{"text": "July correction PLoS Biology, volume 2, issue 8In Nicotine's Defensive Function in NatureAnke Steppuhn, Klaus Gase, Bernd Krock, Rayko Halitschke, Ian T. Baldwin10.1371/journal.pbio.0020217DOI: The Academic Editor was erroneously listed as Michael Levine. The Academic Editor for this paper is Joy Bergelson, Chicago University.This correction note may be found online at DOI: 10.1371/journal.pbio.0020382."}
{"text": "Published February 15, 2005PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.0020449The art credits were missing from the image accompanying this synopsis. The image caption should read as follows:"}
{"text": "PLoS Biology, volume 4, issue 10: DOI: 10.1371/journal.pbio.0040321In The legend to Figure 4 incorrectly identifies the dotted line as representing evolutionary rate and the solid line as representing the size of a morphological character. This identification is reversed in the caption. The dotted line represents the morphological trait's magnitude and the solid is the rate of evolution."}
{"text": "PLoS Biology, volume 5, issue 4: doi: 10.1371/journal.pbio.0050072In On page 0801, in the last sentence of the first paragraph of the Results section, \u201cisoleucine\u201d should be replaced with \u201cthreonine.\u201dThe corrected version is:\u201cNonetheless, the overall feature emerging from our analysis is that syntaxins with conserved threonine at the +7 layer appear to be selectively involved in regulated secretion at synapses or neurosecretory cells.\u201d"}
{"text": "PLoS Biology, volume 5, issue 3: doi: 10.1371/journal.pbio.0050065In In Figure 5A, the sequence of the subpanels was inadvertently inverted. The correct Figure 5 is shown here."}
{"text": "E. coli reveals that although these viruses don't age, there is a trade-off between mortality and growth rate, which parallels that observed in many other species.A comparison of life-history traits of 16 phages infecting PLoS Biology, volume 4, issue 7: DOI:10.1371/journal.pbio.0040193InIn the abstract, the mortality rate is described as being negatively correlated with multiplication rate, whereas it is positively correlated."}
{"text": "PLoS Biology, volume 4, issue 8: DOI: 10.1371/journal.pbio.0040255In The figure caption is missing a credit. The caption should read as follows: A model based on growth trajectories estimated from fossils provides evidence that dinosaurs were reptiles whose body temperatures increased with body size. (Painting: Skye White)."}
{"text": "PLoS Biology, volume 4, issue 5: DOI:10.1371/journal.pbio.0040174InThe caption of the image should be \u201cTranscription foci/factories (in blue) are found within areas of intermingling between Chromosome 3 (in red) and the remaining genome (in green), and may mediate functional interactions between them.\u201d"}
{"text": "PLoS Biology, volume 5, issue 5: doi: 10.1371/journal.pbio.0050097In The ninth author's name was incorrectly given as Janet Thonton; it should be Janet Thornton."}
{"text": "A new melanopsin gene, identified in fish, bird, and amphibian genomes, is the true ortholog of the melanopsin gene previously described in mammals. PLoS Biology, volume 4, issue 8: DOI: 10.1371/journal.pbio.0040254In There was an error in the legend for Figure 4. The text describing part A should read \u201cchicken chromosome 6\u201d rather than \u201cchicken chromosome 4.\u201d"}
{"text": "PLoS Biology, volume 3, issue 4, DOI: 10.1371/journal.pbio.0030127In The figure credits for Figures 1 and 2 are missing from the PDF versions of the article. For Figure 1, images are courtesy of Langdon Quetin and Robin Ross, researchers at the Marine Science Institute, University of California, Santa Barbara, and funded by the Office of Polar Programs, National Science Foundation. For Figure 2, images are courtesy of Donna Fraser."}
{"text": "PLoS Biology, volume 4, issue 10: doi:10.1371/journal.pbio.0040325In The \u201cfemale population\u201d part of the x-axis label in Figure 2 has an error: the labels sym and allo are switched in two places. Attached is a PDF of Figure 2 as it should appear. The legend should remain the same."}
{"text": "PLoS Biology, volume 4, issue 2: DOI: 10.1371/journal.pbio.0040031In In the first paragraph of the Materials and Methods subsection \u201cRetroviral infection of INS1 and MIN6 cells,\u201d \u201cpSUPERretro SiRNA-T1 (5\u2032-GCTGCATCCAAGGGCCATG-3\u2032)\u201d should be \u201cpSUPERretro SiRNA-T1 (5\u2032-gatgaagttgacctcctca-3\u2032)\u201d."}
{"text": "PLoS Computational Biology, volume 2, issue 6: DOI:  10.1371/journal.pcbi.0020055In The Figure 5 title and legend should read:Figure 5 Variance of the Natural Logarithm of Size of Memory Lineages Other parameters are the same as in Figure 2B and 2D, and the mean cross-reactivity is kept the same at"}
{"text": "PLoS Biology, volume 3, issue 3: DOI: 10.1371/journal.pbio.0030068In In the caption for Figure 5A, the formula for the lognormal fit was given as follows:.The second squaring exponent should have been inside the parentheses, so it reads as follows:."}
{"text": "PLoS Biology, volume 2, issue 11: DOI: 10.1371/journal.pbio.0020363In http://www.microrna.org/ contains the updated information with changes flagged. We thank Dr. Denman for pointing out this error to us.We incorrectly named the products of two genes, gamma actin and betaAPP, as FMRP ligands because of a misreading of Table 1 from ["}
{"text": "Correction for:10.1371/journal.pbio.0050163Brennand K, Huangfu D, Melton D (2007) All \u03b2 cells contribute equally to islet growth and maintenance. PLoS Biol 5(7): e163. doi:The labels in Figure 2B indicating the \u201cPulse\u201d and \u201cNo Pulse\u201d FACS conditions for the Rosa26-rtTA;tetO-H2BGFP cells were switched. Labeled properly, the middle left panel should be titled \u201cNo Pulse,\u201d and the bottom left panel should be titled \u201cPulse.\u201d"}
{"text": "Correction for:10.1371/journal.pbio.0050219Steiner CC, Weber JN, Hoekstra HE (2007) Adaptive variation in beach mice produced by two interacting pigmentation genes. PLoS Biol 5(9): e219. doi:Figure S2 was erroneously labeled. The caption should read:Agouti Gene Including the Known Cis-Regulatory and Coding Regions Figure S2. Genomic Structure of the In addition, there was an omission in the acknowledgments. The following sentence should be included:T. Glenn, N. Schable, and C. Hagen produced microsatellite libraries and provided PmBW clone sequences that were funded by National Institutes of Health grant R01Gm069601 (to M. Dewey and T. Glenn)."}
{"text": "Microcebus murinus), the golden brown mouse lemur (M. ravelobensis) and the Goodman's mouse lemur (M. lehilahytsara). The first two species occur sympatrically, the latter lives allopatrically to them.A central question in evolutionary biology is how cryptic species maintain species cohesiveness in an area of sympatry. The coexistence of sympatrically living cryptic species requires the evolution of species-specific signalling and recognition systems. In nocturnal, dispersed living species, specific vocalisations have been suggested to act as an ideal premating isolation mechanism. We studied the structure and perception of male advertisement calls of three nocturnal, dispersed living mouse lemur species, the grey mouse lemur (M. murinus from the field using advertisement calls and alarm whistle calls of all three species. Individuals responded significantly stronger to conspecific than to heterospecific advertisement calls but there were no differences in response behaviour towards statistically similar whistle calls of the three species. Furthermore, sympatric calls evoked weaker interest than allopatric advertisement calls.A multi-parameter sound analysis revealed prominent differences in the frequency contour and in the duration of advertisement calls. To test whether mouse lemurs respond specifically to calls of the different species, we conducted a playback experiment with Our results provide the first evidence for a specific relevance of social calls for speciation in cryptic primates. They furthermore support that specific differences in signalling and recognition systems represent an efficient premating isolation mechanism contributing to species cohesiveness in sympatrically living species. Cryptic species are closely related species which are morphologically similar, but differ genetically ,2. The rA fundamental problem for sympatrically living, cryptic mammalian species is the coordination of reproduction between conspecifics in time and space, especially when individuals of a species forage solitarily. Under these circumstances mating partners do not only have to detect, localise and find each other, they also have to discriminate between conspecifics and remarkably similar heterospecifics. Current evolutionary theory ,9-11 sugThe Malagasy mouse lemurs, small nocturnal primates which inhabit the fine branch niche of forests provide an excellent model to explore the significance of vocal communication for species recognition and discrimination. Mouse lemurs have large mobile ears, exhibit a high auditory sensitivity , are higAt present 15 cryptic species are known which are difficult to distinguish in body characteristics ,27-29. IMicrocebus rufus) and their perception by the grey mouse lemur. These three species are genetically distinct from each other but share a large number of morphological features [We studied the structure of male advertisement calls of the grey, the golden brown and the Goodman's mouse lemur, formerly belonging to the rufous mouse lemur  whereas alarm calls do not . Until nThe present study gives the first account of the relevance of communication calls for species recognition and discrimination in cryptic primates in an area of sympatry combining a call structure analysis and playback experiments. Three questions were addressed:1. To what extent do advertisement calls of sympatric cryptic mouse lemurs differ in structure?2. Do mouse lemurs discriminate between advertisement calls of different species and do they show stronger discrimination between conspecific and sympatric than between conspecific and allopatric calls?3. Do mouse lemurs discriminate between call types of different species which are irrelevant for species recognition in the reproductive context?To answer these questions we recorded male advertisement calls of grey and golden brown mouse lemurs and measured several time and frequency parameters for an interspecific statistical comparison of the sympatrically living species. Per individual, we analysed 3 to 21 (median 5) calls and calculated individual median values for each acoustic parameter. On the basis of these values we conducted a Kruskal-Wallis analysis of variance (ANOVA) to test for species-specificity.In addition we conducted playback experiments with 16 grey mouse lemurs from the field. Six categories of playback stimuli were presented during a single experimental session: conspecific male advertisement calls (referred to as conspecific advertisement), heterospecific male advertisement calls of the golden brown mouse lemur (referred to as sympatric advertisement), heterospecific male advertisement calls of the Goodman's mouse lemur  and male whistle alarm calls ,38 of alThe behavioural responses of the tested individuals were classified into two different response categories: (1) no orientation, not involving any orientation response including no reaction, ear movement, interruption of activity or startle without turning towards the speaker and (2) orientation, including turning towards the speaker and approaching the speaker, sometimes accompanied by antiphonal vocalisation.For statistical comparison of call categories, an individual-based analysis was conducted comparing individual response indices for all call categories of advertisement calls and short whistles, respectively. The individual response index towards a call category was defined by the number of orientation responses divided by all responses of an individual towards stimuli of the respective call category, that isFriedman-ANOVA and Wilcoxon tests with a serial Bonferroni correction procedure  were perThe frequency contour of the harmonically structured advertisement calls from the three species was remarkably different Figure . The gref0 min: H2 = 3.470, p = 0.176; f0 max: H2 = 0.928, p = 0.629; f0 band: H2 = 2.566, p = 0.278; N = 14 for all tests; see Table H2 = 11.623, p = 0.003, N = 14). The calls of the grey mouse lemurs were the longest, those of the Goodman's mouse lemur the shortest and those of the golden brown mouse lemur took an intermediate position.No measured frequency parameter showed any species specificity . In addition, response strength was independent of the presentation number of stimuli . This shows that inter-stimulus intervals were sufficient to avoid any habituation effects owing to the consecutive stimulus presentation design.Neither the sound pressure level nor the signal-to-noise ratio of stimuli had a significant effect on the stimulus response indices .Individual response indices revealed remarkable differences for conspecific, sympatric and allopatric stimuli  and maximum (f0 max) frequency of the fundamental and calculated the bandwidth of the fundamental (f0 band = f0 max - f0 min).Male advertisement calls were recorded in the presence of oestrous females ,60. The Statistics were made using Statistica 6.0 (StatSoft Inc.), the level of significance was 0.05 for all statistical tests.Playback experiments were conducted in the Ankarafantsika National Park , about 110 km south-east of Mahajanga, Madagascar during the dry season from September to October 2000 and from July to October 2001 covering the mating period of the mouse lemurs. They were performed in a part of the dry deciduous forest where the grey and the golden brown mouse lemur occur sympatrically.Sixteen  grey mouse lemurs were subjects of our playback experiments. The experiments were conducted under temporary captivity conditions in the field. A stationary setup under controlled conditions was necessary because mouse lemurs communicate in the ultrasonic range which requires special playback and recording equipment. To test for differences in the perception of sympatric and allopatric calls, we needed animals from the field which were experienced with their sympatric species.The intervention on the individual and population level by the experimental study was reduced to a minimum by the following procedure: we trapped the animals using Sherman Live Traps  by setting them in the late afternoon in trees and bushes . Traps wDue to the fact that mouse lemurs are seasonal breeders , it was For the playback experiment a playback stimulus consisted of one call for the categories conspecific and sympatric advertisement, two calls for the category allopatric advertisement and three calls for the three whistle categories, respectively. By using this setup we accounted for the different duration and repetition rates of male advertisement calls and short whistles from the different species. We used two different advertisement stimuli from each of four conspecific males, two from the Hannover population and two from a Madagascar population. In addition, two different stimuli from each of two sympatric and allopatric males were taken. As whistle stimuli, we used two short whistles each of two males of the grey, two males of the golden brown and one male of the Goodman's mouse lemur.With these stimuli, we produced a playback tape based on the original analogue recordings from the NAGRA tape recorder. To minimise background noise the stimuli were highpass filtered at a frequency of 7 to 15 kHz depending on the minimum frequency of the call. The playback tape was started at a random position using a NAGRA IV-SJ tape recorder , a custom-made amplifier and a speaker (Leaf Tweeter EAS-10Th400A). Stimuli ranged between 70.5 and 83.0 dB sound pressure level at a distance of 1 m , that is, sound pressure levels corresponded to the naturally occurring ranges. The loudspeaker was placed between 0.6 and 0.8 m above the ground at a distance of about 0.5 m from the cage to ensure a sufficient presentation quality of the highly directional ultrasonic calls at any position in the cage.In each playback session seven different stimuli were played back in a random order: two of the category conspecific advertisement (one each of the two different populations), and one stimulus each of the other five categories. To avoid a habituation to playback stimuli, the inter-stimulus interval was kept between 1 and 10 minutes. Each individual took part in one to three playback sessions in which each of them received every stimulus only once.Behavioural responses to playback stimuli were observed at a distance of about 5 m from the observation cage using a headlamp and binoculars and reported to a dictaphone for further analysis. We recorded the behavioural responses belonging to the categories 'orientation' and 'no orientation' within 10 seconds just after the onset of a stimulus. In all cases, response behaviour had finished within this period.Cases were excluded in which animals were not visible to the observer because they went into their bamboo trunk or were hidden by cage enrichment. We were able to analyse 186 responses to playback stimuli. The frequencies of no orientation and orientation responses were determined per stimulus and per individual, respectively. We recorded 5 to 13 (median 8) responses for each stimulus. Each individual contributed between 3 and 20 responses (median 11.5). The behavioural responses were counted for the respective response categories and visualised within each call category.N = 22) with their sound pressure level and their signal-to-noise ratio, respectively. A stimulus response index was defined by the number of orientation responses divided by all responses towards a stimulus. To make sure that the consecutive presentation of playback stimuli resulted in independent responses we conducted a Spearman rank correlation for the response indices with the order of stimulus presentation. The order response index was defined by the order number of the orientation responses divided by all responses for the respective presentation number.We conducted Spearman rank correlations to exclude the effects of stimulus quality by correlating the response indices of all stimuli for which we saw behavioural responses . Therefore, responses of an individual towards conspecific advertisement stimuli were averaged for further analysis.Furthermore, to test for habituation effects we analysed the response strength towards the first and the second stimulus of the two conspecific advertisement stimuli during a given playback session. A chi-square test revealed that the distribution of no orientation and orientation responses did not differ significantly between the first and the second conspecific advertisement stimulus (chi-square-test: \u03c7PB participated in the design of the study, conducted the experiments, performed the statistical analysis and prepared the manuscript. SS mentored the study, contributed to the technical design of the study and the preparation of the manuscript. EZ initiated, financed, mentored and contributed to the design of the study and the preparation of the manuscript. All authors read and approved the final manuscript."}
{"text": "Upright faces are thought to be processed holistically. However, the range of views within which holistic processing occurs is unknown. Recent research by McKone  suggests By necessity, we must be able to generalize across many natural sources of image variation in order to recognize a face, including changes in distance and viewpoint as we move around each other and changes in lighting as we move in and out of different environments. We rarely view faces from directly in front (known as the \u201cfull face\u201d or 0\u00b0 view). Most commonly, our visual experience of faces falls within a range of viewpoints rotated away from 0\u00b0 by up to 45\u00b0 to the left or right , above or below , and clockwise or anticlockwise . Within this range of variation in viewpoint, face recognition is remarkably good, even for unfamiliar faces  is the observation that the inversion (180\u00b0 roll or picture-plane rotation) of faces dramatically impairs recognition compared to upright faces, and that this impairment is disproportionately larger for faces than objects Yin, . BecauseIt is now generally accepted that turning a face upside-down disrupts face-specific holistic processing. For example, if one creates a composite face by aligning the photographs of the top and bottom halves of two different faces, the obligatory holistic processing of the new \u201cwhole\u201d face will impair naming accuracy and increase reaction times (RTs) for each half face  in a similar manner. During picture-plane rotations, Rossion and Boremanse  found a To date there has been little investigation of holistic processing in faces rotated in pitch and yaw , configural information appears to be more useful. Second, pitch rotation disrupts configural information to a greater degree than yaw or roll rotation. Compared to rotations about other axes, pitch camera rotations result in a greater foreshortening and occlusion of features as well as a general reduction in the amount of available \u201cface\u201d information. Thus, Favelle et al.\u2019s  findingsMcKone  found evThis study was approved by the University of Wollongong Human Research Ethics Committee, and written consent was obtained from all participants.Participants were 20  volunteer undergraduate students attending the University of Wollongong. The average age of participants was 21.6\u2009years (age range: 18\u201329\u2009years). All participants had normal or corrected-to-normal vision and none were familiar with the faces used as stimuli.d\u2032, defined below) and RT to correct same matches.Participants made \u201csame/different\u201d judgments of two sequentially presented faces, shown centrally, and both upright or inverted. On each trial, these two faces were typically seen from different views. There were 10 levels of viewpoint  crossed with orientation (upright and inverted), all manipulated within subjects. The dependent measures were matching sensitivity  as well as hair were removed digitally. Faces were illuminated by an ambient light source located directly above the model plus four directional light sources located 1\u2009m in front of the model . The two lights located just above the face were oriented horizontally , whereas the lights located just below the face were oriented 45\u00b0 below the horizontal. Lighting was held constant across all viewpoints. In addition to a full-face (0\u00b0) view, each face was presented from three different viewpoints rotated 15\u00b0, 45\u00b0, and 75\u00b0 away from 0\u00b0 in three different directions: yaw leftAll images were viewed in the center of the computer screen against a white background. The on-screen height of 0\u00b0 faces was approximately 16\u2009cm with a width of 10\u2009cm, which produced a visual angle of 14.7\u00b0\u2009\u00d7\u200919.2\u00b0. For yaw viewpoints the height of the face image remained constant, however face width increased as the viewpoint was rotated further away from 0\u00b0. Face width remained constant for pitch camera rotations, however face height decreased as the viewpoint was rotated further away from 0\u00b0 (for both pitch-up and pitch-down conditions). The 75\u00b0 pitch-up camera condition had the shortest image, which was 12\u2009cm high and produced a visual angle of 11\u00b0\u2009\u00d7\u20099.2\u00b0. The rectangular patterned mask used in the experiment subtended a visual area of 18\u00b0\u2009\u00d7\u200922\u00b0 and was composed of various elements taken from the stimuli used in the task.www.tarrlab.org) guided the trial sequence. Responses were made via key presses on a keyboard placed in front of the participant.Full color images were presented to participants on a 48\u2009cm flat-screen monitor with a resolution of 1024\u2009\u00d7\u2009768 pixels. Trials were run on a Macintosh G4 computer and RSVP experimental software  was presented first followed by a mask, and then a test face  was presented followed by the mask again. Participants were first verbally instructed how to complete the task, with emphasis placed on both speed and accuracy in responding. Written instructions on how to complete the task were also provided on the computer screen. After reading the instructions participants completed 10 practice trials to familiarize them with the task. Stimuli used in the practice trials were different from the stimuli used in the task. Following the practice trials participants were given a chance to ask any questions about the procedure, should they have any, before continuing on with the experiment.Orientation (upright or inverted) was blocked and the order of the blocks counterbalanced. Each block consisted of 180 trials, giving a total of 360 experimental trials. In half of the trials the two faces presented were the same, regardless of viewpoint . In the other half of trials the two faces were different; the different face was randomly selected from the remaining eight faces . Trial type was presented in random order within each block. Participants were given five self-timed rest periods spaced equally throughout each block. The experiment lasted approximately 30\u2009min.Each trial began with a fixation cross displayed for 500\u2009ms. This was followed by the presentation of face 1 for 250\u2009ms. Then the mask was presented for 500\u2009ms. Face 2 was then presented for 250\u2009ms, followed by a second presentation of the mask for 500\u2009ms. Following the second mask the screen remained blank for 2\u2009s or until a response was made by the participant. If a response was not made within this time, the trial ended . The interval between trials was 1\u2009s. Participants were required to respond by pressing clearly labeled \u201csame\u201d and \u201cdifferent\u201d keys on a keyboard depending on whether they judged face 1 and face 2 to be the same face or two different faces.z-scores and then used to calculate d\u2032  and RT (ms) scores were analyzed in a series of two-way (viewpoint\u2009\u00d7\u2009orientation) repeated measures ANOVAs. Pitch viewpoints  were analyzed separately to yaw viewpoints . The alpha level was 0.05. Greenhouse\u2013Geisser corrections were made whenever the assumption of sphericity was violated. Bonferroni adjustments were made where necessary to control for family wise error.Participants\u2019 responses were converted into hit (H) and false alarm (FA) rates, where a hit was a correct \u201csame\u201d response to a face, and a FA was an incorrect \u201csame\u201d response to a \u201cdifferent\u201d face. These H and FA rates were converted into d\u2032 data revealed a main effect of orientation, F\u2009=\u200925.75, p\u2009<\u20090.001, MSE\u2009=\u200930.12, F\u2009=\u200943.07, p\u2009<\u20090.001, MSE\u2009=\u200930.85, F\u2009=\u20093.17, p\u2009<\u20090.01, MSE\u2009=\u20092.19, Post hoc comparisons showed a significant FIE (upright\u2013inverted) at the full-face (0\u00b0) view and at pitch viewpoints of 15\u00b0 and 45\u00b0 above and below 0\u00b0  with no evidence of a FIE at either of the 75\u00b0 viewpoints (both p\u2009>\u20090.84).As can be seen in Figure F\u2009=\u20094.77, p\u2009<\u20090.05, MSE\u2009=\u2009381809.7, F\u2009=\u20098.15, p\u2009<\u20090.001, MSE\u2009=\u2009156663.29, F\u2009=\u20090.74, p\u2009=\u20090.56, MSE\u2009=\u200912112.8. Post hoc comparisons showed significantly faster response times to 0\u00b0 than to either 45\u00b0 or 75\u00b0 pitch viewpoints above (both p\u2009<\u20090.01).The pattern of the RT data approximately reflected that of the sensitivity data except at pitch +75\u00b0 \u2009=\u200939.76, p\u2009<\u20090.001, MSE\u2009=\u200926.71, F\u2009=\u200915.73, p\u2009<\u20090.001, MSE\u2009=\u200910.28, F\u2009=\u20091.86, p\u2009=\u20090.15. Post hoc comparisons showed significantly higher sensitivity to 0\u00b0 viewpoints than to any other yaw viewpoint . There was no difference in sensitivity to matching faces between 15\u00b0, 45\u00b0, and 75\u00b0 viewpoints .As can be seen in Figure F\u2009=\u20094.8, p\u2009<\u20090.05, MSE\u2009=\u2009212161.9, F\u2009=\u20095.98, p\u2009<\u20090.001, MSE\u2009=\u200956426.8, F\u2009=\u20090.16, p\u2009=\u20090.93. Post hoc comparisons showed significantly faster responses to 0\u00b0 viewpoints than to either 45\u00b0 or 75\u00b0 yaw viewpoints (both p\u2009<\u20090.05).The RT data reflects the pattern of the sensitivity data with a FIE evident at all viewpoints . We found that recognition performance was most sensitive for the upright full-face views and declined as these upright faces were rotated away from the full-face view. While sensitivity continued to decline for upright face views following pitch rotations of up to \u00b175\u00b0 , no further decline was evident following yaw rotation beyond 45\u00b0.As in previous experiments from this lab  the absence of inversion effects for these viewpoints was unlikely to have arisen due to floor effects.Interestingly, inversion impaired face recognition sensitivity  for some viewpoints, but not for others. As Hills et al.  found, rThe advantage of the face inversion manipulation is that the low-level stimulus properties present in an upright face are identical to those in an inverted face. Thus, any differences between the two conditions have to be accounted for by higher-level, probably face-specific, processes. As noted in the Section The current experiment is the first to investigate the holistic processing of pitch rotated face views. While the FIE was observed at pitch \u00b115\u00b0 and \u00b145\u00b0 viewpoints, the absence of a FIE at pitch \u00b175\u00b0 viewpoints suggests that there is a fixed limit to the range of upright face views in which holistic processing occurs. That is, there appears to be a qualitative difference in the way that upright faces are processed at pitch \u00b175\u00b0 viewpoints compared to the other pitch and yaw-rotated viewpoints tested in this experiment. Face recognition performance at these viewpoints could be explained in two ways: (i) holistic processing occurs much less efficiently at 75\u00b0 pitch rotations because we have little to no expertise with these views  and hence cannot be explained in terms of response biasRecently, Richler et al.  have argRossion and his colleagues Rossion,  on the oMore recently, Van Belle et al.  found emFollowing on from this, the current results might be accounted for in the same way. According to this view, all faces  are handled in a similar way by the early visual system. However, it may not be possible to match 75\u00b0 pitch rotated views of faces to the experience-derived face template/s and so as a result they do not appear to initiate holistic processing. If this is the case, since neither 75\u00b0 pitch rotated views of faces nor any inverted faces are processed holistically, then there is no basis for the FIE. Importantly, what this suggests is that a visual stimulus simply being categorized as a face, as we assume at least the 75\u00b0 pitch view from below would, is not enough to trigger holistic processing.Changes in view can result in substantial variations in the visual appearance of a face. Despite this, our results show that holistic processing occurs for a large range of views around the upright full-face view. Specifically, the range of views within which upright faces are processed holistically extends to rotations of 90\u00b0 in yaw McKone,  and \u00b145\u00b0The 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": "AbstractAchillea millefolium L.) accessions was reported. Cytological analyses on four Achillea millefolium accessions, indicated that two accessions were diploids (2n=2x=18) and two tetraploids (2n=4x=36). Cluster analysis based on chromosomal characteristics and karyotype asymmetry, categorized the four accessions separated into two groups. In terms of the Stebbins\u2019 system, the karyotype of diploid accessions grouped in 2A class. The average value of the total form percentage (TF%) in the group one (diploid accessions) and two (tetraploid accessions) were 40.85 and 41.15, respectively. The group one had the highest mean value for the symmetry index (S%=57.5). Consequently, it can be inferred that diploids belonging to the group one are the earlier evolutionary forms.In this study, a new chromosome number for Iranian yarrow ( Achillea is one of the most recent genera of the Asteraceae family which exists throughout the world  to find any relationship between the karyotype characteristics and asymmetrical index with ploidy levels.he world . Three che world . The aimAchillea millefolium accessions were collected from three provinces in north, west and south of Iran , mean chromosome length (MCL), and mean arm ratio (MAR) were calculated using MICROMEASURE (Version 3.3) Software . Stebbinn = 4x = 36 chromosomes, while Achillea millefolium  that were collected in northern parts of Iran.Am1 and Am2 accessions were diploids (2n= 2x= 18) whereas the two other accessions (Am3 and Am4) showed tetraploid (2n= 4x= 36) level . AccordiAchillea millefolium  in the group one and two were 40.85 and 41.15, respectively. The TF% index has frequently been used to explain karyotype asymmetry  and karyotype asymmetry (TF% and S%)  and agreAchillea millefolium accessions. Cluster analysis indicated that accessions can be classified based on ploidy levels.The results of the present study illustrated a new ploidy level (2n= 2x= 18) in Iranian"}
