant. We used the Charlson comorbidity index (CCI) to estimate a 10-year mortality risk. The OPAT duration was the number of weeks from hospital discharge until the physician discontinued antibiotics. Laboratory values at initial discharge from the hospital were considered baseline. Clinically significant laboratory values included creatinine, sodium, potassium, aspartate aminotransferase, alanine transaminase, alkaline phosphatase, total white blood count, platelet count, absolute eosinophil count, vancomycin trough, CK and urinalysis.p value < 0.05 from the univariate analysis were included for the multivariable logistic regression model. We considered a p value \u2009< \u20090.05 statistically significant for our regression model. IBM SPSS 26 was used for the analysis.Statistical Analysis: Continuous variables are reported in medians and interquartile ranges (IQRs). Nominal and categorical variables are reported as proportions and frequencies. Univariate analysis with Chi square test of independence was used to identify the risk factors of OPAT complications. Variables with a Complications are not uncommon in OPAT patients. Recognizing predictors of OPAT complications will help determine strategies to optimize care, increase patient satisfaction and potentially further decrease cost. The less frequent monitoring of laboratory values could be useful in OPAT patients at a lower risk of complications. Less-frequent laboratory testing could be important to reduce costs  and potentially increase patient satisfaction ."}
{"text": "Staphylococcus aureus bacteremia enrolled in outpatient parenteral antibiotic therapy monitoring program (OPAT-MP) upon hospital discharge with patients not enrolled. OPAT-MP patients were more likely to attend infectious diseases follow-up appointments. OPAT-related emergency room visits and/or readmissions were more common among non\u2013OPAT-MP patients, but differences were not statistically significant.We compared patients with  Outpatient parenteral antibiotic therapy (OPAT) has become an integral part of the treatment of deep-seated infections. Advantages of OPAT include cost-effectiveness, decreased exposure to hospital-acquired infections, and improved patient satisfaction; disadvantages include risk of readmission, catheter-related complications, and adverse drug events.Staphylococcus aureus bacteremia, which requires 2\u20136 weeks of intravenous antibiotic therapy. Many patients are therefore discharged from the hospital on intravenous anti-staphylococcal treatment. However, in the absence of appropriate laboratory monitoring, the available antibiotics  for S. aureus bacteremia are associated with significant and potentially life-threatening side effects. OPAT monitoring is therefore particularly applicable for this patient group. Given the persistent high morbidity and mortality of S. aureus bacteremia, new strategies to improve clinical outcomes are imperative.A notable indication for OPAT is We conducted a retrospective study to investigate whether a structured OPAT monitoring program (OPAT-MP) with dedicated faculty support improved outcomes among patients discharged after an episode of staphylococcal bacteremia.A formal OPAT-MP was established in July 2016 by the Division of Infectious Diseases at Columbia University Irving Medical Center (CUIMC), an academic, multicampus, acute-care hospital center in New York City. As part of the program, dedicated infectious diseases (ID) clinicians review weekly laboratory results collected by home infusion services (1) to adjust antibiotic levels and monitor antibiotic toxicity, (2) to troubleshoot issues regarding long-term intravenous lines to prevent emergency room (ER) visits and hospital readmissions, and (3) to coordinate with the primary ID physician to ensure proper completion of therapy.S. aureus bacteremia. Patients admitted between July 2016 and December 2017 and enrolled in the OPAT-MP at discharge were compared to those admitted between January 2015 and December 2017 who were not enrolled in OPAT-MP at discharge. Patients discharged to a short- or long-term rehabilitation center or nursing care facility were excluded. Outcomes included ID follow-up appointment attendance, OPAT-related hospital readmissions and ER visits, microbiological recurrences, and death. Readmissions and ER visits were deemed OPAT related if they were due to intravenous catheter complications , adverse drug reactions, new Clostridium difficile infection, or worsening of the primary infection after source control had previously been achieved.We conducted a retrospective chart review of patients discharged home on intravenous antibiotic therapy after a hospital admission at CUIMC for  tests or Fisher exact tests, as indicated. SAS version 9.4 software  was used for all analyses.Statistical measures included \u03c7This study was approved by the Institutional Review Board of Columbia University Irving Medical Center.S. aureus bacteremia during the study period: 45 patients were included in the OPAT-MP cohort and 106 patients were discharged on OPAT without formal monitoring. The median age of patients was 55.8 years and did not significantly differ between the 2 groups (P = .34). Sex and race did not significantly differ between groups; more patients in the OPAT-MP group were Hispanic  (Table\u00a0P = .03) and 27% versus 7% (P < .01), respectively. Also, 80% of the overall study cohort had an inpatient ID consultation; consultation was more common in the OPAT-MP group than in the unmonitored group . In the OPAT-MP group, at time of bacteremia, Pitt bacteremia scores ranged from 0 to 11 . In the non\u2013OPAT-MP group, Pitt bacteremia scores ranged from 0 to 6 .Overall, 151 patients were discharged on intravenous anti-staphylococcal therapy for S. aureus (MSSA) bacteremia and 13 (29%) had methicillin-resistant S. aureus (MRSA) bacteremia. In the non\u2013OPAT-MP group, 79 patients (75%) had MSSA bacteremia and 27 (25%) had MRSA bacteremia. Discharge regimens and sources of bacteremia are described in Table\u00a0P = .55). At the time of discharge, the median planned duration of therapy was 4 weeks for the OPAT-MP group and 3 weeks for the non\u2013OPAT-MP group.The median duration of admission was 16.1 days in the OPAT-MP group and 16.8 days in the unmonitored group. In the OPAT-MP group, 32 patients (71%) had methicillin-sensitive P = .04) (Table P = .11). Similarly, although OPAT-related readmissions within 30 days were more common in the non\u2013OPAT-MP cohort, the trend was not statistically significant . Microbiological recurrence within 30 days was low . There were no deaths in the study cohort within the 30 days after discharge. Most patients completed therapy as planned: 64% in the OPAT-MP cohort and 57% in the non-OPAT-MP cohort (P = .37). There was no significant difference between the 2 groups regarding stopping therapy early (P = .23), changing or extending therapy (P = .08), or lack of documentation of therapy outcome (P = .07).Patients in the OPAT-MP cohort were more likely to attend an ID follow-up appointment  Table . AlthougAmong the 13 patients in the OPAT-MP cohort with MRSA bacteremia, 4 (31%) required changes in vancomycin dosing over the course of treatment. Of 23 patients in the OPAT-MP cohort with MSSA bacteremia on penicillin-based therapy, 4 (17%) developed drug rash, transaminitis, and/or acute interstitial nephritis. Of these 4 patients, 3 patients required antibiotic therapy changes, and 1 patient was monitored with continuation of therapy.S. aureus bacteremia, those who were followed by a formal OPAT-MP were significantly more likely to attend an ID follow-up appointment. We detected a nonsignificant trend toward fewer ER visits and readmissions among the patients in the monitored cohort.Among patients discharged on OPAT for This study had several limitations. The sample size was modest. This study was likely underpowered to detect differences in readmissions and ER visits between the 2 cohorts. Many patients who were not enrolled in OPAT-MP had limited follow-up documentation in the medical record after discharge. We detected a bias toward underdetection of adverse drug events and outside hospital readmissions and ER visits in this group. This group also had a higher percentage of patients with chronic or end stage renal disease, which may have predisposed them to having more readmissions or ER visits. Conversely, because patients in the monitored group were followed until the end of their course, they were more likely to have all adverse events captured appropriately. In our study, although we did not detect a significant difference in readmissions between the groups, patients in the OPAT-MP group were more likely to attend an ID appointment, and ID follow-up itself has been associated with a lower rate of 30-day readmission among OPAT patients.Although OPAT-related admissions were relatively low, overall readmissions within 30 days approached 30%, indicating that this is a high-risk group. Multiple studies have sought to elucidate the risk factors for readmission among patients on OPAT.Given the advantages of OPAT regarding efficient, effective, and patient-centered care, it is imperative to understand how best to keep patients safe while on OPAT and to further explore the potential benefits of OPAT-MP."}
{"text": "To determine whether a structured OPAT program supervised by an infectious disease physician and led by an OPAT nurse decreased hospital readmission rates and OPAT-related complications and whether it affected clinical cure. We also evaluated predictors of readmission while receiving OPAT.A convenience sample of 428 patients admitted to a tertiary-care hospital in Chicago, Illinois, with infections requiring intravenous antibiotic therapy after hospital discharge.2 test. Factors associated with readmission for OPAT-related problems at a significance level of P < .10 in univariate analysis were eligible for testing in a forward, stepwise, multinomial, logistic regression to identify independent predictors of readmission.In this retrospective, quasi-experimental study, we compared patients discharged on intravenous antimicrobials from an OPAT program before and after implementation of a structured ID physician and nurse-led OPAT program. The preintervention group consisted of patients discharged on OPAT managed by individual physicians without central program oversight or nurse care coordination. All-cause and OPAT-related readmissions were compared using the \u03c7P = .003). OPAT-related readmission reasons included infection recurrence or progression (53%), adverse drug reaction (26%), or line-associated issues (21%). Independent predictors of hospital readmission due to OPAT-related events included vancomycin administration and longer length of outpatient therapy. Clinical cure increased from 69.8% before the intervention to 94.9% after the intervention (P < .001).In total, 428 patients were included in the study. Unplanned OPAT-related hospital readmissions decreased significantly after implementation of the structured OPAT program (17.8% vs 7%; A structured ID physician and nurse-led OPAT program was associated with a decrease in OPAT-related readmissions and improved clinical cure. OPAT is not without limitations, however; unplanned 30-day hospital readmission rates as high as 26% have been reported. Moreover, intravascular catheters used for OPAT are associated with an increased risk for deep venous thrombosis and catheter-related infections.Outpatient parenteral antimicrobial therapy (OPAT) is a treatment option for medically stable patients who require intravenous (IV) antimicrobial therapy in an outpatient setting. It has been utilized since the 1970s. OPAT permits decreased length of hospital stay in patients who require IV antimicrobial therapy, reduces cost to both the patient and the healthcare system, and reduces the risk of nosocomial infections. Due to the need for multiple providers and institutions to be involved in the care of patients on OPAT, communication and care coordination is critical to the feasibility, safety, and success of OPAT programs in successful treatment and in reducing hospital readmission rates for OPAT-related complications. According to the Infectious Diseases Society of America (IDSA) guidelines on OPAT, an effective OPAT team encompasses the physician, nurse, pharmacist, and the patient. Each individual in this collaborative team plays a unique role in the planning and delivery of high-quality care for patients discharged home on OPAT. The presence of a formal OPAT care team has been reported as one of the most important quality indicators in optimal OPAT care. However, not every institution with an OPAT program has dedicated personnel or a complete OPAT team as recommended by IDSA guidelines. In a 2018 survey of 672 ID physicians in the Emerging Infectious Network (EIN), only 36% had a structured OPAT program at their institution. An explanation for this may be inadequate hospital administrative support of OPAT programs (as reported by 59.8% of providers) or financial support (as reported by 64.6%). Lack of evidence demonstrating the value of dedicated staff for a successful OPAT program may explain why more resources are not allocated to OPAT programs. Studies that compare OPAT outcomes in OPAT systems, which utilize various team models, provide much needed evidence and justification that these models are effective.OPAT programs require the involvement of multiple providers including infectious disease (ID) physicians and often pharmacists and nurses, in addition to care coordination between multiple institutions including hospitals, outpatient clinics, home health agencies, infusion companies, rehabilitation facilities, and skilled nursing facilities. ID consultation was not required for OPAT; however, most OPAT patients (69%) described in a cohort from 2012 to 2013 at UI Health had an ID physician involved in their care at the time of OPAT initiation. Individual physician management was problematic for several reasons: nonstandardized structure of the OPAT program, significant time coordinating care, competing responsibilities of physicians, and an increasing number of OPAT patients in the program. In August 2017, a dedicated  registered nurse with nursing education, health utilization management, and discharge planning expertise was hired to provide centralized and standardized care coordination, to support patient communication, and to facilitate and document laboratory and pharmaceutical management for our OPAT program. In addition, an ID physician was designated to oversee the program (0.1 FTE) and a requirement was enacted that all OPAT orders and patient management would be directed by the OPAT team and an ID physician. An inpatient ID pharmacist was available to support the OPAT program as needed, but no pharmacist FTE support was dedicated. In this study, we examined risk factors for readmission of patients receiving OPAT and assessed readmission rates before and after the implementation of a structured OPAT program as a key programmatic indicator of OPAT functioning at a large, academic, tertiary-care hospital.The University of Illinois Hospital and Health Sciences System  is a 463-bed tertiary-care academic hospital in Chicago. The evolution of the OPAT program at UI Health captures the spectrum of OPAT programs across the United States. Prior to hiring dedicated staff for the UI Health OPAT program, OPAT was managed by individual physicians who coordinated care individually without administrative support, dedicated care coordination, nor standardized management protocols. The postintervention group included patients discharged through the new structured OPAT program between October 1, 2017, and January 1, 2019. The structured OPAT program consisted of a full-time, dedicated nurse who provided care coordination and communication with home health agencies, infusion companies, patients, ID attending physicians and fellows, the ID clinic, and nursing and rehabilitation facilities. In addition, a dedicated ID physician served as the medical director by providing oversight for the program. This medical director developed laboratory monitoring protocols and management, and enhanced documentation standards. Although the medical director supervised the management of the entire program and oversight and support for the OPAT nurse, clinical decisions after hospital discharge were made by the supervising ID physician, often with an ID fellow, who cared for the patient while in the hospital and created the OPAT treatment plan. In most instances, the medical director and the supervising ID physician were not the same individual. The OPAT nurse utilized laboratory monitoring protocols for laboratory review, actions, and adjustment of dose and frequency, in coordination with the supervising ID physician. The primary end point of this study was unplanned, OPAT-related readmissions during receipt of OPAT. Readmissions deemed related to OPAT included treatment toxicities, complications of venous access devices , and relapse of infection being treated. Secondary end points included all-cause readmission during OPAT and treatment outcome (successful versus failed treatment). Successful treatment was defined as completion of planned duration of IV antimicrobial therapy or a transition from IV antimicrobials to oral antimicrobials. Failed treatment was defined as clinical signs or symptoms of infection with or without readmission to UI Health Hospital due to infection progression or recurrence while on OPAT. Predictors of OPAT-related readmission were also evaluated. Initial review was performed by a study team member, and an ID physician verified categorization of whether a readmission was related or not related to OPAT. The University of Illinois at Chicago Institutional Review Board approved this research study.This retrospective quasi-experimental study included patients aged \u226518 years who received an ID consultation and were discharged from UI Health on OPAT. The preintervention group included patients discharged from UI Health with OPAT recommendations from the ID consultation service between January 1, 2012, and August 1, 2013. This cohort was a subgroup of patients from our previously published study.Patients included in this study were retrospectively identified through the following sources: billing codes, orders for outpatient IV antimicrobials, a registry of patients who had a peripherally inserted central catheter (PICC) placed while in the hospital, and a registry of patients enrolled in the structured OPAT program. Patients must have received their medication through a PICC line for a minimum of 2 days for treatment of an infection as an outpatient to be included. Patients were included if they were receiving IV antimicrobials after hospital discharge in any setting except for those who were receiving IV antimicrobials during scheduled hemodialysis sessions who were excluded. Each patient was followed for the entire duration of their IV antimicrobial therapy even if it was completed outside the enrollment period.The UI Health electronic medical record  was utilized to collect patient information and data was stored using REDCap (Research Electronic Data Capture). Data abstracted included patient general characteristics such as age, sex, comorbidities, insurance status, presence of assigned primary care physician. In addition, healthcare utilization measures were collected, including hospital length of stay, previous hospitalizations for any cause within past 12 months, intensive care unit (ICU) stay during hospitalization, primary clinical team upon discharge, presence of ID consultation during hospitalization. Additional data included ID diagnosis, microbiology cultures, treatment information , total duration of antimicrobial therapy including number of inpatient days , and outpatient days . Further data were collected including frequency of dosing, location of OPAT administration , and outpatient follow-up appointment including with which specialty. Outcome data