{"text": "SHR using quantitative real-time PCR. Kidney cortex, but not liver or heart Mt-gene expression was decreased ~2\u20135 fold in 12 of 13 protein encoding genes of HT BN/SHR-mtSHR. Kidney cortex but not liver mRNA expression of the nuclear transcription factors Tfam, NRF1, NRF2 and Pgc1\u03b1 were also decreased in HT BN/SHR-mtSHR. Kidney cortical tissue of HT BN/SHR-mtSHR exhibited lower cytochrome oxidase histochemical staining, indicating a reduction in renal oxidative phosphorylation but not in liver or heart. These results support the hypothesis that renal cortex of rats with SHR mitochondrial genome has specifically altered renal expression of genes encoding mitochondrial proteins. This kidney-specific coordinated reduction of mitochondrial and nuclear oxidative metabolism genes may be associated with heritable hypertension in SHR.Mitochondrial (Mt) dysfunction contributes to the pathophysiology of renal function and promotes cardiovascular disease such as hypertension. We hypothesize that renal Mt-genes derived from female spontaneously hypertensive rats (SHR) that exhibit hypertension have reduced expression specific to kidney cortex. After breeding a female Okamoto-Aoki SHR (SAP = 188mmHg) with Brown Norway (BN) males (SAP = 100 and 104 mmHg), hypertensive female progeny were backcrossed with founder BN for 5 consecutive generations in order to maintain the SHR mitochondrial genome in offspring that contain over increasing BN nuclear genome. Mt-protein coding genes  and nuclear transcription factors mediating Mt-gene transcription were evaluated in kidney, heart and liver of normotensive (NT: n = 20) vs. hypertensive (HT: n = 20) BN/SHR-mt Hypertension constitutes a primary and significant factor in the development of cardiovascular disease. Despite major gains in the long-term treatment of hypertension, cardiovascular disease remains the number one cause of death and disability in developed countries. Primary or essential hypertension is regarded as a multi-factorial disease, influenced by both genetic inheritance and environmental conditions that influence gene expression. The genetic basis of hypertension has been focused primarily on inheritance and expression of nuclear genes (nDNA) , despitelle gene (GenBank accession no. NC_001807) and hypertension. The genetic association of mtDNA variants and tRNA mutations [Mitochondrial dysfunction has been implicated in a wide variety of genetic disorders , 5, and utations  to type utations . Taken tSHR) [The impact of the kidney on long-term blood pressure regulation is well known. It has been hypothesized that the \u201cset-point\u201d for the long-term control of BP resides in the kidney \u201315. In tSHR) . HyperteAll experiments were carried out in strict accordance with the recommendations provided in the AAALAC Guide to the Care and Use of Laboratory Animals and the National Institutes of Health. All protocols in this study were specifically approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC #000649L2003). All surgeries were performed following complete anesthesia of individual animals. Tissue collection for these studies also was conducted under full anesthesia. At the time of any change in protocol (minor or major) amendments were submitted to the IACUC and approved prior to conducting any individual experiment. Since this was a long term breeding study, the original IACUC approval served for a significant time period. All individual studies for this protocol approval were reevaluated at 3 year intervals as required by AAALAC standards. A \u201cconplastic\u201d colony using phenotypic selection was employed. The development and phenotypic characterization have been described in detail elsewhere . The AokAfter repeated blood pressure recordings that assured consistent determination of arterial pressure, rats not scheduled for rebreeding were euthanized with an overdose of sodium pentobarbital (60 mg/kg i.p), decapitated, and organ tissues were rapidly frozen in acetone super-cooled on dry ice and stored for later analysis.SHR rats  were utilized for the current study  was evaluated in parents and offspring beginning no earlier than 10\u201312 weeks of age. Phenotypes were assigned as either normotensive (NT: SAP \u2264 124mmHg), borderline hypertensive (BHT: 125 \u2264 SAP \u2264149 mmHg) or hypertensive  as described above. Total RNA was extracted with Trizol reagent  and purified using RNeasy minicolumns  according to the manufacturer\u2019s protocol. Possible genomic DNA was digested with DNase I . Concentration and purity of all RNA samples was determined by the Nanodrop ND-1000 spectrophotometer . Extracted RNA was reverse-transcribed into complementary DNA (cDNA) using qScript cDNA Supermix  in a total volume of 20\u03bcl using a MyCyler Thermal Cycler .Renal cortex, liver and left ventricular cardiac tissue were selected from age and sex-matched HT and NT BN/SHR-mtmt-ND1- Rn03296764_s1, mt-ND2- Rn03296765_s1, mt-ND3- Rn03296825_s1, mt-ND4- Rn03296781_s1, mt-ND4L- Rn03296792_s1, mt-ND5- Rn03296799_s1, mt-ND6- Rn03296815_s1, mt-CO1- Rn03296721_s1, mt-CO2- Rn03296737_s1, mt-CO3- Rn03296820_s1, mt-CYB- Rn03296746_s1, mt-ATP6- Rn03296710_s1, mt-ATP8- Rn03296716_s1, NRF1- Rn01455958_m1, NRF2a- Rn01767215_m1, NRF2b-Rn01514289_g1, Pgc1\u03b1-Rn00598552_m1, Tfam-Rn00580051_m1, Cyc1-Rn01504159_g1, Cox6c-Rn00820983_gH, GAPDH- Rn01775763_g1). Primers and probes were verified as operating at similar efficiencies. Target gene and endogenous control amplicons were labeled with either FAM or VIC. The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression were measured in all samples to normalize gene expression for sample-to-sample differences in RNA input, RNA quality and reverse transcription efficiency. Each sample was analyzed in triplicate, and the expression was calculated according to the 2\u2212\u0394\u0394Ct method [Quantitative Real-Time PCR was performed on a StepOnePlus Real-time PCR system . Real-time quantitative PCR amplifications were performed in triplicate on a 96-well plate. Pre-designed TaqMan primers and hydrolysis probes for all genes of interest were purchased from Applied Biosystems  . Total mCytochrome oxidase (CO) activity was determined in fresh frozen sections (20\u03bcm) in kidney, liver and heart tissue, as described previously . BrieflyU Test comparisons. The 0.05 level of probability was utilized as the minimum criterion of significance. All statistical analyses were performed using GraphPad Prism 4.0 .Blood pressures and citrate synthase activity among animals were initially analyzed by 1-way analysis of variance (ANOVA) followed by post-hoc comparisons using the Bonferroni t-test. Tissue mRNA expression levels were analyzed using Mann-Whitney SHR, including five complex I, one complex III, three complex IV and both subunits of ATP synthase. Additionally, the nuclear trans-factors Tfam, NRF1, NRF2a, NRF2b and Pgc1a were all downregulated in the kidney, but not elsewhere, of HT BN/SHR-mtSHR.Multiple mtDNA encoded genes of the mitochondrial respiratory chain were significantly reduced in renal, but not liver or cardiac tissue of HT BN/SHR-mtSHR. mt-ND1 was reduced ~3.7 fold in HT BN/SHR-mtSHR (P = 0.0317). mt-ND3 was reduced ~2.6 fold in HT SHR/BN-mtSHR (P = 0.0077). mt-ND4 was reduced ~10.8 fold in HT SHR/BN-mtSHR (P = 0.0221). mt-ND4L was reduced ~7.7 fold in HT SHR/BN-mtSHR (P = 0.05). mt-ND5 was reduced ~2.7 fold in HT SHR/BN-mtSHR (P = 0.00167). mt-ND6 was reduced ~1.7 fold in HT SHR/BN-mtSHR (P = 0.0822) (mt-ND2 was not different between the two phenotypes (P = 0.5994).The renal expression of five of the seven mt-encoded genes of complex I were significantly reduced in hypertensive versus normotensive BN/SHR-mt 0.0822) . mt-ND2 mt-CYB) was significantly reduced in hypertensive versus NT BN/SHR-mtSHR. mt-CYB was reduced ~5 fold in HT BN/SHR-mtSHR (P = 0.0103)  .SHR. mt-CO1 was reduced ~3.2 fold in HT BN/SHR-mtSHR (P = 0.0164), mt-CO2 was reduced ~3.6 fold in HT BN/SHR-mtSHR (P = 0.0063), and mt-CO3 was reduced 4.1 fold in HT BN/SHR-mtSHR (P = 0.0085) (All three mt-encoded genes of complex IV were significantly reduced in hypertensive versus normotensive BN/SHR-mt 0.0085) .mt-ATP6 was reduced ~2.3 fold in HT BN/SHR\u2014mtSHR (P<0.05), while mt-ATP8 was reduced ~3.1 fold in HT BN/SHR-mtSHR (P = 0.0255)  .SHR as described above. mt-CYB, mt-CO2 and mt-ND1, mt-ATP6 were shown to be not different (P>0.05) between the HT and NT BN/SHR-mtSHR liver or heart tissues. This is in contrast to renal tissue wherein each of these genes exhibited reduced expression in HT vs. NT animals  in the kidney, but not the liver or heart (P>0.05) in HT BN/SHR-mtSHR  in NT (n = 6) and HT (n = 6) BN/SHR-mtHR-mtSHR .PGC-1\u03b1 regulates NRF-dependent transcription, increases expression of both mitochondrial and nuclear encoded genes of oxidative phosphorylation and induces mitochondrial biogenesis. HT BN/SHR-mtSHR exhibited ~2.5 fold reduction in PGC-1\u03b1 mRNA in kidney tissue compared with NT BN/SHR-mtSHR (P = 0.0098) ( 0.0098) .Tfam, whose expression is regulated by NRF1. All three NRFs were reduced in the kidney, but not liver of HT compared to NT BN/SHR-mtSHR. Renal NRF1 mRNA expression was reduced ~1.8 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0307) (NRF2a mRNA expression was reduced ~2.3 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0083) (NRF2b mRNA expression was reduced ~1.9 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0377)  . Renal N 0.0083) . Renal N 0.0377) .Tfam is a key activator of mammalian mitochondrial transcription. Kidney, but not liver exhibited reduced Tfam mRNA expression ~2.5 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0281) ( 0.0281) .CYC or Cox6c between NT (n = 10) and HT (n = 10) BN/SHR-mtSHR (P>0.05)  .SHR  and HT (n = 10) BN/SHR-mtSHR . There wSHR compared with NT BN/SHR-mtSHR , also exhibited reduced expression in kidney cortex, but not liver, compared with NT BN/SHR-mtSHR  subunits and the potential for disease has been previously evaluated , 30. Lopet al.  reported [et al.  reportedet al. [SHR compared with NT BN/SHR-mtSHR. It is possible that mitochondrial activity is elevated in renal proximal tubules of very young SHR prior to the onset of hypertension. As the development of hypertension progresses in the maturing SHR, renal mitochondrial gene expression may then decline as a compensatory mechanism to, for instance, decrease sodium transport along various segments of the nephron. This is plausible, as altered renal sodium handling and renal function are a hallmark of hypertension. Ongoing and future studies are being conducted to address this possible relationship and mechanism specific to the SHR.Data presented here poses the important and critical question: Does the reduction of renal mitochondrial gene expression and function contribute significantly to the etiology of hypertension? Or does hypertension cause the downregulation of mitochondrial gene transcription and function? Recently, Lee et al.  postulattrans-factors that regulate them. As shown in SHR, hence, it appears that mitochondrial function was decreased in the kidneys of HT BN/SHR-mtSHR. Altered renal function has been well recognized as a key factor in the development and maintenance of hypertension [et al. [Agtr1a gene resulted in the reduced age-related cardio-renal complications, improved mitochondrial biogenesis, and increased longevity in mice. Treatment with antioxidants, mitochondrial superoxide dismutase mimetics, and AT1r blockers decreased vascular O2.- production and attenuated development of hypertension in SHR [SHR exhibit elevated AT1r mRNA (Agtr1a) expression compared to NT BN/SHR-mtSHR, while the renal and systemic expression of renin, angiotensinogen, and angiotensin-converting enzymes were not different [SHR also had elevated AT1r protein compared with NT BN/SHR-mtSHR, and this increase was positively correlated with elevated systolic BP [et al. [1r blockade with Losartan. These data support the hypothesis that hypertensive phenotype derived from the SHR is driving the reduced mitochondrial gene expression, which may lead to decreased OXPHOS.One of the more interesting aspects of this study is a potential ETC dysfunction driven by transcript differences in the kidneys, but not in other tissues, of mt-genes and the nuclear rtension , 33\u201338.  [et al.  Deletionn in SHR , 41. In ifferent . Renal ctolic BP . De Cava [et al.  demonstrPGC-1\u03b1 plays a central role in regulating mitochondrial content and function within cells, because of its ability to co-activate and augment several promoters of nuclear-encoded mitochondrial genes, as well as regulating mitochondrial transcription via the NRF-Tfam pathway [PGC-1\u03b1 regulates NRF-dependent transcription, increases expression of both mitochondrial and nuclear encoded genes of oxidative phosphorylation and induces mitochondrial biogenesis [PGC-1\u03b1 in a tissue specific manner in brown fat, muscle and liver [PGC-1\u03b1 and its effectors is unknown. Results from this study show a coordinated reduction of the kidney-specific expression of nuclear and mitochondrial genes vital to OXPHOS in hypertensive BN/SHR-mtSHR, which may play a significant role in the development and maintenance of hypertension  nor cytochrome c oxidase subunit Vic (Cox6c) were differentially expressed in kidney cortex of HT and NT BN/SHR-mtSHR .None of the 7 mitochondrial encoded genes of complex 1 exhibited gene expression differences (P>0.05) between the HT and NT BN/SHR-mt(TIF)Click here for additional data file.S2 FigA: Superoxide dismutase 1 (SOD1) nor B: superoxide dismutase 2 (SOD2) were different (P>0.05) between NT and HT BN/SHR-mtSHR .Neither renal (TIF)Click here for additional data file."}
{"text": "DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 \u03bcg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg\u2013 1 \u03bcg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95\u00b0C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system. Furthermore, an a posteriori Tukey test showed that DNA recovery using 1 M NaOH and 95\u00b0C formamide were significantly different than recoveries using buffer EB, water, and 50 mM Tris-Cl (pH 8.5) at \u03b1 = 0.01. In addition, there was a significant difference in DNA recovery using 1 M NaOH versus 95\u00b0 formamide at \u03b1 = 0.01.The elution curve data is shown in The DNA loss data suggests that DNA adsorption on silica is mainly dependent on pH. DNA has an inherently greater affinity for the silica surface in acidic environments. However, the addition of the chaotrope (GuSCN) increased the surface affinity for DNA at pH 3 and 5.2. We expected this result because the isoelectric points of DNA and silica are 5 and 1.5\u20133.6 respectively. At pH 3, we did not expect electrostatics to interfere with the chaotropic mechanism that increased DNA adsorption, resulting in the highest surface affinity. Both the DNA and the silica surface become more negative at pH 5.2, and the corresponding increase in electrostatic repulsion resulted in more DNA loss. At pH 8, the loss was maximized. It has been theorized that chaotropes increase DNA-silica affinity by dehydrating the surface and promoting hydrogen bonding between the DNA and silica surface . Our DNAFocusing on events downstream of the DNA adsorption process, our elution results suggest that an increase in DNA adsorption capacity at low pH does not necessarily leads to a higher DNA recovery rate. Instead, the best DNA recovery results were obtained by taking advantage of a weaker DNA-silica-chaotrope adsorption complex facilitated at the intermediate pH of 5.2. Buffer EB, the gold-standard, was not able to disrupt the DNA-silica-chaotrope complex created at pH 3 since DNA recovery did not exceed 10.1%. Conversely, when the DNA-silica-chaotrope complex was formed at pH 8, weak DNA adsorption events resulted in massive 48.8% DNA loss prior to the EB elution steps. Thus, a DNA-silica-chaotrope adsorption event at pH 5.2 represents an optimum situation where: (A) The initial DNA loss due to a weaker solid-phase complex is offset by (B) An ease in complex dissociation with buffer EB at pH 8.5, thus resulting in a multi-factor local maximum in total DNA recovery.Diving deeper into 2 in the presence of a chaotrope, depending on the pH of the adsorption solution [2. In this regime when using GuSCN, no appreciable DNA was adsorbed at pH 8 and no appreciable DNA will be eluted when adsorbed at pH 3. This is in contrast to other experimental designs in literature that have demonstrated recovery of significant amounts of DNA after adsorbing at pH 4 or 8, with the caveat that the amount of unrecoverable DNA within the system was not addressed. To elaborate, these studies were conducted using large concentrations of input DNA, such that the net unrecoverable amount of DNA was negligible compared to the net recovered [The experiments were conducted such that DNA, rather than the available surface, was the limiting factor for adsorption. Existing literature has shown that the adsorption capacity for DNA per unit surface of silica is 240\u2013800 \u03bcg/msolution ,19,26. Oecovered ,26. ThesOur results suggest that the standard method for eluting DNA using buffer EB from silica following chaotrope-mediated adsorption may not be ideal for low concentration of DNA. Only two conditions allowed for the recovery of more than 30% of input DNA with buffer EB. Based on these results, DNA recovery depends on 1) the concentration of DNA in the adsorption solution, 2) the adsorption solution pH, 3) the presence of a chaotrope, and 4) the elution buffer. It also depends on the ratio of input DNA to the available surface area of silica when comparing to previous results. This however was controlled for in these experiments. Thus, designing a device to use this technology requires not only an understanding of the DNA-silica-chaotrope interaction, but the dynamic range of characteristics associated with clinical samples and how sample preparation steps affect these characteristics. Our data suggests that we can increase the range of initial sample conditions for which this technology can be implemented.We developed a protocol to assess the adsorption and elution of DNA from silica particles while emulating POC DNA extraction conditions for samples containing \u2264 1 \u03bcg DNA. Controlling for presence of a chaotrope, DNA adsorption increased with increasing acidity of the aqueous adsorption solution. Adsorption was further enhanced by adding 5 M GuSCN to the adsorption solution. Elutions exceeded 30% DNA recovery only when DNA was adsorbed at pH 5.2 with 5 M GuSCN for inputs of \u2265 100 ng net DNA. Adsorption at pH 3 resulted in DNA-silica-chaotrope complex that was too strong for buffer EB to disrupt effectively, while adsorption at pH 8 was not strong enough for DNA to adsorb sufficiently. Due to the poor recover of DNA with buffer EB at pH 3, we attempted to disrupt DNA-silica-chaotrope complex by varying the eluting solution. Hot formamide and 1 M NaOH resulted in increased recovery of 27.5% and 71.9% respectively, leading to the conclusion that the interaction was dominated by hydrophobic interactions and hydrogen bonding. However, neither of these buffers is practical for POC use. The optimal adsorption condition for POC use studied here was achieved using pH 3 with 5 M GuSCN resulting in up to 99.998% DNA adsorbed, while the optimal DNA recovery with buffer EB was 53.5% DNA using pH 5.2 with 5 M GuSCN. As our knowledge of these interactions improve, so will the range of clinical applications for this technology."}
{"text": "N- and O-linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N-glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids  in the N-glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N-glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation  of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins  was used to explore charge- and polarity-based mechanistic constraints in N-glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N-glycosylation at preferred and non-preferred sites.The structural and functional diversity of the human proteome is mediated by  O- and N- linked sugar moieties (N-glycosylation) and hydroxyl groups of serine and threonine (O-glycosylation) generates a large number of glycoforms that are credited for the modulation of diverse cellular functions , the oligosaccharyl transferase (OT) mediates the co-translational transfer of a lipid-linked tetradecasaccharide (GlcNAc2-Man9-Glc3) from a dolichol phosphate to an asparagine included in a NXS/T sequon. The selective recognition by OT of the consensus sequence (NXS/T) has enabled investigation of the structural requirements for N-glycosylation. The rapid increase of substrate data for protein N-glycosylation has led to the development of different databases and prediction tools: dbPTMs, UniProt, NetNGlyc and MAPRes  (Proteins that undergo cations) , 12\u201315.N-glycosylated to perform key biological functions (N-glycosylation (preferred and non-preferred motifs) is needed to explore the biological relationships between sequence, structure, and function of glycoproteins. MAPRes is a valuable tool to define the significantly preferred and non-preferred amino acids in the vicinity of a N-glycosylation site by resorting to the association rule mining technique (N-glycosylation (N+) and non-N-glycosylation (N\u2212) sites on the basis of potential score and consensus sequences within the target protein  in 1,117 human proteins in which the majority (96.5%) of N + sites is followed by the canonical motif of NXS/TY. According to our MAPRes analysis for general protein sequence analyses, Val at +1, Ser/Thr at 2, Leu/Val at 3 and Leu at \u22125 positions were found significantly preferred residues to mediate the glycosylation of Asn residues in the human proteome. After classifying amino acids\u2019 charge and polarity according to properties of their side-chain R-groups, significant preference for N-glycosylation was found for non-polar, uncharged R-groups  at position 1, polar R-group (Met/Thr/Ser/Cys/Asn/Gln: L) at position 2, polar, negatively charged acidic R-groups  at position 3/5/\u22124 and aromatic amino acids: Phe/Trp/Tyr: A, at position 3/\u22125/\u22121. Furthermore, we validated the MAPRes-predicted preferred association pattern for the wider N-glycosylation sequence contexts by using the NetNGlyc 1.0 server and 130 literature-reported UniProt proteins, and provided further evidence that charge and polarity of O amino acids  at position 1, A amino acids (Phe/Trp/Tyr) at positions \u22126, \u22125, \u22122, \u22121,1,3, and 10, P-amino acids (Lys/Arg/His) at positions \u22129, \u22123, 9, 10, and N amino acids (Asp/Glu) at positions \u22124/3/5, in combination with L amino acids at position 2, is likely to generate significantly preferable environments for the N-glycosylation of human proteins. Any change in charge and polarity environments may, therefore, result in aberrant N-glycosylation on both preferred and non-preferred sites.In this study, we have identified 2,909\u2009N-glycosylated (N+) and non-N-glycosylated (N\u2212) residues by using MAPRes. Then, preferred amino acids and association patterns were determined on the basis of polarity and charge of the surrounding amino acids of N+ and N\u2212 residues. Next, association patterns mined by MAPRes were validated with the NetNGlyc 1.0 server (see text footnote 1) for 15 biologically important proteins. The second level of validation was performed by using 130 literature-reported human proteins with 438\u2009N-glycosylated motifs from the UniProt database.As a first step, preferred amino acids and association patterns were found around N-glycosylated proteins were downloaded from dbPTM, version 3.0,N-glycosylation included 16,915 entries in total and provided all required information for MAPRes analysis except for the protein primary sequences (from UniProt), which were added manually in the final dataset. All entries other than human proteins were removed. In the final dataset, only 1,117 proteins and 2,909 asparagine residues were found modified.The primary sequences for The data cleaning of the final dataset was essential for estimating modified residues on vicinal amino acids. Several types of errors that can be generated during the compilation of the dataset such as incorrect position of the modified residues, presence of non-standard characters in the sequence, incorrect sequence length and repetitions of entries. These inconsistencies were identified and removed by utilizing the Data Inconsistency and Duplication Check module of MAPRes.N-glycosylation proteins (Table To the environments of the N+ (positive) and N\u2212 (negative), the negative sites were annotated in the final dataset. There were in total 31,052 negative sites (non-modified Asn) in the final dataset of human The new version of MAPRes can mine association patterns for neighboring amino acids of modified residues on the basis of their specific biophysical and biochemical properties. In this study, the association patterns mined on the basis of polarity and charge of the amino acids were distributed into five different groups Table . SpecifiN-glycosylated and non-N-glycosylated datasets. MAPRes supplied the charge and polarity information to estimate the significantly preferred amino acids and association rule mining for neighboring amino acids for all datasets (Table N-glycosylated \u201cN+\u201d and other is non-N-glycosylated \u201cN\u2212\u201d) and two for classified/encoded sequence of proteins (N-glycosylated and non-N-glycosylated) for which MAPRes mined association rules  found by NetNGlyc from the selected 15 proteins. The rest of the 642 Asn were considered to be N-residues. Peptides of 21 amino acids were generated for both N+ and N\u2212 residues and patterns mined by MAPRes for N-glycosylated and non-N-glycosylated, and found in both datasets. The confirmatory percentage for association patterns in the peptide dataset was found to be 94% for the N-glycosylated dataset and 59% for the non-glycosylated dataset  and sequence-based statistical analysis of N-glycosylated motifs validated the association rules of MAPRes. From the general dataset, around 99% of N-glycosylated motifs were found in the NXS/TY sequon . From classified datasets, among 438 residues, 99% were L amino acids at position 2 , and 30% of total were O-amino acids at position 1, in combination with L amino acids at position 2, to validate the <O,1><L,2> rule. Therefore, a strong correlation was observed between polar and non-polar R-groups in the NXS/TY sequon. The presence of O amino acids  at position 1 and of L amino acids at position 2 were characteristic for N-glycosylation  at 5, \u22124, and 3 positions. In addition to O and N amino acids, A amino acids also showed preference with at \u22122, 10, 3, \u22121, and \u22125 positions, in combination with L amino acids at 2 , N73 (NYDL), N128 (NKTG), N175 (NMSM), N196 (NGSC), N352 (NATN), N361 (NCTS), N413 (NRTD), N444 (NITS), N528 (NVSR), N568 (NITC), and N603 (NNTL)], localized in the extracellular EGFR , another key regulator of neoplastic cells, the extracellular N-glycosylation sites \u201cN558 (NSTY), N570 (NGSP), N622 (NTSP), N637 (NWTI)\u201d were modeled to validate the MAPRes association pattern  found by NetNGlyc from the selected 15 proteins. The rest of the 642 Asn were considered to be N-residues. From the second level of validation, we selected the 438 experimentally defined glycopeptides from 130 reviewed proteins and found validation of MAPRes rules in all 438 glycoepitopes. We further modeled two cell membrane proteins, epidermal growth factor receptor (EGFR), and E-cad, because these proteins are heavily decorated with N-glycans and involved in cancers as a result of aberrant or excessive glycosylation  inhibitor erlotinib, a frequently utilized drug to downregulate EGFR activation supporting constitutive proliferation of non-small cell lung cancer (N-glycosylation (a modification of the extracellular portion of EGFR) on the intracellular TK enzyme strongly suggests that the allosteric organization of EGFR TK is dependent on extracellular N-glycosylation events and that EGFR functions are indeed linked to the N-glycosylation status of EGFR.In the first level of validation, the association patterns mined by MAPRes for sylation , 44, 45.d breast \u201348. Actug cancer . This efN-glycosylation sites  in its extracellular domain were reported in the UniProt database  achieves the conformation favoring N-glycosylation of N56 -based structural modification of E-cad N-glycosylation in epithelial-to-mesenchymal transitions and susceptibility to colorectal cancer (N-glycosylation sites \u201cN558 (NSTY), N570 (NGSP), N622 (NTSP), N637 (NWTI)\u201d , and N637 (as Asn633) were found aberrantly decorated with complex-type high-mannose N-glycans , and critical for regulating the biological functions of E-cad in cancer \u201d , supportn cancer , 34. ForN-glycosylation site, favor the normal N-glycosylation process. However, the mutational changes at N-glycosylation site and/or vicinal preferred sites may impact the oncogenicity of proteins  and reinforces the neoplastic phenotype. Thereby, the genetic alterations in many transmembrane receptors and adhesion proteins are important to determine the success or failure of glycosylation in these transmembrane proteins. This is particularly critical in neoplastic cells where disease-associated risks need to be assessed for many transmembrane oncogenic TKs.Glycans remodel the protein backbone by reducing its conformational freedom and the loss of configurational entropy upon folding. To unravel the phenomenon of N-glycosylation. Moreover, remote parts of the protein chain rich in aromatic amino acids (A) support protein folding and promote the glycan\u2013protein hydrophobic and nucleophilic interactions at positions 3/\u22125/\u22121  and O amino acids at position 1  was found significantly preferred for 1 Tables , in the 1 Tables . The N aThis publication is dedicated to the memory of Professor Dr. Nasir-ud-Din (1937\u20132016), founder and chairman of Institute of Molecular Sciences and Bioinformatics and Fellow of Pakistan Academy of Sciences.MM designed the basic research theme, and contributed for data collection, analysis and validation. He also contributed significantly in manuscript writing. ZI defined and designed the research theme, methods, and generated various results. WQ contributed to generating results and participated in the improvement of the results interpretations. DH provided suggestions for improving the structure of the basic theme of the research and writing of the manuscript. He also helped to improve the scope of the study.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": "Background: Endobronchial and endotracheal metastases from extra-pulmonary solid tumors are rare.Patients and methods: We reported the case of a patient diagnosed with endobronchial and endotracheal metastases from rectal adenocarcinoma.Case report: Patient P.G., 62 years old, was diagnosed with a rectal tumor in 2011, for which, a surgical intervention was performed . Afterwards, she underwent adjuvant chemotherapy and concomitant radiochemotherapy.In September 2013, the chest CT showed 2 nodules for which, an incomplete surgical resection was done and which were histopathologically diagnosed as metastases from rectal cancer. The patient continued the treatment with chemotherapy associated with Bevacizumab and after 6 months only Bevacizumab for maintenance.In June 2015, the chest CT pointed out a nodule in the right upper lobe and the bronchoscopy highlighted a 4-5 mm lesion at the level of the right primary bronchus, whose biopsy proved the rectal origin. Afterwards, another surgical intervention was performed. Unfortunately, the postoperative chest CT revealed an intratracheal tissue mass (11/ 7mm) and multiple metastases in the right lung. The bronchoscopy showed 2 endotracheal lesions, out of which one was biopsied . Despite the fact that chemotherapy was continued, other endobronchial lesions appeared. All of them were removed and the patient started radiotherapy on the tracheal area. Afterwards, she refused to continue chemotherapy. The last bronchoscopy highlighted one endobronchial and two endotracheal secondary malignant lesions.Conclusion: Endobronchial and endotracheal metastases must be taken into consideration in all the patients with a history of extra-pulmonary cancer.Abbreviations: CT = computed tomography, MRI = magnetic resonance imaging, IMRT = intensity-modulated radiotherapy, ESMO = European Society for Medical Oncology, NCCN = National Comprehensive Cancer Network, iv = intravenous, PET \u2013 CT = Positron Emission Tomography \u2013 Computed Tomography Endobronchial and endotracheal metastasis secondary to extra-pulmonary solid tumors or to primary lung tumors are rare, but can be life-threatening [1]. The most frequent solid primary non pulmonary tumors which determine the appearance of endobronchial/ endotracheal metastasis are breast cancer, colorectal cancer, renal cell carcinoma and malignant melanoma .Among the different localizations of the tracheobronchial tree, the trachea represents an extremely rare site for metastasis from solid extra-pulmonary cancers, being involved in only 5% of the cases , have occurred in the evolution of a rectal adenocarcinoma. Usually, they appear late after the diagnosis of a primary tumor, at a median of 50 months, being associated with other metastases . Our patient presented tracheobronchial tree metastases after 43 months from the diagnosis of a rectal cancer.The particularity of this case was represented, firstly, by the fact that endobronchial and endotracheal metastases, which are a rare condition . Through histopathology, it is very important to make the differential diagnosis with a primary lung cancer, because the treatment and prognosis differ [5-7]. As in the presented case, the symptoms that can appear in this condition can be life-threatening and are characteristic for an upper airway obstruction: cough, hemoptysis, dyspnea and stridor .In this association with rectal cancer it is possible that they are underdiagnosed because bronchoscopy is the most important test through which the physician establishes the diagnosis of certitude, but it is not seldom used between the investigations that are done in a rectal cancer . It can relief the symptoms which are due to the upper airway obstruction and improve the quality of life . It is represented by surgery, chemotherapy, external beam radiotherapy, cryotherapy, and brachytherapy [4]. Regarding the treatment of endobronchial and endotracheal metastasis, usually it is palliative . The goals of neoadjuvant radiochemotherapy are to increase local control, pathologic complete response and the rate of sphincter preservation; in addition, it is less toxic than administered postoperative [9]. Maybe it would have been better for this patient, whose tumor was cT3 on the MRI performed in August 2011, to receive neoadjuvant radiochemotherapy and according to the postoperative pathological result, adjuvant treatment.Neoadjuvant treatment (radiotherapy +/ - chemotherapy) represents the standard for locally advanced rectal cancers tumor (T3-4 N0 or Tany N+) [The tracheobronchial tree should be taken into consideration as a metastatic site for primary solid tumors localized extrapulmonary.Source of foundingThis work received financial support through the project entitled \u201cCERO \u2013 Career profile: Romanian Researcher\u201d, grant number POSDRU/159/1.5/S/135760, co-financed by the European Social Fund for Sectoral Operational Programme Human Resources Development 2007-2013.DisclosuresAuthors declare that there is no conflict of interest regarding the publication of this paper."}
{"text": "Enterococcus faecalis in a chemostat of our own design with limiting nutrient in the inflow set near saturation constant at three dilution rates . The highest dilution rate was near the critical rate calculated by the model. The one-day total biofilm buildup was 21\u00d7 larger and its estimated growth rate 2.4\u00d7 higher at highest dilution rate than at the lowest one. This increased biofilm formation with increased dilution rates is in agreement with previously published data on pure and mixed continuous flow cultures.We revisited the mathematical model of the chemostat and examined consequences of considerably decreasing the concentration of limiting nutrient in the inflow for the growth of both the planktonic and biofilm cells in the chemostat tank (fermenter). The model predicts a substantially lower steady-state biomass of planktonic cells in response to decreasing inflowing nutrient concentration. Contrarily, the steady-state concentration of nutrient inside the fermenter is expected to remain the same, as long as the inflowing concentration does not fall below its value. This allows the biofilm cells to grow at a rate regulated only by the exchange rate of the medium (dilution rate). We maintained a strain of  A large amount of both experimental data and mathematical description of biofilm buildup has accumulated in the past , yet litA chemostat as a physical instrument allows bacterial populations to grow at a constant rate for an indefinite period of time. As a mathematical model, the chemostat is simple, realistic, and thoroughly tested against the physical instrument by several generations of researchers .Even though the chemostat model itself does not include a biofilm component, it calculates a \u201ccommon currency\u201d for both plankton and biofilm, i.e., concentration of substrate limiting the growth of the strain in question. As soon as a cell attaches to a submerged surface , the current concentration of substrate ceases to be the limiting factor for its survival which creates a competitive advantage for attached cells over the cells still in suspension. The difference between the two types of cells is in prerequisites for surviving in the system: The suspended cells will be washed from the tank unless they divide at a rate dictated by its dilution rate. The biofilm cells can adjust to lower concentration of substrate by lowering their growth rate without punishment, their challenge, however, is to stay attached. Adhesion is a key trait that biofilm cells need to succeed in competition. It is accomplished using attachment factors and extracellular polymers. The production of extracellular matrix varies among strains and may result in eliminating the less producing cells from the systems by sloughing .The mathematical model of the chemostat devised by A part of the following analysis reiterates the structure of the theory of the chemostat and a reader not fully familiar with the topic is recommended to read more comprehensive texts such as by \u03bcmax), its saturation constant (Ks), and a biomass yield coefficient , all for a given limiting substrate.The dynamic budget of the chemostat model is determined by three constants: the bacterial strain\u2019s maximum growth rate (\u03bc) from the increment of the natural logarithm of the biomass (X) plotted against the increment of time (t).For probing the applicability of the chemostat model to biofilm growth rates, we need to presume that the growth kinetics of both planktonic and biofilm cells of a given microbial species in relation to their nutrients (substrates) is similar, despite the differences in gene expression between the two phenotypes . We willS): The maximum specific growth rate \u03bcmax and the saturation constant Ks . The growth rate at a given limiting nutrient concentration is approximated by the function:Equations (1) and (2) are sufficient to characterize growth in a closed system (static culture) with high initial nutrient concentrations to support it. In order to describe the growth of populations under changing nutrient conditions, we must use kinetic parameters to characterize the relationship between growth rates, and the availability of nutrients. Experimental data typically follow the hyperbolic Monod\u2019s function , a saturX, in the chemostat, substrate-dependent growth \u03bc(S) increases the biomass, while dilution at rate D (volumetric flow rate per volume of the container) decreases the biomass, leading to the rate equation:For the planktonic biomass, \u03bc(S) \u2013 D = 0; both values have the same physical dimension (h-1)]. The steady state value of the planktonic biomass, , depends on how efficiently the substrate can be converted into cells, and this is typically characterized with a strain-specific empirical yield coefficient, Y: = Y (Sr), where  is the steady-state limiting substrate concentration and Sr is the in-flowing (\u201creservoir\u201d) concentration of the limiting substrate.When they reach a steady-state concentration, the cells in the chemostat will have adjusted their substrate-controlled growth rate so that it matches the dilution rate  and after 15 min carefully rinsed with PBS for the same period in another petri dish. The surface treated with the fluorescent probe was then covered with a coverslip and the edges sealed with nail polish to prevent evaporation. The bottom side of the slide was cleaned with 95% ethanol.The slide was transferred to a confocal laser scanning microscope  and processed promptly. Scanning was performed in inverted position (coverslip at the bottom) with a 63\u00d7 oil objective. Green fluorescence of Syto 9 was excited with a laser wavelength of 488 nm , while tImages of individual bacteria acquired with CLSM using DNA probes are often poorly defined for the purposes of quantification with \u201cclassical\u201d image analysis, as the cell size nears the resolution limits of optical microscopy including CLSM . GrowingE. faecalis (ATCC 29212) for estimating initial rates of exponential growth at serial dilutions of its common culture medium, Trypticase Soy Broth (TSB). The initial concentrations of the medium were divided in half in each consecutive dilution. When we plotted the highest growth rate reached in the respective batch culture against dilutions of the original complex medium, the graph looked like the classical Monod\u2019s saturation curve. Even though the original function had been designed for a single limiting substrate, it allowed estimating the concentration of sterile medium just sufficient to support population of bacteria growing at a given rate  values. This way of estimating the above parameters is not regarded as the most accurate . To apply the model, we used two constants, \u03bcmax (1.07 h-1) and Ks (0.019) derived from our E. faecalis static cultures. The third constant, the yield coefficient, was set as 40%, Y = 0.4 based on typical literature data for bacteria. Lastly, the fourth parameter  had to be dramatically adjusted upward  so that the model did not produce negative values at high settings of dilution rate, D ( (D), suspended cell biomass , and its production ( =  \u22c5 D) covering the range of dilution rates equivalent to 0 \u2013 \u03bcmax. The arbitrary units of substrate and biomass could be easily converted to physical units, such as dry weight, carbon, etc.We exploited the potential of the  rate, D . The modmax and the planktonic cells are no longer able to grow quickly enough to compensate for the dilution rate; the level of available substrate rises, until it eventually reaches a physically constrained maximum of Sr; and the production of planktonic biomass increases to a maximum where the effects of increased dilution rate and decreased biomass are optimized, before falling as the planktonic biomass begins to drop.As the plot  indicateKs ) concentration of substrates in the inflow (rS ), we applied the same model at 1/16 of the full TSB medium (rS = 0.06). These conditions generated positive values of cell biomass in planktonic cells, (D), only forIn order to address the effect of low (near D < 0.8 ( (D) accompanying extremely low values of  (D) and , when the dilution rate (D) approached the highest value (0.8 h-1). However, comparing the same scale of  (D) concentrations in both graphs shows that that the calculated values were identical. In D that were implemented in the subsequent E. faecalis chemostat experiments. Corresponding values are marked in the graph on the limiting nutrient  (D) \u2013 green line . Three different dilution rates (D) were used to establish steady-state cultures: 0.09, 0.28, and 0.81 h-1. In each run, a sterile biofilm slide was immersed in the chemostat culture for 24 h. Before the slide was inserted in the culture, the flow was kept constant for a period of at least 9 residence times  for inoculation and sampling of biofilms. All additional operations were performed in a sterile biosafety cabinet. Back-flow contamination was prevented by a protective stream of sterile gas. The constancy and homogeneity of nutrient supply to the fermenter was improved by instant dispersion of medium by gas bubbles. A precision pump allowed control of a precise and stable dilution rate. Lastly, cell quantification was carried out by the combination of CLSM and 3-dimensional image analysis allowing adjustment of the algorithm to image condition.rS value used in the chemostat culture lies near the junction of the two branches of the curve. Therefore, the steady-state substrate values [ (D) \u2264rS] were located on a steep part of the function, where small changes in nutrient concentration (S) seem to be correlated with large changes of specific growth rate (\u03bc) and dilution rate . Thus we re-calculated the \u201cstarting\u201d number of cells per cm2 from the one-day estimates for the assay reversing equation (2)  was much slower than the growth rate of plankton biomass .As opposed to the assay with the middle and highest dilution rate, in the run at  minimal . We prestion (2) . We can o assays . Based o-1h-1  with highest P had the lowest proportion of dead cells (0.8%). Therefore, the noticeable lack of significant difference between cell density at D = 0.28 h-1 and 0.81 h-1 could also be caused by a higher rate of attachment at D = 0.28 h-1 due to a higher production of plankton. It has been previously demonstrated , represents the biomass that can be \u201charvested\u201d , its physical unit being g L-1h-1 . Using t0.81 h-1 . The midnstrated  that thenstrated .Apparently, the tools used in this study allowed only a rough comparison between the mathematical and physical model. Even over standardized time, the biofilm biomass increase was not a direct measure of its growth rate or production. On the other hand, we have not yet fully utilized the versatility of our chemostat design. If employed in multiple tanks, it would enable researchers to dissect the plankton/biofilm interplay owing to its capability to transfer independently the biofilm-covered slide and/or the entire tank contents (plankton) to a new, sterile tank in real time. It would allow us using markers  and third assay . As the exposure would be shortened roughly in proportion to the plankton population doubling time  than at faster dilution rates . E. faecalis developed a 21-fold denser biofilm in response to a ninefold increase in the chemostat dilution rate  after evaluating a series of batch cultures and applying the function of the saturation curve (S). According to the mathematical chemostat model (rS ). However, there is a restriction of the S value by the fact that S \u2264rS at all times. We used this rule for setting the limits for calculation of variables in continuous culture fed by medium with limiting nutrient concentration. Using the Monod\u2019s function, we estimated the highest  dilution rate still allowing a steady-state planktonic growth.We established the on curve . Then, wat model , this va\u03bcmax, hence, the equations held only for a range of dilution rates (D) lower than its critical value. In our physical continuous culture, there was a trend for an increased rate of biofilm formation over that range. Since the calculated steady-state substrate concentration (S) was the only variable that increased with D at rates approaching its critical value, the rate of biofilm formation may be predominantly driven by the growth rate of attached cells due to increased ambient substrate concentration (S). Thus, the biofilm formation is controlled by the dilution rate (D) through the growth rate (\u03bc) of the planktonic component. Higher planktonic growth rates resulted in increased biofilm growth while the planktonic biomass decreases.We fed the system with a concentration of nutrients supporting a growth rate lower than E. faecalis cultures predicted the maximum planktonic biomass production when D approached the critical value (either due to availability of substrate in our \u201cnutrient restricted\u201d chemostat or approaching limits of the strain\u2019s capacity for growth \u2013 \u03bcmax). This could also provide insight into the contribution of the planktonic biomass to the biofilm buildup. The calculated \u201cproduction\u201d variable , D = 50 h-1), and D between 65 and 260 h-1). While, due to  plug flow in the flow cells, the actual dilution rates at any point would be even higher, the calculated values of D are far beyond the highest known growth rates for bacteria. Research of these systems underscores the understanding that in addition to the biological behavior of biofilms as a result of nutrient concentration and fluid flow, their physical properties determine their mechanical response to the flow  organisms including humans.Even though a certain range of shear stress values has been found beneficial for building the biofilm architecture, a wider scale of shear stress spans from high values causing detachment, i.e., destruction of biofilms to low values, where no influence, detrimental, or beneficial could be observed. Both the Although the two parameters are positively correlated with the rate of flow, their effective values depend on a set of additional conditions, such as a container shape and its dimensions, fluid viscosity, etc. Yet, we suggest that in the same hydraulic space, two separate boundary values of flow rate can be distinguished that control the biofilm development. Dilution rate appears to enhance the rate of biofilm formation up to a certain value, beyond which the acceleration of biofilm growth does not happen. Similarly, in the opposite way, the shear stress seems to restrict the biofilm formation down to a value, beyond which its influence is negligible.Thus, for biofilm microorganisms, there may be three zones of flow involvement in their biofilm building capability:(a)-1.Slow flow domain of positive dilution rate influence that we addressed in this study, limited to dilution rates slower than 4 h(b)Intermediate flow domain where dilution rate or shear stress fluctuations have virtually no effect.(c)-1 and shear stress alone selects the organisms capable of attachment to the substratum under the given conditions. Within this domain values around 0.01 Pa allow for biofilm buildup, whereas the shear stress 1 Pa and higher generally causes biofilm destruction.Fast flow domain where dilution rates are substantially higher than 4 hvice versa.The intermediate domain, by definition, represents the conditions where the biofilm buildup is maximized. Hence, those conditions should be met, where biofilms may be beneficial and avoided wherever biofilm formation  is undesirable. The behavior and interactions of biofilm organisms reported in the flow cell studies are derived from the fast-flow domain, thus they do not necessarily characterize the same organisms growing under conditions of the slow-flow domain, and For the proposed boundary between the slow and intermediate domain, the implication of results of the present study is that the borderline value of flow/dilution rate is further controlled by the concentration of limiting nutrients.E. faecalis can be achieved with a more data intensive study, our results demonstrated that in a defined range of the fluid exchange rate, the chemostat theory is a good predictor of the biofilm buildup.Experimental data describing the positive correlation between biofilm formation and dilution rate have been previously published referring to both clonal  and mixeML contributed to the conception and study design; acquisition, analysis, data interpretation, and application of the mathematical model; and performed the experiments and wrote the manuscript. DC contributed to the study materials, analysis tools and reagents, and wrote the manuscript. DM contributed to the application of the mathematical model and wrote the 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": "When using nuclear magnetic resonance (NMR) to assist in chemical identification in complex samples, researchers commonly rely on databases for chemical shift spectra. However, authentic standards are typically depended upon to build libraries experimentally. Considering complex biological samples, such as blood and soil, the entirety of NMR spectra required for all possible compounds would be infeasible to ascertain due to limitations of available standards and experimental processing time. As an alternative, we introduce the in silico Chemical Library Engine (ISiCLE) NMR chemical shift module to accurately and automatically calculate NMR chemical shifts of small organic molecules through use of quantum chemical calculations. ISiCLE performs density functional theory (DFT)-based calculations for predicting chemical properties\u2014specifically NMR chemical shifts in this manuscript\u2014via the open source, high-performance computational chemistry software, NWChem. ISiCLE calculates the NMR chemical shifts of sets of molecules using any available combination of DFT method, solvent, and NMR-active nuclei, using both user-selected reference compounds and/or linear regression methods. Calculated NMR chemical shifts are provided to the user for each molecule, along with comparisons with respect to a number of metrics commonly used in the literature. Here, we demonstrate ISiCLE using a set of 312 molecules, ranging in size up to 90 carbon atoms. For each, calculation of NMR chemical shifts have been performed with 8 different levels of DFT theory, and with solvation effects using the implicit solvent Conductor-like Screening Model. The DFT method dependence of the calculated chemical shifts have been systematically investigated through benchmarking and subsequently compared to experimental data available in the literature. Furthermore, ISiCLE has been applied to a set of 80 methylcyclohexane conformers, combined via Boltzmann weighting and compared to experimental values. We demonstrate that our protocol shows promise in the automation of chemical shift calculations and, ultimately, the expansion of chemical shift libraries.The online version of this article (10.1186/s13321-018-0305-8) contains supplementary material, which is available to authorized users. The compounds \u2014remains ompounds \u20137. The compounds  can be pompounds , 10. Altompounds \u201313 and mompounds \u201316 have ompounds \u201321, deteompounds \u201325.The most practical approach expand reference libraries for comprehensive identification of compounds detected in metabolomics studies is through in silico calculation of molecular attributes. Molecular properties that can be both accurately predicted computationally and consistently measured experimentally may be used in \u201cstandards free\u201d metabolomics identification approaches. The metabolomics community has made many advances in calculations of measurable chemical attributes, such as chromatographic retention time , 27, tanMetabolomics researchers unfamiliar with DFT or similar calculations may find the application of quantum chemical calculations complicated or challenging to apply quickly, and thus avoid these techniques. To this end, and to help bring DFT calculations to large sets of small organic molecules relevant to the mainstream metabolomics community, we have developed a Python-based workflow and analysis package, the ISiCLE  NMR chemical shift module employs DFT methods through use of NWChem , a high-ISiCLE is a Python module that provides straightforward automation of DFT using NWChem, an open source, high-performance computational quantum chemistry package, developed at Pacific Northwest National Laboratory (PNNL), for geometry optimization and chemical shift and solvent effect calculations. Figure\u00a0Here, we describe each step of a typical ISiCLE run . File A must contain all input molecules either as (i) International Union of Pure and Applied Chemistry (IUPAC) International Chemical Identifier (InChI) strings , 53 or  and the reference molecule, respectively. i and the reference molecule, respectively.For each molecule, ISiCLE generates MDL Molfiles (.mol)  that conISiCLE also calculates errors in NMR chemical shifts if experimental data is provided in the MDL Molfiles in a required way as explained in the tutorial. The errors are quantified in terms of mean absolute error (MAE) Eq.\u00a0, correctEmpirical scaling of isotropic shieldings or NMR chemical shifts is the most common approach to remove systematic errors. If experimental data is provided, ISiCLE uses two optional approaches for its linear regression method, where slope and intercept values are derived from (i) regression of computed NMR chemical shifts versus experimental NMR chemical shifts using Eq.\u00a0, and/or Alternatively, if the user does not provide experimental NMR chemical shifts, ISiCLE can scale NMR chemical shifts using provided intercept and slope values. The scaled NMR chemical shifts are appended to MDL Molfiles.A detailed description of InChIs and InChIKeys, and why they were chosen, can be found in the Additional file Furthermore, installation details for OpenBabel and other required Python packages are provided in the tutorial  hybrid functional [1H and 13C NMR chemical shifts were computed relative to TMS using the Gauge Including Atomic Orbitals (GIAO) formalism [As a first demonstration of ISiCLE, a benchmark study was performed with 8 different DFT methods to predict nctional \u201396 and tnctional . This lenctional , 99. Isonctional , 95, B3Lnctional \u201399, B35Lnctional . DFT metnctional  or triplnctional ). All banctional \u2013104. Forormalism . Chlorofin vacuo molecular dynamics (MD) simulations, using the sander MD software program from AmberTools (version 14) [For a second demonstration of ISiCLE, the NMR chemical shifts, along with frequency calculations (and subsequent Boltzmann weighting), two sets of axial and equatorial conformers (40 conformers each) of methylcyclohexane were processed. We performed sion 14) , to genesion 14) . Second,sion 14) , 107\u2013109All results shown in this manuscript were generated using the Cascade high-performance computer .To test ISiCLE, we generated a set of 312 molecules. This set is large relative to other metabolomic molecule sets found in the literature, which in our literature survey averaged 34 molecules  is higher than those with cc-pVDZ (5\u20136\u00a0ppm). MAE of methods with a larger basis set deviate more compared to those with a smaller basis set. The smallest deviations are observed for B3LYP and B35LYP, both in MAE and MAXAE results. The same situation is observed for 1H NMR chemical shifts as well: MAE of each method with cc-pVTZ (~\u20090.35\u00a0ppm) is higher than those with cc-pVDZ (~\u20090.30\u00a0ppm). In contrast to 13C NMR chemical shifts, 1H NMR chemical shifts are better predicted with methods using larger basis sets (cc-pVTZ). Although the error differences among each method may be too low to confidently identify the outperforming method, B3LYP/cc-pVDZ is the most successful combination in the calculation of 13C and 1H NMR chemical shifts for our application shown here.A total of 2494 carbon nuclei and of 3127 hydrogen nuclei were calculated for all 312 molecules of the demonstration set and compared with experimental data. Deviation bars indicating MAE and MAXAE are plotted for each method in Fig.\u00a013C and 1H NMR chemical shifts was an acceptable compromise between accuracy and computational performance, compared with the larger cc-pVTZ basis. This finding is similar to a recent benchmark study [13C chemical shifts calculation. The larger basis set (cc-pVTZ) took 2\u20133 times longer to complete than cc-pVDZ . The computational times of the isotropic shielding and chemical shift calculations for this demonstration set are given in the file of DemonstrationSet_CPUtimes.xlsx in the Additional file Figure\u00a0rk study  that sho13C and 1H NMR chemical shifts, respectively. It provides confidence for applying linear regression effectively as it reduces the possibility of overfitting. Empirical scaling was applied to the data obtained with the best combination, B3LYP/cc-pVDZ, using two different relationships: computed shifts versus experimental chemical shifts  by 0.02 for both 13C and 1H NMR chemical shifts. Linear fits with correlation coefficients of 0.99  magnetic shieldings and chemical shifts derived from the various DFT methods are highly correlated, as shown by a correlation coefficient of 0.99  and sp3- (745 carbons) hybridized carbons  chemical shifts are significantly affected by intermolecular interactions, particularly in aqueous states, especially compared to 13C chemical shifts. Agreement with experimental values improves as empirical linear scaling is performed for 1H chemical shifts. GIAO/B3LYP/cc-pVDZ//B3LYP/6-31G(d) yields scaled 1H chemical shifts in chloroform solution having a MAE of 0.30\u00a0ppm in comparison with solution experimental values. The 1H chemical shifts in the range of 10\u201317\u00a0ppm show the largest deviation, occurring higher than 5\u00a0ppm.Proton , nitrogen (n\u2009=\u200917), and oxygen (n\u2009=\u200941). Oxygen-bound hydrogen nuclei have the largest errors (up to 10\u00a0ppm), which is to be expected due to the electronegative property of oxygen atoms, as discussed in the previous section. It is followed by less electronegative nitrogen-bound hydrogen atoms, with an MAE of 0.71\u00a0ppm and a MAXAE of 2.25\u00a0ppm. About 95% of the 1H NMR chemical shifts calculated for this set are from H\u2013C attachments. These chemical shifts had a MAE of 0.27\u00a0ppm and a MAXAE of 4.41\u00a0ppm. The high occurrence of outliers could be evidence of how 1H NMR chemical shifts are sensitive to intermolecular interactions.In Fig.\u00a01H NMR chemical shifts in the presence of H\u2013O attachments.H\u2013O attachments are highly sensitive , and it is non-trivial to determine the NMR chemical shift value of arbitrary protons experimentally as well as predict them by using a single, \u201ccatch all\u201d DFT method, which explains the relatively low correlation coefficient of 0.93 Fig.\u00a0c\u2013d. For Application of empirical scaling to functional groups are given in detail in the Additional file As a final assessment for the data collected with the demonstration set, we assessed the stability and accuracy of the linear regression approach using cross-validation . Cross-v13C and 1H NMR chemical shifts generalize well to the groups that are not represented in the training fold.We observed that the estimated linear model parameters (i.e. slope and intercept) from the training set do not differ from that of the entire set. Therefore, the predictive linear model is stable to be accurately estimated and the subsets of Metabolites were experimentally interrogated using solution-state NMR, where the observed signal arises from the combined signals of present conformers. It is routine that NMR chemical shifts calculations are carried out on a single dominant conformer. However, it is well known that metabolites do not comprise a single conformer in solution and are instead found in a collection of various conformers , and the1H and 13C chemical shifts  to experimental values reported by Willoughby et al. [1H chemical shifts  and writes the data to appended MDL Molfiles. It also quantifies the error in calculated NMR chemical shifts if the user provides experimental values.The functionality of ISiCLE is demonstrated on a molecule set consisting of 312 molecules, with experimental chemical shifts reported in chloroform solvent. 1H and 13C NMR chemical shifts were calculated using 8 different levels of DFT , referenced to TMS in chloroform by carrying initial geometry optimizations out at B3LYP/6-31G(d) for all molecules. The optimal combination for this set was found to be B3LYP/cc-pVDZ//B3LYP/6-31G(d) with mean absolute error of 0.33 and 3.93\u00a0ppm for proton and carbon chemical shifts, respectively. We show that DFT calculations followed by linear scaling do in fact provide an analytically useful degree of accuracy and reliability. Finally, we used ISiCLE for the calculation of NMR chemical shifts of 80 Boltzmann-weighted conformers of methylcyclohexane and compared our results with earlier studies in the literature.ISiCLE is a promising automated framework for accurate NMR chemical shift calculations of small organic molecules. Through this tool, we hope to expand chemical shift libraries, without the need for chemical standards run in the laboratory, which could lead to significantly more identifiable metabolites. Future work includes wrapping individual steps of the ISiCLE NMR module into a formal workflow management system such as Snakemake, to include better fault tolerance, modularization, and improved data provenance. Furthermore, additional chemical properties will be included, such as ion mobility collision cross section and infrared spectra. Finally, ISiCLE will be adapted to run seamlessly on cloud computer resources such as Amazon AWS, Microsoft Azure, and Google Cloud. ISiCLE is a promising tool contributing to standards-free metabolomics, which depends on the ability to calculate properties for thousands of molecules and their associated conformers.Additional file 1. Supporting information document.Additional file 2. Supporting information files. Including tutorial, code, and all other files."}
{"text": "We estimated mean intake of key iodine sources by race and Hispanic origin. We present the first national estimates of mUIC for non-Hispanic Asian persons and examine the intake of soy products, a potential source of goitrogens. One-third of National Health and Nutrition Examination Survey (NHANES) participants in 2011\u20132014 provided casual urine samples; UIC was measured in these samples. We assessed dietary intake with one 24-h recall and created food groups using the USDA\u2019s food/beverage coding scheme. For WRA, mUIC was 110 \u00b5g/L. For both non-Hispanic white (106 \u00b5g/L) and non-Hispanic Asian (81 \u00b5g/L) WRA mUIC was significantly lower than mUIC among Hispanic WRA (133 \u00b5g/L). Non-Hispanic black WRA had a mUIC of 124 \u00b5g/L. Dairy consumption was significantly higher among non-Hispanic white (162 g) compared to non-Hispanic black WRA (113 g). Soy consumption was also higher among non-Hispanic Asian WRA (18 g compared to non-Hispanic black WRA (1 g). Differences in the consumption pattern of key sources of iodine and goitrogens may put subgroups of individuals at risk of mild iodine deficiency. Continued monitoring of iodine status and variations in consumption patterns is needed.We estimated iodine status (median urinary iodine concentration (mUIC (\u00b5g/L))) for the US population (6 years and over;  Iodine is a trace element that is required for the synthesis of thyroid hormones, which are necessary for adequate growth, development, and metabolism . DeficieThe world\u2019s oceans are the main reservoir of iodine, containing iodide ions, and organic and inorganic iodine species which are volatized from seawater to the atmosphere and then eventually deposited on land as components in rain water . Iodine Dietary supplements and iodized salt  are alsoRoughly 90% of all iodine consumed is excreted in the urine  and thusp = 0.001) [The U.S has been considered iodine sufficient for decades ,25. Howe= 0.001) . Similar= 0.001)  and amon= 0.001) . Iodine = 0.001) ,29,30, k= 0.001) .Beginning in 2011\u20132012, for the first time, NHANES has nationally representative data of urinary iodine status and food group consumption for non-Hispanic Asian persons. The goal of this analysis is to update estimates of iodine status for the US population and women of reproductive age (WRA). We present estimates of iodine status by subgroups of race and Hispanic origin, and examine dietary patterns associated with iodine status by race and Hispanic origin using the most recent data from NHANES 2011\u20132014.NHANES is a complex, stratified, multistage probability sample of the U.S. noninstitutionalized population, conducted by the National Center of Health Statistics (NCHS). Since 1999, NHANES has continuously collected and publicly released data on roughly 10,000 participants every 2 years. Participants receive a detailed in-home interview, followed by a dietary interview and a physical examination, which includes sample collection for laboratory tests, at a mobile exam center (MEC). Participants aged 18 and over provide consent, children and adolescents aged 7 to 17 years provide documented assent, and parental permission is obtained for those younger than 18 years. Non-Hispanic Asian, non-Hispanic black, and Hispanic individuals were oversampled for the 2011\u20132014 survey years . The NHA\u00ae DRC II)  [Spot urine samples were collected from a one-third sample of participants aged 6 years and over in the MEC. UIC was assessed using an Inductively Coupled Plasma Mass Spectrometer with Dynamic Reaction Cell Technology  ,34,35. BCT, USA) . The WHOCT, USA) . Method CT, USA) ,26,33,34Trained interviewers, using a computer-assisted dietary interview system that included a multiple-pass format with standardized probes , collectThe USDA\u2019s Food and Nutrient Database for Dietary Studies (FNDDS) ,40 was uSince 2007\u20132008, NHANES has included a 24-h dietary supplement recall, administered at the same time as the dietary recall. We used the day 1 24-h supplement recall to identify participants who had reported consuming a supplement that contained iodine in the previous 24-h. We dichotomized participants based on this reported consumption.Responses to the salt use questions administered during the 24-h recall allowed us to group participants by usage frequency for food preparation and at the table . These questions do not ask specifically about iodized salt.Age is categorized into 8 groups for the US population  and 4 groups for WRA . We also used sex , race and Hispanic origin  to describe the data. Individuals with race and Hispanic origin classified as \u2018other\u2019 include those reporting multiple races, and are included in the overall estimates but not shown separately in the results. Pregnancy status for women aged 15\u201319 years, and 45 years and above is not included in the public use NHANES data files due to disclosure risk. We obtained this information through the Research Data Center .Statistical analyses were performed using SAS version 9.4  and SUDAAN version 11.0 . Survey design variables and sample weights, which account for differential probabilities of selection, nonresponse, noncoverage, and sample design, were used to obtain estimates representative of the noninstitutionalized US population. We used the day 1 dietary weights and adjusted for non-response in the urinary data (see below).Guidance regarding selecting the most appropriate sample weight advise selecting \u201cthe weight of the smallest analysis subpopulation\u201d . The curp-values from adjusted Wald tests were less than 0.05. The hypothesis of no linear trend across ordinal variables was tested by using orthogonal contrast matrices (p < 0.05).Using our adjusted weights, we calculated