included unplanned OPAT-related readmission during OPAT, all-cause readmission during OPAT, reason for readmission, infection cure, infection relapse, and OPAT discontinuation due to OPAT related complication. The Charlson comorbidity index was calculated for each patient using collected clinical information. test. Continuous data were evaluated using the Mann-Whitney U test. Predictors of readmission were also evaluated; factors associated with readmission at a significance level of P < .10 in univariate analysis were eligible for testing in a forward, stepwise, multinomial, logistic regression to identify independent predictors of readmission. Data analysis was conducted with SPSS version 25 software .Comparisons of nominal data, such as patient demographics and readmission rates, were evaluated using the Fisher exact test or the \u03c7This study included EMR data for 428 patients: 73 from the preintervention period and 355 from the postintervention period. Table\u00a0P = .003). Reasons for readmissions due to OPAT-related complications included infection recurrence or progression , adverse drug reaction , or line-associated issues . The all-cause unplanned readmission rate decreased after the implementation of the structured program  compared to the preimplementation period ; however, this difference was not statistically significant (P = .165).After implementation of the structured OPAT program, the unplanned readmission rate due to OPAT-related complications significantly decreased from 17.8% (13 of 73) to 7.0% .Clinical cure or treatment failure was documented for 385 (90%) of 428 patients. Among them, clinical cure occurred in 91.4%. Clinical cure increased from 69.8% before the intervention to 94.9% after the intervention  for OPAT patients. Future studies should revisit the independent risk of readmission and vancomycin when monitored by AUC because AUC-based dosing may improve therapeutic target attainments and is now recommended instead of troughs.Beta-lactams and vancomycin were the 2 most-used OPAT drugs before and after the intervention. Patients who received vancomycin were more likely to be readmitted, which may in part be explained by the increased rates of adverse outcomes associated with vancomycin compared to other antimicrobials used in OPAT.In our study, longer OPAT duration was associated with a significant increased risk of unplanned readmission  compared with a relatively shorter OPAT duration . A strength of our study was that we were able to collect the completed duration of outpatient treatment, which may differ from the planned duration set at the time of hospital discharge. This finding reinforces the importance of regular monitoring for adverse events, especially as outpatient treatment length increases. we also found older age to be protective for readmission, supporting recommendations that elderly patients can be safely treated with OPAT. Several studies have found no association between age and readmission. We are uncertain why the younger age group  was more likely to be readmitted; we agree with Keller et al that younger patient populations may be more noncompliant with therapy. A survey of 65 adults discharged from a tertiary-care hospital on OPAT revealed that patient self-reported nonadherence to therapy was associated with younger age  as well as lack of time and low income. In addition to considering patient age and the potential risks of noncompliance, complexity of dosing regimen, social support, and convenience should all be assessed when prescribing OPAT.Similar to Keller et alWe did not find significant associations between readmission and Charlson comorbidity index, socioeconomic factors (utilizing insurance as a proxy), aminoglycoside use, or the presence of multidrug-resistant organisms. Inconsistent conclusions have been drawn regarding the association between readmission and these risk factors. and a follow-up OPAT clinic visit has been associated with a lower 30-day hospital readmission rate compared to no follow-up visit.Our study had several limitations. The retrospective study design may have introduced recall and selection bias. Our data only can capture what is documented in the medical record, and adverse events may have occurred that were not recorded. Our patient population was discharged from an urban, tertiary-care, academic medical center and may not reflect patients in other settings. Moreover, we had a greater number of patients with bone and joint disease which may not be generalizable to other patient populations. Several differences in baseline characteristics of the 2 groups could have affected outcomes, including more bone and joint infections, less culture positivity and culture-directed therapy, and more vancomycin and less \u03b2-lactam therapy in the preintervention group compared to the postintervention group. To mitigate these differences, our multivariate analysis included all factors potentially predictive of readmission that were identified in the univariate analyses. However, we were unable to capture readmissions to other hospitals. We did not include analysis of subsequent readmissions after the first readmission to allow for better comparison with our preintervention cohort and to avoid confounding factors. We defined successful treatment as completion of IV therapy regardless of whether the patient stopped all antibiotics at that point or was transitioned to oral therapy. Some patients may have relapsed with infection while on oral therapy after completion of IV therapy. Finally, we did not assess the impact of our structured OPAT program on outpatient ID follow-up rates or the availability of monitoring laboratories while on OPAT. Multidisciplinary OPAT teams have been associated with increased ID clinic follow-up rates,The implementation of an ID physician and nurse-led OPAT program significantly reduced unplanned OPAT-related hospital readmission rates and improved clinical cure rates, underscoring the importance of multidisciplinary teams. Enrollment in our strengthened OPAT program and older age were protective factors from readmission, whereas vancomycin administration and a longer outpatient treatment duration were associated with an increased risk of readmission."}
{"text": "Rapidly progressive crescentic glomerulonephritis associated with anti-neutrophil cytoplasmic antibodies (ANCA-GN) is a major cause of renal failure. Current immunosuppressive therapies are associated with severe side effects, intensifying the need for new therapeutic strategies. The activation of Mas receptor/Angiotensin-(1-7) axis exerted renoprotection in chronic kidney disease. Here, we investigated the effect of adding the lanthionine-stabilized cyclic form of angiotensin-1-7 [cAng-(1-7)] to cyclophosphamide in a rat model of ANCA-GN. At the onset of proteinuria, Wistar Kyoto rats with ANCA-GN received vehicle or a single bolus of cyclophosphamide, with or without daily cAng-(1-7). Treatment with cAng-(1-7) plus cyclophosphamide reduced proteinuria by 85% vs. vehicle, and by 60% vs. cyclophosphamide, and dramatically limited glomerular crescents to less than 10%. The addition of cAng-(1-7) to cyclophosphamide protected against glomerular inflammation and endothelial rarefaction and restored the normal distribution of parietal epithelial cells. Ultrastructural analysis revealed a preserved GBM, glomerular endothelium and podocyte structure, demonstrating that combination therapy provided an additional layer of renoprotection. This study demonstrates that adding cAng-(1-7) to a partially effective dose of cyclophosphamide arrests the progression of renal disease in rats with ANCA-GN, suggesting that cAng-(1-7) could be a novel clinical approach for sparing immunosuppressants. Pauci-immune crescentic glomerulonephritis is the most common form of rapidly progressive crescentic glomerulonephritis (RPGN), characterized by the lack of significant IgG deposits within glomeruli ,2. It isANCA-vasculitis is a life-threatening disease; its management consists of inducing remission with cyclophosphamide or rituximab, combined with glucocorticoids, followed by maintenance of remission to prevent relapse. However, this immunosuppressive approach is often associated with serious side effects, and 30\u201340% of patients may experience refractory disease or relapses , intensiob/ob mice with type 2 diabetic nephropathy by limiting albuminuria, inflammatory cell infiltration, podocyte injury and glomerular and peritubular capillary rarefaction [Angiotensin-converting enzyme 2 (ACE2)/angiotensin 1-7 (Ang-1-7)/Mas receptor axis is the counter-regulatory pathway of the harmful action of angiotensin II (Ang II), which is one of the major contributors to the progression of chronic kidney disease . ACE2 liefaction . In addiefaction .Here, we investigated the effect of treatment with cAng-(1-7) on top of cyclophosphamide in a rat model of MPO-ANCA-associated GN, which replicates the characteristic features of the disease in humans . CyclophMale Wistar Kyoto (WKY/NCrlBR) rats were purchased from Charles River Laboratories Italia  and maintained in a specific pathogen-free facility with a 12 h dark/12 h light cycle, in a constant temperature room and with free access to a standard diet and water.n = 5\u20136 rats/group) the following: vehicle ; the lanthipeptide cAng-(1-7)  plus cyclophosphamide (single bolus 50 mg/kg i.p.); or cyclophosphamide (50 mg/kg single bolus 50 mg/kg i.p.). Cyclophosphamide was administered in a single bolus on day 21. A group of unimmunized WKY rats (n = 5) served as controls. All animals were followed until day 35, when they were euthanized by CO2 inhalation.ANCA-associated crescentic glomerulonephritis was induced in WYK rats (180\u2013220 g), as previously described . BrieflyTwenty-four-hour urine samples were collected using metabolic cages on days 21, 28, 35 post-immunization. Hematuria was assessed using dipstick Multistix . Urinary protein excretion was measured using the Coomassie method with a Miura one auto-analyzer . Renal function was assessed as blood urea nitrogen (BUN) in whole blood samples using the Reflotron test . Systolic blood pressure was measured with a computerized tail-cuff system in conscious rats .Duboscq-Brazil-fixed, paraffin-embedded renal sections (3 \u03bcm) were stained with periodic acid-Schiff reagent (PAS) , and Masson\u2019s trichrome staining to analyze glomerular and tubular interstitial injury. The presence of at least two layers of proliferating cells in the Bowman\u2019s space of the glomerular cross-sectional area was defined as a crescentic glomerular lesion. The mean percentage of glomeruli with crescents was estimated through the analysis of all glomeruli for each section. Tubular damage (atrophy and dilatation) was graded from 0 to 4+ . The number of tubular casts was counted in an average of 15 fields of interstitial areas (HPF/\u00d7200). All biopsy specimens were examined by two pathologists blind to treatment groups.For immunofluorescence experiments, OCT-frozen kidney sections (3 \u03bcm thick) were fixed with cold acetone and incubated with 1% BSA to block nonspecific sites. For the quantification of neutrophils, CD4+ T cells and endothelial rarefaction, the following primary antibodies were used: mouse anti-rat granulocytes (HIS48) , mouse monoclonal anti-rat CD4 , mouse monoclonal anti-rat RECA-1  followed by the specific Cy3-conjugated secondary antibodies. Claudin-1/NCAM double stainings were performed through incubation with rabbit anti-claudin-1 , mouse monoclonal anti-rat NCAM  followed by the specific FITC- or Cy3-conjugated secondary antibodies. Nuclei were stained with DAPI, and the renal structure with FITC-wheat germ agglutinin (WGA). Negative controls were obtained by omitting primary antibodies on adjacent sections. For the detection of claudin-1 and NCAM stainings, antigen retrieval with citrate buffer was performed to enhance the reactivity of antibodies to antigens. Fluorescence was examined using an inverted confocal laser-scanning microscope . At least 30 glomeruli were examined for each animal, and the extent of altered distribution of claudin-1 in the glomeruli was expressed as a score from 0 to 3 related to the percentage of the glomerular tuft area occupied by the claudin-1-positive cells . HIS48-positive neutrophils in the glomeruli, and CD4+ cells within glomeruli, were counted in an average of 25 glomeruli in each kidney sample and expressed as the average number of cells per glomerulus. RECA-1 staining of capillaries in the glomerular tuft was scored as previously described  as folloED-1 and CD8 stainings were performed using the immunoperoxidase method. Duboscq-Brazil-fixed, 3 \u03bcm paraffin-embedded kidney sections were incubated for 5 min with Peroxidazed 1  to quench endogenous peroxidase, after antigen retrieval in Rodent Decloaker . After blocking for 10 min with Rodent Block R , sections were incubated with mouse monoclonal antibody to monocyte/macrophage ED-1 surface antigen  or mouse anti-rat-CD8  followed by Rat HPR-Polymer  for 30 min. The stainings were visualized by the addition of the betazoid 3,3\u2032diaminobenzidine chromogen kit solutions . Finally, slides were counterstained with Mayer hematoxylin and observed through light microscopy . Negative controls were obtained by omitting the primary antibody on adjacent sections. ED-1-positive monocytes/macrophages and CD8+ T cells within glomeruli were counted in an average of 25 glomeruli and expressed as the average number of cells per glomerulus.Fragments of kidney tissue were fixed overnight in 2.5% glutaraldehyde  in 0.1 M sodium cacodylate buffer (pH 7.4)  and washed repeatedly in the same buffer. After postfixation in 1% OsO4, specimens were dehydrated through ascending grades of alcohol and embedded in Epon resin. Ultrathin sections were stained with Uranyless (Electron Microscopy Sciences) and lead citrate (Electron Microscopy Sciences) and examined using a transmission electron microscopy .p < 0.05.Data are presented as the mean \u00b1 SEM. Data analysis was performed using Prism Software . Comparisons were made using ANOVA with the Tukey post hoc test or two-way ANOVA for repeated measures. Statistical significance was defined as Data on the systemic parameters evaluated at the end of the study (day 35) are reported in ANCA-GN was associated with the development of systemic hypertension, which was reduced significantly by treatment with cAng-(1-7) plus cyclophosphamide, but not with cyclophosphamide alone.Rats with ANCA-GN given vehicle exhibited kidney hypertrophy. A decrease in the kidney weight/body weight ratio was observed in both the combination therapy and the cyclophosphamide group, compared to the vehicle group. Notably, the kidney weights of rats treated with cAng-(1-7) plus cyclophosphamide were similar to those of control rats.p < 0.05). Indeed, the combined therapy afforded for a 60% reduction in proteinuria levels with respect to cyclophosphamide alone  (p < 0.05) lower compared to the vehicle group, but higher than the combination therapy group (p < 0.001) (p < 0.05 vs. cAng-(1-7) + cyclophosphamide)  B. Importvehicle) C. The pevehicle) B. ANCA-G< 0.001) B. These phamide) B,C.According to our previous observations , the glo+ T cells and CD8+ T cells in the glomerular tuft (+ T cells was prevented by the combination of cAng-(1-7) and cyclophosphamide, while CD8+ T cells were numerically limited compared to the vehicle group  plus cyclophosphamide was associated with an amelioration of glomerular endothelial damage. Electron microscopy analysis of glomeruli from rats with ANCA-GN that were on vehicle showed crescentic lesions closely resembling human pathology, as previously described . Fibrin p < 0.05) reduction in the number of proteinaceous casts (1.74 \u00b1 0.16) was observed compared to vehicle; tubular dilatation (score: 0.60 \u00b1 0.19) and atrophy (score: 0.70 \u00b1 0.12) were no different from vehicle. In animals treated with a single bolus of cyclophosphamide, the number of proteinaceous casts averaged 2.37 \u00b1 0.62 (NS vs. vehicle). Next, given the striking effect of the combined treatment on the glomerular capillary wall, we investigated whether a protective effect could be exerted on ultrastructural changes in post-glomerular vessels. The analysis of interstitial areas in rats with ANCA-GN on vehicle revealed swelling and loss of fenestrations of endothelial cells of peritubular capillaries associated with the accumulation of inflammatory cells, mainly monocytes/macrophages and lymphocytes , accompanied by tubular casts  A. In thephocytes B. In thephocytes B.In this study, we provide experimental evidence that daily treatment with the cyclic form of Ang-(1-7) on top of a single injection of cyclophosphamide has a deep renoprotective effect on rats with MPO-ANCA-associated crescentic GN. This therapeutic protocol blocked the rise of proteinuria and ameliorated renal function, dramatically reducing the number of glomeruli affected by crescents and limiting glomerular inflammation. Conversely, the administration of cyclophosphamide alone had partial effects on the course of the disease and pathology.ob/ob mice with diabetic nephropathy, in which the addition of cAng-(1-7) to ACE inhibitor therapy had a superior effect to ACE inhibitor alone, thanks to better preservation of podocytes and an improvement in glomerular endothelial damage [Here, the first remarkable finding is that combined therapy had a stable and pronounced effect on proteinuria, while evidence of a further rise in proteinuria at the end of the study in rats treated with cyclophosphamide alone indicates a tendency for the disease to relapse in the absence of co-administered cAng-(1-7). This may reflect the clinical condition of patients with ANCA-GN who do not completely respond to cyclophosphamide. Interestingly, the antiproteinuric effect of the combined treatment may be reminiscent of the data collected by our group from BTBR l damage . LikewisThe improvement in proteinuria after treating ANCA-GN rats with c-(Ang-1-7) plus cyclophosphamide was associated with a very strong reduction in the number of glomeruli affected by crescents and an increased number of normal glomeruli. The trigger event in crescent formation in ANCA-GN is the development of necrotizing and inflammatory injury to the glomerular capillary wall, leading to gaps in the GBM, extravasation of plasma proteins and red cells, and a severe inflammatory reaction ,24,25.Studies reported a pivotal and interconnected role for neutrophils and monocytes/macrophages in the acute phase of the disease ,27. ANCAIn the glomeruli of animals treated with combination therapy, we also found a reduced number of CD4+ T cells, which may be a consequence of the reduced number of neutrophils recruited to the glomerular tuft in response to therapy. Indeed, MPO released by activated neutrophils is known to act as planted antigen for anti-MPO effector CD4+ T cells  and, in Ultrastructural analysis also revealed that, according to our previous study , rats wiIn conclusion, the addition of cAng-(1-7) to cyclophosphamide enhanced the renoprotective effect of cyclophosphamide alone in an experimental model of ANCA-GN. The combination therapy, but not cyclophosphamide alone, interrupted the vicious cycle of inflammation initiated by neutrophil recruitment and degranulation, resulting in reduced glomerular accumulation of macrophages and CD4-positive T cells . This an"}
{"text": "Arum maculatum is particularly well-known for its potent dung-like volatile organic compound emissions and specialized floral chamber, which traps pollinators\u2014mainly Psychoda phalaenoides and Psychoda grisescens\u2014overnight. However, little is known about the genes underlying the production of many Arum maculatum volatile organic compounds, and their influence on variation in pollinator attraction rates. Therefore, we performed de novo transcriptome sequencing of Arum maculatum appendix and male floret tissue collected during anthesis and postanthesis, from 10 natural populations across Europe. These RNA-seq data were paired with gas chromatography\u2013mass spectrometry analyses of floral scent composition and pollinator data collected from the same inflorescences. Differential expression analyses revealed candidate transcripts in appendix tissue linked to malodourous volatile organic compounds including indole, p-cresol, and 2-heptanone. In addition, we found that terpene synthase expression in male floret tissue during anthesis significantly covaried with sex- and species-specific attraction of Psychoda phalaenoides and Psychoda grisescens. Taken together, our results provide the first insights into molecular mechanisms underlying pollinator attraction patterns in Arum maculatum and highlight floral chamber sesquiterpene (e.g. bicyclogermacrene) synthases as interesting candidate genes for further study.Deceptive pollination often involves volatile organic compound emissions that mislead insects into performing nonrewarding pollination. Among deceptively pollinated plants,  Angiosperms have evolved to produce a wide array of specialized metabolites to mediate their interactions with other organisms and their environment, including volatile organic compounds (VOCs) emitted from flowers, leaves, fruits, and roots. While VOCs represent a relatively small proportion of all plant metabolomic diversity , they plArum maculatum L. (Araceae).Pollinator-mediated selection is known to influence floral morphology  and coloA. maculatum is a common European woodland flower with a long history of study, due to its deceptive pollination strategy which involves olfactory deception through brood-site mimicry  during anthesis which are also present in cow manure  grisescens (Tonnoir) , which is surrounded by a leaf-like bract (the spathe). The spathe surrounds the fertile flowers, creating a basal trap chamber with only a narrow opening at the top of the chamber, which is surrounded by a ring of hair-like sterile flowers. The scent of the floral chamber during anthesis appears to be dominated by 1 sesquiterpene, bicyclogermacrene  and not terpenes, which form a proportionally larger part of the floral scent bouquet in A. maculatum  use these results to identify differentially expressed transcripts putatively related to VOC biosynthesis, and (3) to characterize and compare differential expression in these transcripts across Europe, with a focus on populations with divergent pollinator attraction patterns. We predicted that during anthesis, AAA metabolism would be highly expressed in appendix tissue, while sesquiterpene synthases would be highly expressed in male floret tissue. We further expected to observe differential expression of transcripts associated with AAA metabolism and TPSs in inflorescences with dung-like  vs. sesquiterpene-dominated floral bouquets, respectively. Finally, given that the A. maculatum population in For\u00eat du G\u00e2vre, France, appears to attract almost exclusively P. grisescens  investigate variation in A. maculatum between April and May 2019 , and pumping air over them for 30\u2009min at an air flow rate of 200\u2009ml/min. All samples were analyzed using a gas chromatography (Agilent 7890a) mass spectrometry (Agilent 5975c) system. Desorption of the VOCs was carried out in a thermal desorption unit  with an initial temperature of 50\u00b0C, followed by a ramp of 60\u00b0C/min until 250\u00b0C (followed by a 3.5-min hold time). Before injection, the VOCs were cryo-focused  at \u221280\u00b0C, then released with a ramp of 12\u00b0C/min until 260\u00b0C (hold time 6\u2009min). PTV solvent vent at 14 PSI and 50\u2009ml/min flow was used for injection into a HP5-MS column in constant flow mode (0.9\u2009ml/min) with helium as the carrier gas. The Mass Selective Detector (MSD) transfer line was set at 280\u00b0C. Scan mode was used for the MS acquisition with a solvent delay (1\u2009min), over a mass range of 33\u2013300 m/z; MS source and quadrupole temperatures were set at 230 and 150\u00b0C, respectively. Electron ionization (70\u2009eV) was used. Major ions were recorded for each integrated peak using Agilent Chemstation, and compound identifications were derived from NIST 2.3 (library v.17) and published Kovats retention indices; all names used in our analyses should therefore be considered hypotheses. Appendix temperatures were recorded at the time of VOC sampling using FLIR thermographic imaging, and ambient air temperature was recorded using a thermistor.Before collecting tissue samples, we noninvasively sampled the dynamic headspace VOCs emitted by each Immediately following VOC sampling (i.e. during anthesis), we used sterile scalpel blades to collect 1-mm thick slices of appendix and/or male floret tissue. As a control, male floret tissue samples were also collected from inflorescences the following morning, after pollinator attraction had ended. All samples were individually preserved in RNAlater (Thermo Fischer Scientific) and stored at \u221280\u00b0C until extraction. After collecting each during-anthesis male floret tissue sample, we closed and sealed the small window we cut in floral chamber, so that pollinator trapping was not affected. During the morning following anthesis, we collected all insects trapped within inflorescences, preserved them in 70% ethanol and identified them to at least the suborder level. Psychodidae were further identified to species level (including sex) following taxonomic descriptions and illustrations .All samples and tissue types were extracted using identical methods; approximately 100\u2009mg of tissue was rinsed in sterile RNase-free water, flash frozen in liquid nitrogen, powdered in a QIAGEN TissueLyser II, and immediately extracted using a QIAGEN RNeasy Plant Mini Kit, following the manufacturer\u2019s protocol. Extraction quality and concentration were verified using an Agilent Fragment Analyzer. Three Illumina TruSeq Stranded mRNA polyA libraries were prepared from these samples, following the manufacturer\u2019s recommended protocol. The first library contained the appendix RNA samples, and the second and third each contained a mix of during anthesis and postanthesis male floret tissue samples from all populations. Each library was then sequenced on its own lane  using an Illumina HiSeq 4000 (150-bp paired-end sequencing) at the Lausanne Genomic Technologies Facility (Switzerland).FilterUncorrectablePEfastq.py (https://github.com/harvardinformatics/TranscriptomeAssemblyTools).We performed an initial quality filtering of all raw read files using fastp  to removA. maculatum transcriptome  using Trinity v2.11.0  to identify candidate coding regions and searches the OrthoDB, Pfam-A, Rfam, and uniref90 protein databases for transcript annotations, with an E-value threshold of 1\u2009\u00d7\u200910\u22125. Unannotated transcripts were not removed from the final A. maculatum reference transcriptome. We also uploaded the EvidentialGene output to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server  term analysis with the R package TopGO  and male floret (n\u2009=\u200916) tissue collected during anthesis . Second, we compared male floret tissue collected during anthesis (n\u2009=\u200914) against their paired control samples from the morning following anthesis (n\u2009=\u200914). Paired control samples were not collected for 2 inflorescences from Sokobanja, Serbia; these individuals were excluded from this analysis. Finally, we compared transcript expression in during-anthesis male floret tissue sampled in the For\u00eat du G\u00e2vre population against all other populations, to identify transcripts putatively linked to this population\u2019s exclusive attraction of P. grisescens. To account for some of the effects of isolation by distance, we further split this analysis along the 2 main neutral genetic clusters (n\u2009=\u20095) against during-anthesis male floret tissue from (1) France, Switzerland, and Italy (n\u2009=\u20097) and (2) Serbia (n\u2009=\u20094).We performed 3 sets of differential expression analyses, each addressing one of the main aims of our study. Our first comparison was between appendix  our complete reference transcriptome and (2) sets of significantly differentially expressed genes. Since the genes responsible for the production of 2-heptanone and p-cresol in A. maculatum are unclear and unknown respectively, we also used homology searches to identify additional candidate genes. Here, we performed BLASTp searches of genes known involved in the biosynthesis of the aforementioned VOCs in other plants and bacteria, against a database of all of our Transdecoder predicted peptide sequences using, with an e-value cutoff of 1\u2009\u00d7\u200910\u22125. After all candidate transcripts were identified, we visualized variation in their average expression across all tissue types and populations using the R package pheatmap , performed a multiple sequence alignment of these sequences using Clustal Omega . Then, weMemoire , and adeBetween 27,933,352 and 66,825,898  paired-end reads from individual tissue samples passed all quality filtering steps . These wA total of 49,779 transcripts remained in our expression matrix following prefiltering in DESeq2 (i.e. >0.75 CPM in at least 7 samples). Principal components analysis of filtered transcript expression  revealedP-values <0.05; log2fold change \u00b11), after controlling for population effects. Furthermore, we found that VOC biosynthetic activity is significantly elevated in A. maculatum appendix tissue during anthesis. Many major biosynthetic pathways  were significantly enriched in the appendix tissue during anthesis, whereas transcripts related to pollen production were significantly enriched in male floret tissue  revealed 8,683 transcripts with significantly greater expression in appendix tissue and 6,581 transcripts with significantly greater expression in male floret tissue . While tryptophan (i.e. indole) biosynthesis was elevated in male florets during anthesis, we did not identify increased expression of other putative VOC biosynthetic pathways  and (2) 327 and 175 transcripts were, respectively, differentially expressed in For\u00eat du G\u00e2vre vs. Serbian populations . However, no transcripts putatively linked to VOC biosynthesis were identified among these transcripts.Our third and final set of differential expression analyses, comparing male floret tissue during anthesis among populations, revealed that (1) 84 and 9 transcripts were, respectively, differentially expressed in For\u00eat du G\u00e2vre vs. all other French, Swiss, and northern Italian populations  subunit, which catalyzes the conversion of indole-3-glycerolphosphate to indole.Consistent with recent large-scale surveys of al scent . We furtr\u2009=\u20090.988, P <\u20090.001). This enzyme may further catalyze the conversion of 4-hydroxyphenyllactate to p-coumaric acid. However, we did not identify any homologs of bacterial proteins linked to the production of 4-hydroxyphenylacetate and/or p-cresol  and hydroxyphenylpyruvate reductase (HPPR). Pearson correlation tests further identified several transcripts correlated with HPPR expression in appendix tissue during anthesis ; notablyp-coumaric acid\u2014appear to be produced by A. maculatum, transcripts responsible for the production of benzenoid and phenylpropanoid VOCs generally appeared to be absent or weakly expressed in A. maculatum. Finally, we identified putative homologs of methylketone synthases (MKS1 and MKS2), which are known to be involved in 2-heptanone biosynthesis  compounds\u2014particularly A. maculatum transcripts putatively encoding all proteins in both the mevalonate and MEP pathways. During anthesis, the MEP pathway appears to be more highly expressed than the mevalonate pathway, particularly in appendix tissue (d-xylulose-5-phosphate synthase (DXS), which was highly expressed in appendix tissue, also featured the greatest isoform diversity among all terpene backbone synthesis genes. Furthermore, we identified 108 putative TPS transcripts expressed in appendix and male floret tissue during anthesis. A phylogeny of all putative A. maculatum TPSs is given in We identified x tissue . The firA. maculatum VOCs (e.g. humulenes and germacrenes); others, such as 2-methylisoborneol (2-MIB) synthase, catalyze the production of terpenes not previously described in A. maculatum. However, in the case of the latter, the transcript we identified likely encodes a novel TPS, given that we identified an uncharacterized Colocasia esculenta (Araceae) gene, which shares 92.5% identity with our transcript, as opposed to approximately 22.8% identity with the bacterial 2-MIB synthase  in appendix tissue samples emitting high quantities of an unnamed sesquiterpene with a nonpolar Kovats Retention Index of 1681, which is known to be a strong predictor of P. grisescens attraction . However, we did identify significant concordance between putative TPS expression in male floret tissue and Psychodidae pollinator composition trapped within inflorescences ; the first coinertia axis split TPS expression based on species , while the second coinertia axis split TPS expression based on sex  via a pathway homologous to tyrosine degradation in bacteria  has also been reported in p-coumaric acid from phenylalanine, where also abundantly expressed in the appendix during anthesis. However, downstream proteins producing common FBP VOCs  would further modify p-coumaric acid into precursors for the aforementioned FBP VOCs, but this gene was expressed at relatively low levels in appendix tissue. Interestingly, experimental knock-outs of C3H in Petunia \u00d7 hybrida have demonstrated that downregulation of this gene also leads to unexpected production of p-cresol  and cinnamate 4-hydroxylase (C4H), which produce A. maculatum VOC with a \u201cfermented\u201d scent. In this study, we were able to identify several methylketone synthase homologs (MKS1 and MKS2), which produce 2-heptanone and related compounds in tomato plants , which can further modify volatile terpenes through oxidation, methylation, or acylation  homolog appears to be correlated with the production of at least 1 unnamed A. maculatum sesquiterpene (Kovats RI 1681). This unnamed sesquiterpene was the single strongest predictor of P. grisescens attraction in a large-scale survey of A. maculatum pollinators  to attract females . Such terpene emissions in the floral chamber could stimulate the movement of trapped Psychodidae over the male and female florets, aiding in pollination and pollen dispersal; however, further experiments are needed to test these hypotheses. A recent gas chromatography\u2013electroantennography , and the aforementioned trimethyltridecatetraene synthase homolog, which may produce an unnamed sesquiterpene with a Kovats Retention Index of 1681 (covarying with P. grisescens). Notably, these same compounds were highlighted as key predictors of pollinator species trapped by A. maculatum inflorescences in a random forest analysis with a larger sample size . Our results highlight a need for further focused study of VOCs within the trap chamber, given that specific sesquiterpenes appear to be an important aspect of this highly specialized lure-and-trap pollination system.Our data provide a deeper understanding of the relationships between transcript expression, floral scent, and pollinator attraction in https://www.ncbi.nlm.nih.gov/bioproject/PRJNA856436). Additional data, including the files and R code needed to reproduce all analyses and figures, have been archived in the Zenodo repository 6806366 (https://doi.org/10.5281/zenodo.6806365).G3 online.jkac175_Appendix_S1Click here for additional data file.jkac175_Appendix_S2Click here for additional data file."}
{"text": "The use of tumor markers aids in the early detection of cancer recurrence and prognosis. There is a hope that they might also be useful in screening tests for the early detection of cancer. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for \u201cideal\u201d tumor markers, digging very deep into plasma proteomes. The situation in this area can be improved in two ways\u2014by attempting to find an ideal single tumor marker or by generating panels of different markers. In both cases, proteomics certainly plays a major role. There is a line of evidence that the most abundant, so-called \u201cclassical plasma proteins\u201d, may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but also proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected bibliographic information about the connection of cancers with levels of \u201cclassical plasma proteins\u201d. Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step, similar profiles representing protein perturbations in plasma produced in the case of different cancers will be generated. Additionally, based on this information, different test systems can be developed. In a broad sense, tumor biomarkers are components that are either produced directly or indirectly because of a tumor. Moreover, these biomarkers can be common cellular products that are overproduced by cancer cells or the products of genes that are expressed only during malignant transformation. Thus, a tumor marker that is present in significant quantities indicates the presence of cancer. The marker can be present inside the tumor or enter the bloodstream ,2. This https://www.hupo.org/plasma-proteome-project accessed on 10 September 2022). Now, this initiative has achieved great success in plasma protein analysis (http://plasmaproteomedatabase.org/index.html accessed on 10 September 2022)  th th265] ter 2022) . Accordihttps://glyconnect.expasy.org/browser/proteins/780 accessed on 10 September 2022). In SWISS-2DPAGE, alpha-1-B glycoprotein is represented as a chain of six spots  [The two-dimensional electrophoresis pattern of alpha-1-B glycoprotein represents a long chain of spots in the pI-range 4\u20136 and Mw ~ 54,000  that is https://world-2dpage.expasy.org/ accessed on 10 September 2022 [https://glygen.org/protein/P01023#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of alpha-2-M represents a chain of heavy Mw spots (mostly in the pI-range 5.8\u20136.3) . This pahttps://glygen.org/protein/P08697#glycosylation accessed on 10 September 2022). In SWISS-2DPAGE, alpha-2-antiplasmin is represented as a chain of seven spots .The two-dimensional electrophoresis pattern of \u03b12AP represents a chain of spots in the pI-range 4\u20136 with Mw ~ 50,000  that is https://world-2dpage.expasy.org/ accessed on 10 September 2022 and has a characteristic for multiple glycosylation profiles, where acidic spots have higher Mw [https://www.uniprot.org/uniprot/P02750 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of LRG1 represents a chain of spots in the pI-range 3.5\u20135.0 with Mw ~ 40,000\u201350,000 . This pahttps://glygen.org/protein/P01011#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ACT represents a chain of spots in the pI-range 4.0\u20135.0 and Mw 50\u201360,000 . This pahttps://glygen.org/protein/Q15848#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of adiponectin represents a chain of spots in the pI-range 5.0\u20135.5 and Mw 26,000 . There aThe two-dimensional electrophoresis pattern of adiponectin represents a chain of spots in the pI-range 4.5\u20136.0 and Mw ~ 70,000 . There ahttps://glygen.org/protein/P02768#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of albumin represents a chain of spots in the pI-range 5.5\u20136.5 and Mw ~ 70,000 . Its pathttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of AMBP represents two groups of spots in the pI-range 4.0\u20136.5: alpha-1-microglobulin with Mw ~ 26,000 and inter-alpha-trypsin inhibitor light chain/bikunin that is assembled in a high Mw complex by a chondroitin-like glycosaminoglycan (GAG) cross-linking with Mw 120,000 . In the https://glygen.org/protein/P01019#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of angiotensinogen represents a chain of spots in the pI-range 4.0\u20136.4 and Mw ~ 50,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ATIII represents a chain of eight spots in the pI-range 4.5\u20136.0 and Mw ~ 