estimates for mUIC and Wald 95% confidence intervals. Neither SAS nor SUDAAN directly test the difference between medians. Using SUDAAN to account for complex survey design and weighting, we indirectly tested whether differences in mUIC by sociodemographic characteristics, salt, and supplement use were significant by first categorizing individuals as above or below the overall median. We then tested whether a below-overall-median proportion for any group differs from 0.5 with logistic regression. This indirect method is considered to be equivalent to testing whether medians varied by group . We alsoIn NHANES 2011\u20132014, 14,489 participants aged 6 years and over provided a complete and reliable dietary interview. Of these, 4744 participants were also in the 1/3 subsample who were eligible for urine iodine measurement. We excluded 68 participants because they did not have a value for urinary iodine, although they were eligible for urine collection , and 64 women who were pregnant or lactating, leaving a final analytical sample of 4613 participants, aged 6 years and over. For the analysis of WRA, the final analytic sample size was 901, after restricting to non-pregnant, non-lactating women, aged 15\u201344 years.p < 0.05), with the lowest median among adults aged 40\u201349 years , and highest medians observed among children aged 6\u201311 years  and older adults aged 70 years and over . Women had a significantly lower mUIC compared to men, 122 \u00b5g/L  vs. 147 \u00b5g/L  (p < 0.05). The only significant difference in mUIC by race and Hispanic origin in the US population was between non-Hispanic Asian individuals, ), and non-Hispanic blacks,  (p < 0.05)). The mUIC for non-Hispanic white and Hispanic individuals was 134 \u00b5g/L  and 133 \u00b5g/L , respectively. Among those who consumed a dietary supplement containing iodine, mUIC was significantly higher: 174 \u00b5g/L  compared with 127 \u00b5g/L  among those with no consumption (p < 0.05). We found that mUIC did not vary by salt use at the table. However, mUIC was significantly higher among those reporting never using salt in food preparation: 172 \u00b5g/L  compared to all other patterns of salt use: rare, 138 \u00b5g/L , occasional, 128 \u00b5g/L , and very often, 128 \u00b5g/L  (p < 0.05).The mUIC in the US population was 133 \u00b5g/L   in 2011\u2013p < 0.05). Among WRA, non-Hispanic Asian women had the lowest mUIC: 81 \u00b5g/L , significantly lower than Hispanic  women (p < 0.05). Differences between non-Hispanic Asian and non-Hispanic white women  and non-Hispanic black women 124 \u00b5g/L  were not significant. Differences in mUIC by supplement use were not significant. Among WRA with rare salt use at the table, mUIC was 122 \u00b5g/L  significantly higher than those with occasional salt use . WRA who reported rare salt use in food preparation has a significantly higher mUIC  compared with occasional  and very often use of salt in food preparation  (p < 0.05).The mUIC for US WRA, 15\u201344 years, in 2011\u20132014 was 110 \u00b5g/L  . We founp < 0.05). Grain consumption was highest among non-Hispanic Asian persons ), followed by Hispanic ) and non-Hispanic black ) and non-Hispanic white individuals ) . There were no statistical differences in egg consumption by race and Hispanic origin; the average consumption was 23 g . Both non-Hispanic blacks ) and non-Hispanic Asian individuals ) had higher fish consumption compared with non-Hispanic white ) and Hispanic individuals ) . Consumption of soy products was highest among non-Hispanic Asian individuals ) compared to non-Hispanic white ), Hispanic ), and non-Hispanic black individuals ) .p < 0.05). Differences in dairy consumption between Hispanic WRA  and other race and Hispanic ethnic groups and between non-Hispanic Asian women  were non-significant. The only significant difference in mean grain consumption was between non-Hispanic Asian women ), compared to 293 g  by non-Hispanic white women, (p < 0.05). Non-Hispanic black and Hispanic WRA consumed 308 g  and 343 g , respectively. There were no statistical differences in egg consumption by race and Hispanic origin; the average consumption was 22 g  among WRA. Non-Hispanic black WRA consumed significantly more fish ) compared with non-Hispanic white  and Hispanic WRA ) . Non-Hispanic Asian WRA consumed 24 g  of fish. Non-Hispanic black WRA consumed significantly less soy ) compared with Non-Hispanic Asian 18 g  and non-Hispanic white WRA ) . Hispanic WRA consumed 9 g  of soy.Among WRA , non-HisIn this analysis of NHANES 2011\u20132014 we found that the general US population aged 6 years and above had a mUIC of 133 \u00b5g/L and WRA had a mUIC of 110 \u00b5g/L, suggesting iodine sufficiency, according to WHO criteria . For theDespite the lack of a publicly available national nutrient database with iodine values for foods and beverages consumed in the US, there is consistent agreement that dairy, grains, eggs, and fish provide the bulk of iodine in the US diet ,30,48, aA novel finding of the current analysis is that non-Hispanic Asian individuals, who tended to have lower mUIC than other race/Hispanic origin groups, had higher consumption of soy products. Soymilk typically does not contain high amounts of iodine  comparedSalt iodization is considered the central strategy for eliminating iodine deficiency . In the The use of a dietary supplement containing iodine was positively associated with mUIC in the US population aged 6 years and above. A similar association between mUIC and use of a dietary supplement containing iodine was suggested for WRA; however, a significant difference was not detected, possibly because so few women in this age group consumed a dietary supplement with iodine. Like folic acid, iodine is critical for brain development in the early weeks of pregnancy when many women do not yet know they are pregnant . Unlike While our analysis did not include pregnant women, the Recommended Dietary Allowance for iodine increases from 150 \u00b5g/day when not pregnant to 220 \u00b5g/day during pregnancy. Thus, it is possible that without some changes in dietary intake, some women may not have sufficient iodine intake if they were to become pregnant. Severe iodine deficiency during pregnancy is detrimental for child survival and development, but the effects of mild-to-moderate iodine deficiency are less well defined. While challenging methodologically to assess this relationship because there is no established method for assessing an individual\u2019s iodine status, mild iodine deficiency in pregnancy has been associated with poorer educational outcomes at 8\u20139 years among cohorts in Tasmania  and the This study is not without limitations. It is well documented that self-reported measures of dietary intake are subject to random and systematic error due to day-to-day differences in intake, systematic underreporting, difficulty remembering foods, and errors in the food and nutrient database that intakes are linked to ,47. As windividual intake, neither does a single spot urine sample capture an individual\u2019s iodine status, as measured by UIC [The determination of iodine status as assessed by UIC also has challenges. The WHO definition of population iodine status does not include adjustment for creatinine . Howeverd by UIC . We therDespite merging two survey cycles, there was limited statistical power to identify differences in food group consumption by race and Hispanic origin among WRA. A separate issue is the considerable heterogeneity within race and Hispanic ethnic groups. For example, the non-Hispanic Asian group includes south Asian, Korean, Japanese, and Chinese, and eating patterns are likely to vary greatly among these subgroups, and the moderate iodine deficiency seen for among non-Hispanic Asian WRA may be more or less problematic in some racial subgroups.Some analyses were beyond the scope of this study. Seaweed is a known source of iodine; however, the iodine content in seaweed is highly variable  and it iThe strengths of this analysis include the most recent, nationally representative estimates of iodine intake and mUIC for the US population, WRA, and for the first time, non-Hispanic Asian persons. We estimated mUIC by the important sources of iodine in the US diet: food and beverages, supplements, and salt use. Another novel contribution of this work is the exploration of soymilk and soy products as a source of goitrogens in the US diet that may block the utilization of iodine and contribute to mild iodine deficiency.There is no overt sign of widespread iodine deficiency in the US, based on the observed mUIC from NHANES 2011\u20132014. However, non-Hispanic Asian WRA had a mUIC of 81 mg/L, indicating mild iodine deficiency in that subgroup. We found that mUIC did not follow the expected pattern of association with dietary intake of key food and beverage sources of iodine among all race and Hispanic origin groups. In the context of decreasing iodine status, variation in iodine status by race and Hispanic origin, and variation in dietary intake of key food sources of iodine, continued monitoring of iodine status and factors associated with iodine status is needed."}
{"text": "Tobacco smoking is a highly prevalent, addictive behaviour and a key public health priority. However available cessation therapies have low quit and high relapse rates, indicating an urgent need for more effective treatments. Predicated on promising preclinical and pilot clinical data, this paper presents a rationale and protocol for the trial of N-acetylcysteine (NAC) as a novel anti-craving smoking cessation aid.n\u2009=\u2009120) of at least 10 cigarettes a day are recruited through online advertisements, print publications and dissemination of flyers. Participants are randomised on a 1:1 ratio to receive either 16-week treatment of 1.8\u2009g/day of NAC or placebo with all participants receiving quit support from the online QuitCoach tool. Participants are attending visits at baseline, 8 and 16\u2009weeks with a 42-week post-discontinuation follow-up. The primary outcome measure is sustained abstinence at six\u2009months after treatment based on self-reported rating scales and confirmed by exhaled carbon monoxide and salivary cotinine levels. Secondary outcomes are timing of the first lapse and relapse, between-group cigarette consumption, withdrawal symptoms, general wellbeing and mood/anxiety symptoms. Between-group differences in adverse events and subgroup analyses for variables including gender and Diagnostic Statistics Manual 5 diagnostics will also be investigated.Current smokers , The online version of this article (10.1186/s13063-019-3628-5) contains supplementary material, which is available to authorized users. Tobacco smoking is the leading cause of preventable morbidity and mortality globally  and killIt is essential to trial new strategies and medications to make further significant gains in reducing tobacco smoking. Developing a safer, better tolerated pharmacotherapy for smoking cessation is a critical step towards making further gains. Vulnerable groups and special populations may especially benefit from a low-cost, over-the-counter, effective pharmacotherapy with a better safety profile than existing pharmacotherapies such as varenicline and bupropion \u20138.N-acetylcysteine (NAC) has antioxidant properties, both increasing glutathione and modulating glutamatergic, neurotropic, mitochondrial and inflammatory pathways . NAC is n\u2009=\u200922), treated for 3.5\u2009days with NAC (3.6\u2009g/day) or placebo, identified that NAC-treated participants reported that their first cigarette was significantly less rewarding when compared with the first cigarette of placebo-treated smokers [n\u2009=\u200919) treated with NAC and varenicline reported a reduction in cigarettes smoked per day [n\u2009=\u200933, compared with placebo), there was a significant decrease in the number of cigarettes smoked, but no reduction in craving, withdrawal symptoms or level of carbon monoxide.There are four small published pilot studies investigating the effects of NAC in nicotine dependence. A short-term abstinence study of heavy smokers ( smokers . A four- per day . A randon\u2009=\u200917) or placebo (n\u2009=\u200917) adjunctive to addiction-focused cognitive behavioural therapy. NAC treatment significantly reduced the number of cigarettes smoked daily, exhaled carbon monoxide and improved depression scores. Quit rates were 47.1% in those treated with NAC versus 21.4% of placebo-treated patients. There were no significant differences in treatment emergent adverse events (AEs) between both groups. These pilot data are very promising [In another pilot trial, participants were randomised to 12\u2009weeks of 3\u2009g/day NAC n\u2009=\u2009 or placeThe aim of this study is to investigate the efficacy of NAC (1.8\u2009g/day) for smoking cessation in a randomised, placebo-controlled trial of current smokers who wish to quit smoking. The primary outcome measure will be 24\u2009weeks of continuous abstinence from end of treatment  in tobacco smoking, confirmed by biological measures. Secondary outcome measures include point prevalence abstinence, time to relapse and total cigarette consumption. Safety, tolerability and subgroup analyses will also be conducted.It is hypothesised that: (1) treatment with NAC will be superior to placebo for smoking cessation at follow-up (week 42), confirmed by assaying exhaled carbon monoxide, salivary cotinine (COT); and (2) treatment with NAC will be superior to placebo for smoking cessation at treatment endpoint (week 16), confirmed by assaying exhaled carbon monoxide, salivary COT.This is a double-blind, randomised, placebo-controlled trial, to compare 1.8\u2009g/day of NAC with placebo for smoking cessation, conducted over a period of 16\u2009weeks. A six-month post-treatment discontinuation (week 42) follow-up is assessing ongoing abstinence. Data are collected by interview at baseline and weeks 8 and 16, and at the follow-up visit, six\u2009months after completion of the trial or 42\u2009weeks in total. The trial has approval from the relevant research and ethics committees and is being conducted in accordance with the Good Clinical Practice (GCP), Australian Clinical Trial guidelines and the National Ethical guidelines for Human Research. The trial is registered on the Australian New Zealand Clinical Trials registry (ANZCTR), ACTRN12617001478303. This study was approved by the Barwon Health Human Research Ethics Committee (reference: 17/15).n\u2009=\u2009120) are randomised to 16\u2009weeks of treatment with NAC (1.8\u2009g/day) or placebo effervescent tablets taken with water. The first participant was recruited in November 2017 and recruitment is ongoing. The study is being sponsored by Barwon Health and is supported by the ARC Harry Windsor Research Grants Scheme\u00a02017.The study is being conducted at two sites, Barwon Health, Geelong and the Deakin University Burwood campus. Participants  use before the baseline visit. Female participants are required to be utilising effective contraception if of childbearing age and sexually active.Participants are withdrawn from the trial under the following conditions: failure to take the trial medication for seven consecutive days; pregnancy; emergence of serious AEs suspected to be associated with the trial medication; or commencement of a different pharmacotherapy for smoking cessation during the 16-week treatment phase. Participants will be questioned about these events and their adherence to the study protocol at the bi-weekly telephone interviews during the treatment phase of the study. Withdrawal of consent at any time in the study will result in immediate withdrawal from the trial.Recruitment is anticipated to occur mainly through targeted online advertising. Other recruitment techniques will include the use of flyers, which will be displayed in the waiting rooms of health services and other conspicuous locations. Print and radio advertisements will be included where possible. Following initial contact by potential participants, a researcher will establish that inclusion and non-inclusion criteria are satisfied. Written informed consent for the trial will be obtained from all participants at the first meeting.All participants will provide informed written consent before they undergo a baseline face-to-face interview with a trained researcher. Participant contact will be conducted using procedures adapted from the treatment manual for the Healthy Lifestyles Program , 19. ConSaliva will be collected at baseline and at weeks 8, 16 and 42 for COT measurements. Before the baseline meeting, participants will be provided with the following instructions: (1) avoid foods with high sugar or acidity, or high caffeine content immediately before sample collection; (2) do not eat a major meal within 60\u2009min of sample collection; (3) do not consume alcohol within 12\u2009h of sample collection; (4) participants should not brush their teeth within 45\u2009min before sample collection; (5) dental work should not be performed within 24\u2009h before sample collection. The trial clinician will document consumption of alcohol, nicotine, caffeine and medications in the 12\u2009h before collection, as well as vigorous physical activity and the presence of oral disease or injury. Saliva collection is performed by instructing participants to allow saliva to pool in the mouth and then drool through the Salimetrics Saliva Collection Aid into a cryovial. This sample is stored in a\u00a0\u2013\u200980 \u00b0C freezer until it is time for analysis. An enzyme-linked immunosorbent assay (ELISA) will be performed to determine levels of COT in the samples. Samples are collected at each participant meeting.Individuals will be assigned in a 1:1 ratio using permutated block randomisation to treatment with NAC or placebo, in a double-blind fashion. The randomisation code is generated by an independent researcher and held by the pharmacy to maintain blinding. The randomisation code remains concealed until all data analysis has been completed (triple-blind design). Unblinding for emergency situations is undertaken by the pharmacy, with the approval of the Principle Investigator.The trial medications will be supplied by the trial pharmacist; participants will be instructed to return all containers to allow capsule counts. Concordant with SPIRIT guidelines, adherence will be assessed by pill counts of returned medication packs. Use of QuitCoach will be monitored by participant self-report  <\u200910\u2009ppm measured using a Micro+ Smokerlyzer (Bedfont Scientific) and salivary COT <\u200950\u2009ng/L, which is a sufficient margin to allow for passive tobacco smoking. COEXH may be <\u200910\u2009ppm in non-abstinent participants who have not smoked within 2\u2009h [EXH levels are <\u200910\u2009ppm and COT is <\u200950\u2009ng/mL. Where participants self-report use of non-tobacco nicotine, COEXH will be used to confirm abstinence. Secondary outcomes will include the timing of the first lapse and first relapse, based on a dichotomous variable and defined as self-reported smoking seven\u2009days in a row at any time between two\u2009weeks after quitting and the trial end. Prolonged abstinence will also be reported and defined as any slips or lapses following the quit date [The primary outcome of this aid-to-cessation trial will be between-group differences in continuous abstinence for the 26-week period between weeks 16 and 42. The primary outcome is measured at six\u2009months post-treatment discontinuation (week 42) rather than at treatment discontinuation (week 16), in line with the Russell Standard for international smoking cessation research . To assethin 2\u2009h . Smokinguit date .EXH and COT measures will be taken.At the baseline visit, the Fagerstr\u00f6m Test for Nicotine Dependence and a smoking history questionnaire will be administered. The smoking history questionnaire will explore how long the participant has smoked for , their average consumption of cigarettes and the number of previous attempts to quit smoking. Data will also be collected regarding how determined they are to give up smoking on this attempt  and to rate their chances of permanently quitting on this attempt . The Minnesota Nicotine Withdrawal Scale will be administered to assess urge to smoke among other withdrawal symptoms, depressed mood, irritability, anxiety, difficulty concentrating, restlessness, increased appetite and sleep. Participants will also complete the Brief Questionnaire of Smoking Urges, which assesses cravings related to desire to smoke and expectations of positive effects anticipation of relief from negative affect. Demographic data, lifetime history of smoking , level of social support, self-reported medical and psychiatric history will also be collected at baseline. A psychiatric diagnostic interview (SCID-5-RV), based on the DSM-5 will be conducted at baseline. The WHO ASSIST and AUDIT will be collected at baseline and visits at weeks 8 and 42. The Standardised Assessment of Personality \u2013 Abbreviated Scale (SAPAS), a brief screen for personality disorder, will be used at baseline. The WHO-5, Depression, Anxiety, and Stress Scale (DASS-21) and K10 will be administered at baseline and at weeks 8, 16 and 42 , will be examined separately as well as pooled as treatment failures. Non-cigarette tobacco use, but not non-tobacco nicotine use, will be categorised as smoking.Intended use of bupropion, varenicline or NRT during the 42-week trial period will not be permitted but is only a withdrawal criterion if used during the 16-week treatment period. Some participants may decide to use NRT during the 42-week study. Although this is contrary to the directions given to participants in the study, it is not a study withdrawal criterion and these participants may remain in the study. COT levels will be measured using chromatographic techniques.Records of participation in this study will be held confidential so far as permitted by law. This will include de-identification of all documents relating to the trial by assigning a random number that will identify participants in terms of a dataset and not an individual. However, researchers, regulatory agencies and the Barwon Health Human Research Ethics Committee (HREC) will be able to inspect and have access to confidential data that identify participants by name.The measures listed under \u2018Variables and instruments\u2019 in the study design section will be co-rated by trained trial clinicians. Inter-rater reliability will be checked before commencement of the trial and repeated every six months during the trial, using a standardised videotaped patient interview. Raters will need to achieve an intraclass  correlation score of 0.8 and will undergo further training as necessary during the study to maintain inter-rater reliability.The sample size was initially determined to be 360. In order to detect a 7% difference between the groups , the sample size has been calculated for a Cochran-Mantel-Haenszel (CMH) test to acknowledge a cluster randomised design with three recruitment centres as strata . A sample size of 180 in the NAC group and 180 in the control group  achieves 80% power to detect an odds ratio\u00a0(OR) =\u20097, assuming a two-sided type I error of 0.05. The sample size also achieves 80% power to detect a 7% difference in six-month smoking cessation between NAC and placebo groups assuming two-sided alpha\u2009=\u20090.05 and an 8% inflation of the required sample size due to clustering. However, the current trial has a recruitment target of 120 in total, with the intention of scaling up as more funding is acquired.A modified \u2018intention-to-treat\u2019 approach, following CONSORT Statement guidelines, will be used. Missing values will be scrutinized to check for non-random distribution and analyses that utilize baseline data will be executed twice: once using observed data and once using multiple imputation under multivariate normal assumptions. The CMH test will be used to compare the proportions of relapse between the treatment groups (control versus intervention). The common OR and its 95% confidence interval will also be reported as well as the results of the Breslow-Day test for homogeneity of the ORs across the strata. In further supportive analyses, generalized linear mixed models (GLMMs) will be used to compare the rates of relapse after adjusting for baseline measurements and to explore possible interactions between baseline factors and the intervention. Average treatment group differences for the continuous secondary outcomes will be examined using a likelihood based mixed-effects model, repeated measures approach (MMRM). The MMRM models include the fixed categorical effects of treatment, visit and treatment-by-visit interaction, as well as the continuous fixed covariates of baseline score and baseline score-by-visit interaction. Effect sizes will be calculated using Cohen\u2019s guidelines. All comparisons will be conducted using a two-sided alpha level of 0.05. There will be no interim analysis.Barwon Health is the sponsor of this study. The sponsor is indemnified for any harms arising from trial participation and it approves protocol amendments when ethics approval has been obtained. Principal investigators and trial clinicians manage the day-to-day running of the trial. Principal investigators are three well-established and experienced researchers in relevant fields. Itemised lists of various trial staff members are as follows:Trial designPublication of study reportsStudy planningProvision of expertise to problem resolutionMedia promotionFunding application and managementCase report form creation and managementResearch staff managementParticipant recruitment, contact and interviewingLiaison with external bodies Maintenance of trial equipmentTraining of research staffOrganisation of research team meetingsThere is no Data Management Committee for this trial due to the small sample size; however, independent data integrity will be assessed by independent researchers as required. Trial progress and processes are assessed every two weeks by the principal investigators at a research team meeting.A summary of the data will be provided to each participant along with whether they took the active or placebo medication. This information will be provided at the soonest reasonable opportunity after trial completion and will be provided electronically. The results will be published and disseminated throughout both formal and informal collegiate settings.There is currently no plan to share participant-level data with external individuals.Tobacco smoking is a major preventable cause of morbidity and mortality. NAC may provide a safe, novel and effective treatment for smoking cessation, at low cost. The tolerability and safety of NAC as well as its efficacy in co-morbid psychiatric symptoms may have advantages in special populations compared to other pharmacotherapies for smoking cessation. If positive, of the use of NAC could be translated immediately into practice and established as a new treatment for smoking cessation. NAC additionally has a clearly elucidated potential mechanism of action in addiction. The planned trial, if positive, may result in substantial changes to the management of smoking with overt public health implications.Additional file 1:SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents. (DOCX 18 kb)"}
{"text": "Furthermore, HDAC4 was targeted for proteasomal degradation early after infection with VACV, and VACV protein C6, an inhibitor of type I IFN signaling, was necessary and sufficient for this degradation. In summary, HDAC4 is a restriction factor for large DNA viruses.Histone deacetylases (HDACs) are regulators of host gene expression. HDAC4 is shown here to have an important role in type I interferon (IFN) signaling. Here, multiple cell lines lacking HDAC4 had impaired responses to IFN-\u03b1 and were rescued by reintroduction of HDAC4. The biological significance of HDAC4 was demonstrated by the enhanced replication and spread of two DNA viruses, vaccinia virus (VACV) and herpes simplex virus type I, in HDAC4 \u2212/\u2212 cells was rescued by reintroduction of HDAC4 or catalytically inactive HDAC4, but not HDAC1 or HDAC5. ChIP analysis showed HDAC4 was recruited to ISG promoters following IFN stimulation and was needed for binding of STAT2 to these promoters. The biological importance of HDAC4 as a virus restriction factor was illustrated by the observations that (i) the replication and spread of vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1) were enhanced in HDAC4\u2212/\u2212 cells and inhibited by overexpression of HDAC4; and (ii) HDAC4 is targeted for proteasomal degradation during VACV infection by VACV protein C6, a multifunctional IFN antagonist that coprecipitates with HDAC4 and is necessary and sufficient for HDAC4 degradation.Interferons (IFNs) represent an important host defense against viruses. Type I IFNs induce JAK-STAT signaling and expression of IFN-stimulated genes (ISGs), which mediate antiviral activity. Histone deacetylases (HDACs) perform multiple functions in regulating gene expression and some class I HDACs and the class IV HDAC, HDAC11, influence type I IFN signaling. Here, HDAC4, a class II HDAC, is shown to promote type I IFN signaling and coprecipitate with STAT2. Pharmacological inhibition of class II HDAC activity, or knockout of HDAC4 from HEK-293T and HeLa cells, caused a defective response to IFN-\u03b1. This defect in HDAC4 Histone deacetylases (HDACs) can repress gene expression by deacetylating histones leading to strengthened histone\u2013DNA interactions and chromatin condensation . HDACs cIn humans there are 18 HDACs that are classified in four subfamilies: class I ; class II HDACs, subdivided into class IIa  and class IIb (HDACs 6 and 10); class III (sirtuins 1\u20137); and class IV (HDAC11) (Type I interferons (such as IFN\u03b1/\u03b2) are secreted glycoproteins that induce antimicrobial states in cells, orchestrate the inflammatory response to invading pathogens, and activate the adaptive immune system , 6. HDACHDAC activity also affects type I IFN-stimulated gene (ISG) expression as shown by impairment of ISG transcription by addition of TSA  or the a+ T cells  and Epstein-Barr virus (EBV) \u201324. Simi T cells , 26. Dur T cells . In huma T cells , 29. IntAlthough interplay between viruses and class I HDACs is well established, less is known about viral interactions with members of the class II HDAC family, such as HDAC4. HSV-1 protein ICP0 was reported to coprecipitate with HDAC4, however, the functional consequence of this is unknown . EBV nucThis study reports that HDAC4 is required for type I IFN signaling and restricts the replication of HSV-1 and vaccinia virus (VACV). Mechanistically, HDAC4 coprecipitates with STAT2 and is recruited to IFN-stimulated response element (ISRE)-containing promoters following addition of type I IFN. Without HDAC4, binding of STAT2 to these promoters after IFN-\u03b1 addition is greatly reduced. Lastly, HDAC4 is targeted for proteasomal degradation during VACV infection. VACV expresses many inhibitors of innate immunity and IFN signaling , 35 and To investigate further the roles of HDACs in type I IFN signaling, the effect of TSA on type I ISRE-dependent reporter gene expression was assessed in HeLa cells. Consistent with previous reports, type I IFN signaling was largely inhibited by TSA A. Next, IFIT3, IFIT1, and 2\u20325\u2032-OAS (OAS1)] . These data indicate that class II HDAC activity is needed for ISRE-dependent gene expression in response to IFN-\u03b1 but not for the expression of IL-1\u03b2\u2013induced genes that require NF-\u03baB activation.To explore this further, the effect of LMK235 on endogenous gene expression in response to IFN-\u03b1 and IL-1\u03b2 was analyzed in HeLa cells by reverse transcription-quantitative PCR (RT-qPCR). LMK235 inhibited the induction of mRNA of three IFN-\u03b1\u2013responsive genes [ (OAS1)] D\u2013F but n\u2212/\u2212 cell lines generated by CRISPR/Cas9 genome editing , and two HDAC4\u2212/\u2212 HeLa cell lines, genome edited with gRNA1, were selected as described in SI Appendix, Fig. S2A. Sequencing of the genomic region targeted by the gRNAs confirmed frameshift mutations had been introduced into each allele and that no wild-type (WT) allele remained . Consistent with this, immunoblotting showed loss of HDAC4 expression . These cell lines showed no significant difference in growth rate compared with the parental cell lines, indicating that although HDAC4\u2212/\u2212 mice displayed skeletal defects and a very limited life span  were then used in reporter gene assays. ISRE-luciferase expression after IFN-\u03b1 stimulation was significantly diminished in H4KO1 and H4KO2 cells compared with parental HEK-293T cells (Left), whereas the NF-\u03baB response to TNF-\u03b1 was normal (Right). So, consistent with the results using pharmacological inhibitors of HDACs, two independent HDAC4\u2212/\u2212 cell lines showed impaired type I IFN signaling. Similar analysis in HDAC4\u2212/\u2212 HeLa cells (H4KO3 and H4KO4) showed a greater deficiency in response to IFN-\u03b1 stimulation (Left). In contrast, IFN-\u03b3\u2013activated sequence (GAS)-luciferase and TNF-\u03b1\u2013induced NF-\u03baB\u2013luciferase were not significantly different in these HDAC4\u2212/\u2212 cell lines (Center and Right). The role of HDAC4 in type I IFN signaling was also investigated by RT-qPCR of endogenous ISGs  in HeLa HDAC4\u2212/\u2212 cells. For each ISG there was a significant reduction in the response to IFN-\u03b1 compared with control cells , and the effect analyzed. Immunoblotting demonstrated dose-dependent FLAG-HDAC4 expression. In HDAC4\u2212/\u2212 cells there was a dose-dependent increase in ISRE-luciferase activity following type I IFN stimulation .Four HDAC4ll lines A and WT mulation A. Howeve\u2212/\u2212 cells and found to complement HDAC4 deficiency as efficiently as WT HDAC4, indicating that interaction with 14-3-3 is not necessary for type I IFN signaling .Given that HDAC4 deacetylase activity was not needed for type I IFN signaling, the inhibition of this pathway by the HDAC inhibitor LMK235 suggests that enzymatic activity of another class II HDAC is required for type I IFN signaling. Consistent with this, LMK235 still inhibited type I FN signaling in two HDAC4IFIT1, IFIT3, and ISG15) were enriched by the other anti-HDAC4 antibody after IFN-\u03b1 stimulation compared with mock-treated cells, and, as expected, there was no enrichment in H4KO3 cells. Similar analysis with two different anti-STAT2 antibodies showed enhanced binding of STAT2 following addition of IFN-\u03b1 to WT cells, but binding of STAT2 to the IFIT1, IFIT3, and ISG15 promoters was greatly reduced in H4KO3 cells compared with WT cells  assays were undertaken with antibodies against HDAC4 and STAT2. HeLa and H4KO3 cell lines were treated with IFN-\u03b1 and then fixed with formaldehyde to crosslink chromatin-associated proteins. After chromatin fragmentation, samples were immunoprecipitated with two antibodies against HDAC4 or STAT2 and the enriched chromatin was analyzed by qPCR. One anti-HDAC4 antibody did not work in this assay. However, as shown in WT cells B and C. The reduced STAT2 binding to the IFN-\u03b1\u2013stimulated promoters suggested that HDAC4 might interact with components of the ISGF3 complex  and this was investigated by immunoprecipitation. FLAG-tagged HDAC4 coprecipitated with STAT2 but not STAT1, while FLAG-tagged TANK did not coprecipitate with either STAT1 or STAT2 A. The knSI Appendix, Fig. S6). HDAC4 expression was then induced (+dox) or mock induced (\u2212dox) and cells were infected with either VACV  , 46 were/+ cells A and B. /\u2212 cells C. Furthentivirus  reduced ntivirus C. In sumC6L gene . It was shown that pharmacological inhibition of HDACs by TSA reduced the response to type I IFN  and thatectively , inhibitectively . But neiPharmacological inhibition of HDAC activity by TSA, or of class II HDAC activity by LMK235, and knockout of HDAC4 in four independent cell lines, each caused a defective response to type I IFN using reporter gene assay and RT-qPCR of endogenous ISGs. This defect was rescued by reintroduction of HDAC4, but not HDAC5 or HDAC1. In addition, ChIP analysis of HDAC4 and STAT2 showed that HDAC4 is recruited to multiple ISG promoters following IFN-\u03b1 stimulation, and HDAC4 is needed for the recruitment of STAT2 to ISG promoters. Given that HDACs are needed for IRF9-mediated recruitment of RNA polymerase II to ISG promoters , the ChI\u2212/\u2212 cell lines . LMK235 is most potent against HDAC5, but it inhibits several other class II HDACs. HDAC6 up-regulates IFN-\u03b2 expression after stimulation with dsRNA . The second study knocked out HDAC4 from Hep-2 cells and reported that this caused reduced HSV-1 replication  were maintained in MEM (Gibco) supplemented with 10% FBS and 50 \u03bcg/mL P/S.Immortalized primary human fetal foreskin fibroblasts (HFFF-TERTs) , human HPlasmids used in this study were from the following sources: pcDNA3.HDAC4-FLAG , pcDNA3.HDAC4 3SA-FLAG , pcDNA3.HDAC5-FLAG , and pcDNA3.HDAC1-FLAG . pcDNA3.HA-STAT2, pcDNA3.HA-STAT1, pcDNA3.IRF9-TAP, pcDNA3.IRF9-S2C, pcDNA3.TAP-C6, and pcDNA3.N1-TAP were described . The repC6L were described (WT VACV strain Western Reserve (WR) and derivative strains expressing GFP fused to the capsid protein A5 (A5GFP VACV) , or lackescribed . HSV-1 sescribed .The following antibodies were used: Rabbit (Rb) anti-HDAC4 , Rb anti-FLAG , Rb anti-STAT2 , Rb anti-STAT2 , Rb anti-STAT1 , Rb anti-C6 (described in ref. 6) were seeded in a 10-cm dish on day 1. On day 2, the cells were transfected with 3 \u03bcg pLKOneo.EGFPnlsTetR together with 3 \u03bcg of each pMD-G and pCMV.dR8.91 plasmids. Three hours posttransfection, the cell culture medium was removed and replaced with DMEM with 30% FBS and 50 \u03bcg/mL P/S. On day 3, the cell culture supernatant was collected and replaced with 5 mL DMEM containing 30% FBS. The collected supernatant was passed through a 0.45-\u03bcm filter and 2 \u03bcg/mL Polybrene  was added. This lentivirus stock was used to infect U2OS cells that were seeded on day 2. On day 4, the same lentivirus infection was repeated. Following the lentivirus infection, transduced cells were selected with 500 \u00b5g/mL neomycin , and thereafter neomycin was maintained in the transduced cell culture medium. The same method was used to prepare HDAC4-FLAG expression lentivirus with plasmid pLKO.TetO.HDAC4-FLAG. Following the HDAC4-FLAG expression lentivirus infection, cells were selected with 1 \u00b5g/mL puromycin .HEK-293T cells .HDAC4g) for 1 min. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) alongside protein molecular mass markers . Proteins separated by SDS/PAGE were transferred onto nitrocellulose membranes  by using Trans-Blot Turbo transfer system (Bio-Rad) in 25 mM Tris\u22c5HCl, 250 mM glycine, and 20% methanol. Membranes were blocked with 5% skimmed milk in PBST or 5% BSA in TBST at 4 \u00b0C overnight. Blocked membranes were then incubated with primary antibodies overnight at 4 \u00b0C with consistent agitation, washed three times in PBST or TBST, and then incubated with secondary antibodies for 2 h at room temperature. The membranes were then washed three times with PBST or TBST, dried at room temperature, and imaged using the LI-COR Odyssey imaging system.Cells were lysed in passive lysis buffer  and lysates were boiled at 98 \u00b0C for 5 min. The samples were cooled to room temperature and the insoluble debris was collected by centrifugation at . The cell lysate was kept at \u221220 \u00b0C and the firefly and Renilla luciferase activity were measured within 2 wk. To measure the luciferase activity, 50 \u03bcL firefly luciferase reagent , or 50 \u03bcL Renilla luciferase reagent (2 \u03bcg/mL coelenterazine in PBS)  was added to 10 \u03bcL cell lysate. The luminescence value was measured with a microplate reader (BMG Labtech). Firefly luciferase values were normalized to the Renilla luciferase values and the fold inductions in each reporter gene assay were calculated relative to the unstimulated controls. Experiments were performed in triplicate and conducted at least three times.HEK-293T, HeLa, or derivative HDAC48 cells for each condition (with/without IFN-\u03b1 treatment). Cells were stimulated or mock treated with IFN-\u03b1  for 3 h and processed as described with slight modification  by qPCR. Primer sequences used in the above qPCR were reported (HeLa and H4KO3 cells were seeded at 1 \u00d7 10fication . Brieflyreported  previous6) were seeded on six-well plates 1 d before stimulation with 1,000 units/mL IFN-\u03b1 or 100 ng/mL interleukin (IL)-1\u03b2 for 4 h. The cells were harvested and the total mRNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer\u2019s instructions. Thereafter, 500 ng of RNA was used to synthesize cDNA using SuperScript III reverse transcriptase (Invitrogen).Cells . ViiA 7 real-time PCR system (Thermo Fisher Scientific) was used to determine the cycle threshold of each reaction and determine the fold induction of the investigated genes. Gene amplification was normalized to GAPDH amplification and the fold induction was determined relative to control cells that had not been stimulated with IFN-\u03b1.RT-qPCR analysis of the level of mRNA for specific ISGs was performed on HDAC4C6L were described (13 cells and titrated by plaque assay on BSC-1 cells. HSV-1 strain s17 expressing GFP fused to virus protein 26 (VP26GFP) was provided by Prashant Desai (n = 20) was measured at 50\u00d7 magnification using AxioVision 4.8 software and a ZEISS Axio Vert.A1 fluorescent microscope.WT VACV strain WR and derivative strains expressing GFP fused to the capsid protein A5 (A5GFP VACV)  or lackiescribed . VACV stnt Desai . This vin = 4).To measure virus replication, monolayers of HeLa or H4KO3 were infected at 0.001 pfu per cell and the yield of infectious total virus present at 2 d (A5GFP VACV) or 3 d (VP26GFP HSV-1) postinfection was determined by plaque assay on BSC-1 cells or U2OS cells for VACV and HSV-1, respectively. Measurements were made from multiple independent experiments , P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Unpaired Student\u2019s Supplementary File"}
{"text": "Its greenhouse effect will cause serious environmental problems, such as the global warming and climate change. Therefore, the worldwide scientists have devoted great efforts to control CO2 emissions through various strategies, such as capture, resource utilization, sequestration, etc. Among these, the catalytic conversion of CO2 to methane is considered as one of the most efficient routes for resource utilization of CO2 owing to the mild reaction conditions and simple reaction device. Pioneer thermodynamic studies have revealed that low reaction temperature is beneficial to the high catalytic activity and CH4 selectivity. However, the low temperature will be adverse to the enhancement of the reaction rate due to kinetic barrier for the activation of CO2. Therefore, the invention of highly efficient catalysts with promising low temperature activities toward CO2 methanation reaction is the key solution. The Ni based catalysts have been widely investigated as the catalysts toward CO2 methanation due to their low cost and excellent catalytic performances. However, the Ni based catalysts usually perform poor low-temperature activities and stabilities. Therefore, the development of highly efficient Ni based catalysts with excellent low-temperature catalytic performances has become the research focus as well as challenge in this field. Therefore, we summarized the recent research progresses of constructing highly efficient Ni based catalysts toward CO2 methanation in this review. Specifically, the strategies on how to enhance the catalytic performances of the Ni based catalysts have been carefully reviewed, which include various influencing factors, such as catalytic supports, catalytic auxiliaries and dopants, the fabrication methods, reaction conditions, etc. Finally, the future development trend of the Ni based catalysts is also prospected, which will be helpful to the design and fabrication of the Ni catalysts with high efficiency toward CO2 methanation process.With the development and prosperity of the global economy, the emission of carbon dioxide (CO In 2017, the total CO2 emissions reached 41 billion tons, attracting the increasing worldwide attention. Therefore, how to control and reduce the amount of CO2 in the atmosphere has become an urgent issue  due to the exothermic feature of this process. Therefore, when the reaction temperature is higher than 627\u00b0C, it is supposed to obtain lower CO2 conversion and CH4 selectivity. However, the change in Gibbs free energy will be >0 according to thermodynamic calculation and the reverse reaction will take place, namely, CH4 reacts with H2O to form CO2  with strong C = O bond into CH4 (\u22124). Low temperature will reduce the reaction rate based on the dynamic theory  , tetragonal (t-ZrO2), and cubic structure  catalyst was affected by the composition of the Ce/Zr ratio  catalyst was higher than other catalysts. Specifically, the addition of ZrO2 increased the oxygen mobility in the lattice of cerium oxide and promoted the formation of vacancies, which in turn affected the consumption of H2 and the activation of CO2 rapidly  among all the catalysts.As regard to the Al2-Al2O3 has excellently sintering-resistant and thermally stable properties  was obviously higher than that of 20Ni/Al2O3. When the ZrO2 content of the support was <40%, the weakening effect of ZrO2 on the NiO-Al2O3 interaction was more obvious, and the reduction was easier to proceed with the increase of ZrO2 content  exhibited higher catalytic activity and better stability than non-mesoporous materials(NPA) and Ni/\u0264-Al2O3 due to its outstanding structural property as shown in 2 , which could enhance the metal-support interaction and improve the high temperature stability of Ni. Furthermore, Xu et al.  was obviously higher than that of Ni/OMA. In addition, Guo and Lu (2 and CO2). However, excessive Mg doping can also cause blockage of the active center of the catalyst, resulting in the decrease of the catalytic activity  and microwave assisted methods (M). The CO2 conversion rates of NiAl2O3-M were higher than that of NiAl2O3-I at the equal amount of Ni loadings in 2O3-M catalyst, indicating that the Ni particles were highly dispersed in nano size state, lower than the XRD detection limit (3.0 nm). As a comparison, the Ni20Al2O3-I catalyst presented much higher metallic Ni diffraction peaks, suggesting the worse dispersion of metallic Ni particles. Therefore, the microwave method could facilitate the uniform distribution of Ni on the support. In addition, microwave assisted method contributes to greater catalytic stability and sintering resistance of the catalyst  was beneficial to the enhancement of the CO2 methanation reaction. As shown in 2 methanation reaction gradually dominated with the increase of the Ni loading amount. They found that the catalyst could achieve the best activity for methanation and low yield of CO when the Ni loading was 20 wt%. Therefore, it could be concluded that the increase of active sites (Ni0) was beneficial to the achievement of high CO2 conversion. However, when the nickel loading amount exceeded a certain range, further increasing the loading amount would cause seriously thermal agglomeration of the metallic nickel active centers, thereby destroying the structure of the catalyst  reaction and thereby promoting the conversion of CO2 to CH4  reaction between intermediate CO and the excess of water. In addition, water vapor may cause dilution of the gaseous reactants, inhibiting the interaction of H2 with CO2  analysis. As observed in 2 molecules during the process of its activation. The activated CO2 dissociated on the surface of Ni, forming carbonate intermediate species. Finally, the atomic hydrogen reacted with the carbonate intermediate to form methane. Takano et al. (2 catalyst were observed and the carbonate was the main adsorbate in the reaction during the process of the DRIFTS analysis. Thus, carbonates were the reaction intermediate for the formation of CH4 on the Ni/ZrO2 catalyst. They found that the bidentate carbonates were probably the most important intermediates adsorbed on these highly active Y doped catalysts. The combination of Y3+ and Ni2+ with ZrO2 created oxygen vacancies in the oxide, which would help to form a stable bidentate carbonate adsorbate on the catalyst.Another viewpoint that CO is not the COo et al.  also fou2 methanation processes (Solis-Garcia and Fierro-Gonzalez, 2 methanation reaction over Ni-based catalysts supported on different metal oxides. Although almost no CO2 methanation reaction occurred over Ni/La2O3 catalyst at 250\u00b0C, the chemisorption of CO2 by Ni/La2O3 was more obvious than other samples. In contrast, the strong chemisorption of CO2 was not observed over the Ni/Y2O3 and Ni/Sm2O3 catalysts. For Ni/Y2O3 catalysts, the main reaction intermediates of the CO2 methanation reaction were carbonate and formate species. The carbonate species was the initial reaction intermediates and then it was gradually converted into monodentate formate and bidentate formate through hydrogenation process. The main reaction pathway for CH4 production was hydrogenation of monodentate formate because the reaction rate of monodentate was faster than that of bidentate. CO2 methanation process over the Ni/La2O3 catalyst did not experience bicarbonate and formate intermediates because of the high desorption temperature of CO2. CH4 was formed by experiencing the formate intermediate rather than the CO pathway over the Ni/ZrO2 catalyst. Furthermore, Pan et al. (2O3 and Ni/Y2O3 were similar. As regard the Ni/Al2O3, CO2 reacted directly with surface hydroxyl groups and surface oxygen to form bicarbonate and monodentate carbonate intermediates. The reaction between CO2 and the surface hydroxyl group could be attributed to the nucleophilic attack of the oxygen atom of the hydroxyl group on the CO2 carbon atom (Solis-Garcia and Fierro-Gonzalez, 2. As the temperature increased, methane characteristic peaks began to form and the intensity of formate slowly decreased. According to previous reports (Guilera et al., 2 methanation catalysts commonly possess weak basicity site, medium basicity site, and strong basicity site. The weak basicity sites are usually derived from surface hydroxyl groups and the moderate and strong basicity sites are derived from surface oxygen. Moderate basicity sites promote the decomposition of formate salts. Strong alkaline sites have a negative effect on the activation of carbonates, thereby inhibiting the generation of CH4. Therefore, the hydrogenation of monodentate carbonates was hindered by the strong basicity sites of surface oxygen in Ni/Al2O3. The hydrogenation of bidentate carbonates was the main reaction pathway for CH4 formation. The methanation reaction of Ni/CeO2-ZrO2 was different with that of Ni/Y2O3. The hydrogen carbonate, bidentate carbonate and monodentate carbonate intermediates appeared over Ni/CeO2-ZrO2 during the methanation reaction. As the temperature increased, the number of carbonate species gradually decreased and the number of bidendate species gradually increased. This indicated that the bidentate species were main intermediates in the reaction (Pan et al., 2 was mainly converted to 2 catalyst while CO2 was converted to bicarbonate on Ru/\u03b1-Al2O3 catalyst. Besides, Wei et al. (Liu J. et al., 2 methanation. As shown in 2 molecule to carbonate/hydrocarbonate species and Ni/CNT lacked base sites. Further, CO2 was mainly converted into methane via the formate intermediate over the Ni-based catalyst prepared by the ammonia evaporation method. However, the formate intermediate could not be formed over the Ni/CeO2-ZrO2 catalyst prepared by wet impregnation method (Ashok et al., 2.It was reported that different supports also could cause different reaction intermediates during the COn et al.  also fou2, some scholars believe that the activation of CO2 occurs over the metallic Ni active site (Aziz et al., 2 methanation on Ni/MSN. Specifically, CO2 and H2 molecules were firstlyadsorbed on Ni active sites, followed by dissociation to form CO, O, and H atoms, and migrated onto the MSN surface. The CO then interacted with oxide surfaces of the MSN and bidentate formate was also formed through the interaction with atomic hydrogen. Meanwhile, the O atom spilt over onto the surface of the MSN and was stabilized in the oxygen vacancy site near the Ni sites. However, the viewpoint of Jangam et al. (2 with a surface basic oxyanion on the metal oxide. Therefore, the activation of CO2 occurred on the support. Besides, Aldana et al. (2 was activated on the CeO2-ZrO2. Therefore, up to now, the real activation site of the CO2 molecule has still been the controversy of the CO2 methanation reaction.As for the activation site of COm et al.  was oppoa et al.  also fou2 emissions are increasing, resulting in a series of global environmental problems. Therefore, it is of greatly practical significance to study CO2 methanation technology in order to solve the problem of energy shortage and reduce the concentration of CO2 in the atmosphere. Besides, it is of great urgency to develop highly efficient Ni based catalyst to accelerate this process because of the kinetic limitations of CO2 methanation.Nowadays, CO2 methanation process. In terms of reaction conditions, such as temperature, pressure, humidity, etc., they are also considered as important factors affecting the catalytic behavior of the catalyst, which have been carefully summarized in this review. In addition, the reaction mechanism of Ni based catalysts toward CO2 methanation are also carefully reviewed. It was believed that the nature of the catalytic support, additive and preparation strategy of the catalyst could affect the dominant reaction pathway and intermediates during the process of the methanation of CO2.In recent years, Ni-based catalysts have been widely used due to their good catalytic performance and low price. In order to develop Ni-based catalysts with good low-temperature activity, the global scientists have made great efforts to investigate different influencing factors, such as catalytic support, dopant, preparation method. This review generally summarizes different ways to improve catalyst performance. From the perspective of preparation methods, the traditional preparation methods of Ni-based catalysts, such as impregnation and coprecipitation, have become basically mature technologies. Therefore, the researchers have begun to develop new methods and strategies, such as microwave assisted method and plasma method. The catalytic performance of the catalyst prepared by these methods is usually superior to that by the conventional preparation method. In the viewpoint of dopants, their addition can adjust the acidity-alkalinity and electronic/redox property of the catalyst, further improving the dispersion and coordination environment of the metallic active sites. As a result, the additive-modified Ni-based catalyst has the advantages of good reactivity and long lifespan. In this review, the additives are divided into four categories, namely rare earth metals, alkaline earth metals, transition metals and precious metals, and their effects on the catalyst are described and summarized in detail. As for the catalytic supports, they can obviously influence the textural property of the catalyst, thereby affecting the catalytic performances toward CO2 methanation should still be placed on the improvement of both low-temperature catalytic performance and anti-sintering property of the metallic Ni active site. The development of new supports ought to be the main research line. Besides, the investigation of CO2 methanation mechanism on the Ni-based catalyst is beneficial to finding routes to improve the activity of catalysts. Meanwhile, it is of great significance to optimize the preparation method in order to scale up the synthesis of the catalysts in the future industrialization process. Finally, taking the cost of preparing catalysts into consideration is necessary in order to achieve green catalysis and catalysts should be applied to industrial CO2 methanation by improving reactors and other methods.In future research, the hot spots of research on Ni-based catalysts toward COMC, LX, and CL: conceptualization. YC and XH: software. MC, LX, and CW: validation. XW, YL, and BY: investigation. MC, XH, and QS: resources. ZM, BY, and CW: data curation. CL: writing\u2014original draft preparation. CL, LX, and MC: writing\u2014review and editing. MC and LX: supervision, methodology, and project administration.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": "This process can achieve the efficient resource utilization of CO2 and CH4 and reduce greenhouse gases. Therefore, CRM has been considered as a significantly promising route to solve environmental problems caused by greenhouse effect. Ni-based catalysts have been widely investigated in CRM reactions due to their various advantages, such as high catalytic activity, low price, and abundant reserves. However, Ni-based catalysts usually suffer from rapid deactivation because of thermal sintering of metallic Ni active sites and surface coke deposition, which restricted the industrialization of Ni-based catalysts toward the CRM process. In order to address these challenges, scientists all around the world have devoted great efforts to investigating various influencing factors, such as the option of appropriate supports and promoters and the construction of strong metal-support interaction. Therefore, we carefully summarized recent development in the design and preparation of Ni-based catalysts with advanced catalytic activity and enhanced anti-coke performance toward CRM reactions in this review. Specifically, recent progresses of Ni-based catalysts with different supports, additives, preparation methods, and so on, have been summarized in detail. Furthermore, recent development of reaction mechanism studies over Ni-based catalysts was also covered by this review. Finally, it is prospected that the Ni-based catalyst supported by an ordered mesoporous framework and the combined reforming of methane will become the future development trend.CO They found that the catalytic performance of the catalyst was affected by the size of the Ni nanoparticle and the metal\u2013support interaction between Ni and SiO2. Besides, the shape of SiO2 nanospheres did not obviously change even under high calcination and CRM reaction temperatures, demonstrating good thermal stability. Li et al. . The interaction of Ni and NiPhy materials on the Ni@Niphy@SiO2 catalyst increased because of the presence of the SiO2 shell, which inhibited the growth of Ni nanoparticles. Thus, the growth of CNTs was eliminated and high carbon-resistant performance could be obtained. Das et al.  CRM reaction because of the confinement effect of the sandwich structure and the bifunctional mechanism of dry reforming.SiOi et al.  synthesis et al.  synthesi2O3 support, it can effectively disperse Ni particles because of its large surface area, favorable thermal stability, and strong metal\u2013support interaction  as a support to prepare the NI/CF-LA2O3 catalyst. The addition of La2O3 improved the activity of the Ni/CF catalyst because La2O3 could improve the dispersion of Ni and enhance the metal\u2013support interaction between Ni and support. In addition, the conversion of La2O3 to La2O2CO3 affected the removal rate of deposited carbon and improved the catalyst activity of Ni/CF-La2O3.As an alkaline support, Lai et al.  tested to et al.  doped th2 support possesses excellent redox properties; however, its thermal stability was relatively poor, especially under CRM conditions  over the CeO2 surface has been considered as an effective strategy  is a sort of zeolite with weak acidity owing to no alumina component in the zeolite framework  , well-known as a new nano-carbon catalyst support, possesses many advantages, such as large surface area, unique electronic properties, tubular structure, chemical inertness, thermal stability, and high mechanical strength  to the Ni/\u03b3-Al2O3/ACF catalyst was beneficial to improvement of the CRM catalytic activity. ACF could reduce the grain size of NiAl2O4 spinel in Ni-based catalysts, which was conducive to the conversion of methane. The addition of ACF could provide abundant functional groups on the catalyst surface and effectively promote the catalytic performance of the CRM reaction.As for carbon fiber, it has a similar thermal stability and high specific surface area to the activated carbon. Besides, their fibrous properties make them more beneficial to producing structurally defined catalysts , performing an effect toward CRM. It was found that MgO could greatly improve the dispersion and metal area of the metallic Ni active site. The Ni/5MgO-Al2O3 catalyst performs the best anti-coke property and highest H2/CO ratio among these catalysts. Singha et al. , high oxygen capacity, and abundant activated oxygen species  catalysts by co-precipitation and impregnation methods toward CRM reaction. The CeO2-Ni-MgO-Al2O3 catalyst exhibited much higher activity, better stability, and more excellent anti-coking performance than the Ni-MgO-Al2O3 catalyst because of the improvement of Ni dispersion and the metal\u2013support interaction by CeO2 promotion. Wang et al. , which contributed to the elimination of the carbon deposition on the surface of the support  and plasma (Ni/ZrO2-P) methods for the CRM reaction. Compared with NiZrO2-C, the Ni/ZrO2-P catalyst displayed more Ni(111) planes, smaller metallic Ni particles, and more oxygen vacancies. As a result, the Ni/ZrO2-P catalyst displayed better catalytic activity than Ni/ZrO2-C toward CRM reaction. Fang et al. 4 nanosheets in the catalyst precursor. Among them, the strong electrolytic capacity of NaOH made the most Ni3Si2O5(OH)4 nanoflakes formed in the Ni-MSC-1 catalyst, which results in the highest Ni dispersity and highest catalytic activity. Park et al.  the gradual decomposition and dehydrogenation of CH4 to *, (2) the recombination of H* to generate H2, and (3) the desorption of the catalyst surface at high temperature  sites to generate carbon atom and hydrogen atom. Finally, the oxygen atom reacted with the carbon atom to produce CO. Furthermore, it could be observed in 2O2CO3 by inhibiting the oxygen supply from the filamentous carbon chains to the metal surface. The metal nanoparticles were kept in close contact with the supporting site to obtain a stable oxygen supply and CO generation. When a large amount of coke was formed, the metal particles were removed from the scaffold by filamentous carbon. As a result, there was no oxygen supply from La2O3. The carbon atoms formed on the metal surface could not be effectively eliminated and the polymerization of the carbon atoms accelerated the coking process. Therefore, the close contact between the metallic active site and the support had an important impact on the high activity and stability of the CRM catalyst because the CO formation reaction might take place at the metal\u2013support interface. In summary, so far there has not been a single reaction mechanism that can summarize all CRM reactions. However, extensive research on CRM reaction mechanism contributes to a deeper understanding of CRM reaction and then design of the catalyst with better catalytic activity.Chong et al.  reported. et al.  found thu et al.  preparedm et al.  studied 2 has been considered as one of the major incentives of many environmental problems, such as global warming and extreme weather. CRM not only effectively utilizes the CO2 resource and reduce carbon emissions but also produces the value-added syngas, which can be further employed as the building unit of the synthesis of alcohols, olefins, and other valuable products. For now, the main challenge of the CRM reaction is to develop a highly efficient catalyst with high catalytic activity, sinter proof, coke resistance, and low cost.CONi-based catalysts have been widely regarded as promising alternatives to noble metal catalysts. However, their industrial application is limited due to the serious sintering of Ni nanoparticles and rapid catalyst deactivation caused by coke deposition. Various strategies of improving the performance of catalysts are carefully and comprehensively summarized in this review. In order to reduce carbon deposition on Ni-based catalysts, researchers have tried to add promoters to change the alkalinity of the support, such as alkaline earth metal oxides and rare-earth metal oxides, which can enhance the stability and decrease carbon formation. It is found that the catalytic performances can be affected by the preparation methods. Besides, the choice of the support is also very crucial. Various materials, such as metal oxides, ordered mesoporous silica gel materials, zeolite materials, and carbon materials, can be used as the supports of the Ni-based catalysts.2/CO ratio of syngas by changing the feedstock gas ratio, which will provide different approaches for large-scale industrial applications of CRM.In future research, it can be prospected that the research hotspot of Ni-based catalysts in the field of CRM will focus on the Ni-based catalysts supported or confined by novel materials, such as ordered mesoporous materials and hollow zeolites. Specifically, the mesoporous framework in these catalysts facilitates the dispersion of active sites, and the confinement effect of mesoporous channels or hollow cavity can effectively control the size of Ni nanoparticles during CRM reactions. Besides, the combination of CRM with other reactions, such as SRM and POM, can alter the HLX conceived of the presented idea. LX encouraged XWu to investigate the project and supervised the whole progress of this work. CL, YC, XWen, CW, BY, and ZM assisted XWu to carry out the literature survey and summary. XWu, LX, and MC wrote the manuscript with support from XH. LX, MC, and XH supervised the whole project. All authors discussed the results and contributed to 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": "Recently, exploring brain activity based on functional networks during naturalistic stimuli especially music and video represents an attractive challenge because of the low signal-to-noise ratio in collected brain data. Although most efforts focusing on exploring the listening brain have been made through functional magnetic resonance imaging (fMRI), sensor-level electro- or magnetoencephalography (EEG/MEG) technique, little is known about how neural rhythms are involved in the brain network activity under naturalistic stimuli. This study exploited cortical oscillations through analysis of ongoing EEG and musical feature during freely listening to music. We used a data-driven method that combined music information retrieval with spatial Fourier Independent Components Analysis  to probe the interplay between the spatial profiles and the spectral patterns of the brain network emerging from music listening. Correlation analysis was performed between time courses of brain networks extracted from EEG data and musical feature time series extracted from music stimuli to derive the musical feature related oscillatory patterns in the listening brain. We found brain networks of musical feature processing were frequency-dependent. Musical feature time series, especially fluctuation centroid and key feature, were associated with an increased beta activation in the bilateral superior temporal gyrus. An increased alpha oscillation in the bilateral occipital cortex emerged during music listening, which was consistent with alpha functional suppression hypothesis in task-irrelevant regions. We also observed an increased delta\u2013beta oscillatory activity in the prefrontal cortex associated with musical feature processing. In addition to these findings, the proposed method seems valuable for characterizing the large-scale frequency-dependent brain activity engaged in musical feature processing. Understanding how our brain perceives complex and continuous inputs from the real-world has been an attractive problem in cognitive neuroscience in the past few decades. Brain imaging technology provides an opportunity to address this issue. However, revealing brain states is generally more difficult during real-word experiences than those recorded brain activities during resting-state or simplified abstract stimuli like controlled and rapidly repeated stimuli   is a well-established data-driven approach increasingly used to factor resting-state fMRI data into temporally covarying, spatially independent sources or networks. By contrast, in the analysis of EEG/MEG data, ICA has mainly been applied for artifact rejection. However, spatial Fourier\u2013ICA was proposed for data-driven characterization of oscillatory brain activity using EEG/MEG data. Compared with other ICA method applied to the context of music listening, spatial Fourier\u2013ICA used in the current study can automatically extract narrowband oscillations from broadband data without having to manually specify a frequency band of interest. So far, spatial Fourier\u2013ICA has already been proved to be fruitful in gaining insights into electrophysiological underpinnings of networks  for each source point while a relative conductivity coefficient was assigned to each tissue (with default MNI MRI template).To solve the inverse problem, minimum-norm estimate . Now the challenge is to determine which one of these represents the genuine brain responses. In all ICA based methods, it is a general question that which independent components need to be retained or which component just reflects noise. Here, we examine which components were modulated significantly by the musical features. We computed the correlation (Pearson\u2019s correlation coefficient) between the time courses of musical features and the time courses of those ICs  in order to select stimulus-related activations. We used the Monte Carlo method and permutation tests presented in our previous research  for initializing the algorithm. We used the minimum description length (MDL) to determine the number of clusters M. Afterwards we countered the number of subjects involved in ICs in each cluster. If the number of subjects in one cluster is less than half of the all subjects, this cluster would be discarded for the reason that such a cluster does not reveal information shared among enough participants. For the retained clusters, the spatial-spectral-temporal information was obtained, which was represented by the centroid of the cluster, the spectra of ICs and the numbers of subjects whose temporal courses were involved in this cluster.Five musical features were extracted by MIRtoolbox  firstly need to be extracted from the MEG data and were concatenated in certain dimensionality; ICA was then performed to concatenated data (Nugent et al. In this study, we introduced a novel framework with several techniques including Fourier ICA, source estimation, acoustic feature extraction, and clustering for exploiting the spectral\u2013spatial structure of brain during naturalistic stimulus. A complex-value ICA applied to source-space time\u2013frequency representation of EEG data. Following this, a modified ICASSO was performed to evaluate the stability of ICA estimate and a cluster analysis was applied to examine the inter-subject consistency. The identified networks involved in music perception were in line with those previous studies. Further, we found that brain networks under music listening were frequency-specific and three frequency-dependent networks associated with processing musical features were observed."}
{"text": "Blueberry wine residues produced during the wine-brewing process contain abundant anthocyanins and other bioactive compounds. To extract anthocyanins from blueberry wine residues more efficiently, a novel procedure of ultrasound-assisted deep eutectic solvent extraction (UADESE) was proposed in this work. The extraction process was optimized by response surface methodology coupled with genetic algorithm. The optimum extraction parameters to achieve the highest yield of anthocyanins (9.32 \u00b1 0.08 mg/g) from blueberry wine residues by UADESE were obtained at water content of 29%, ultrasonic power of 380 W, extraction temperature of 55 \u00b0C, and extraction time of 40 min. The AB-8 macroporous resin combined with Sephadex LH-20 techniques was used to purify the crude extract (CE) obtained under optimum extraction conditions and analyze the anthocyanins composition by HPLC-ESI-MS/MS. The cyanidin-3-rutinoside with purity of 92.81% was obtained. The HepG2 antitumor activity of CE was better than that of the purified anthocyanins component. Moreover, CE could increase the intracellular reactive oxygen species levels and the apoptosis, and arrest HepG2 cells in the S phases. These findings provided an effective and feasible method for anthocyanins extraction, and reduced the environmental burden of this waste. Vaccinium spp.), as a good source of bioactive compounds, includes polyphenols, anthocyanins, superoxide dismutases and flavanols +) corresponded to the loss of a glycoside on m/z 449.0. The mass spectra obtained were similar to those obtained by other authors in the case of cyanidin-3-rutinoside from litchi pericarp and tartary buckwheat + at m/z 331.0 corresponded to the loss of the glucoside from malvidin-3-glucoside. This anthocyanin was identified in grape skins [Rhodomyrtus tomentosa [Vitis vinifera red grape [The ESI/MS profile of peak 4 . However, the viability of HepG2 cells treated with CE at different concentrations was significantly lower than that of the cells treated with component \u2160 (p < 0.05). Moreover, the viability of the HepG2 cells treated with CE at 48 h was lower than that at 24 h. Results indicated that the anti-tumor effect of CE on HepG2 cells was better than that of component \u2160, and this effect was observed in a time-dependent manner. Based on the above results, the 48 h exposure of CE was chosen for later studies. In addition, the normal human hepatocytes (HL-7702 cells) were treated by using different concentrations of CE and component \u2160 at 24 h and 48 h. Results are described in p > 0.05). Results implied that CE and component \u2160 had no toxic effect on HL-7702 cells at a range of 0.1\u201310.0 \u00b5g/mL. Subsequently, the cytotoxicity of the CE and the component \u2160 to HL-7702 cells increased significantly with increasing concentrations at 24 and 48 h (p < 0.05). Consequently, 0.1\u201310.0 \u00b5g/mL of CE were used in subsequent experiments.First, the effects of the CE and component \u2160 on cell viability were determined using the MTT assay. Experimental results are shown in p < 0.05). Results indicated that CE could inhibit the growth of HepG2 cells by improving the intracellular ROS levels. Similar experimental results were found in the case of purple rice and bilberry anthocyanins extracts [The production and the clearance of ROS in normal cells were in a dynamic equilibrium. The appropriate amount of ROS can promote macrophages to play their phagocytotic and enzymatic immunophysiological functions. However, the generation of excessive ROS can inhibit tumor cell activity and cause apoptosis. Thus, the fluorescent probe DCFH-DA was utilized to monitor the intracellular ROS levels and clarify whether the growth inhibition of CE on HepG2 cells was related to intracellular ROS levels. As illustrated in extracts ,31.The Annexin V/PI double staining and the flow cytometry analysis were carried out to elucidate the effect of CE on the apoptosis of HepG2 cells. As shown in p < 0.05). The CE treatment (0.1\u201310.0 \u03bcg/mL) notably enhanced the percentage of cells in the S phases in a dose-dependent manner compared with the vehicle-control treatment (p < 0.05). Compared with the vehicle treated group, 0.1\u201310.0 \u03bcg/mL CE treatment significantly increased the percentage of cells in the G2/M phases (p < 0.05). Nevertheless, no remarkable difference in the percentage of cells in the G2/M phases was observed between the 0.1 \u03bcg/mL and the 10.0 \u03bcg/mL CE treated groups (p > 0.05). These results revealed that CE could arrest the HepG2 cells in the S phases.The effect of different concentrations of CE on the cell cycle distribution was analyzed using the commercial cell cycle detection kit to clarify whether the growth inhibition of CE on HepG2 cells was related to cell cycle arrest. As shown in Blueberry wine residues were collected from Heilongjiang blueberry manor Biotechnology Co.; Ltd. . The squeezed blueberry wine residues weighing 50 kg were dried in the FD-1A-50 freezing-vacuum dryer  at \u221218 \u2103 until the moisture content was less than 5%, and then the dehydrated blueberry wine residues were stored at \u221218 \u2103 for 12 h before crushed. The frozen samples were thawed and milled into powdered particles smaller than 0.45 mm using an electric mill , sealed in a brown reagent bottle and stored at 4 \u2103 for the follow-up experiments.Cyanidin-3-O-glucoside (C3G) was offered from Chengdu Gelip Biotechnology Co.; Ltd. . Choline chloride, glycerol, 1,3-butanediol, 1,4-butanediol, glycol, acetonitrile, and sodium acetate buffer of analytical purity were purchased from Shanghai McLean Biochemical Technology Co.; Ltd. . AB-8 macroporous resin and Sephadex LH-20 were offered from Tianjin Tianxin Fine Chemical Factory . Human hepatoma cells (HepG2 cells) and Human normal hepatic cell (HL-7702 cells) were afforded from Concorde Cell Repository . The fetal bovine serum (FBS) was from GIBCO-BRL . MTT was obtained from Beijing Baileibo Technology Co.; Ltd. . RMPI-1640 medium, penicillin, and streptomycin were provided from Beijing Shengmu Biotechnology Co.; Ltd. . Trypsin was obtained from Beijing Fubo Biotechnology Co.; Ltd. . Annexin V-FITC/PI cell apoptosis detection and ROS kits were from Shanghai Yisheng Biotechnology Co.; Ltd. . Formic acid, ethanol, and potassium chloride buffer were purchased from Shanghai Jinjinle Industrial Co.; Ltd. .DESs were prepared following the previous research with some modifications . DESs cog and 25 \u00b0C for 15 min by using the KH20R-\u2161 type centrifuge  to obtain the anthocyanins extract. The extract was concentrated using the DZFY-2L type vacuum rotary evaporator  at 40 \u00b0C and freeze-dried using the FD-1A-50 dryer for 48 h. After drying, the crude anthocyanin powder was sealed in a brown reagent bottle and stored at \u221218 \u00b0C.Anthocyanins were extracted using UADESE in accordance with a previously described method with some modifications . The lyo3COONa-HCl buffer at pH 4.5). Each of these mixtures was stored away from light for 30 min at 25 \u00b0C. The absorbance of each sample was determined at 510 and 700 nm, respectively. The anthocyanins yield was calculated by Equation (2) [\u03c9M is the molecular weight of C3G, 449.2 g/mol; DF is the dilution factor; \u03b5 is the molar extinction coefficient of C3G, 26,000 L/mol\u00b7cm; L is the path length, cm; V is the total volume of extraction solvent, mL; m is the weight of blueberry wine residues powder, g.The yield of anthocyanins in blueberry wine residues was measured by a pH differential method using a UV-3802S visible spectrophotometer . Briefly, 3 mL of anthocyanins extracts were diluted with 5 mL of two different buffers (0.025 M KCl-HCl buffer at pH 1.0 and 0.4 M CHtion (2) .(2)c=[.X1), ultrasonic power (X2), extraction temperature (X3), and extraction time (X4) are selected as independent variables, whereas the anthocyanins yield (Y) is regarded as the response. The level and code of these factors are shown in Y is the anthocyanins yield, mg/g; iX and jX are the coded variables (i and j range from 1 to k); \u03b20, j\u03b2, jj\u03b2 and ij\u03b2 are regression coefficients of intercept coefficient, linear, quadratic, and the second-order terms, respectively; k is the number of independent parameters (k = 4) and ie is the error.According to the single-factor experimental results, the Box\u2013Behnken design (BBD) of 4-factor-3-level was conducted to investigate the influence of different variables and their interactions on the yield of anthocyanins from blueberry wine residues. The following four independent factors were considered: water content in DESs (R2) and the percentage absolute error of deviation (AED) between the experimental (Yexp) and the calculated (Ymod) results were used to assess the effectiveness of the model in this paper. R2 and AED were calculated using Equations (3) and (4), respectively.The regression coefficient  Determination of decision variablesX1), ultrasonic power (X2), extraction temperature (X3), and extraction time (X4). The process could be represented by Equation (5).According to the single-factor experimental results, four factors were selected as decision variables: Water content in DESs  Determination of objective functionidK is the design value of the i-th parameter; imK is the target value of the i-th parameter; iK is value of i-th parameter processing.Deviation method has the advantage of avoiding the influence of unit dimension on optimization model. The process could be described by Equation (6)iK is equal to 1, the function exactly satisfies the design aim of the objective parameter i. Euclidean distance is employed to express the deviation of each parameter from the target value, which is calculated by Equation (7).ik is the deviation of parameter i from the target value. Each design parameter has a big optimization objective. The following objective function is confirmed to achieve overall optimization.iw is the target weight of the i-th parameter, iw is larger, parameter i is more important, and the goal of parameter i is satisfied first in design.When (3) Determination of constraint functionConstraints of GA optimization: Select the upper and lower limits of each factor level, and the constraints of the highest anthocyanins yield are as follows:The optimization mathematical model of UADESE anthocyanins from blueberry wine residues is a mixed programming problem with inequality constraints. According to the required optimization objective function and related constraints, the GA toolbox of MATLAB version R2018b was employed for optimization studies. The objective function can be detected by Equation (8).v/v) of pH 3 at 2 BV/h. The eluent was collected, and ethanol was recovered using the rotary evaporator at 40 \u00b0C to obtain an anthocyanin-rich solution. The anthocyanin-rich solution was further separated using Sephadex LH-20 medium pressure column chromatography (2.9 \u00d7 25 cm) and washed with 70% ethanol  at 1.5 BV/h when anthocyanins were completely absorbed in the Sephadex LH-20. The eluent was collected 1 tube for pre 3 mL and freeze-dried via the FD-1A-50 dryer. Finally, the purified anthocyanin component (component \u2160) was obtained.The separation and the purification of anthocyanins were described in a previous report . CE (200v/v)) and filtered through a 0.45 \u03bcm filter membrane. The 1100 series liquid chromatography system equipped with a DAD and Zorbax Eclipse XDB-C18 column  was used to determine the anthocyanins contents. Two solvents, including 5% (v/v) formic acid (mobile phase A) and 1% (v/v) formic acid acetonitrile (mobile phase B), were used in the mobile phase. The gradient elution was as follows: 5%\u201320% B (0\u20135 min), 20%\u201325% B (5\u201315 min), 25%\u201330% B (15\u201320 min), 30%\u201333% B (20\u201335 min), and 33%\u20135% B (35\u201340 min). The mobile phase was pumped by the system at a rate of 0.8 mL/min. The injection amount of the sample was set to 20 \u00b5L. Moreover, the column temperature and the wavelength set 25 \u00b0C and 520 nm, respectively. The purity of the anthocyanins component can be calculated using Equation (10).iw and iA are purity and peak area of component i, respectively. if is the correction factor.The HPLC-DAD and HPLC-MS methods for the analysis of anthocyanins components in CE and component \u2160 were consistent with our previous study . Before m/z in the positive mode. The voltages of the capillary, sampling cone, and extraction cone were 2.0 kV, 40 V, and 2.0 V, respectively. The temperatures of source and desolvation were set to 115 and 350 \u00b0C, respectively. The time of scan and interscan were 13 min and 0.28 s, respectively. The gas flow of the cone and the desolvation were 50 and 900 L/h, respectively. The collision energy was set 20.0\u201345.0 eV. The Mass-Lynx TM V 4.1 software was used to collect experimental data.The mass spectra were obtained at a range of 100\u20131000 2, and the media were replenished every two days. Logarithmic cells were used in this study. The RMPI-1640 medium was employed to prepare the sample solutions of CE and component \u2160. Samples were filtered through a 0.22 \u00b5m membrane for sterilization before the experiment.HepG2 and HL-7702 cells were cultured in the RMPI-1640 medium with 10% FBS and 1% penicillin-streptomycin and placed in an incubator maintained at 37 \u00b0C and 5% CO3 cells/mL) were grown in 96-well plates at 37 \u00b0C in a humidified 5% CO2 incubator for 24 h. Cells were treated with different concentrations of CE  and component \u2160 for 24 h and 48 h. After the above treatment, the supernatant was removed, and each well was added with 20 \u00b5L MTT (5 mg/mL) and incubated for another 4 h. The culture medium was then replaced with 150 \u00b5L DMSO in each well to dissolve the formed blue formazan crystals. Subsequently, the absorption value of each well was determined at 490 nm by using the WD-2102b automatic enzyme labeling instrument . Cell viability was calculated using Equation (11). All experimental results were described as the mean \u00b1 SD of three experiments with six wells per treatment group.Cell viability was measured by using the MTT method in accordance with the previously described method . The Hep5 cells/mL) were seeded into 6-well plates and incubated overnight. Cells were treated with different concentrations of CE  for 48 h. Cells were then washed with PBS and subsequently incubated with 5 \u03bcM DCFH-DA in PBS at 37 \u00b0C for 30 min. HepG2 cells were washed with PBS, and collected by centrifugation, and re-suspended in PBS. Finally, the ZE5 multicolor flow cytometer  was used to determine the fluorescence intensity.In accordance with the results of MTT, CE was selected for subsequent experiments. The effect of CE on ROS generation in HepG2 cells was monitored by the ROS kit in accordance with its instructions. In short, HepG2 cells (1 \u00d7 10The apoptosis of HepG2 cells was determined using the Annexin V-FITC and propidium iodine (PI) dual staining, which was performed using the ZE5 multicolor flow cytometer . The tre5 cells/mL and incubated overnight. The cells were exposed to different concentrations of CE . After 48 h, HepG2 cells were harvested and fixed with cold 70% ethanol  at \u221220 \u00b0C for 24 h. Subsequently, the treated HepG2 cells were washed with PBS and incubated with RNase (50 \u03bcg/mL) at 37 \u00b0C for 20 min. Finally, the cells were stained with 50 \u03bcg/mL PI at 4 \u00b0C for 15 min in the dark and subjected to the ZE5 multicolor flow cytometer. The experimental data were analyzed using the Modfit LT software .The cell cycle analysis was conducted using a previously published method . Brieflyp < 0.05 represented the experimental data with statistical significance. Design-Expert software (version 8.0.6) for BBD analyses was employed to design combinatorial experiments. All analyses were performed with SPSS Statistics 19 (version 13.0). Origin software (version 9.0) was used for drawing in this study.All experimental data were represented as mean \u00b1 SD. The one-way analysis of variance was used to analyze statistical differences between groups. This study was performed to investigate the UADESE anthocyanins from blueberry wine residues and optimize the extraction conditions by GA based on RSM. The optimum extraction parameters to achieve the highest yield of anthocyanins of (9.32 \u00b1 0.08 mg/g) from blueberry wine residues via UADESE were obtained at water content of 29%, ultrasonic power of 380 W, extraction temperature of 55 \u00b0C, and extraction time of 40 min. The cyanidin-3-rutinoside with purity of 92.81% was obtained. The HepG2 antitumor activity of CE was better than that of component \u2160. In addition, CE could increase the intracellular ROS levels and the apoptosis, and arrest the HepG2 cells in the S phases. Finally, findings confirmed that the UADESE was an efficient, reliable, and environmentally friendly extraction method. Further studies will focus on more detailed identification and quantification of other phenolic compounds available in the extract and to investigate their antitumor mechanism."}
{"text": "Complying with individual privacy perceptions is essential when processing personal information for research. Our specific research area is performance development of elite athletes, wherein nutritional aspects are important. Before adopting new automated tools that capture such data, it is crucial to understand and address the privacy concerns of the research subjects that are to be studied. Privacy as contextual integrity emphasizes understanding contextual sensitivity in an information flow. In this study, we explore privacy perceptions in image-based dietary assessments. This research field lacks empirical evidence on what will be considered as privacy violations when exploring trends in long-running studies. Prior studies have only classified images as either private or public depending on their basic content. An assessment and analysis are thus needed to prevent unwanted consequences of privacy breach and other issues perceived as sensitive when designing systems for dietary assessment by using food images.The aim of this study was to investigate common perceptions of computer systems using food images for dietary assessment. The study delves into perceived risks and data-sharing behaviors.We investigated the privacy perceptions of 105 individuals by using a web-based survey. We analyzed these perceptions along with perceived risks in sharing dietary information with third parties.We found that understanding the motive behind the use of data increases its chances of sharing with a social group.In this study, we highlight various privacy concerns that can be addressed during the design phase. A system design that is compliant with general data protection regulations will increase participants\u2019 and stakeholders\u2019 trust in an image-based dietary assessment system. Innovative solutions are needed to reduce the intrusiveness of a continuous assessment. Individuals show varying behaviors for sharing metadata, as knowing what the data is being used for, increases the chance of it being shared. Food images are highly relevant for use in medical research and sport science. They can capture continuous and accurate measurement of diets, and therefore are imperative in understanding the relationship between food intake and athletic development  or betweAlthough research on human subjects is already strictly regulated by local, national, and international boards and procedures, the increased usage of personal information recorded automatically through new technology comes with new concerns for the security and privacy of the subjects. Little attention has been given to the specific individual privacy requirements related to the design of IBDA systems ,18, and survey wherein subjects were asked about their perceptions of privacy with regard to capturing food images by using a smartphone camera. Taking food pictures is already a trend on many social networks, where people typically post images of their meals during vacations and special events [To improve our understanding of the privacy requirements in population-based research data, we conducted a web-based l events , and thel events ,24; our Another important lesson from previous epidemiological-related work is that the data capturing should not be intrusive. We have previously attempted to use, for instance, 24-hour dietary recall\u2013inspired methods with pictures taken during meals, but this showed to be too intrusive and too time-consuming for elite athletes. Moreover, the dropout rates of these elite athletes were very steep with these methods. Hence, we developed this survey by using alternative schemes for data assessment, wherein pictures of meals were captured similar to that captured in social media engagement.From a more general perspective on privacy, food images offer an interesting case to study, as few food images might not carry much sensitive information. However, a large individual data set of images that is continuously recorded over long periods (>2 weeks) and linked to an individual\u2019s identity might disclose information that many find too sensitive to share. Such disclosure is a growing public concern ,26 and tSystematic reviews of dietary assessment methods ,27 have Zerr et al  exploredThe work on IBDA methods by Boushey et al ,11 and OTo improve our understanding of the privacy perception related to the capture and use of food images, we conducted a study by using a web-based questionnaire  hosted bThe survey was designed to record participants\u2019 perceptions in the following scenarios: (1) Scenario A, the participant having to record and share dietary data as an athlete; (2) Scenario B, the possibility and severity of privacy leak from one\u2019s dietary data; and (3) Scenario C, sharing dietary data and reports among different social groups.The scenarios were designed to be familiar to our participants and to cover various angles on data sharing. For Scenario A, sharing is both internally and externally motivated and controlled by the subject; however, the subject is not in control of the processing. For Scenario B, the subject is not in control of the data processing but has concerns about the processing outcomes, and for C, the sharing is consensual but is based on external motivation from different social groups, for example, receiving feedback from a doctor or sharing with family/friends as part of social behavior/interaction.We used these scenarios to record our participants\u2019 perceptions and attitudes toward sharing data. Note that we considered the perceptions on a scenario valid even for participants who never encountered that scenario in real life beforehand. One\u2019s perceptions can affect one\u2019s participation in a study if the concerns are not addressed at the beginning of a study.Likert item, ranging from strongly disagree to strongly agree. These include perceptions toward responsibility for privacy, intrusiveness, and general attitude toward dietary practices [Our questionnaire starts by familiarizing participants with food pictures on social networks (Scenario A). It then asks about the use of social networks and experience with taking food pictures. Additionally, participants are asked about their preference of IBDA methods over other similarly used techniques for dietary assessment. Attitudes toward privacy and personal control over data were collected on a 5-level ractices .Next, the participants are asked to consider Scenario A\u2014an athlete who records his/her diet by taking pictures. We specify that every meal is recorded by taking a picture, including drinks, and even at events outside training by using a mobile app. We specify that the app allows the team owner, manager, coach, and doctor to monitor his/her diet and recommend diet plans. Continuing with questions from Scenario A, we further obtain responses toward the privacy and usability of metadata collected through such a system. tag the location of a restaurant when posting a food picture. Further, we presented the hypothetical situation of a third party that gains control of a participant\u2019s diet data of 1 year. Based on that data, a few aspects of the individual might be inferred. We presented Scenario B and collected responses on what the participants thought can be inferred. The inferred information examples were hypothetical, and to our knowledge, no such work exists. It was designed to evaluate perceptions toward what is possible and how sensitive particular information is to the participant. The responses were collected on a 3-item likelihood and sensitivity Likert scale.Regarding metadata collected through an IBDA system, we presented existing social network jargon that many are familiar with. For example, some users Family, Friends, Doctor, Team, and Fans. Participants indicated their binary responses by checking corresponding blocks in the questionnaire. Additionally, we provided an option if they thought the information is sensitive and they do not wish to share with anyone. At the end of the questionnaire, the participants were asked a series of demographic questions, including some additional ones about their diet and allergy. The collected data from Nettskjema were downloaded and analyzed after the end of the study. The results are presented in the Results section.In addition to perceived threats with sharing food pictures and subsequently data set, we collected responses about which social groups an individual was voluntarily willing to share information about their diet with. The information as food images and attached metadata typically associated with an image was considered for sharing. In addition to food images, we added additional parameters such as medications and diet plans. The groups provided were P>.05 supporting the hypotheses that the responses are uniform across the participants. The actual P values that we obtained were P=.14, P=.15, P=.56, P=.18, and P=.78. We performed another set of Kruskal-Wallis tests to determine whether the responses among European and non-European participants had statistically significant differences. For E1-E5, the observed P values, that is, P=.20, P=.14, P=.28, P=.92, and P=.50 indicated that they were not different. Therefore, we proceeded with reporting ordinal variables in our results by using compound bar charts.We performed the Kruskal-Wallis test  to deterlikelihood did not indicate a strong correlation with the concern. The maximum correlation coefficient we observed was between the likelihood and severity of concern for \u201cfinancial status\u201d with r=0.41 (P<.001). However, it was still a low correlation. We report our observed ordinal values later in the Results section. We did not use a prediction model in our analysis as we did not find any predictor variables to be significantly related to the outcome in our analysis (P>.05).Additionally, we measured consensus among the reported ordinal values by using the Tastle and Wierman\u2019s consensus measure . We reposnowball sampling methodology [Chung at al  showed thodology  to recruOf the 105 participants, 99 (94.2%) indicated having an account on a social network. Of the 99 participants, 95 (96%) reported seeing food pictures at least once over social networks. In this section, we discuss the perception of privacy and related attitudes based on the findings in our study. We divided our results into expectations and concerns. The expectations cover general perception toward privacy, IBDA methods, and data use. The concerns cover information that can be inferred from their dietary data. Additionally, we discuss the concerns toward exposing such information to a third party from their mobile Food Records (mFRs). Finally, we present our findings regarding the sharing of collected dietary information with different social groups.We present the general expectations that participants have indicated toward IBDA methods. We also explored their attitudes toward data collection and use.Approximately 80.9% (85/105) of the participants agreed that capturing diet records by using a phone camera is easier than writing down their dietary intake see E1, . Individterms of use.\u201d While Europeans have a legal right to demand such explanations, we excluded data from them to see what the participants from outside Europe prefer. Even among non-Europeans , we observed a similar interest in the participants in knowing what their data are being used for.Individuals tend to have very little or no information about how their data are being used. Many individuals feel that they have no control . In 2018Trust is important for participation in epidemiological research . The earWith regard to food allergies, only a fraction of the participants  were very concerned about it being exposed to a third party . About oAbout the possibility of deriving allergies from their mFRs, more than two-thirds  thought that allergies can be inferred. A little more than a quarter  thought that it was not very likely to be derived from the mFR of an individual. This trend was very similar among participants with allergies. Approximately 30% (12/39) of the participants with allergies thought that it was not likely that it can be inferred from their mFRs.Religious belief is often considered sensitive information in regional laws . It can very concerned about a third party learning about their religion. Participants following religious diets (n=17) were slightly more concerned about a third party learning about their religion. Approximately 65% (11/17) of the participants following religious diets indicated that they were concerned about a third party learning about their religion from the mFRs.With regard to a third party learning about their religion, a little more than half  of the participants were not concerned. Only 17.1% (18/105) were As personalized dietary interventions are more effective , IBDA syInformation collected by an IBDA system about an athlete\u2019s diet provides insights into dietary habits and can guide toward a proper diet. There might be additional metadata collection through an IBDA system. The information collected in the form of food images along with metadata can be mined for other purposes as well. For example, the time of dietary intake can be useful for maximizing performance on the field or predicting burnout. Similar to trends on social networks , an athlfamily, the participants were more willing to share food images with their doctors . Only a quarter  of the participants showed willingness to share food pictures with their sports team while nearly half  showed willingness to share food pictures with friends. In terms of the metadata associated with dietary data, such as the time of the meal, the willingness to share further drops. Only 68.6% (72/105) of the participants agreed to share the time of the meal with their families in comparison to 82.9% (87/105) sharing the time of the meal with their doctors. Time of food intake is in fact an important consideration for elite athletes and coaches with respect to restitution and training planning.About three-fourths  of the participants showed willingness to share food pictures continuously with family. In comparison to the social group family and also with doctor. Unsurprisingly, participants were not very keen on sharing location with their fans . About 16.2% (17/105) of the participants did not want to share their location of places they eat with anyone.Individuals are more cautious about sharing their location. Only 70.5% (74/105) expressed willingness to share the location associated with diet records with doctor. There was a weak correlation between willingness to share location along with the food image with a doctor. Within the family group, there was a strong correlation in sharing diet plan and time. In terms of sharing metadata with one\u2019s team, we observed a strong correlation between diet plan and food image. The willingness to share time and location with one\u2019s team was also strongly correlated.In this paper, we present our findings on the perception of privacy for an IBDA system. Our findings provide a coarse view of privacy attitudes toward conducting dietary assessments with food images. Expanding upon prior works ,18, thesPersonalized dietary interventions are more effective than universal recommendations . HoweverOur initial assumption from the study by Chung et al  was thatSharing an athlete\u2019s data with fans can be an interesting opportunity to engage with followers, such as for crowd support using HeartLink . HoweverElite sport clubs, particularly in our elite soccer domain, have nutritional experts hired as part of their management and support team. Such experts are involved in providing detailed dietary plans for their athletes, and they know in detail about most of the common meals provided on-premise for the athletes. For instance, our main elite soccer clubs involved in our cooperation, as a rule, have breakfast and lunch together in their training facilities. Involving such experts using our proposed scheme means that they receive the needed data from their athletes when outside the training facilities to complete the picture.We investigated the privacy perceptions and concerns for conducting long-running studies using IBDA methods. For epidemiological studies, it is important for users to continuously record diets without any biases. In this study, individuals preferred recording diets using a digital camera over other methods. However, taking a picture of every meal is still perceived as intrusive for some users. For long-running studies, prediction models can be employed to reduce the labor of taking pictures . In summOur study has the following limitation. A questionnaire-based study fails to identify the causation of behavior. For privacy reasons, we did not collect the contact information from the participants. Hence, any further study with the same set of participants is not possible.In conclusion, we conducted a questionnaire-based study to understand the privacy perceptions and concerns for building IBDA systems. The privacy concerns can be addressed during the design phase to mitigate risks and strengthen participants\u2019 and stakeholders\u2019 trust in a system. We find a growing interest to know what the collected data are being used for. While IBDA methods are preferred for ease of use, continuous assessment is still seen as intrusive. GDPR compliance is an attractive feature for individuals worldwide. While uncertain about the inferences from mFRs, identity remains a top concern with regard to privacy for individuals. Knowing what the data is being used for, increases the chances of it being shared. Individuals are concerned about metadata sharing with third parties. We recently started a large interdisciplinary study involving computer scientists, sports scientists, psychologists, mathematicians, and medical experts . Our select cohort includes over 400 female elite soccer athletes, from Norway to Portugal, and we intend to conduct our next study in this cohort."}