50,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoA-I represents a chain of spots in the pI-range 4.5\u20136.5 and Mw ~ 26,000 . In the The two-dimensional electrophoresis pattern of apoA-II represents a chain of spots in the pI-range 4.5\u20136.0 and Mw ~ 9000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoA-IV represents a chain of spots in the pI-range 4.5\u20136.0 and Mw ~ 40,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo B-100 represents a long chain of spots in the pI-range 3.5\u20137.5 and heavy Mw > 120,000 . Apo B-1https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-CI represents a chain of spots in the pI-range 7.8\u20138.5 and Mw ~ 6000 . This prhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (3 O-linked glycans at 4 sites) (https://www.glygen.org/protein/P02655 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-CII represents a chain of spots in the pI-range 4.8\u20135.2 and Mw ~ 8000 . In the https://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (4 O-linked glycans at 1 site) (https://www.glygen.org/protein/P02655 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoC-III represents a chain of spots in the pI-range 3.8\u20136.1 and Mw ~ 9000 . In the https://www.uniprot.org/uniprotkb/P05090/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoD represents an unusual set of spots in the pI-range 3.5\u20136.5 and Mw from ~15,000 to ~26,000 and 80,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoE represents a chain of spots in the pI-range 4.5\u20136.5 and Mw ~ 35,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-F represents a set of spots in the pI-range 3.5\u20134.2 and Mw from ~15,000 to ~32,000 . ApoF cahttps://glygen.org/protein/P02749#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-H represents a chain of spots in the pI-range 6.2\u20138.4 and Mw ~ 52,000 , which ihttps://glygen.org/protein/O95445#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoM represents a chain of spots in the pI-range 4.5\u20136.5 and Mw ~ 22,000 . There aThe results of several studies suggest that changes in the complement system can not only promote an antitumor response but can also influence tumor development through proliferation, survival, angiogenesis, and invasiveness ,268. Thehttps://glygen.org/protein/P00736#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern represents only a chain of spots of the complement C1r subcomponent in the pI-range 4.5\u20136.2 and Mw ~ 80,000  that corhttps://glygen.org/protein/P09871#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis-pattern represents a chain of five spots of C1s in the pI-range from 4.0 to 4.9 and Mw ~ 80,000 . The clehttps://glygen.org/protein/P02747#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of C1q represents a long horizontal chain of spots in the pI-range 3.0\u20139.5 with Mw ~ 23,000 and a vertical chain of heavy complexes  with pI ~ 9.0 . It was https://glygen.org/protein/P05156#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor I represents the chains of many spots in the pI-range 4.5\u20136.8 from Mw ~ 64,000 (complement factor I) to Mw ~ 30,000 (the complement factor I heavy and light chains) . In the https://glygen.org/protein/P00751#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor B represents the chains of spots in the pI-range 4.5\u20136.8 with Mw ~ 90,000 . The clehttps://www.phosphosite.org accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor D represents two spots  . The prohttps://glygen.org/protein/P08603#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor D represents a long chain of spots in the pI-range 5.5\u20137 with Mw ~ 140,000 . It was https://glygen.org/protein/P06681#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C2 represents a chain of spots in the pI-range 6\u20137 with Mw ~ 80,000 . It was https://glygen.org/protein/P01024#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C3 represents a cluster of spots in the pI-range 3.5\u20137.5 and Mw from ~30,000 to 180,000 . In the https://glygen.org/protein/P0C0L4#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C4-A represents a wide cluster of spots in the pI-range 3.0\u201310.0 and Mw from ~25,000 to 190,000 . There ahttps://glygen.org/protein/P0C0L5#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C4-B represents a wide cluster of spots in the pI-range 3.0\u201310.0 and Mw from ~35,000 to 19,0000 . In the https://glygen.org/protein/P01031#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C4-B represents a wide cluster of spots in the pI-range 5.0\u20136.8 and Mw ~ 70,000\u2013190,000 . It was https://glygen.org/protein/P13671#glycosylation accessed on 10 September 2022), and 5 sites of phosphorylation (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C6 represents a chain of spots in the pI-range 4.0\u20136.5 and Mw ~ 100,000 . It was https://glygen.org/protein/P10643#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C7 represents a chain of spots in the pI-range 4.5\u20136.5 and Mw ~ 100,000 . It was https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C9 represents a chain of spots in the pI-range 4.5\u20135.5 and Mw ~ 60,000 . The prohttps://glygen.org/protein/P00915#glycosylation accessed on 10 September 2022), phosphorylation (11 sites), acetylation (5 sites), and ubiquitylation (2 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CAB represents a chain of spots in the pI-range 5\u20137 and Mw ~ 28,000 . It was https://glygen.org/protein/P08185#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CBG represents a chain of spots in the pI-range 3.7\u20135.1 and Mw ~ 50,000 . It was https://glygen.org/protein/P08185#glycosylation accessed on 10 September 2022.The two-dimensional electrophoresis pattern of CBPN represents a chain of spots in the pI-range 4.5\u20137 and Mw ~ 50,000 . It was https://glygen.org/protein/P08571#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CD14 represents a chain of spots in the pI-range 4.5\u20135.8 and Mw ~ 40,000 . It was https://glygen.org/protein/P00450#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ceruloplasmin represents a chain of spots in the pI-range 4.0\u20136.2 and Mw ~ 120,000 . In the https://glygen.org/protein/P06276#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of cholinesterase represents a chain of five spots in the pI-range 4.5\u20135.2 and Mw ~ 65,000 . There ahttps://glygen.org/protein/P10909#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ceruloplasmin represents a chain of 18 spots in the pI-range 4.5\u20136.5 and Mw ~ 35,000 . In the https://glygen.org/protein/Q96KN2#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of beta-Ala-His dipeptidase represents two spots around pI 5.0 and Mw ~ 54,000 . It was https://www.glygen.org/protein/P22792 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of carboxypeptidase N subunit 2 represents nine spots with pI 3.5\u20135.5 and Mw ~ 65,000 . It is khttps://www.uniprot.org/uniprotkb/P02741/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CRP represents a single spot  . In the https://www.glygen.org/protein/Q16610 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ECM1 represents a spot  . It was https://www.glygen.org/protein/P23142 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of FIBL-1 represents a chain of four spots  . It was https://www.glygen.org/protein/O75636 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of ficolin-3 represents a chain of five spots  . It was https://www.glygen.org/protein/P02765 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of fetuin-A represents a set of proteoforms  . In the https://www.glygen.org/protein/Q9UGM5 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of fetuin-B represents a chain of proteoforms  . The prohttps://www.glygen.org/protein/P02671 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of FBA represents several sets of chains with pI 5.0\u20137.5  . In the https://www.glygen.org/protein/P02675 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of FBB represents a chain of spots  . In the https://www.glygen.org/protein/P02679 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of FGG represents a chain of spots with pI 4.5\u20137  . In the https://www.glygen.org/protein/P02751 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of fibronectin represents a chain of spots with pI 4.5\u20136.7  . The prohttps://www.phosphosite.org accessed on 10 September 2022).The two-dimensional electrophoresis pattern of gelsolin represents a chain of spots with pI 4.5\u20136.5  . The prohttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 4.9\u20136.9  . It is phttps://www.glygen.org/protein/P69905 accessed on 10 September 2022), phosphorylated (17 sites), acetylated (4 sites), and ubiquitinated (8 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 7.5\u20139  . In the https://www.glygen.org/protein/P68871 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots with pI 6.5\u20136.9  . In the https://www.glygen.org/protein/P02790 accessed on 10 September 2022)).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 5\u20136.9  . In the https://www.glygen.org/protein/P05546 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots with pI 4.9\u20136.5  . The prohttps://www.glygen.org/protein/P00738 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of Hp represents ~16 spots of beta chains with pI 4.8\u20136.0  and 3 spots of alpha 2 chain . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 4.6\u20136.5  . The prohttps://www.glygen.org/protein/P04196 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots with pI 4.5\u20137.8  and spots around pI/Mw: 5.5/53,000 . In the https://www.glygen.org/protein/P05155 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a long chain of spots with pI 3.2\u20135.2 . The protein is heavily glycosylated  (https://www.phosphosite.org/ accessed on 10 September 2022).In our experiments, 2DE patterns of these proteins are presented by the chains of the precursor proteoforms and the mature ITIH1 . The prohttps://www.glygen.org/protein/P29622 accessed on 10 September 2022, https://www.phosphosite.org/ accessed on 10 September 2022.In our experiments, the 2DE pattern of this protein is presented as a cluster of proteoforms around pI/Mw: 6\u20137/40\u2013120,000 . The prohttps://www.glygen.org/protein/P01042 accessed on 10 September 2022), phosphorylated, acetylated, and ubiquitinated (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents multiple spots  . In the https://www.glygen.org/protein/P04180 accessed on 10 September 2022), phosphorylated, and ubiquitinated (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents three spots  . The prohttps://www.uniprot.org/uniprotkb/P51884/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents chains of spots  . The proThe two-dimensional electrophoresis pattern of this protein represents just a single spot  .https://www.uniprot.org/uniprotkb/P36955/entry accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots  . The prohttps://www.uniprot.org/uniprotkb/Q96PD5/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . The prohttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . The prohttps://www.uniprot.org/uniprotkb/P00747/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents two chains of multiple spots  and  . In the https://www.uniprot.org/uniprotkb/P27169/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the The two-dimensional electrophoresis pattern of this protein represents two spots  . The prohttps://www.uniprot.org/uniprotkb/P07225/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . The prohttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents two spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated  (https://www.glygen.org/protein/P02743 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022) and glycosylated .The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (1 O-linked glycan) (https://www.uniprot.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents one spot  . Howeverhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (1 O-linked glycan) (https://www.uniprot.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of S100-A9 represents one spot  . This prhttps://www.uniprot.org/uniprotkb/P05452/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . This prhttps://www.uniprot.org/uniprotkb/P00734/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022). The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the Tumorigenesis leads to multiple variations in the human plasma proteome that can be dynamic and alterable during the progress of the disease. Practically all major, so-called \u201cclassical\u201d, plasma proteins change abundances or PTMs. The majority of these proteins are secreted by the liver, so it could be anticipated to see these changes only in the case of liver cancer. However, they can be observed with other tumors as well as cancer induces disturbances in the blood homeostasis that is supported by \u201cclassical plasma proteins\u201d. It follows that it is possible to search the specific/unspecific ways of tumor prediction not only through the detection of products of the tumor but also by analyzing the changes in \u201cclassic plasma proteins\u201d. It is relevant to mention that plasma analysis by a very different approach, differential scanning calorimetry (DSC), can give us a hint. Typically, DSC is used to determine the partial heat capacity of macromolecules as a function of temperature, from which their structural stability during thermal denaturation can be assessed. The method is very sensitive and allows precise determination of thermally-induced conformational transitions of proteins present in plasma. There are already quite a few publications showing that DSC can be used to distinguish between normal and cancerous plasma samples ,274. Morhttps://vermillion.com/ova-products accessed on 10 September 2022). The observation of enhanced levels of clusterin, ITIH4, antithrombin-III, and C1RL in sera of endometrial cancer patients allowed a mathematical model to be built to detect cancer samples [It follows that there is a possibility of building test systems based on these major  proteins. What is important is that many examples of such systems have been introduced already. For example, the relationship between inflammation and clinical outcome is described using the Modified Glasgow Prognostic Scale (mGPS), which includes levels of C-reactive protein (CRP) and albumin . The com samples . Accordi samples . The dec samples , NSCLC [ samples , nasopha samples , esophag samples , and BC  samples , but it  samples . The lev samples ,87 but i samples ,89. A si samples .Another aspect that should be considered is the appearance of proteoforms produced by genetic polymorphisms, alternative splicing, PTMs, etc. These events change the charge (pI) and the weight (Mw) of the protein. Because of that, the experimental pI/Mw of the proteins can be different from the theoretical ones. This leads to the production of sets of proteoforms that in our case are detected as 2DE patterns. There is a belief that some 2DE patterns can be different between norm and cancer and could be used as specific biomarkers. Thus far, there are not many such examples, but progress in proteomics methods should improve the situation ,278. Prohttp://2de-pattern.pnpi.nrcki.ru/ accessed on 10 September 2022 [Our aim is to build a comprehensive proteoform database containing norm and cancer samples ber 2022 . GlioblaThe pooled human plasma was from healthy male donors (age 20\u201347 years) ,279. Depv/v) ampholytes, pH 3\u201310, protease inhibitor cocktail) and then with 100 \u03bcL of rehydrating buffer  buffer, pH 3\u201311 NL, 0.001% bromophenol blue). Immobiline DryStrip 3\u201311 NL (7 cm) was passively rehydrated by this solution for 4 h at 4 \u00b0C. IEF was run on Hoefer\u2122 IEF100 . After IEF, strips were incubated 10 min in the equilibration solution  glycerol, 1% DTT), following in the same solution with 5% IAM instead of DTT. The strips were sealed with a hot solution of 0.5% agarose prepared in electrode buffer  on top of the polyacrylamide gel (14%), and run in the second direction [2) was shredded and treated with trypsin. Tryptic peptides were eluted from the gel by extraction solution (5% (v/v) ACN, 5% (v/v) formic acid) and dried in Speed Vac. In the case of a semi-virtual 2DE, the 18-cm Immobiline DryStrip 3\u201311 NL was cut into 36 equal sections after IEF. For complete reduction, 300 \u03bcL of 3 mM DTT and 100 mM ammonium bicarbonate were added to each section and incubated at 50 \u00b0C for 15 min. For alkylation, 20 \u03bcL of 100 mM IAM were added and samples were incubated in the dark at r.t. for 15 min. The peptides were eluted with 60% acetonitrile and 0.1% TFA and dried in Speed Vac.The detailed process was described previously . In shorirection . Gels stv/v) formic acid. Tandem mass spectrometry analysis was conducted in duplicate on an Orbitrap Q-Exactive mass spectrometer . The data were analyzed by Mascot \u201c2.4.1\u201d  or SearchGui [A detailed procedure was described previously ,280. PepearchGui  using thOnly 100% confident results of protein identification were selected. Two unique peptides per protein were required for all protein identifications. Exponentially modified PAI (emPAI), the exponential form of protein abundance index (PAI) defined as the number of identified peptides divided by the number of theoretically observable tryptic peptides for each protein, was used to estimate protein abundance .w/v) BSA) or rabbit polyclonal anti-Hp . Secondary goat anti-mouse immunoglobulins G labeled by horseradish peroxidase  were used in TBS containing 3% (w/v) nonfat dry milk (1/5000 dilution). The reaction was developed using ECL  and X-ray film (Amersham Hyper film ECL).Plasma proteins (0.5 mg) were run by 2DE . Proteins were transferred  from the gel onto PVDF membrane  using two sheets of thick paper , saturated with 48 mM Tris, 39 mM glycine, 0.037% SDS, 20% ethanol. The membrane was treated following a protocol of Blue Dry Western  and treahttp://plasmaproteomedatabase.org/index.html accessed on 10 September 2022 [For now, proteomics is collecting big data about the human plasma proteome ber 2022 . These dber 2022 ,285. In"}
{"text": "Total hip arthroplasty is a widely performed surgical procedure, which enables patients to regain mobility, alleviates pain, and improves overall quality of life. Periarticular multimodal drug infiltration (PAI) is increasingly being used as an effective postoperative pain management, decreasing the systemic consumption of opioids. Extensive postoperative skin necrosis without a deep joint infection as a complication of total hip arthroplasty with PAI has not yet been described.A 71-year-old patient who underwent total hip arthroplasty of the right hip for primary osteoarthritis through the Direct Anterior Approach presented postoperatively a large area of necrotic skin at the incision. Joint infection was excluded. An extensive debridement was performed and the tissue defect was reconstructed by a pedicled anterolateral thigh flap. The skin maintained a satisfactory appearance at 1 year postoperatively, and the hip was pain-free with restored ranges of motion. The patient was able to walk with no support and without limitation.We address the possible risk factors, discuss the use of epinephrine in PAI and explore possible treatment options for such a complication.The online version contains supplementary material available at 10.1186/s12891-023-06643-z. Total hip arthroplasty (THA) is a common surgical treatment which provides reliable functional outcomes in patients suffering from hip osteoarthritis. Effective postoperative pain control is most important to enable prompt rehabilitation, reduce postoperative complications, and reduce the length of hospital stay. Pain control is achieved by several methods, including systemic treatments with Non-Steroidal Anti-inflammatory Drugs, opioids, and regional treatments such as epidural anaesthesia, peripheral nerve blocks, and periarticular multimodal drug infiltration (PAI). Up until recently, the efficacy of PAI in pain relief following total hip arthroplasty during rest and activity, and its impact on the length of hospital stay and total opioid consumption was not fully established. A recent meta-analysis concluded that PAI showed better pain relief and less opioid consumption and is safe and effective following total hip arthroplasty . HoweverThis case is presented in accordance with the 2013 CARE checklist.A 71-year-old male, non-smoker, with a previous history of cardiac ischemia was admitted for a THA. The patient was positioned in the supine position on a traction table. Surgery was performed under general anaesthesia through the Direct Anterior Approach. The patient received a fully hydroxyapatite-coated uncemented stem , an uncemented cup , and a ceramic-highly-crosslinked polyethylene bearing surface Fig.\u00a0. The patConsidering the presence of a large skin necrosis and the high risk of infection of the underlying hip joint, we decided to perform a single-staged surgical debridement and reconstruction of the skin and soft tissue defect using a pedicled fasciocutaneous anterolateral thigh flap (ALT) of about 15\u2009\u00d7\u20096\u00a0cm Fig.\u00a0; video. Wound complications after joint replacement can result in significant morbidity to the patient with increased risk on infections, further surgeries, increased recovery period and hospital stay . Wound cInterestingly, in our patient, the skin necrosis was first noted on day 8 postoperatively. The dressings had been changed at day 3 and day 6 and a clean wound with no oozing or redness was described. In other reported cases of skin necrosis following local anaesthetics infiltration with epinephrine, skin changes only appeared around day 5. This difference could be due to the site of injection, which was the penoscrotal junction in one case , and theBased on expertise from our department of infectious diseases, we excluded the possibility of acute periprosthetic joint infection as the bacterial culture of synovial fluid was negative with a WBC count of 4650 cells/\u00b5l (<\u200910 000 cells/\u00b5l) in the joint fluid . A broadMalnutrition compromises wound healing as decreased protein reserves affects collagen synthesis and fibroblast proliferation and decreased albumin levels promotes tissue oedema formation . It has PAI is a multimodal drug solution comprised of three active agents, long-acting local anaesthetics such as ropivacaine and bupivacaine, nonsteroidal anti-inflammatory drugs (NSAIDs) and epinephrine. Local anaesthetics block voltage-gated sodium channels in nociceptors, thereby decreasing pain transmission. Injection of NSAIDs such as ketorolac reduces pain by inhibiting the production of proinflammatory mediators such as prostaglandins. Epinephrine is a nonspecific alpha- and beta-adrenergic agonist, widely used in local anaesthesia with the rationale that through local vasoconstriction it decreases absorption thereby increasing the duration of analgesia. While some studies have reported that the inclusion of epinephrine in PAI prolongs the duration of local anaesthetics, other studies have reported no significant increase in the duration when epinephrine is added to ropivacaine due to the intrinsic vasoconstrictive effect of ropivacaine . A recenOther possible causes for wound complications include the use of retractors and their pressure effects on skin and soft tissues. However, we have routinely performed total hip arthroplasty through the Direct Anterior Approach (DAA) for more than 12 years (approx. 400 hips/year), and we have not observed this issue previously . Also, gThe DAA has dramatically gained in popularity over the last 15 years because it is a muscle sparing.approach, with a potential reduction of postoperative pain and length of stay. As the skin is thinner in the anterior aspect of the hip, the risks for wound complications might be increased. However, there have been several studies, reviews and meta-analyses which compared early outcomes of total hip replacement through the main different surgical approaches, and reported similar rates of wound complications  16]. Wi. Wi16]. In this case, we performed surgical debridement and reconstruction of the skin and soft tissue defect using a pedicled fasciocutaneous anterolateral thigh flap. This method is a reliable and versatile option for loco-regional reconstruction due to the large skin and soft tissue availability, reliable blood supply, long vascular pedicle and wide arc of rotation . FurtherTo the best of our knowledge, this case represents the first evidence of a non-infectious and non-warfarin related extensive skin necrosis as a complication following total hip arthroplasty. In such cases, further testing should be performed to rule out possible causes such as infection and malnutrition. We achieved a good outcome with extensive debridement and wound reconstruction using a pedicled fasciocutaenous anterolateral thigh flap. As the exact cause of skin necrosis cannot be determined from a single case report, larger studies are required. Additionally, further clinical trials are needed to establish the risk and benefits of using epinephrine in PAI. Meanwhile, we decided to change our practice and not to use epinephrine anymore.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2"}
{"text": "Nature Communications 10.1038/s41467-023-38746-5, published online 25 May 2023Correction to: The Supplementary Information associated with this Article contained text highlights corresponding to tracked changes that were made during revision. The HTML has been updated to include the correct version of the\u00a0Updated Supplementary InformationIncorrect Supplementary Information"}
{"text": "Nature Communications 10.1038/s41467-023-38087-3, published online 22 April 2023Correction to: The original version of the\u00a0Updated Supplementary Information"}
{"text": "Nature Communications 10.1038/s41467-022-35768-3, published online 19 January 2023Correction to: Updated Supplementary Information"}
{"text": "Proteinuria and glomerular endotheliosis are characteristics of glomerular injury in preeclampsia, a hypertensive disorder in human pregnancy. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are biomarkers of acute/chronic renal tubule injury. To determine if tubule injury occurs in preeclampsia, we determined maternal plasma and urine NGAL and KIM-1 levels and evaluated NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia.n = 100), preeclampsia (n = 83), and pregnancy complicated with chronic hypertension (n = 20). Plasma and urine levels of NGAL and KIM-1 were measured by ELISA. Kidney biopsy tissue sections from patients with preeclampsia (n = 5) were obtained from Pathology Archives and processed to determine NGAL and KIM-1 expression by immunostaining and high kidney solution images were assessed by electron microscopy (EM).Prenatal and postpartum maternal blood and urinary samples were collected from three groups of pregnant women: normal pregnancy (p < 0.01. In normal pregnancy, both plasma and urine levels of NGAL and KIM-1 at 24\u201348 h after delivery and 6\u20138 weeks postpartum were relatively comparable to that of antenatal levels. In preeclampsia, urine, but not plasma, NGAL levels were reduced at 6\u20138 weeks postpartum compared to the antenatal levels, p < 0.05. Although maternal and urine KIM-1 levels were reduced at 6\u20138 weeks postpartum compared to the antenatal levels in preeclampsia, the levels were still higher than those in normal pregnancy. Positive expression of NGAL and KIM-1 was detected in proximal tubule epithelial cells in kidney tissue specimens from preeclampsia but not in non-pregnancy controls. EM examination showed glomerular and tubular injury in preeclampsia.Prenatal plasma and urine levels of NGAL and KIM-1 were significantly higher in preeclamptic than in normal controls, Our findings of increased maternal levels and urine secretion of NGAL and KIM-1, along with the upregulation of NGAL and KIM-1 expression in tubular epithelial cells in preeclampsia, provide plausible evidence that tubular injury exists in preeclampsia. The higher postpartum NGAL and KIM-1 levels in preeclamptic pregnancies indicate that tubular injury would not resolve within 2\u20133 months after delivery and suggest that proper follow-up and management of kidney function in women with preeclampsia would be necessary to reduce chronic kidney diseases in those women later in life. Proteinuria and glomerular endotheliosis are the classic hallmarks of renal dysfunction/pathology in preeclampsia, a hypertensive disorder in human pregnancy. Recent studies also indicate that other than glomerular endotheliosis, podocyte injury is present in preeclampsia, as demonstrated by increased podocyte shedding and podocyte protein shedding in women with preeclampsia \u20134. IncreIn addition to glomerular and podocyte injury in preeclampsia, emerging evidence also suggests that this pregnancy disorder is a risk factor for kidney tubular injury in women during pregnancy . There an = 100), pregnant women complicated with chronic hypertension (n = 20), and normotensive pregnant controls (n = 83). Institutional Review Board's (IRB) approval was obtained and informed consent was obtained from all the study subjects. Plasma and urinary samples were aliquot and stored at \u221270\u00b0 until assay. Normotensive pregnancy is defined as pregnancy with a maternal blood pressure of <140/90 mmHg, absence of proteinuria, and medical and obstetrical complications. Chronic hypertension (CHT) in pregnancy is defined as either a documented history of high blood pressure before pregnancy or with a blood pressure of \u2265140/90 mmHg on two occasions more than 24 h apart before the 20th week of gestation. Diagnosis of preeclampsia is based on the American College Obstetricians and Gynecologists (ACOG) criteria: sustained systolic blood pressure of \u2265140 mmHg or a sustained diastolic blood pressure of \u226590 mmHg on two separate readings; proteinuria measurement of 2+ or more on the dipstick, or 24 h urine protein collection with \u2265300 mg or protein creatinine ratio > 0.3 in the specimen after 20 weeks of gestation. Smokers or patients with signs of infection were excluded. To avoid clinical phenotypic differences in preeclampsia, patients complicated with nephritic syndrome, diabetic mellitus, or gestational diabetes were also excluded from the study. Patient demographic and clinical information was obtained via medical record review and is presented in A total of 203 pregnant women were recruited for the study. Maternal venous blood and urine specimens were collected either in a clinic or Labor and Delivery in University Health hospital, Louisiana State University Health Sciences Center-Shreveport (LSUHSC-S), including patients diagnosed with preeclampsia  and human KIM-1 DuoSet ELISA kit (Cat# DY1750) were purchased from R&D Systems . The range of the standard curve for NGAL is 4.9\u20135,000 pg/ml and for KIM-1 is 0.95\u20132,000 pg/ml. For the NGAL assay, plasma was 1:20 diluted and urine was 1:10 diluted. For the KIM-1 assay, both plasma and urine specimens were 1:2 diluted. All assays were performed following the manufacturer's instructions. All specimens were measured in duplicate. Between-assay and within-assay variations were <8% for both assays.n = 5) and control cases (n = 5) were obtained retrospectively from the Department of Pathology Archives at LSUHSC-Shreveport. For the preeclamptic cases, a kidney biopsy was done for diagnostic purposes due to persistent hypertension, massive proteinuria, or other complications after clinical treatment. For the controls, kidney tissue was taken from a normal portion of kidney surgical cases that were performed for excluding neoplasia. Control cases (non-pregnant) were anonymous, without hypertension or renal disease. IRB approval was obtained. Clinical information on the five preeclamptic cases is presented in Kidney biopsy tissue sections from preeclamptic  for 2 h, tissue sections were incubated with anti-NGAL or anti-KIM-1 antibody overnight at 4\u00b0C and then followed by incubation of biotinylate-conjugated secondary antibody (anti-rabbit IgG) for 2 h. NGAL (rabbit mAb D4M8L) and KIM-1 (rabbit mAb E1R9N) antibodies were purchased from Cell Signalling Technology . Biotinylate-conjugated goat anti-rabbit secondary antibody was obtained from Vector Laboratories . Slides without primary antibody staining served as a negative control. Nuclei counterstain was done by hematoxylin. Stained slides were reviewed under an Olympus microscope . Images were captured in the area containing glomerular and/or tubules by a digital camera.post-hoc test. Unpaired t-test, Mann\u2013Whitney test, or Chi-square test were used to compare results between preeclamptic and normotensive or pregnancy complicated with chronic hypertension groups. Paired t-test was used to compare biomarker concentrations within each group before delivery and 6\u20138 weeks postpartum. Pearson product\u2013moment correlation coefficient (Pearson r) was used to analyze the correlation between (1) maternal KIM-1 and NGAL, (2) urine KIM-1 and NGAL, (3) urine and maternal NGAL, and (4) urine and maternal KIM-1. A probability level of <0.05 was considered statistically significant.Clinical data are presented as mean \u00b1 SD . Data foPatient demographic data and clinical characteristics including maternal age, body mass index (BMI), blood pressure, gestational age at specimen collection and delivery, and medical history with chronic hypertension or preeclampsia are presented in n = 100) than those in normotensive controls (n = 83), p < 0.01. While maternal NGAL, but not KIM-1, levels were also significantly higher in pregnant women complicated with CHT (n = 20) than in normotensive controls, p < 0.01.Among these subjects, urine specimens were also obtained from 27 normotensive pregnant women, 20 women complicated with CHT, and 53 women with preeclampsia. Urine NGAL and KIM-1 levels were also significantly higher in women with preeclampsia than in normotensive pregnant controls. Urine NGAL and KIM-1 levels were increased, but not statistically significant, in women complicated with CHT compared to those in normotensive controls .p < 0.05. In comparison, urine NGAL, but not urine KIM-1, levels were reduced at 24\u201348 h after delivery. Although urine NGAL and KIM-1 levels were significantly reduced at 6\u20138 weeks postpartum compared to the antenatal levels in preeclampsia (p < 0.05 for NGAL and p < 0.01 for KIM-1), both urine NGAL and KIM-1 levels were still higher in preeclampsia than in normotensive controls.Among these subjects, maternal blood from 18 normotensive and 39 preeclamptic pregnancies and urine specimens from 21 normotensive and 35 preeclamptic pregnancies were also obtained at 24\u201348 h after delivery and 6\u20138 weeks postpartum. Results of plasma and urine NGAL and KIM-1 levels are shown in r = 0.320, and in preeclampsia, r = 0.317, but not in normal pregnant women  uriwith CHT ; and (4)with CHT .Expression of NGAL and KIM-1 was determined in kidney biopsy tissue sections from five patients who were clinically diagnosed with preeclampsia in the most recent pregnancy. Kidney tissue sections from a normal portion of the kidney surgical cases served as control. Representative transmission electron microscopy micrographs of glomeruli and proximal tubule from a non-hypertensive control and a patient with preeclampsia are shown in This study highlights the kidney tubular injury in preeclampsia, by assessment of maternal and urinary levels of NGAL and KIM-1, and by evaluation of NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia. We found that both maternal and urine levels of NGAL and KIM-1 were significantly higher in women with preeclampsia than in normotensive pregnant controls. We further found that NGAL and KIM-1 expression was upregulated in proximal tubule epithelial cells in kidney biopsy specimens from women with preeclampsia but not in controls. Since NGAL and KIM-1 are considered biomarkers for acute and chronic kidney tubular injury , 9, 11, To determine if tubular injury was restored after delivery in preeclampsia, we measured maternal and urinary NGAL and KIM-1 levels at 24\u201348 h after delivery and 6\u20138 weeks postpartum. It is interesting to note that both maternal and urinary levels of NGAL and KIM-1 at 24\u201348 h after delivery and 6\u20138 weeks postpartum were comparable to those of antenatal levels in normotensive pregnant women. While in preeclampsia, although maternal plasma and urine levels of NGAL and KIM-1 reduced at 6\u20138 weeks postpartum compared to those of antenatal levels, NGAL and KIM-1 levels were still much higher than the levels in normotensive controls, which suggests that kidney injury may not be resolved within 2\u20133 months after delivery in preeclampsia.de novo synthesis of NGAL is markedly increased, levels of NGAL increase rapidly and can be easily detected in urine, blood, and other body fluids  who had a history of CHT in the preeclampsia group, only a few of them had 6\u20138 weeks postpartum specimens collected and measured. Therefore, it is not known if those CHT pregnancies impact the elevated NGAL and KIM-1 levels at 6\u20138 weeks postpartum in the preeclampsia group. Although kidney specimens in the control group were from non-pregnant patients, we believed that immunostaining and electron microscopic results provide convincing information and support the concept that kidney tubule injury is present in preeclampsia. In addition, >75% of subjects were African American in our study. Since the disproportionate population was evenly distributed among the three study groups which represent the demographic and ethnic disparities in the Shreveport community, we do not believe that racial differences would affect our results.In summary, we found that maternal and urinary levels of NGAL and KIM-1 were significantly increased in women with preeclampsia compared to normotensive pregnant controls. We also found upregulation of NGAL and KIM-1 expression in proximal tubule epithelial cells in kidney specimens from women who had preeclampsia. Electron microscopic study further revealed loss of integrity of proximal tubule epithelial cells, showing vacuolization, apical blebbing, and loss of brush borders. All these findings demonstrate that kidney tubular injury exists in preeclampsia. It is well-accepted that preeclampsia is a risk factor for cardiovascular diseases in women later in life. Since preeclampsia is also a risk factor for subsequent end-stage renal diseases , proper The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB: H09-085, Kidney Podocyte Injury in Preeclampsia. The patients/participants provided their written informed consent to participate in this study.YW and DL contributed to the study design. YW, YG, XG, and DC contributed to the sampling process, assays, and data collection. YW and YG performed the statistical analysis. YW wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version."}