{"text": "Given that most evidence-based recommendations for managing type 2 diabetes mellitus (T2DM) are generated in high-income settings, significant challenges for their implementation exist in Latin America and the Caribbean region (LAC), where the rates of T2DM and related mortality are increasing. The aim of this study is to identify the facilitators and barriers to successful management of T2DM in LAC, from the perspectives of patients, their families or caregivers, healthcare professionals, and/or other stakeholders.We conducted a systematic review in MEDLINE, Web of Science, SciELO, and LILACS. We included studies of disease management, prevention of complications and risk factor management. We qualitatively synthesized the verbatim text referring to barriers and/or facilitators of diabetes management according to the Theoretical Domain Framework and described their relative frequencies.We included 60 studies from 1,595 records identified. 54 studies (90%) identified factors related to the environmental context and resources, highlighting the importance of questions related to health care access or lack of resources in the health system, and the environmental context and living conditions of the patients. Issues related to \u201csocial influences\u201d (40 studies) and \u201csocial/professional role and identity\u201d (37 studies) were also frequently addressed, indicating the negative impact of lack of support from family and friends and clinicians\u2019 paternalistic attitude. 25 studies identified patients beliefs as important barriers, identifying issues such as a lack of patients\u2019 trust in the effectiveness of the medication and/or the doctor\u2019s advice, or preferences for alternative therapies.Successful diabetes management in LAC is highly dependent on factors that are beyond the control of the individual patients. Successful disease control will require emphasis on public policies to reinforce health care access and resources, the promotion of a patient-centred care approach, and health promoting infrastructures at environmental level. The global burden of diabetes has been continuously increasing in the past decades and this trend is expected to continue in the coming years , 2. As gIn Latin America and the Caribbean (LAC), the lower consumption of fruit and vegetables, as well as the higher intake of saturated fats, sugar and salt in comparison with other regions in the world, is leading to a remarkable problem of obesity and, consequently diabetes . ImportaMany countries in LAC have developed a universal health insurance program. However, the programs tend to be more focused on patient\u2019s rehabilitation and treatment, rather than on the prevention or diagnosis phases of disease . Like moThe reviews that have so far described barriers to T2DM management also come from high-income countries and have mainly focused on individual behaviours rather than considering the patients\u2019 environment \u201319. The In this systematic review, we evaluate observational studies with qualitative and/or quantitative methodology to identify the barriers or facilitators to the management of T2DM in LAC countries, from the perspectives of patients, their relatives or caregivers, healthcare professionals, and/or any other stakeholders.We conducted a systematic review to identify barriers and/or facilitators in the management of the T2DM disease in LAC countries. We considered T2DM management to include strategies or care protocols for the control of the disease and its risk factors, as well as prevention of complications such as diabetic retinopathy, kidney or heart disease, and diabetic foot. How successful management of the disease was defined depended on the focus of the different studies included. For qualitative studies, it depended on the perceptions of the participants, and how they defined good or poor control of the disease. In quantitative reports the issue was more complex because it was highly influenced by the researcher who designed the data collection tools. In this case, successful care was considered according to measurements such as adherence to care protocols using predefined tools, questionnaires assessing quality of life, or indicators of health care access. We defined facilitator as any factor that supports the management of T2DM and barrier as any factor that limits the disease management. These factors may be socioeconomic, educational, cultural, behavioural, cognitive, structural, or logistical. The source of the data can be patients, patients\u2019 families or caregivers, healthcare professionals or any other stakeholder. We recorded the information related to barriers and facilitators as they were reported in the studies. Depending on the perception of the study participants, the same factor may be perceived as a barrier by some stakeholders and a facilitator by others, e.g. religion.Paired reviewers independently assessed each stage of the review process, from study selection to data synthesis and analysis, and a third reviewer resolved discrepancies. Identification of barriers and facilitators related to disease management was not always explicitly stated as the objective of the research, and the use of different terminology was common. During the abstract review, if it was apparent that barriers or facilitators to diabetes management were reported, we assessed the full text for inclusion.https://www.crd.york.ac.uk/PROSPERO/, protocol number CRD42019134938 on 22 July 2019  AND . We included the condition that the research was carried out at least in one country in the LAC region, according to the World Bank . TherefoWe selected studies that: (1) included individuals with laboratory confirmed or self-reported T2DM who lived in LAC countries (2) described facilitators or barriers related to successful management of the disease; (3) reported original data; (4) were written in English, Spanish, French or Portuguese. Studies that included numerous pathologies were included provided that some participants had T2DM and they their results were reported separately. We excluded studies that included solely patients with other diagnoses, or with healthy individuals focussing on primary prevention of T2DM.We extracted the information of interest using an ad-hoc checklist that included the following variables:Study characteristics: first author, year of publication, study location, study focus , study methods , and whether or not the study was carried out in an urban or rural setting.Participants characteristics: medical issue ; participant type , number of participants, number of people with diabetes by sex, age of people with diabetes, socioeconomic status of people with diabetes, ethnicity of people with diabetes.The facilitators and/or barriers related to the management of T2DM described in the study to improve diabetes care.To evaluate the external validity and risk of bias of the selected studies, we developed a seven-item checklist using the Newcastle-Ottawa Scale  and the We qualitatively synthesized the verbatim text referring to barriers and/or facilitators of diabetes management and described their relative frequencies. We conducted a qualitative analysis of the identified barriers and facilitators according to the Systematic Text Condensation method developed by Malterud . This foWe initially identified 1,595 records, of which 168 full-text articles were assessed for inclusion and 60 studies were finally included \u201386 Fig . A detaiThe studies involved participants from 16 different countries from the LAC region. Nearly half of the studies included participants from Brazil (46.7%), followed by Mexico (20%). Thirty-six studies (55%) used qualitative methods, 17 (28.3%) quantitative methods and 10 (16.7%) mixed methods. Most of the studies were carried out in urban settings . Forty-five (75%) studies targeted diabetes alone, and 15 (25%) studies included diabetes with hypertension or other non-communicable diseases. Forty-nine (81.7%) studies analysed barriers and facilitators from the patients\u2019 perspective, 19 (31.7%) included the health professionals\u2019 perspective, while some studies included other stakeholders, such as family members, caregivers or health managers.The results of the quality assessment are summarised in The barriers and facilitators described in the studies were classified according to 13 different theoretical domains, which contained a further 29 themes .\u201cenvironmental context and resources\u201d, where 54 studies (90%) identified at least one barrier or facilitator Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for the opportunity to review this interesting systematic review. The authors studied barriers and facilitators for diabetes care in LAC. The topic is relevant, and certainly the evidence is much needed in this region. The results are very well organised and easy to follow. I have a few suggestions that hopefully will improve this work.Introduction1. In the second paragraph, gender, socio-economic, and ethnic inequalities are pinpointed. Although relevant, and much related to the topic, the references used to support these statements are from Europe and US. I will encourage the authors to replace or complement these references with other studies conducted in Latin-America.Methods1. In the study selection subheading, it reads that studies were excluded if these did not focus \u201con patients with an established T2DM diagnosis\u201d. This needs further clarification. Does this mean that only patients with a history of diabetes (self-reported diagnosis) were included? In other words, reports that studied patients with diabetes (self-reported) and people newly diagnosed (blood tests as part of the study) were not included? In this line, how was diabetes history defined? Was this based on self-reported diagnosis only, a combination of self-reported or taking drugs for diabetes, or other means?Discussion1. The discussion begins signalling that diabetes is a complex issue and that contextual factors seem to be more relevant than individual profiles. These ideas would benefit from strong references. The findings of this work, although interesting, do not provide evidence to conclude that contextual factors are more important. If there were not any relevant references, these statements should be toned down. This concern would also apply to the first lines of the conclusions; statements appear to be too strong and not fully backed-up by the findings.2. The limitations of the study argued briefly that qualitative original reports rarely study representative samples. Further discussion on this matter -representative and extrapolation of the results- is warranted. Can the findings of the review really apply to all patients in LAC? Should there be any considerations when interpreting the results across countries ?3. The conclusion includes arguments about \u201ckey determinants for successful care\u201d. This seems quite a strong idea that has not been fully defined nor explored in the results. What is successful care? How is this defined? Does this relate to any care metric  or only to the patient\u2019s perspective?Reviewer #2: This is a well written manuscript that aims to fill an important gap in the literature regarding barriers and facilitators in the management of T2DM in Latin American region. As stated by the authors, this region is experiencing a very large burden of T2DM and it is important to understand the challenges and opportunities uniques to the region.The study design, a systematic review is a great choice.Below are concrete comments for authors to consider that I think would improve the paper.Intro: the study is really about understanding barriers and facilitators of diabetes secondary and tertiary prevention; the authors should consider using these commonly used public health terms.Per PRISMA guidelines for the reporting of systematic reviews, introduction should include information about the study designs. Were these mostly qualitative studies or also quantitative studies.Methods:Study selection: include information about the type of study.With respect to 2) The objective of the study was to identify or describe facilitators orbarriers related to the management of the disease.Did the authors rely on the presence of these terms in the stated objectives? If so, please elaborate on the justification for this decision.Data extraction:With respect to 3) The facilitators and/or barriers related to the management of T2DM described in the study to improve diabetes care.This is key given the objective of the study and the statement is vague in light of the types of studies included. Specifically: 1) it is unclear to me whether for quantitative studies, the authors used the results of the studies to identify barriers and facilitators, in other words and for example, did the authors examined results to identify factors associated with a controlled A1c, vs. uncontrolled A1c, first ones were considered facilitators, others barriers. I suspect the authors encountered that type of studies (e.g. registries) and it is important to clarify how they handled/used these results under their own conceptual framework. In fact, some of the characteristics listed in number 2 of the data extraction can fall in the category of barriers (e.g. socioeconomic status). 2) Did they rely on the use of the terms \u201cbarriers and facilitators\u201d in the article text. If so, please elaborate on the justification for this decision and consider doing a validation study, examining a random 1-2% of the 65 studies excluded because not focused on \u201cbarriers/facilitators\u201dDid the authors obtain information about the setting: urban, rural, clinic in the hospital, pharmacies, etc.. I think this information would be very valuable to understand the context.It is recommended to avoid using \u201cdiabetic\u201d to refer to patients with diabetes.Results:In addition to having table 3 as is, I\u2019d recommend to have 2 similar tables subdivided by type of stakeholder with 2 main groups:1- Individual patient, caregiver, relative2- Health care professionals, health managers and other stakeholders.Figure 1.For the 890 studies removed in early phase, please include the reasons for these exclusions. (as you do for the other exclusions).**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0 20 Jul 2020Journal Requirements:When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfWe have checked the manuscript according to the style requirements.2. We note that your search was completed in February 2019.Please update your search to include studies published in the last 12 months.We have updated the search to the 16th June 2020, as requested. 12 new studies have been added to the review. The results section and figure 1 have been updated accordingly. Responses to reviewers\u2019 comments1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: PartlyReviewer #2: YesWe have modified the paper in light of the different comments made and strongly believe that the manuscript has improved.2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes 3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.Reviewer #1: YesReviewer #2: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes We have reviewed the language. The final manuscript has been proofread by an experienced native English researcher.5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for the opportunity to review this interesting systematic review. The authors studied barriers and facilitators for diabetes care in LAC. The topic is relevant, and certainly the evidence is much needed in this region. The results are very well organised and easy to follow. I have a few suggestions that hopefully will improve this work.Introduction1. In the second paragraph, gender, socio-economic, and ethnic inequalities are pinpointed. Although relevant, and much related to the topic, the references used to support these statements are from Europe and US. I will encourage the authors to replace or complement these references with other studies conducted in Latin-America.Thank you for your comments. We appreciate your suggestions for improving this manuscript. We have replaced the previous references 7 to 9 with studies conducted entirely or partially in LAC region. We have included the following papers: \u25cf Emmerick ICM, Luiza VL, Camacho LAB, Vialle-Valentin C, Ross-Degnan D. Barriers in household access to medicines for chronic conditions in three Latin American countries. International journal for equity in health. 2015;14(1):115.\u25cf Rivera-Andrade A, Luna MA. Trends and heterogeneity of cardiovascular disease and risk factors across Latin American and Caribbean countries. Progress in cardiovascular diseases. 2014;57(3):276-85.\u25cf Nieblas-Bedolla E, Bream KD, Rollins A, Barg FK. Ongoing challenges in access to diabetes care among the indigenous population: perspectives of individuals living in rural Guatemala. International journal for equity in health. 2019;18(1):1-10.Beagley J, Guariguata L, Weil C, Motala AA. Global estimates of undiagnosed diabetes in adults. Diabetes research and clinical practice. 2014;103(2):150-160.Methods1. In the study selection subheading, it reads that studies were excluded if these did not focus \u201con patients with an established T2DM diagnosis\u201d. This needs further clarification. Does this mean that only patients with a history of diabetes (self-reported diagnosis) were included? In other words, reports that studied patients with diabetes (self-reported) and people newly diagnosed (blood tests as part of the study) were not included? In this line, how was diabetes history defined? Was this based on self-reported diagnosis only, a combination of self-reported or taking drugs for diabetes, or other means?We agree that the way it was originally worded left this issue unclear. We included studies that were carried out with patients with self-reported type 2 diabetes and/or those with laboratory tests that confirmed their diagnosis. We only excluded studies that included solely patients with other diagnoses, or with healthy individuals focussing on primary prevention of type 2 diabetes. Studies including a mixture of pathologies were included provided that some of the participants had type 2 diabetes, and the results provided the information on type 2 diabetes separately. We have modified the manuscript to make this clearer. This section now reads: \u201cWe selected studies that: (1) included individuals with laboratory confirmed or self-reported T2DM who lived in LAC countries (2) described facilitators or barriers related to successful management of the disease; (3) reported original data; (4) were written in English, Spanish, French or Portuguese. Studies that included numerous pathologies were included provided that some participants had T2DM and they their results were reported separately. We excluded studies that included solely patients with other diagnoses, or with healthy individuals focussing on primary prevention of T2DM.\u201dDiscussion1. The discussion begins signalling that diabetes is a complex issue and that contextual factors seem to be more relevant than individual profiles. These ideas would benefit from strong references. The findings of this work, although interesting, do not provide evidence to conclude that contextual factors are more important. If there were not any relevant references, these statements should be toned down. This concern would also apply to the first lines of the conclusions; statements appear to be too strong and not fully backed-up by the findings.In light of the comments of the reviewer we have toned down out statement in the conclusions section. Furthermore, we have added 2 references to support the observations made. Please see revised text: \u201cWe show here that diabetes management in LAC region is a complex issue. Contextual factors appeared to be highly relevant and in some cases individual factors appeared to be on a more secondary plane. The relative importance of contextual factors in diabetes self-management and in diabetic retinopathy screening has been identified in other studies .\u201d The references that were added are: \u25cf Schmidt-Busby J, Wiles J, Exeter D, Kenealy T. Understanding \u2018context\u2019 in the self-management of type 2 diabetes with comorbidities: A systematic review and realist evaluation. Diabetes research and clinical practice. 2018;142:321-34.\u25cf Piyasena MMPN, Murthy GVS, Yip JL, Gilbert C, Zuurmond M, Peto T, et al. Systematic review on barriers and enablers for access to diabetic retinopathy screening services in different income settings. PloS one. 2019;14(4). 2. The limitations of the study argued briefly that qualitative original reports rarely study representative samples. Further discussion on this matter -representative and extrapolation of the results- is warranted. Can the findings of the review really apply to all patients in LAC? Should there be any considerations when interpreting the results across countries ?The reviewer has made a valid point here. We have added the following text into the discussion/limitations section: \u201cHowever, we can suppose that the barriers and/or facilitators identified are potentially generalizable to similar sociocultural contexts with limited income and similar health coverage. By synthesising the results of 60 studies from the LAC region, this synthesis of the different types of issues affecting diabetes management can help broaden our understanding of the challenge ahead. The studies were mostly qualitative, and although this type of methodology rarely uses representative samples [100] it is clear that qualitatively synthesised information from the purposively selected participants in a specific setting can generate rich and useful information locally, but it may not be generalisable to the entire LAC region. For example, qualitative studies describing views and beliefs of specific indigenous groups. Despite this limitation, the review and synthesis of such studies can help shed light on the complexity of the issue in this region, and generate new lines of query for practitioners and researchers in similar contexts.\u201d We also included some text to acknowledge the bias towards research from certain countries being included: \u201cFurthermore, many of the studies included were from Brazil and Mexico, and other countries were under-represented. This is likely due differences with academic culture and publication of research findings in the different countries in the region.\u201d 3. The conclusion includes arguments about \u201ckey determinants for successful care\u201d. This seems quite a strong idea that has not been fully defined nor explored in the results. What is successful care? How is this defined? Does this relate to any care metric  or only to the patient\u2019s perspective?We agree that this concept is perhaps not well described in the initial version of the manuscript. Our definition of \u201csuccessful care\u201d depended highly on how it was considered in the manuscripts that were included in the systematic review. Given that many of the included studies were qualitative, the definition of successful care followed what was reported or considered to be successful care according to the perspective of the study participants, ie patient or health care providers etc. In the quantitative studies the idea of what is successful care, would be influenced by choice of outcome measurement and the barriers/facilitators highly dependent on which factors and/or characteristics the researchers who conducted each study chose to measure. In some cases the studies were focussed on a specific issue of diabetes care, such as adherence to medication, in which case care was considered to be successful when individuals were adherent . Similarly, some studies used indicators of glycemic/metabolic control, indicators of health service access, or questionnaires to determine whether the living and health conditions of the patients were good (indicated they were successfully managed) or poor (poorly managed). We have modified the description provided in the methods section to make this more clear. Please see the new description in the methods section: \u201cHow successful management of the disease was defined depended on the focus of the different studies included. For qualitative studies, it depended on the perceptions of the participants, and how they defined good or poor control of the disease. In quantitative reports the issue was more complex because it was highly influenced by the researcher who designed the data collection tools. In this case, successful care was considered according to measurements such as adherence to care protocols using predefined tools, questionnaires assessing quality of life, or indicators of health care access.\u201d Reviewer #2: This is a well written manuscript that aims to fill an important gap in the literature regarding barriers and facilitators in the management of T2DM in Latin American region. As stated by the authors, this region is experiencing a very large burden of T2DM and it is important to understand the challenges and opportunities uniques to the region.The study design, a systematic review is a great choice.Below are concrete comments for authors to consider that I think would improve the paper.Intro: the study is really about understanding barriers and facilitators of diabetes secondary and tertiary prevention; the authors should consider using these commonly used public health terms.Firstly, we would like to thank the reviewer for her keen observations. We have used the terms suggested to make the focus of our review clearer. Please see the modification in the introduction section. Inserted text: \u201cLike most regions in the world strengthening the primary care system for improved secondary and tertiary prevention of diabetes, by ensuring the disease and its complications are promptly detected and correctly treated, could reduce the consequences of diabetes in the population.\u201d Per PRISMA guidelines for the reporting of systematic reviews, introduction should include information about the study designs. Were these mostly qualitative studies or also quantitative studies.The reviewer makes a valid point here. This point has now been addressed. Please see the new text: \u201cIn this systematic review, we evaluate observational studies with qualitative and/or quantitative methodology to identify the barriers or facilitators to the management of T2DM in LAC countries, from the perspectives of patients, their relatives or caregivers, healthcare professionals, and/or any other stakeholders.\u201dMethods:Study selection: include information about the type of study.With respect to 2) The objective of the study was to identify or describe facilitators orbarriers related to the management of the disease.Did the authors rely on the presence of these terms in the stated objectives? If so, please elaborate on the justification for this decision.We included studies where the stated objective clearly indicated that they sought to identify barriers and/or facilitators of diabetes care. However, given that these terms are not always used, during the abstract review if it appeared that determinants of diabetes care were reported in the results section, two researchers independently reviewed the full text and assessed each paper for inclusion according to our inclusion and exclusion criteria. We have modified the text in the methods section to make this more clear. Please see text: \u201cIdentification of barriers and facilitators related to disease management was not always explicitly stated as the objective of the research, and the use of different terminology was common. During the abstract review, if it was apparent that barriers or facilitators to diabetes management were reported, we assessed the full text for inclusion.\u201dData extraction:With respect to 3) The facilitators and/or barriers related to the management of T2DM described in the study to improve diabetes care.This is key given the objective of the study and the statement is vague in light of the types of studies included. Specifically: 1) it is unclear to me whether for quantitative studies, the authors used the results of the studies to identify barriers and facilitators, in other words and for example, did the authors examined results to identify factors associated with a controlled A1c, vs. uncontrolled A1c, first ones were considered facilitators, others barriers. I suspect the authors encountered that type of studies (e.g. registries) and it is important to clarify how they handled/used these results under their own conceptual framework. In fact, some of the characteristics listed in number 2 of the data extraction can fall in the category of barriers (e.g. socioeconomic status). 2) Did they rely on the use of the terms \u201cbarriers and facilitators\u201d in the article text. If so, please elaborate on the justification for this decision and consider doing a validation study, examining a random 1-2% of the 65 studies excluded because not focused on \u201cbarriers/facilitators\u201dThis point was also raised by reviewer 1. In some cases the studies were focussed on a specific issue of diabetes care, such as adherence to medication, in which case care was considered to be successful when individuals were adherent . Similarly, some studies used indicators of glycemic/metabolic control, indicators of health service access, or questionnaires regarding the living and health conditions of the patients. In light of these comments, we have made significant changes in the wording of the methods section to clarify the definitions used and how information was managed. Please see text on lines 90-102: \u201cWe considered T2DM management to include strategies or care protocols for the control of the disease and its risk factors, as well as prevention of complications such as diabetic retinopathy, kidney or heart disease, and diabetic foot. How successful management of the disease was defined depended on the focus of the different studies included. For qualitative studies, it depended on the perceptions of the participants, and how they defined good or poor control of the disease. In quantitative reports the issue was more complex because it was highly influenced by the researcher who designed the data collection tools. In this case, successful care was considered according to measurements such as adherence to care protocols using predefined tools, questionnaires assessing quality of life, or indicators of health care access. We defined facilitator as any factor that supports the management of T2DM and barrier as any factor that limits the disease management. These factors may be socioeconomic, educational, cultural, behavioural, cognitive, structural, or logistical. The source of the data can be patients, patients\u2019 families or caregivers, healthcare professionals or any other stakeholder.\u201d Furthermore, we have added a clarification about how barriers and facilitators were defined: \u201cIdentification of barriers and facilitators related to disease management was not always explicitly stated as the objective of the research, and the use of different terminology was common. During the abstract review, if it was apparent that barriers or facilitators to diabetes management were reported, we assessed the full text for inclusion.\u201dDid the authors obtain information about the setting: urban, rural, clinic in the hospital, pharmacies, etc.. I think this information would be very valuable to understand the context.We agree that this point would be very interesting to include. We have integrated this question into the manuscript. Please see table 1, and the comments in the results section lines 205-207: \u201cMost of the studies were carried out in urban settings \u201d . Furthermore we have added this as a limitation in the discussion. See text on lines 606-608: \u201cThe majority of the studies were carried out urban setting so the challenges faced by individuals living in rural areas are likely to be underrepresented.\u201dIt is recommended to avoid using \u201cdiabetic\u201d to refer to patients with diabetes.Given. We have changed this term to \u201cpeople with diabetes\u201d throughout the manuscript.Results:In addition to having table 3 as is, I\u2019d recommend to have 2 similar tables subdivided by type of stakeholder with 2 main groups:1- Individual patient, caregiver, relative2- Health care professionals, health managers and other stakeholders.We agree with the reviewer that this would be a very interesting way to review the results. We have considered at length how to make these changes but concluded that with the study, the way it was originally designed, it is not possible to split the table into 2. The main reason is related to the inclusion of quantitative studies. It is difficult to identify who is really identifying the barrier or facilitator because the questionnaires used or the observations made is highly influenced by the researchers and what they consider may be a barrier etc. For example, if we present a table reflecting barriers identified by patients and their family members and include those identified in this manner (because the patients are the ones answering the questionnaire), we would not be properly the views of the patients, but rather the barriers identified by the observation. For this reason, we feel that only the qualitative reports truly reflect the perceptions of the participants. As suggested by the reviewer, we have prepared 2 tables according to the perspectives using the qualitative studies. At the moment they are included in the supplement but if the editor wishes we can move them to the main body of the article. Figure 1.For the 890 studies removed in early phase, please include the reasons for these exclusions. (as you do for the other exclusions). As it is suggested, we have included in Figure 1 the reasons for exclusion of the studies after screening title and/or abstract.6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: Yes: MARIANA LAZOhttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, Thank you for the suggestion. We have used it. 29 Jul 2020Barriers and facilitators to successful management of type 2 diabetes mellitus in Latin America and the Caribbean: A systematic reviewPONE-D-20-13651R1Dear Dr. Parker,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Cesar Ugarte-Gil, MD MSc PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 18 Aug 2020PONE-D-20-13651R1 Barriers and facilitators to successful management of type 2 diabetes mellitus in Latin America and the Caribbean: A systematic review Dear Dr. Parker:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Cesar Ugarte-Gil Academic EditorPLOS ONE"}