{"text": "These losses are still underperceived by the academic community, and by a global society that is disconnected from biocultural diversity. To reconnect society with biocultural diversity, we integrate temporal and spatial dimensions of seasonal cycles, by combining two conceptual frameworks: ecological calendars and the \u201c3Hs\u201d model of the biocultural ethic . The latter values the vital links between human and other\u2010than\u2010human co\u2010inhabitants, their life habits , and the structure and processes of their shared habitats. This integration enhances an understanding of links between cultural practices and the life cycles of biocultural keystone species. As a synthesis, we use the term biocultural calendars to emphasize their co\u2010constitutive nature that result from interactions between dynamic biophysical and cultural processes embedded in specific ecosystems and cultures. These calendars link astronomical, biological, and cultural seasonal cycles that sustain life and enhance the integration of Indigenous and scientific knowledge to confront challenges of climate change faced from local to global scales. To illustrate this integration, we examine cultural practices and socio\u2010environmental changes across four contrasting ethnolinguistic communities in southwestern South America, from southern to northern Chile along a marked climatic gradient to show the broad application of the concept of biocultural calendars.Since the mid\u201020th century, the so\u2010called Great Acceleration ( Biocultural calendars are co\u2010constitutively generated through interactions between dynamic biophysical and cultural processes3Hs model values the vital links among human and other\u2010than\u2010human co\u2010inhabitants, their habits, and shared habitatsThe biocultural ethic's Biocultural calendars are based on seasonal cycles of keystone species that are part of communities of co\u2010inhabitants This synchrony is particularly relevant in the context of climate change because it makes explicit the inextricable links between cultures and nature. Changes in cultures involve changes in nature, and vice versa. Communities that are locally attuned to biophysical and cultural habitats have an enhanced capacity to adapt their life habits to variabilities of temperature, rainfall, and other climatic events.In this article we complement ecological calendars with the biocultural ethic to develop the concept of biocultural calendars. By interrelating phenological events in the biophysical realm with worldviews and practices in the cultural realm, biocultural calendars help us to understand the links between different life habits in contrasting habitats and annual seasons. This attribute of biocultural calendars is crucial in the context of climate change. To develop this concept, we organize our article in two parts. First, we describe the biocultural calendars of Indigenous and other local communities from four climatically contrasting regions of Chile. Second, we concisely discuss the broader implications of this biocultural systemic, contextual, and co\u2010evolutionary understanding. We propose that biocultural calendars could help set social\u2010environmental priorities, policies, and actions to conserve critical habitats and biocultural diversity not only in Chile but also in other heterogeneous regions of the planet. We show that our biocultural outlook introduces an understanding that transcends the purely descriptive plane, since it implies an ethical responsibility.1.1In Latin America, the history of Indigenous peoples and local communities is one in which their life habits have fostered co\u2010inhabitation with high levels of biodiversity Toledo,\u00a0. Globallbiocultural calendars, an expression recently used by Mariana Landwehr\u00a0(Zea mays L.) in Zinacant\u00e1n is part of the cultural identity and is present in the social, political, religious, ceremonial, ritual and gastronomic organization during a well\u2010defined biocultural calendar  Rozzi,\u00a0. AdditioIn this section we use the concept of biocultural calendars to examine cultural practices and socio\u2010environmental changes in southwestern South America, specifically in Chile Figure\u00a0. We orgaanalemmas (or lemniscates), showing the position of the sun in the sky as seen from a fixed location on Earth at a fixed time ), began to paint the leaves giving rise to autumn. The trees revived months later giving rise to spring. This was the beginning of the annual cycles ) and the exemplary sanction for hoarding this vital element was death  Oerst.) is collected for the construction of canoes  Hook.fil.). Different types of baskets are made with its fibers: steapa with a tight weave are used to collect berries, and keichi with a loose weave are used to collect shellfish  is the season that begins when the leaves of the high\u2010deciduous beech or hanis (Nothofagus pumilio (Poepp. & Endl.) Krasser) turn red (lush). Fall is also marked by the arrival of abundant sardines ) and caiquenes ) are fat, and can be hunted more easily ) inhabiting the intertidal zones are central for the Yahgan diet, because they exhibit low seasonal variation in abundance ), American mink ), and muskrat ), is having a major impact on forest and wetland habitats and is decimating bird populations, especially of ground nesting species  that is acquired from the high\u2010deciduous beech tree (hanis) on the slopes of the mountains (tulara) in the fall. Ina, winter, is marked by long nights and snow, associated with lampia (black) and yakua (white), respectively. Iahakisi (spring) the short (iaha) summer (kisi), is signaled by the shoots of leaves that color this season with arlampia (green). Kisi, the summer, arrives with its interminable days where kurlampia (blue) inundates the sea and sky.To illustrate the biocultural conservation initiative in Cape Horn  inhabited by ca. 12,000 people ) and other seaweeds are harvested during the pilcanes ) and snoeks or \u201csierra\u201d ). During their spawning period, these large fish approach the coast chasing schools of sardines. Fishing weirs are used throughout the year to capture other species such as the Patagonian blennie or \u201cr\u00f3balo\u201d (Eleginops maclovinus). Finally, fishing weirs are a refuge for multiple intertidal species, such as rock fish, crustaceans, and seaweeds that support coastal families. Thus, fishing weirs generate relationships of human\u2010nature reciprocity since these anthropogenic habitats stimulate greater biodiversity at the beaches ) and carnivorous mollusks, such as the Chilean abalone or \u201cloco\u201d ). In 1989, these fishing habitats and habits were included in the Chilean legal decree 18,892 on Benthic Resources Management and Exploitation Areas.3.4Since mid\u2010twentieth century, neoliberal economic policies have instilled private property rights, economic efficiency, and deregulation  offer opportunities for adapting to current social\u2010environmental problems. For example, today local fishers recognize the high intelligence of sea lions and maintain a \u201cmutual understanding\u201d of co\u2010inhabitation. Fishers must vary their fishing practices to avoid interference by sea lions which quickly detect the new sites, baits, and techniques used by fishers FSP,\u00a0. On the 44.14.1.1Lafkenche means people (che) of the sea (lafken)   land (mapu). For them, this territory has both material and symbolic cultural significance ):Millenary bird that works by the light of day, and this it divides into four parts: the daybreak, the morning, the noon\u2010time, and the afternoon; symbol of life, work, results and production, family, planting, harvest and animals ) possesses special powers to connect the human with the divine and nature  land, that is like a reflection of the Mapu; it is inhabited not only by planets and stars, but also by relatives and friends who have died and now are working there just like they did in the Mapu   the Wenumapu and the Mapu.Mapu, the land \u201cof here\u201d where human beings live, suffer, and love. Mapu, also called mother earth (\u00f1ukemapu), is delicate and must be loved. The land is worked freely and fraternally, like brothers (pe\u00f1i). In the \u00f1ukemapu, birds and trees should be treated as equals. To achieve this, the ancestors consumed only what they needed.Minchemapu, the land of below (minche) that encompasses all that is underneath, the rivers and the soil, is like the subsoil , the Lloyka is a \u201cfull\u2010time healer bird\u201d that enhances the growth of medicinal plants. In Aillapan's poem the bird says: \u201cI have always healed my people with pure herbs. For this, my name is full\u2010time healer\u201d  and lawen (remedy). Aillapan explains that this healer bird wishes to cure its own gaudy red colored chest that carries the blood of the Mapuche people  Hanst. (Gesneriaceae), Cissus striata Ruiz & Pav. (Vitaceae), Eccremocarpus scaber Ruiz & Pav. (Bignoniaceae), Elytropus chilensis (A.DC.) M\u00fcll.Arg. (Apocynaceae), Lapageria rosea Ruiz & Pav. (Philesiaceae), Tropaeolum speciosum Poepp. & Endl. (Tropaeolaceae), Lardizabala funaria (Molina) Looser, Boquila trifoliolata (DC.) Decne. , and Berberidopsis corallina Hook.fil. (Berberidopsidaceae)  Hariot, 1892), involves synchronizing harvesting and drying habits with the periods in which the seaweeds grow. In addition, the climate that allows them to be dried on the shore for storage and marketing is considered; otherwise, it would rot.Along the coasts, gathering activities also take place in intertidal and subtidal marine habitats that host a variety of seaweeds, fish, and shellfish  are immediately attended to because their precariousness causes social imbalance  a variety of fungi are gathered. Community activities are reduced giving place to conversations within each family, usually commenting on the experiences they had during the spring and summer. In winter (p\u00fckem), the season of greatest deprivation, community practices include times for collective thought and reflection  implies flowing with the vitality of plants and animals through community activities linked to respectful use of forests  inserted in a matrix of forests and shrublands , the sea urchin Loxechinus albus , the sea snail Tegula atra , and the crab Taliepus dentatus  are four keystone species for the Lafkenche diet  and plants (such as voqui) are salient (see above). Among human families, when faced with unforeseen events, such as periods of drought during spring or summer, Lafkenche and Williche families do not hesitate to exchange seeds that have been culturally modified for centuries and that they know will germinate and grow under stressful conditions  they continue exchanging seeds and begin harvesting fruits. Additionally, young lambs and calves are born, thereby generating a season of opulence. Hence, ecological seasonal patterns and the sequence of social activities among Lafkenche people are closely linked. These biocultural links reinforce knowledge and customs that nourish a sense community, which includes human and other\u2010than\u2010human co\u2010inhabitants  and Eucalyptus L'H\u00e9r. species , which did not acknowledge the presence of the Indigenous peoples on the coasts of Chile. This lack of recognition triggered a social movement by the Mapuche\u2010Lafkenche Indigenous people, producing a shift of the Indigenous demands from the interior lands to the sea. After over a decade of negotiations, in 2008, the government of Chile introduced the \u201cLafkenche Law\u201d (Law 20.249) establishing a new conservation category in Chile: the coastal\u2010marine space of the original peoples   on the northeast border of Chile with Bolivia at 4,000\u00a0m altitude. This area has large wetlands supported by underground saline aquifers that are home to a great diversity of native fauna such as flamingos and camelids  Figure\u00a0. Summer 5.1.2achachila) are ancestors and sacred sites. These sites make the reproduction of life possible and must be respected and remembered in rituals and offerings. Their seasonal changes are the changes of human time; this is how Aymara biocultural calendars have been forged . Manqha Pacha is the world below; it is the place under the Earth where what has already happened is kept, where the gaze is directed toward the past time (Nayra Pacha). It is a sacred space where spirits dwell, and where the waters are lost and the vegetation ends. Aka Pacha is at the center and harmonizes opposites; it is where the Aymara people live. It is where the present time (Jicha Pacha), in which we humans live, work, rest, and dream  and llama (L. glama). Indigenous Aymara communities have sustained models of life coupled with marked seasonal contrasts, which alternate rain and drought, heat and cold  and the Chilean flamingo (Phoenicopterus chilensis), have the world's largest populations in this high Andean region.Central co\u2010inhabitants in the high Andes are the domesticated camelids alpaca . IDAs are understood as territorial units where the Chilean state supports the protection of common resources and access to them by Indigenous communities with three central goals: to overcome poverty, to preserve Indigenous cultures, and to comply with their demands for land and water. The criteria for the designation of IDAs are based on the ancestral occupation of the territory, high density of Indigenous populations, ecological homogeneity, and dependence on natural resources such as land, water, flora, and fauna. IDAs have helped, but often for the defense of these habitats and water resources, Aymara communities have had to appeal to international conventions, such as the Convention 169 of the International Labour Organization (ILO) and/or the Ramsar Convention   S.C. Lindstrom, 2011) is the most eaten red algae . Tsimshian people watch the stalks of the stinging nettle mature and elongate; as they grow in the coastal habitats, so do the seaweed fronds grow in the intertidal zone. The red laver is harvested in late spring, generally in May, so much that in the Tsimshian language this month is called \u2018\u2018the month of the seaweed\u2019\u2019 , but including today cattle of European origin (with higher water requirements). These nomadic habits are intertwined in a complex scenario with other uses of water, principally mining. Mining activities have degraded wetlands and their biodiversity, and triggered accelerated depopulation of Andean rural areas along with disruption of biocultural calendars.Fourth, a conceptual understanding of biocultural conservation approach contributes to balancing the interests and needs among different stakeholders. Just as the Chilean state has created Indigenous Development Areas (IDAs) to protect biological and cultural diversity, worldwide international programs have embraced the concept of including human communities and their habitats in conservation programs (e.g., Man and Biosphere Program, Guevara & Laborde,\u00a0To address this type of conflicts, a biocultural calendars integrates concepts of ecological calendars and the biocultural ethic to cope with social and environmental dimensions of climate change. The drivers of climate change are of two types: direct and indirect (MEA,\u00a0Fifth, the four former concluding remarks show how the concept of ect MEA,\u00a0. Direct ect MEA,\u00a0. In thisThe authors declare no conflicts of interest relevant to this study."}
{"text": "The rate coefficients exhibited a strong negative temperature dependence, reaching (4.62 \u00b1 0.84) \u00d7 10\u221211 cm3 molecule\u22121 s\u22121 at 32 K, and no pressure dependence was observed at 70 K. The potential energy surface (PES) of the CN + CH2O reaction was calculated at the CCSD(T)/aug-cc-pVTZ//M06-2X/aug-cc-pVTZ level of theory, with the lowest energy channel to reaction characterized by the formation of a weakly-bound van der Waals complex, bound by 13.3 kJ mol\u22121, prior to two transition states with energies of \u22120.62 and 3.97 kJ mol\u22121, leading to the products HCN + HCO or HNC + HCO, respectively. For the formation of formyl cyanide, HCOCN, a large activation barrier of 32.9 kJ mol\u22121 was calculated. Reaction rate theory calculations were performed with the MESMER (Master Equation Solver for Multi Energy well Reactions) package on this PES to calculate rate coefficients. While this ab initio description provided good agreement with the low-temperature rate coefficients, it was not capable of describing the high-temperature experimental rate coefficients from the literature. However, increasing the energies and imaginary frequencies of both transition states allowed MESMER simulations of the rate coefficients to be in good agreement with data spanning 32\u2013769 K. The mechanism for the reaction is the formation of a weakly-bound complex followed by quantum mechanical tunnelling through the small barrier to form HCN + HCO products. MESMER calculations showed that channel generating HNC is not important. MESMER simulated the rate coefficients from 4\u20131000 K which were used to recommend best-fit modified Arrhenius expressions for use in astrochemical modelling. The UMIST Rate12 (UDfa) model yielded no significant changes in the abundances of HCN, HNC, and HCO for a variety of environments upon inclusion of rate coefficients reported here. The main implication from this study is that the title reaction is not a primary formation route to the interstellar molecule formyl cyanide, HCOCN, as currently implemented in the KIDA astrochemical model.Rate coefficients for the reaction of CN with CH 2O were measured for the first time below room temperature in the range 32\u2013103 K using a pulsed Laval nozzle apparatus together with the Pulsed Laser Photolysis\u2013Laser-Induced Fluorescence technique.Rate coefficients for the reaction of CN with CH Theoretical calculations of these kinetics parameters require knowledge of the potential energy surface (PES), in particular the energy of transition states and intermediates along the reaction coordinate. At very low temperatures, only reactions with small or no activation barriers are likely to proceed quickly. Calculated rate coefficients are highly sensitive to small changes to the PES, which can be on the order of the uncertainties of the 3 both PESs with small barriers to reaction and/or submerged barriers to reaction can cause sharp increases in rate coefficients at low temperatures until in some cases the collision limit is reached. For the case of small barriers to reaction under low temperature reaction conditions, weakly bound complexes in the entrance channel for reaction are formed with less energy compared with higher temperatures. This causes their lifetime for dissociation back to reactants to become sufficiently long that quantum mechanical tunneling through reaction barriers to products can become competitive, leading to a dramatic increase in the rate coefficient with decrease in temperature.3 Two examples are the reaction of OH with Complex Organic Molecules (COMs) such as CH3OH,3,5\u20138 and the reaction of C(3P) with H2O.9 For the case of submerged barriers, an increase in the rate coefficient with a decrease in temperature is observed consistent with a negative activation energy to reaction. Two examples are the reaction of C2H with O2,10 and the reaction of O(1D) with CH4.11 If a calculated PES is used in conjunction with kinetic models to predict k(T), any uncertainty in the height or width of relatively small potential energy barriers can lead to large uncertainties in the prediction of reaction rate coefficients. Therefore, these reaction-specific PES attributes are important to closely examine alongside robust experimental data spanning a broad temperature range, especially including low temperatures.There is a desire to understand what aspects of the reaction potential energy landscape control the behavior of the reaction rate coefficient and the branching to products as a function of temperature. As discussed in a recent perspective,2O (Reaction 1), which was suggested by astrochemists to be the primary formation route to the interstellar molecule formyl cyanide, HCOCN,12 a suggestion reinforced recently by the ab initio quantum calculations of the PES and master equation calculations of the rate coefficients by Tonolo et al.13 Chemical intuition and results from past studies of similar reactions14\u201316 suggest, however, that CN will likely attack CH2O via a hydrogen abstraction mechanism, forming hydrogen cyanide (HCN) and the formyl radical (HCO). Both reactants and predicted hydrogen abstraction products have already been observed in several astrochemical environments, such as Asymptotic Giant Branch (AGB) stellar winds,17,18 the InterStellar Medium (ISM),19\u201326 and dark clouds,27\u201330 providing further motivation for the current study at low temperatures. Additionally, the CN + CH2O reaction is relevant to combustion processes,31 planetary atmospheres,32 and Titan's atmosphere.33,34 The inclusion of new kinetic data for neutral\u2013neutral reactions at very low temperatures can make a significant difference for the abundance of key species in interstellar environments.3,35The current study focuses on the low temperature reaction of the cyano radical with formaldehyde, CN + CH2O reaction measured overall rate coefficients between 297\u2013673 K by Yu et al.36 and 294\u2013769 K by Chang and Wang37  using the Pulsed Laser Photolysis\u2013Laser-Induced Fluorescence (PLP\u2013LIF) technique in resistively-heated flow cells. In these studies, k1(T) was found to decrease slightly with decreasing temperature, from approximately 4 \u00d7 10\u221211 cm3 molecule\u22121 s\u22121 near 770 K to approximately 1.5 \u00d7 10\u221211 cm3 molecule\u22121 s\u22121 at room temperature. The uncertainties quoted for these studies were on average 2.8% for Yu et al.,36 and 1.2% for Chang and Wang.37 It was predicted that a hydrogen abstraction process, forming HCN, was likely to dominate the chemical mechanism. Indeed, a prior theoretical study (QCISD/6-31G**//UHF/6-31G**) found only a very small (\u223c2.7 kJ mol\u22121) barrier to this H atom abstraction reaction.38 In comparison, a very recent calculation by Tonolo et al.13 (CCSD(T)/CBS+CV) reports a small submerged barrier for the abstraction pathway forming HCN. Further aspects of the reaction potential energy surface were explored in this calculation, with particular attention paid to addition reaction pathways forming CN\u2013CH2O adducts, with bonds formed between the C of formaldehyde and either end of CN. However, the high-pressure limit rate coefficient (2 \u00d7 10\u221210 cm3 molecule\u22121 s\u22121) calculated using transition state theory and based on this new PES13 is close to an order of magnitude larger than the previously measured room temperature studies of YCW. Exothermic product channels for the reaction of CN with CH2O include:13,39,40Previous experimental work on the CN + CH39 for et al.;40 but for et al.13 is given. In the work presented here, low-temperature reaction rate coefficients were measured for the first time below 294 K for the CN + CH2O reaction using the PLP\u2013LIF technique in a pulsed Laval nozzle apparatus. Using CCSD(T)/aug-cc-pVTZ//M06-2X/aug-cc-pVTZ, an ab initio PES was calculated for CN + CH2O and utilized in the Open Source MESMER software package41 to calculate temperature-dependent rate coefficients and product branching ratios. Using a fitting procedure, the parameters within MESMER were optimized to give the best agreement with experimental data over the range 32\u2013769 K. These MESMER rate coefficients were then used to develop a parameterisation via a modified Arrhenius (MA) equation and extrapolated to 4 K. The fits of the rate coefficients were then incorporated into astrochemical simulations of cold dense clouds, AGB stars, and hot molecular cores.Enthalpies for 22.12O were performed in a pulsed Laval nozzle apparatus using the PLP\u2013LIF technique, as shown schematically in Rate coefficient measurements for the reaction of CN with CH14,42\u201347 Gaseous formaldehyde (CH2O) is a difficult reagent to work with and particular care was given to its preparation from paraformaldehyde. Preparation of mixtures with the bath gas, its handling, and quantification of its concentration are necessary for accurate kinetics studies. Generation of the CH2O reagent gas was performed by controllably heating paraformaldehyde powder  in an evacuated 500 mL glass bottle (Duran) with a heat gun  to \u223c70 \u00b0C. During heating of paraformaldehyde, the nascent CH2O gas was then passed through a \u221210 \u00b0C cold trap. The cold trap was submerged in ethanol  which was chilled with a refrigerated immersion probe . The cold trap was utilized to trap any water or other condensable byproducts generated during the heating of paraformaldehyde. The purified CH2O was then allowed to flow into evacuated cylinders until reaching a pressure of \u223c200 torr (\u223c26.7 kPa). The cylinders were then filled with argon  or nitrogen  to \u223c6 atm (\u223c608 kPa) total (generating \u223c4.4% mixtures of CH2O/bath gas). The gas in the cylinders was then allowed to mix for >12 hours. This method of generating CH2O is similar to methods utilized previously by our group and other groups.14,48,49 To generate the cyano radical, CN, the vapor from the solid precursor cyanogen iodide  at 298 K.50,51) was entrained in \u223c2 atm (\u223c203 kPa) of Ar or N2 bath gas.The generation of uniform, thermalized, cold, gas flows for kinetics measurements with this apparatus was described previously in detail and will only be described briefly here.2O, \u223c0.005% ICN, and \u223c99% Ar or N2 bath gas with a total flow rate of \u223c2\u20135 slm. The gases were then allowed to mix in a mixing manifold prior to being pulsed through the Laval nozzle. Since CH2O was found to slowly repolymerize, depositing solid paraformaldehyde in the gas lines and causing the MFC calibration to slowly drift, the concentration of CH2O was determined via UV absorption measurements by sampling gas from the tubing between the mixing manifold and the pulsed valves for each concentration of CH2O utilized in a kinetics experiment.14 Measurements of the concentration of CH2O were made using a custom-made 1 m path length UV/Vis absorption cell filled to \u223c1.2 atm (\u223c122 kPa) with the gas-mixture as measured by a capacitance manometer ). The light source for absorption measurements was a UVB lamp  with continuous output around \u223c290\u2013350 nm. Absorption measurements were performed with a UV/Vis spectrometer  with 0.75 nm resolution. Absorption spectra were integrated for \u223c2 seconds and 4 spectral traces were averaged. Representative averaged UV absorption spectra for CH2O are shown in Fig. S1 in the ESI.2O in an N2 bath in the cylinders was determined to be approximately 4 days.The controlled mixing of reagent gases was accomplished through the use of Mass Flow Controllers (MFC)  such that final mixtures of gases were \u223c0.1\u20131% CH2 bath gases. Impact pressure measurements using a Pitot tube were utilized to determine the Mach number along the flow, and via the Rayleigh equations, the density and rotational\u2013translational temperature of the flow,4 as illustrated for temperature in Fig. S2  to be pulsed at 5 Hz into the pre-expansion region upstream of the Laval nozzle. Gas in the pre-expansion reservoir underwent a controlled expansion through a custom-made, axisymmetric, converging-diverging Laval nozzle such that the flow into the vacuum chamber at 0.3\u20132 torr (40\u2013267 Pa) was a wall-less reactor. The vacuum chamber was continuously evacuated by two Roots blowers operating in parallel: a Roots blower (Leybold RUVAC 251) backed by a rotary pump (Leybold D65B) and a Roots blower (Edwards EH250) backed by a rotary pump (Edwards ED660) and the pressure in the vacuum chamber was monitored by a capacitance manometer ). Flow temperatures between 32\u2013103 K were achieved by switching between Laval nozzles of Mach numbers between 2.49 and 5.00 and/or switching between Ar and Ng. S2 ESI.2O, the ICN precursor was photolyzed at 266 nm (\u223c30 mJ per pulse) using the fourth harmonic of a Nd:YAG laser (Quantel Q-Smart 850), which was directed co-linearly with the supersonic flow along the axis of the Laval nozzle  rotational line of the B2\u03a3 \u2212 X2\u03a3  vibronic transition at 357.893 nm, which is generated by a Nd:YAG  pumped dye laser . This \u201cprobe\u201d laser beam (2\u03a3 \u2212 X2\u03a3) was focused through a series of lenses and through a bandpass filter at 400 nm with a FWHM of 40 nm  onto a Channel PhotoMultiplier (CPM) . The gain of the CPM was controlled with a custom-built time-dependent high voltage gating module in order to block scattered light from the pump laser at 266 nm. Digitization and integration of the CPM signal were performed on an oscilloscope , which is then transferred and saved to a computer for further analysis via a LabVIEW program. The timing of the experiment was also controlled via LabVIEW communication with a digital delay generator , which randomly varied the order of the time delays between the pump and probe laser after each gas pulse.In order to initiate reactions of CN + CHser beam  crossed 2.22O reaction in order to further explore the reaction mechanisms responsible for the behavior of the reaction rate coefficients as a function of temperature. Geometric structures of stationary points ) were first optimized at the BHandHLYP/aug-cc-pVDZ level of theory55\u201358 and further refined using M06-2X/aug-cc-pVTZ.59 Higher-level single-point energy calculations were performed at the CCSD(T)/aug-cc-pVTZ level60,61 to obtain more accurate energies. Vibrational frequency calculations were performed to evaluate zero-point vibrational energies (ZPVE), where TSs were found to have only one imaginary vibrational frequency. The vibrational frequency scaling factors for BHandHLYP/aug-cc-pVDZ and M06-2X/aug-cc-pVTZ are taken to be 0.9589 and 0.956, respectively.62,63 Intrinsic reaction coordinate (IRC) calculations were carried out for all TSs located during the PES search, unless otherwise specified, to verify that they are indeed saddle points on the minimum energy pathways connecting the respective local minima. In order to further explore the long-range reaction PES as the two reactants approach each other, relaxed scans were performed along the reaction entrance channels at the BHandHLYP/aug-cc-pVDZ and M06-2X/aug-cc-pVTZ levels of theory. For a relaxed scan, one of the geometric parameters is selected as the scan coordinate while all the other geometric parameters, unless being specifically frozen , are allowed to be optimised to give the minimum energy geometry. All electronic structure calculations were carried out using the Gaussian 09 program.64Theoretical approaches were used in this work to calculate the PES of the CN + CH41 in order to obtain the rate coefficients of the system. The stationary points of the ab initio calculations provide the energies, rotational constants, and vibrational constants required by the MESMER input file. The energy wells along the PES are divided into energy grains, where each grain couples the reactant, intermediate, and product species to one another via the microcanonical rate coefficients, k(E). The individual grains can be populated/depopulated by exchange with other grains via collisional energy transfer with the buffer gas. The microcanonical rate coefficients were calculated with either Rice, Ramsperger, Kassel, and Marcus (RRKM) theory65 for reactions involving a defined transition state or the inverse Laplace transformation (ILT) method66 for barrierless reactions. Collisional energy transfer probabilities were described using the exponential-down model.67 Corrections for quantum mechanical tunneling were also included using the Eckart expression.68 The set of coupled differential equations that describe each of the energy grains is known as the energy grained master equation (EGME) and can be described by:p is the population density vector containing populations of each grain from each well, and M is the transition matrix that describes the population evolution due to collision energy transfer and reaction. The solution to p(0) contains the initial conditions for each grain, U is the matrix of eigenvectors obtained from the diagonalization of M, and \u039b is the diagonal matrix of corresponding eigenvalues, where the smallest are the chemically significant eigenvalues (CSE). MESMER solves the EGME and obtains the phenomenological rate coefficients from the CSE using the procedure described by Bartis and Widom.69 Using the time dependence of the concentration of all species calculated by MESMER, the branching yield of different products can be determined. In addition, MESMER has a built-in fitting feature using available experimentally measured rate coefficients, whereby input parameters, e.g. the energies of the stationary points, can be adjusted to best fit to the experimental data. The input file for MESMER simulations used in this work are included in the online ESI.\u2020From the generated PES, statistical rate theory calculations were performed using the MESMER software program33.12O were obtained by first measuring the time-evolution of the relative transient LIF signal of CN as the radical reacted with an excess abundance of CH2O under pseudo-first-order conditions. The integrated LIF signal at each time delay between the photolysis laser (pump) and the dye laser (probe) was collected at least 5 times in order to obtain averaged traces of the temporal evolution of CN. Any background signal due to the probe-laser only was subtracted using the average value of data recorded prior to the pump-laser pulse.The temperature-dependent rate coefficients for the reaction of CN + CH52\u201354 CN into the CN  laser-probed state led to a rapid rise in the CN LIF signal (\u22721 \u03bcs) which was not resolved in our experiments. Therefore, traces were analyzed after \u223c5 \u03bcs following the initial rise in the signal, after which there was an exponential decay of CN due to diffusion, reaction with CH2O, and reaction with other possible species. Diffusion of CN out of the volume of supersonic flow through which the pump laser passed is given by:The initial photolytic production of CN and fast collisional relaxation of rotationally-excited2O , and potential reactions with other species  is given by:Xi represents other non-reagent species i. The observed pseudo-first-order rate coefficient for the loss of CN, kobs, is thus given by:N is the total number of species other than CH2O in the cold gas flow and kint represents the intercept of a linear fit of a plot of kobsversus [CH2O], with kint expected to be dominated by kdiff. Each CN integrated LIF decay trace, I(t), which is proportional to [CN]t, was fitted with a single exponential decay function:t is the pump\u2013probe time delay, and I0 is the fitted value of LIF signal at t = 0 for exponential decay fits of the signal. Fits of The reaction of CN with CH2O concentration, the experiment was repeated at least five times and the kobs values obtained were averaged to give k\u0304obs. Second-order (bimolecular) plots of k\u0304obsversus [CH2O] were then generated at each temperature, examples of which are given in kint subtracted for clarity) with a linear least-squares fit of k1 from the gradient.For each CH2O] for which k\u0304obs is linear with [CH2O] to avoid any possible influence from CH2O dimers. Evidence for dimerization of CH2O was found in previous low-temperature studies at higher [CH2O] performed using a Laval nozzle, namely for rate coefficient determinations of the reactions OH + CH2O,15 CH + CH2O,14 and NH2 + CH2O.70 The range of values of the intercept (at [CH2O] = 0 molecule cm\u22123) was kint = 2200\u20137600 s\u22121, and the variation of k\u0304obs with [CH2O] without the subtraction of kint can be found in Fig. S3 of the ESI.2O] used to obtain kobs for a given temperature, always using less than the [CH2O] at which second-order plots have been seen to become non-linear in similar studies of radical + CH2O reactions.14,15 The values of k1(T) determined in this work for the CN + CH2O reaction, together with corresponding experimental conditions, are given in k1 was determined at two overall densities providing evidence that k1 is independent of pressure.For the linear fits of the second-order plots such as shown in k1 for CN + CH2O given in 2O reaction.14 The uncertainty quoted is a statistical error only from linear fits of the type shown in k1(T) is about 10\u2013100 times smaller than k2OCH+CH(T) across the range of temperatures studied . Hence, for the same [CH2O] and available reaction time , the LIF signal from CN does not decay as much as for CH, resulting in a relatively larger uncertainty in the fit of kobs. The values of k1(T) determined in this work for T = 32\u2013103 K for the CN + CH2O reaction are shown in 36,37 reported over the temperature range 294\u2013769 K.The measurement uncertainty for k1(T) exhibits a weak positive temperature dependence above room temperature but a strong negative temperature dependence below \u223c100 K, with a likely minimum somewhere between 100\u2013200 K, suggestive of a change in reaction mechanism between the low and high temperature regimes. The values of k1(T) at low temperatures are less precise than those previously reported at higher temperatures,36,37 due to the challenges for this experiment as discussed above. It is noted though that for the previous data reported at higher temperatures, the absolute concentration of CH2O was not determined by UV absorption spectroscopy (in contrast to the low temperature work reported here), rather manometric/flow methods were used to determine [CH2O].3.22O reaction is shown in 2O entrance channel.The overall potential energy surface for the CN + CHThe geometries of the stationary points, obtained at the M06-2X/aug-cc-pVTZ level of theory, are shown in Fig. S4 ESI. The opt\u2020\u22121) is identified following the approach of CN to CH2O. As seen in 3CH2 (P3) or HC(O)CN + H (P4). The formation of HCN + HCO  is accessible through the submerged barrier TS_VDW/P1 (\u22120.62 kJ mol\u22121) while the formation of HNC + HCO  involves a small positive energy barrier relative to the CN + CH2O entrance channel . The error of these two calculated barrier heights, even with the high level of theory used here, are such that they could both be either positive or submerged barriers if calculated at a different level of theory. At the CCSD(T)/aug-cc-pVTZ level of theory, the error of the energy is estimated to be 3.0\u20134.5 kJ mol\u22121 (250\u2013450 cm\u22121).1A weakly bound van der Waals complex VDW (\u221213.3 kJ mol2O involves surmounting a large barrier TS_VDW/P3-4 (32.9 kJ mol\u22121), leading to the formation of the intermediate H2C\u2013O\u2013CN . H2C\u2013O\u2013CN can dissociate to form NCO + 3CH2  or undergo cyclization through TS_1/2 (\u221244.2 kJ mol\u22121) to form the cyclic intermediate Int2 (\u221281.2 kJ mol\u22121). By going through TS_2/3 (\u221262.5 kJ mol\u22121), the ring opens to form the intermediate H2C(O)CN . Breaking one of the CH bonds gives the products HC(O)CN + H . Although the IRC calculation did not converge successfully for TS_3/P4 (\u221241.0 kJ mol\u22121), judging from the vibrational mode of the imaginary frequency, it is likely that it is the TS connecting Int3 and P4. A dashed line connecting TS_3/P4 reflects the incomplete mapping of the IRC along this coordinate.The addition of CN onto the O atom of CH2O. Two different reactant approaches were investigated: the CN radical approaching from the oxygen side of CH2O, and CN approaching from the hydrogen side. For each scan, the distance between the two reactants was fixed while all other coordinates were allowed to optimize. When CN approaches from the oxygen side of CH2O, as shown in Fig. S5  reaches \u22125 kJ mol\u22121 relative to the entrance channel. In order to further explore this \u201cflat\u201d region of the PES, a relaxed scan from this point has been done using the O PBM data was replaced with SVG by xgml2pxml:<glyph-data id=\"z.dbd\" format=\"PBM\" resolution=\"300\" x-size=\"8\" y-size=\"10\" xml:space=\"preserve\">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform=\"translate scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>C\u22efN angle as the scanning parameter, as shown in In the case where CN approaches from the hydrogen side of CHg. S6 ESI, it is f2O, the potential energy first experiences a fairly flat region of the potential and then falls smoothly into the potential energy well corresponding to the van der Waals structure VDW. Thus, it is suggested that both ways of approach can eventually lead to the van der Waals structure VDW.While the CN radical rotates around CH2O to form HCN + HCO was studied in a previous calculation using the QCISD/6-31G**//UHF/6-31G** level of theory.38 This prior work suggested that the reaction mechanism involved overcoming a small positive \u223c2 kJ mol\u22121 (emerged) barrier relative to the CN + CH2O entrance channel. The current study and a very recent study13 have not identified this pathway on the reaction coordinate, perhaps because of the lower level of theory used for geometry optimization (UHF/6-31G**) in the previous study.13 In the current study, instead of direct abstraction, an indirect channel to form HCN + HCO was found which involves the van der Waals complex VDW and a small submerged barrier TS_VDW/P1. A very recent theoretical study13 by Tonolo et al. also identified the VDW structure on the reaction PES /CBS + CV) leading to hydrogen abstraction to form HCN + HCO through a submerged barrier (\u22121.26 kJ mol\u22121). This pathway is consistent with our current work, with less than 0.5 kJ mol\u22121 difference in energy in the complex and barrier. Our work also details for the first time the presence of the HNC pathway from the VDW complex.A direct H abstraction mechanism of CN from CHet al. identifies several stationary points along the reaction coordinate not used in the current study. For example, Tonolo et al. claim a barrierless route to forming a C\u2013C bond directly from the reactants, resulting in a tetrahedral intermediate which they label 1C in a deep potential well (\u2212153 kJ mol\u22121). Although we agree that this structure can be formed (Int3 in \u22121 (CCSD(T)/aug-cc-pVTZ//M06-2X/aug-cc-pVTZ) higher in energy than the reactants, as shown in Fig. S7 , although we identified a new pathway to HC(O)CN + H through VDW which does not involve roaming and has a barrier (TS_VDW/P3-4).The work from Tonolo  Int3 in , we wereg. S7 ESI. It is l2O, which has been studied extensively in previous work,2,16,71\u201375 the results from the current work on CN + CH2O show that both systems share a similar shape of the PES in terms of the energy profile or mechanism for the H abstraction reaction. Following the approach of the two reacting species, a pre-reaction complex is formed followed by a transition state with a small barrier to form products. The difference in energy which determines whether the barrier is positive or submerged relative to the reactants energies is in the kJ mol\u22121 range, and hence within the uncertainty of most calculation methods, and so whether the transition state is submerged or not will depend on the level of theory used.In comparison with the PES of OH + CH2O computed with more robust methods tend to give lower values,2 decreasing the barrier from being slightly emerged to slightly submerged. The latest value reported from Machado et al.2 obtained at CCSD(T)/CBS level is approximately \u22125.7 kJ mol\u22121.For example, the energy values of the transition state for H abstraction channel of OH + CH3.3ab initio calculations were used as the inputs for the master equation solver, MESMER,41 in order to calculate the rate coefficients for CN + CH2O. From simulations over a wide range of temperatures (4\u20131000 K) and pressures (1015\u20131019 molecule cm\u22123) it was observed that HCN and HNC accounted for greater than 99.99% of the products, under all conditions. The reaction occurs initially via van der Waals complex (VDW) formation followed by transition states TS_VDW/P1 and TS_VDW/P2 to form HCN + HCO and HNC + HCO, respectively. The HNC channel never accounts for more than 1% yield, as shown in Fig. S8(b) (ESI\u22121 at the CCSD(T)/aug-cc-pVTZ level of theory1) can result in a reverse situation for which TS_VDW/P2  ESI. HoweverS_VDW/P2  can be s2O did not show a pressure dependence for k1(T), either experimentally near 1017 molecule cm\u22123 or from MESMER simulations between 1015\u20131018 molecule cm\u22123, the gas density was set at 1013 molecule cm\u22123 (so making sure in a pressure independent region) for the MESMER simulations. Above 1019 molecule cm\u22123 and T < 50 K, a pressure dependence was evident and the VDW species was populated. No pressure dependence was observed in the current calculations, implying that the van der Waals complex is not significantly stabilised, even at the lowest temperatures.As CN + CHTherefore, to a very good approximation, 66 was used to calculate the microcannonical rate coefficients for the barrierless formation of the VDW complex from CN + CH2O. The ILT approach overcomes the problem of explicitly assigning a transition state for a barrierless process, whose position would be varying as a function of temperature. The ILT method is especially convenient when experimental data are available, as for this study. The ILT parameters for VDW formation were assigned by:A\u221eILT,VDW and n\u221eILT,VDW are the ILT parameters which describe the rate coefficient for the formation of the VDW at the high pressure limit (in cm3 molecule\u22121 s\u22121 and unitless respectively). A reference temperature in the expression of 30 K was chosen so that the A factor is representative of the lowest experimental data point at 32 K, making the ILT more suited with the low temperature regime relevant to conditions in the ISM. The MESMER input file for these calculations is given at the end of the ESI.\u2020The inverse Laplace transformation (ILT) method76 to adjust the parameters in order to minimize \u03c72:kcalc(T) is the MESMER calculated rate coefficient, and kexpt(T) and \u03c3 are the experimental rate coefficients and their associated error. For the MESMER fitting exercise, the present low temperature results used the experimental errors given in 36,37 . The MESMER fitting scenarios used in this work and the optimised kinetic parameters are summarised in As well as performing simulations, MESMER can adjust the important rate controlling parameters in ab initio calculated values, as shown in \u03c72/N is a measure of the goodness of fit, where \u03c72 is given by N is the number of degrees of freedom, which is equal to the number of data points minus the number of fitting parameters. A \u03c72/N value close to 1.0 represents a good fit, and from Initially, MESMER data fitting was carried out with energies fixed at the 36,37  is submerged, i.e. is negative with respect to the reagent energies, and a reaction mechanism proceeding via a submerged transition state is going to predict k1 to decrease with increasing temperature, which is at odds with the high temperature literature data, which requires the transition state to have a positive energy with respect to the reagents. The simplest way to account for the variation of k1 across the full range of temperatures is to increase the transition state energy so that it is positive, making it consistent with the high-temperature literature data trends and to increase the imaginary frequency of the tunnelling coordinate in the transition state so that quantum mechanical tunnelling overcomes the effect of the positive barrier, leading to an increase in k1 at lower temperatures, as is observed experimentally. When the model scenario Laval + Lit 2  from our ab initio calculated values at the CCSD(T)/aug-cc-pVTZ level of theory.1 However, this adjustment seems reasonable given it is close to the estimated uncertainty at the CCSD(T)/aug-cc-pVTZ level of theory of \u223c3.0\u20134.5 kJ mol\u22121.1 Similarly, even though the imaginary frequencies has been increased significantly, from 205 cm\u22121 to 806 cm\u22121, the adjusted value of \u223c800 cm\u22121 again is not unreasonable.5 As both transition states emanate from the same pre-reaction complex leading to H abstraction products, it seems reasonable to adjust the energies of each of these by the same amount whilst maintaining the same difference between them. The motivation for changing the initial ab initio results to the fitted results using MESMER is to show that the experimental data can be fitted well if the potential energy surface, specifically for these two transition states, is modified. Just changing the properties of the two initially formed transition states which are formed from the same pre-reaction complex is the most straightforward way to do this.The MESMER simulation model scenario Laval + Lit 2 of the fit to the data is shown in k1 occurs around 150 K, which is the point at which the controlling influences of quantum mechanical tunnelling and the 4.0 kJ mol\u22121 barrier for TS_VDW/P1 become balanced. Also included in k1 is approximately twice that of the Laval + Lit 2 model. This significant difference emphasizes how sensitive k1 is to the parameters used in the models when extrapolating down to very low temperatures, i.e. <10 K, as both the Laval and Laval + Lit 2 models give almost the same quality fits to the experimentally measured k1 using the Laval nozzle.From 3.4k1(T) over a very wide temperature range from 4\u20131000 K, as tabulated in Table S10 (ESIk1(T) together with the MESMER simulations using the Laval + Lit 2 model. As CN + CH2O did not show a pressure dependence for k1(T), neither experimentally near 1017 molecule cm\u22123 nor from MESMER simulations between 1015\u20131018 molecule cm\u22123, the gas density was set at 1013 molecule cm\u22123 for the MESMER simulations; above 1019 molecule cm\u22123 and T < 50 K a pressure dependence was evident and the VDW species begins to become stabilized. From 50\u20131000 K, a 30 cm\u22121 grain size was sufficient to calculate a converged rate coefficient, but it was reduced down to 2 cm\u22121 for the 4\u201350 K simulations. It was not possible to run MESMER simulations at lower temperatures. Over the range of 4\u20131000 K, the MESMER simulations indicate that HCN + HCO are the only significant products formed. The fractional yield of HNC is less than 0.33% for all temperatures between 4\u20131000 K, because the energy of TS_VDW/P2 (forming HNC) is 4.59 kJ mol\u22121 above that of TS_VDW/P1 (forming HCN), and the imaginary frequencies of the both transition states is 806 cm\u22121. The temperature dependence of the fractional yield of the HCN and HNC products is shown in Fig. S8(b)  ESI. The reshttps://udfa.ajmarkwick.net) or KIDA (https://kida.obs.u-bordeaux1.fr78) databases) to represent k(T) is a single modified Arrhenius (MA) equation:\u03b1, \u03b2 and \u03b3 are best-fit parameters. Although Table S10 (ESIk1(T) at a given temperature in the range 4\u20131000 K, a parameterisation in the form of k1(T) using an expression traditionally used by astrochemical modellers. However, a single MA k1(T) for CN + CH2O over the full range of temperatures 4\u20131000 K. It is especially inadequate for the k1 values around 150 K, where the T dependence changes from negative to positive, see k1 is rapidly increasing. Running MESMER simulations down to the lowest temperatures means that no extrapolation of fits using k1(T), and Fig. S10, ESIThe equation utilized commonly as a parameterisation in several astrochemical models  for use in astrochemical modelling.In the parameterization given by . S10 ESI. Also ink1(T) gives values that are realistic at the very lowest temperatures, classical capture theory (CCT) calculations have been carried out for the CN + CH2O reaction to calculate the rate coefficient at the collision limit kcoll(T), using the molecular parameters given in Table S11  is dominated by large dipole\u2013dipole interactions below 600 K. As shown in kcoll at \u223c3 K is equal to 1.3 \u00d7 10\u22129 cm3 molecule\u22121 s\u22121, and from the MESMER simulations k1(T) is still calculated to be a factor of 10 or so less than kcoll(T), and does not get close nor indeed exceed kcoll(T) for all temperatures relevant to conditions used in astrochemical models.As a check of whether the parameterisation for e S11 ESI. As showe S11 ESI for the 4.k1(T)) for the reaction of CN with CH2O over a wide range of temperatures. These were a dark cloud model for a total density of 2 \u00d7 104 cm\u22123, at T = 5, 10 and 30 K, hot cores/corino models at higher densities and temperatures, and C-rich and O-rich AGB outflow models, further details of which are given in the following sections. The network for the model utilised the UMIST Rate12 kinetic database77 with updates to rate coefficients to include recent measurements of SiH with O279 and CH with CH2O.14 Further details are given in Section 3.4, together with the parameterisation used for k1(T).Three different astrochemical environments were considered in order to explore the impact of the newly measured and evaluated rate coefficients (4.1n(H2) = 104 cm\u22123 and temperatures of 5, 10 and 30 K, comparing results using the new Laval + Lit2 MA parameterisation to those obtained with the rate coefficient of the CN + CH2O reaction in the UMIST Rate12  database. The latter adopts an energy barrier of 826 K (6.9 kJ mol\u22121) over the range 297\u20132500 K and, in the lack of low-temperature information, the rate coefficient extrapolates to zero at low temperatures. We find that the inclusion of reaction (1), even at its faster low-temperature fit, makes no difference to the abundances of CN, CH2O, HCO, HCN and HNC  = 104 cm\u22123, T = 10 K).80 Our results indicate that, since CH2O is an abundant reactant, the total production rate of HCOCN is likely to be an order of magnitude less than the observational requirement.80The inclusion of the title reaction does, however, impact on the abundance of HCOCN which our 4.2n(H2) = 5 \u00d7 107 cm\u22123, T = 225 K) and the Plateau (n(H2) = 106 cm\u22123, T = 125 K). In these sources, initial abundances are molecular and reflect those in dust grain ice mantles which evaporate in the hot gas surrounding a massive young star. Our initial fractional abundances are taken from Doddipatla et al.81 Although these values are specific to these objects, our results are likely appropriate to other hot core/corino sources. Fig. S12 (ESI77) rate coefficient. The major reason for this is that neither CN nor CH2O are abundant species in these evaporating interstellar ice mantles nor are they produced in significant fractions in the hot gas chemistry. We note in passing that the difficulty mentioned above, in reproducing the observed HCOCN abundance in hot sources, is likely to persist until kinetic data for any new significant HCOCN production reactions are measured or calculated.We also investigated higher density, higher temperature models appropriate for hot core/corino sources. In particular, we modeled two of the hot sources in the Orion Molecular Cloud, the Orion Hot Core . Following Van de Sande et al.,82 the temperature of the outflows is parameterised as  where T* and R* are the stellar temperature and radius, respectively, and \u03b5 is the power law exponent. We assumed a value of R* = 5 \u00d7 1013 cm, T* = 2300 K and \u03b5 = 0.7. The outflow is assumed to be spherically symmetric with an expansion velocity of 15 km s\u22121, while varying the mass-loss rate between 10\u22125, 10\u22126, and 10\u22127 Msun per year. Similar to West et al.,14 we considered both an O-rich and C-rich outflow, with the abundance of parent species taken from Ag\u00fandez et al.83The AGB outflow model is based on the publicly available UMIST CSE model  are not changed. Fig. S13 and S14 , for the reaction CN + CH2O have been measured for the first time below room temperature in a pulsed Laval apparatus using the PLP\u2013LIF technique. A negative temperature dependence of k1(T) was observed below \u223c100 K, in contrast to the positive temperature dependence of k1(T) reported previously in experiments performed above 300 K. Both ab initio calculations of the potential energy surface (PES) for the reaction and MESMER rate-theory simulations making use of this PES were performed in order to explore the overall mechanism that is consistent with the observed k1(T). Two low energy pathways were found on the PES through a VDW complex (binding energy of 13.3 kJ mol\u22121) with either a small positive barrier (3.97 kJ mol\u22121) to form HNC + HCO, or a submerged barrier (\u22120.62 kJ mol\u22121) to form HCN + HCO. This calculated PES can explain the negative temperature dependence of the rate coefficients at low temperatures but not the previously measured literature rate coefficients at room temperature and above. For the formation of formyl cyanide, HCOCN, a large activation barrier of 32.9 kJ mol\u22121 was calculated. Increasing both the energy of the transition state for the formation of HCN + HCO products from \u22120.62 to +4.0 kJ mol\u22121 (which is within the accuracy of the theory used) and also its imaginary frequency to 806 cm\u22121 in the MESMER simulations, enabled the temperature dependence of k1(T) across the entire experimental data to be reproduced very well. The energy and imaginary frequency of the other transition state for formation of HNC + HCO products were changed by the same amounts so their relative values were the same, ensuring that only the HCN + HCO remains the important one for this reaction. The postulated mechanism is similar to previous reactions of OH with volatile organic compounds where the formation of a weakly bound van der Waals complex, whose lifetime is extended at low temperature, is then followed by quantum mechanical tunneling through a small activation barrier to products.3Low-temperature rate coefficients, k1(T) from 4\u20131000 K, and this dataset was used to recommend a parameterization from best-fit modified Arrhenius expressions for k1(T) for use in astrochemical modelling. Even at 4 K the simulated values of k1(T) were a factor of 10 less than the collision limit. This parameterization of k1(T) was used as input to astrochemical models of dark clouds, hot core/corinos, and Asymptotic Giant Star (AGB) stellar outflow environments using the UMIST Rate12 (UDfa) network of reactions. For a range of temperatures, the models yielded no significant changes in the abundances of HCN, HNC, and HCO upon inclusion of their formation using the rate coefficients reported here. It was found that the new rate coefficients for the reaction CN + CH2O do not have a significant effect on the modeled abundances of reagents and products of this reaction due to several other competing reactions with large rate coefficients. However, we note that the uncertainty at the CCSD(T)/aug-cc-pVTZ level of theory, (\u223c3.0\u20134.5 kJ mol\u22121) is similar to the difference in energy (4.59 kJ mol\u22121) calculated for the transition states forming HCN+HCO and HNC+HCO products. Hence, although HCN+HCO are the most likely products, it is possible that the HNC channel may also be the dominant one, as within the calculation errors the HNC+HCO transition state might be the lower one. In order to examine the impact of the scenario in which HNC is the dominant product of the reaction, the rate coefficient k1 was maintained at the same value, but it was assumed that HNC+HCO formed 100% of the products. For a 10 K rate coefficient and 100% formation to HNC, the title reaction provides less than 1% of the overall HNC production rate for cold environments such as molecular clouds. For the Orion Hot Core case at higher temperatures, the HNC+HCO reaction provides around 1% of the total HNC production rate. So, assuming 100% HNC production rather than very little production from the CN+CH2O reaction made a negligible difference to the modelled HNC abundance.Using the MESMER package the product branching ratio was calculated and the formation of HNC was not found to be important as a reaction channel. MESMER was then used to simulate the rate coefficients The main astrochemical implication of this paper is that the title reaction cannot contribute to the formation of formyl cyanide, HCOCN, in interstellar clouds, as has been suggested previously and currently implemented in astrochemical models.DEH and LD conceived the experiment, NAW and ER performed the experiments, LHDL and MAB performed the theoretical calculations, TJM and MVdS performed the astrochemical calculations, NAW, DEH and JHL wrote the manuscript with contributions from all authors.There is no conflict of interest to declare.CP-025-D2CP05043A-s001"}