{"text": "ATP channels in the vasculature composed of Kir6.1 regulate vascular tone and may contribute to the pathogenesis of endotoxemia. We used mice with cell-specific deletion of Kir6.1 in smooth muscle (smKO) and endothelium (eKO) to investigate this question. We found that smKO mice had a significant survival disadvantage compared with their littermate controls when treated with a sub-lethal dose of lipopolysaccharide (LPS). All cohorts of mice became hypotensive following bacterial LPS administration; however, mean arterial pressure in WT mice recovered to normal levels, whereas smKO struggled to overcome LPS-induced hypotension. In vivo and ex vivo investigations revealed pronounced cardiac dysfunction in LPS-treated smKO, but not in eKO mice. Similar results were observed in a cecal slurry injection model. Metabolomic profiling of hearts revealed significantly reduced levels of metabolites involved in redox/energetics, TCA cycle, lipid/fatty acid and amino acid metabolism. Vascular smooth muscle-localised KATP channels have a critical role in the response to systemic infection by normalising cardiac function and haemodynamics through metabolic homeostasis.KATP channels are more susceptible to death from infection.\u2022 Mice lacking vascular KATP channels depresses cardiac function during infection.\u2022 Absence of smooth muscle K\u2022 Cardiac dysfunction is accompanied by profound changes in cellular metabolites.ATP channels in response to systemic infection.\u2022 Findings from this study suggest a protective role for vascular KThe online version of this article (10.1007/s00109-020-01946-3) contains supplementary material, which is available to authorized users. ATP channels in endotoxemia is unclear. Initially, KATP channel hyperactivity was thought to contribute to the profound hypotension seen in patients with endotoxemia. KATP channels are K+ selective ion channels that respond to and are regulated by changes in the metabolic status (changes in the ATP/ADP ratio) of the cell. They are expressed in many tissues and play a critical role in coupling cellular metabolism to membrane excitability. Functional KATP channels are composed of a hetero-octomeric complex of 4 pore-forming subunits (Kir6.1 or Kir6.2) and 4 SURs  [ATP channels regulate vessel tone and therefore blood pressure by opening or closing in response to vasoactive substances such as adenosine or noradrenaline and/or metabolic stress such as ischaemia. Opening of the channel leads to membrane hyperpolarisation, closure of voltage-dependent Ca2+ channels (VDCC) and subsequent relaxation [Endotoxemia is a component of the pathophysiology of gram-negative sepsis though the relative clinical importance is contentious . The admr SUR2B) , 4. In Vlaxation , 5.ATP blocker glibenclamide reversed an LPS-induced drop in arterial blood pressure, support the hypothesis that hypotension may be due to increased KATP channel activity [ATP may in fact be protective during infection. For example, global deletion of Kir6.1 in mice results in a poor survival outcome following LPS exposure [ATP [ATP channels against infection is preserved across species, for example in insects where suppression and activation of KATP is detrimental and protective, respectively [ATP channels in endotoxemia-induced myocardial metabolic changes are lacking.Studies in animal models of endotoxic shock, where the Kactivity \u20138. Furthactivity , 9. Howeexposure . Furtherure [ATP , 11. Intectively . Endotoxectively . StudiesATP channels can modulate immune cell function [ATP channel in endotoxemia is scarce. In this study, we have investigated the role of vascular  KATP channels in response to endotoxic shock using cell-specific and global Kir6.1 KO mice. We delineate the role of VSM KATP channels in preserving cardiac metabolism during endotoxin-induced shock.Though the studies in the global knockout mice are revealing, their interpretation is complicated by a number of factors. Kir6.1 is known to be ubiquitously expressed in many cells and tissues in addition to VSM. Evidence from mice with conditional deletion of Kir6.1, in endothelium and cardiac tissues, reveals important physiological functions complicating the understanding of pathophysiological data from the global knockout mouse , 15. In function . In geneAll experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the British Home Office regulations, in accordance with the EU Directive 2010/63/EU, and by the US National Institutes of Health .We used 2 models of endotoxemia/sepsis\u2014administration of lipopolysaccharide  , 18 and In vivo cardiac function and morphology were assessed by M-mode echocardiography conducted under anaesthesia . Echocardiography was performed using a VisualSonics Vevo 770 or 3100 imaging system . Short-axis M-mode images were acquired for analysis to provide heart chamber dimensions and calculate percentage fractional shortening.1H-NMR metabolomics and data analysis can be found in the Details of the methods for BP telemetry, mouse generation and genotyping, quantification of renal dysfunction and liver damage, isolated heart experiments, TUNEL assay, P\u2009<\u20090.05 compared WT) and, globally, gKO  were more predisposed to LPS-induced mortality . Global KO mice started to die within 3\u00a0h of LPS administration with 60% of deaths occurring by 18\u00a0h and less than 10% surviving at 48\u00a0h. In the smKO cohort, mice had an improved long-term survival outcome compared with gKO but worse than WT mice with the majority of deaths occurring after 24\u00a0h. These data suggest an important role for vascular KATP channels in the response to endotoxic shock. There were no deaths of untreated mice over the same time period (data not shown).In response to a sub-lethal dose of LPS, all genotypes of mice showed signs of endotoxemia including decreased activity, piloerection and periocular discharge. WT mice were more tolerant to LPS than Kir6.1 KO mice Fig.\u00a0. The majATP channels in the recovery from hypotension during endotoxemia. There was also a significant drop in heart rate (HR) in all 3 cohorts . Left ventricular end-diastolic volume (LVEDV) and left ventricular internal dimensions (LVID) were also significantly increased . In addition, fractional shortening (FS) and fractional area change (FAC) were also compromised suggesting progression towards LV failure in smKO mice . To ascertain if cardiac dysfunction was as a result of cell apoptosis, we used the TUNEL assay on heart sections from WT and smKO LPS-treated mice  in smKO mouse hearts . Furthermore, LV developed potential (peak systolic pressure\u2013end-diastolic pressure) (LVDP) was significantly reduced in smKO hearts (P\u2009<\u20090.001). These data show a significantly reduced LV compliance in LPS-treated smKO hearts. Furthermore, in smKO hearts, the pressure-volume relationships were uncoupled with increasing pre-load of the LV. The LV of smKO hearts had reduced compliance with increased volumes as reflected in the significantly enhanced LVEDP and this occurred together with a minimal change in LVDP (P\u2009<\u20090.001). Overall, these data indicate increased LV stiffness leading to reduced compliance and impaired developed pressure at a given LV volume.We investigated LV function further using the Langendorff isolated heart method Fig.\u00a0. IsolateP\u2009<\u20090.01, Fig. If left untreated, endotoxic shock can lead to multi-organ dysfunction. We investigated possible multi-organ dysfunction by analysing blood markers of kidney dysfunction and liver damage 18\u00a0h post-LPS. There was no difference in the levels of the kidney damage markers, urea and creatinine in smKO mice and their littermates Fig. . Liver fATP channels are also expressed in vascular endothelium [P\u2009=\u20090.35, Fig.\u00a0ATP channels in endothelium make little or no contribution to the cardiac dysfunction in endotoxemia.The phenotype of gKO mice following LPS treatment was comparatively more severe than in smKO mice. Kir6.1-containing Kothelium . To inve1H NMR spectroscopy ."}
{"text": "Characterizing the future risks of climate change is a key goal of climate impacts analysis. Temperature binning provides a framework for analyzing sector-specific impacts by degree of warming as an alternative or complement to traditional scenario-based approaches in order to improve communication of results, comparability between studies, and flexibility to facilitate scenario analysis. In this study, we estimate damages for nine climate impact sectors within the contiguous United States (US) using downscaled climate projections from six global climate models, at integer degrees of US national warming. Each sector is analyzed based on socioeconomic conditions for both the beginning and the end of the century. The potential for adaptive measures to decrease damages is also demonstrated for select sectors; differences in damages across adaptation response scenarios within some sectors can be as much as an order of magnitude. Estimated national damages from these sectors based on a reactive adaptation assumption and 2010 socioeconomic conditions range from $600 million annually per degree of national warming for winter recreation to $8 billion annually per degree of national warming for labor impacts. Results are also estimated per degree of global temperature change and for 2090 socioeconomic conditions. Emissions or concentrations from these scenarios are used as inputs to climate models with the goal of producing comparable results. However, when using climate model output to drive impacts analyses, the differences in climate sensitivity between different models can have a dominant effect, obscuring the role of other structural differences between the models  . An addi2-equivalent concentrations to warming.\u201d In the case of Arctic sea ice, the NRC assessment showed that in some cases, there is value to presenting hazards and impacts on an absolute temperature scale, rather than a degree-change scale. The IPCC 1.5 degrees assessment presented a comparison of impacts at 2\u00b0 and 1.5\u00b0 in order to inform global temperature targets  \u201cClimate Stabilization Targets\u201d assessment  presente targets . The IPC targets . Pattern targets . Wobus e targets . Hsiang Temperature binning aids comparability of independent analyses by producing an estimate of damages for a given amount of warming, without consideration of when that warming occurred or which scenario or climate model was used to develop the estimate. This approach generally reduces the spread of results when showing uncertainty ranges by eliminating the contributions of global climate sensitivities or transient climate responses to variations in estimates of sectoral impact . In a waIn this study, we apply a temperature binning approach to nine US sectors. This approach combines several key features of previous studies: providing quantified, monetized damages in a consistent fashion for a number of different impact sectors as in 2Monetized damages resulting from warming in the US at integer national temperatures from 0 to 6 \u00b0C relative to 1986\u20132005 were calculated for nine sectors  and six downscaled GCMs \u2014see the \u201cThose climate impact sectors where damages are most closely related to national annual average temperatures will generally have the least dispersion between GCMs, as the bins are defined based on that metric (such as winter recreation and electricity demand and supply). GCMs can vary in terms of temporal and regional variability of temperature even for the same national annual average temperature. While precipitation patterns can differ more between models, these changes are often still proportional to large-scale temperature changes. This variation among GCMs can drive differences between the resulting relationship between temperature and damages. The agreement across GCMs in climate damages for a given temperature varies by sector\u2014for winter recreation, there is little difference from one GCM to the next (the standard error is less than 2% of the slope of the temperature/damage relationship) . With thThe results presented focus on the final, monetized outputs for each sector: however, for a number of these sectors, impacts can also be reported in their native physical impact units, such as deaths for the health-related sectors, percent change in hours worked for labor, or number of skiing visits for winter recreation. For some purposes, these native units may facilitate better communication of the results.To illustrate some of the differences between temperature binning and the traditional scenario-based representations of damages, Another option for producing damage functions for climate impact sectors is to use an ensemble of opportunity e.g., . However3The results shown in the previous section can serve as the backbone for several objectives. The first is communication. The graphs can stand alone in order to communicate the relationship between US national temperatures and climate damages expected in various sectors. Alternatively, collating results at each degree point can inform a \u201crisk by degrees\u201d communication effort: for example, looking at the difference between 2 and 3\u00b0 of warming  in terms of damages on the US, which can also be related to temperature targets. The second objective is to develop damage functions for generating reduced form models. The linear fits from Timing is an important part of climate impacts analysis. The temperature binning approach develops functions by initially removing the time element. This time element can then be reintroduced in at least two ways. The first is to provide a mapping function showing the probability of temperature exceedance for key time periods for each scenario of interest . These more complex approaches would be useful for reduced complexity modeling and benefits analysis, but might not be as amenable for general communication purposes.Sectors where the impacts are a function of cumulative exposure will be more challenging to represent in a temperature binning context. For example, sea level rise is a function of the integration of heat absorption by the ocean and melting of land ice and so is a more complex function of temperature over time than impacts such as heat mortality. Similarly, carbon storage in managed forests would be a difficult sector to model in this fashion, both because of the integrative nature of storage and the dependence of the rate on CO2 fertilization, or ozone resulting from methane oxidation in the atmosphere. Impacts that are sensitive to non-GHG factors, such as aerosol emissions or land-use changes, will also be challenging to emulate. Inter-sectoral interactions (such as the land-water-energy nexus) and cascading risks would also be difficult to capture in this framework. Some of these challenges are surmountable\u2014for example, 2 concentration as a complement to the temperature-based reasons for concern\u2014but require more complexity in approach.This approach does not capture impacts that are a function of rate of change rather than absolute change , nor does it capture impacts that are a direct function of greenhouse gas concentrations, such as ocean acidification, COSix GCMs are used in this study . For tho4The framework described in this manuscript builds on approaches demonstrated in numerous previous studies in order to produce quantified, monetized damage estimates for nine different impact sectors across a range of temperatures for two different socioeconomic conditions. The temperature binning approach has several advantages over scenario-driven approaches: improved comparability due to standardizing results by temperature; more accessible communication by moving away from the ever-changing alphabet soup of climate models and scenarios; and increased flexibility of scenario analysis through the development of reduced-form tools. The strong relationship between increased temperatures in the US and monetized damages is also demonstrated by the analyses of the nine sectors analyzed here. While the authors of this manuscript will continue to add new sectors and improve the analysis of existing sectors, this approach would be greatly strengthened by more consistent adoption of similar approaches by the wider impacts modeling community. If future impacts papers were to consider presenting their damage estimates as a function of temperature, whether as the central thrust of the paper or in the 5At its core, temperature binning relies on calculating sectoral impacts for multiple future temperatures while using constant socioeconomic parameters. In this manuscript, we maximize consistency by using a standard set of six climate models , one downscaling approach applied to the contiguous US , this analysis used climate projections from the Geophysical Fluid Dynamics Laboratory coupled General Circulation model (GFDL_CM3) in addition to the five GCMs used in CIRA2.0 . The original five GCMs were chosen based on criteria described in All but one of the sectoral impact analyses required downscaled climate data. The temperature binning approach presented here relies primarily on the LOCA  approach to produce daily temperature (maximum and minimum) and precipitation data at a 1/16 degree scale (approximately 6.25 km). The one exception is the sectoral analysis from the coastal property model which requires sea level rise projections produced by a separate method, described in the method for that sector below.For this manuscript, the decision was made to select time slices based on average warming in the continental US compared to the baseline (1986\u20132005) by integer degrees, where the first 11-year period to have an average temperature equal to that of the warming degree was chosen. A subset of nine of the sectoral impact models from CIRA2.0 were used for the temperature binning analysis. These sectors were chosen based on large magnitudes of the monetized damages, high visibility or common interest, and/or amenability to the temperature binning analysis method. The nine sectors chosen were extreme temperature mortality, labor impacts, road infrastructure, electricity demand and supply, rail infrastructure, coastal property impacts, electricity infrastructure, Southwest dust health effects, and winter recreation.Climate projections from each of the temperature bins  realized after a delay. In these cases, it is possible that the trajectory of estimated adaptation costs may not align temporally with an 11-year temperature bin. To improve the alignment, we perform a \u201cfinancial smoothing\u201d of capital costs, essentially annualizing capital costs over the useful life of the adaptation investment, using a discount rate of 3%. Details of the financial smoothing are provided in the To estimate global mean temperature from the six sea level rise projections  used in Five of the sectoral models have the capability of modelling future impacts with and without adaptation . ProjectBecause damages in the temperature binning approach are not time dependent, two methods are used to isolate damages from socioeconomic drivers. Five sectors were run with static socioeconomic inputs, relying on 2010 and 2090 population and GDP projections. The other four sectors were run with a static assumption\u2014that is, constant socioeconomic inputs for the entire time series\u2014as well as a dynamic assumption, where socioeconomic inputs change across the century in a continuous fashion. The difference in damage outputs between the two runs can be used as an indicator of the impact of socioeconomic drivers. These two methods allow for exploration of the potential effect of socioeconomic changes for any given temperature. Socioeconomic projections are drawn from EPA\u2019s Integrated Climate and Land Use (ICLUSv2.0) model for population, and MIT\u2019s Emissions Prediction and Policy Analysis (EPPAv5) model for GDP, as described in more detail in 1As noted above, impact damages are scaled to average national temperatures rather than average global temperatures. This relationship can be used to build a direct computational framework. Such a framework might use reduced complexity models, such as FaIR, MAGICC, or Hector , to prodFor any impact model that resolves the US into smaller sub-national regions , the temBinningSI"}
{"text": "Soil erosion can affect the distribution of soil nutrients, which restricts soil productivity. However, it is still a challenge to understand the response of soil nutrients to erosion under different soil types.2O3, CaO, Fe2O3, K2O, Na2O, MgO, TiO2, and SiO2), were analyzed in the profiles from yellow soils, red soils, and lateritic red soils in an erosion region of Southeast China. Soil erodibility K factor calculated on the Erosion Productivity Impact Calculator (EPIC) model was used to indicate erosion risk of surface soils (0\u223c30 cm depth). The relationships between these soil properties were explored by Spearman\u2019s rank correlation analysis, further to determine the factors that affected the distribution of SOC, SON, and soil major elements under different soil types.The distribution of soil nutrients, including soil organic carbon (SOC), soil organic nitrogen (SON), and soil major elements (expressed as Al2O3 and SiO2, in the yellow soils, were significantly larger than those in the red soils and lateritic red soils. Moreover, the concentrations of major metal elements positively correlated with silt proportions and SiO2 concentrations positively correlated with sand proportions at the 0\u223c80 cm depth in the yellow soils. Soil major elements depended on both soil evolution and soil erosion in the surface layer of yellow soils. In the yellow soils below the 80 cm depth, soil pH positively correlated with K2O, Na2O, and CaO concentrations, while negatively correlated with Fe2O3 concentrations, which was controlled by the processes of soil evolution. The concentrations of soil major elements did not significantly correlate with soil pH or particle distribution in the red soils and lateritic red soils, likely associated with intricate factors.The K factors in the red soils were significantly lower than those in the yellow soils and significantly higher than those in the lateritic red soils. The SON concentrations in the deep layer of the yellow soils were twice larger than those in the red soils and lateritic red soils, while the SOC concentrations between them were not significantly different. The concentrations of most major elements, except AlThese results suggest that soil nutrients and soil erodibility K factor in the yellow soils were higher than those in the lateritic red soils and red soils. The distribution of soil nutrients is controlled by soil erosion and soil evolution in the erosion region of Southeast China. We hypothesized that both soil erosion and soil evolution control the distribution of soil nutrients in the erosion region of Southeast China. This study is closely associated with the conservation of soil nutrients, which is critical for ecological stability . Therefo2  is located in Fujian Province, with a drainage area of 14,741 km2 . The ave2 . The ori2 . Over 902 . These s2 .Soil samples were collected in the winter season of January 2018. The three soil sites were selected according to different soil types, including red soils, lateritic red soils, and yellow soils, respectively . The soi4, and three mL HNO3 at 120 \u00b0C for 3 days  in the soils  were removed by treating with 0.5 mol\u00a0Lfor 24 h , and inofor 24 h . The tret acre h/100 acre/ft/tanf/in. For brevity, the unit of the K factor will not be noted.Soil erodibility can be used to indicate the erosion risk of surface soils because of the significant quantitative relationship between the amount of soil loss and the K factor . Many mo2O3, CaO, Fe2O3, K2O, Na2O, MgO, TiO2, and SiO2), and the K factor values in the red soils, lateritic red soils, and yellow soils. One-way ANOVA with the least significant difference (LSD) test was performed to determine the differences in the proportions of different-sized particles, soil pH values, the concentrations of SOC, SON, and soil major elements, and the K factor values among the three soil types at the level of P <\u00a00.05. All data were calculated via the Kolmogorov\u2013Smirnov test, which is one of the non-parametric tests commonly applied to analyze the normal distribution of the sample data set  in the upper layer. It is can be inferred that the re-assimilation of 15N-depleted inorganic N through microbial immobilization into microbial biomass  than those in the red soils and lateritic red soils . The larN values . The C/NN values , but it N values . The C/N biomass , resultiep soils .+, K+, Ca2+, and Mg2+ are preferentially dissolved and lost under strong leaching conditions, while the iron and aluminum, these have a low mobilization, are gradually accumulated with soil evolution . In the deep soils (below 80 cm depth), the SON concentrations in the yellow soils were twice higher than those in the red soils and lateritic red soils, which was mainly attributed to the re-assimilation of inorganic N through microbial immobilization. The concentrations of soil major elements in the three soil types were almost lower than the background values of major elements in the mean soils of Fujian Province. Moreover, the concentrations of most major elements in the yellows were significantly larger than those in the red soils and lateritic red soils. The variation of soil major elements in the surface layer of the yellow soils was affected by both soil erosion and soil evolution, while it in the deep soils was mainly affected by soil evolution. These results suggest that yellow soils have a higher level in soil nutrients and soil erodibility compared to the red soils and lateritic red soils under the erosion region of Southeast China.10.7717/peerj.11630/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11630/supp-2Supplemental Information 2t acre h/100 acre/ft/tanf/in. YS, yellow soil; LRS, lateritic red soil; RS, red soil.The unit of the K factor is Click here for additional data file."}
{"text": "Mycobacterium ulcerans, is a devastating necrotizing skin disease. Key to its pathogenesis is mycolactone, the exotoxin virulence factor that is both immunosuppressive and cytotoxic. The discovery that the essential Sec61 translocon is the major cellular target of mycolactone explains much of the disease pathology, including the immune blockade. Sec61 inhibition leads to a loss in production of nearly all cytokines from monocytes, macrophages, dendritic cells and T cells, as well as antigen presentation pathway proteins and costimulatory molecules. However, there has long been evidence that the immune system is not completely incapable of responding to M. ulcerans infection. In particular, IL-1\u03b2 was recently shown to be present in BU lesions, and to be induced from M. ulcerans-exposed macrophages in a mycolactone-dependent manner. This has important implications for our understanding of BU, showing that mycolactone can act as the \u201csecond signal\u201d for IL-1\u03b2 production without inhibiting the pathways of unconventional secretion it uses for cellular release. In this Perspective article, we validate and discuss this recent advance, which is entirely in-line with our understanding of mycolactone\u2019s inhibition of the Sec61 translocon. However, we also show that the IL-1 receptor, which uses the conventional secretory pathway, is sensitive to mycolactone blockade at Sec61. Hence, a more complete understanding of the mechanisms regulating IL-1\u03b2 function in skin tissue, including the transient intra-macrophage stage of M. ulcerans infection, is urgently needed to uncover the double-edged sword of IL-1\u03b2 in BU pathogenesis, treatment and wound healing.Buruli ulcer (BU), caused by  Mycobacterium ulcerans. It typically presents as painless, ulcerative skin lesions or as pre-ulcerative nodules, plaques and in oedematous forms (Buruli ulcer (BU) is a neglected tropical disease resulting from subcutaneous infection by us forms . This enus forms , 3. The us forms . Untreatus forms . More seus forms .M. ulcerans  by the Sec61 translocon  Figure, 38, 39,M. ulcerans infections , and iNOS, the inducible nitric oxide synthase that generates bactericidal NO in macrophages, both increase susceptibility to infection. Interestingly, SNPs in three genes involved in the autophagy pathway also affect BU. Two polymorphisms have been identified in the E3 ligase PARK2 which increase susceptibility to BU while one in NOD2 increases the risk of severe disease   . This lad lipids .M. ulcerans, or mycolactone-containing EV exposure, it has been demonstrated that priming of macrophages is probably via activation of TLR2, since genetic knockout of this receptor ablated the response  (We now confirm that mycolactone induces IL-1\u03b2 release by macrophages  mycolacttis (RA) , 71\u201376, tis (RA) ]. ConseqOnce produced, several mechanisms for secretion of IL-1\u03b2 have been proposed. All of these are independent of the canonical secretory pathway and Sec61-dependent translocation of proteins into the ER, and are known as unconventional secretory pathways  Figure. Recent Hence, the recent discovery of IL-1\u03b2 in BU lesions, and its induction by mycolactone in macrophages is entirely in line with our current knowledge of the cell biology and biochemistry of Sec61 inhibition by mycolactone.M. ulcerans infection, by enhancing the activation state of surrounding cells, including innate immune macrophages and neutrophils. In the mouse footpad model of M. ulcerans infection, treatment with the non-specific steroid anti-inflammatory dexamethasone reduced footpad swelling  and down-regulating anticoagulant proteins such as thrombomodulin and the EPCR. IL-1\u03b2 also induces procoagulant PAI-1, enhances tissue factor expression, and promotes angiogenesis via upregulation of adhesion molecule and vascular endothelial growth factor (VEGF) expression . This work was also supported by a grant from the Kennedy Institute Trustees.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": "CD40 is a costimulatory molecule that is key for the activation of antigen-presenting cells and other innate immune cells. It plays an important role in anti-tumor immunity, and agonists of CD40 have been shown to eliminate tumors in both pre-clinical and clinical settings, alone and in combination with other treatment modalities. Here we assess the expression of CD40 and associations with other mediators of immunity in a variety of tumor types and review the potential of CD40 agonists for cancer treatment, given the promise of enhancing the interplay between innate and adaptive immunity.CD40 is expressed on a variety of antigen-presenting cells. Stimulation of CD40 results in inflammation by upregulation of other costimulatory molecules, increased antigen presentation, maturation (licensing) of dendritic cells, and activation of CD8+ T cells. Here we analyzed gene expression data from The Cancer Genome Atlas in melanoma, renal cell carcinoma, and pancreatic adenocarcinoma and found correlations between CD40 and several genes involved in antigen presentation and T cell function, supporting further exploration of CD40 agonists to treat cancer. Agonist CD40 antibodies have induced anti-tumor effects in several tumor models and the effect has been more pronounced when used in combination with other treatments . The reduction in tumor growth and ability to reprogram the tumor microenvironment in preclinical models lays the foundation for clinical development of agonistic CD40 antibodies  that are currently being evaluated in early phase clinical trials. In this article, we focus on CD40 expression and immunity in cancer, agonistic human CD40 antibodies, and their pre-clinical and clinical development. With the broad pro-inflammatory effects of CD40 and its ligand on dendritic cells and macrophages, and downstream B and T cell activation, agonists of this pathway may enhance the anti-tumor activity of other systemic therapies. In the past decade, oncologic care has changed dramatically as immunotherapies have been developed for multiple tumor types. Immune checkpoint inhibitors targeting the programmed cell death protein 1(PD-1)/programmed cell death ligand 1 (PD-L1) pathway have significantly increased survival in randomized trials. Although durable responses are observed, most patients are inherently resistant or develop resistance to checkpoint inhibition over time . CombinaCD40 is constitutively expressed as a transmembrane receptor on DCs, myeloid cells, and B cells, as well as several types of non-hematopoietic cells, such as endothelial cells, fibroblasts, epithelial cells, and certain types of malignant cells ,17. The Engagement of CD40 by CD40L promotes the necessary clustering of CD40 receptors on the cell surface that initiates downstream signaling by one of two mechanisms: (a) activated CD40 binds Janus kinase 3 (JAK3) that in turn activates signal transducer and activator of transcription 5 (STAT5)-mediated transcription or (b) activated CD40 engages TNF receptor-associated factors (TRAFs) that can initiate nuclear factor-kappa B (NF-\u03baB), mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways in immune cells. The specific downstream signaling resulting from CD40 activation is cell-type dependent ,15,17,21JAK-STAT pathway signaling is involved in cytokine-mediated signal transduction. CD40-CD40L binding on monocytes recruits JAK3 that phosphorylates and activates STAT5 to initiate transcription of target genes . JAK3-STInduction of NF-\u03baB signaling pathway by binding of CD40 to CD40L induces activation of NF-\u03baB inducing kinase (NIK). NIK activates I\u03baB kinase \u03b1 (IKK\u03b1), and IKKa in turn phosphorylates p100 to produce p52. P52 associates with relB and translocate to the nucleus to induce gene expression . NF-\u03baB sMAPK signaling plays a role in the initiation of innate immunity in various cells to activate adaptive immunity (. p38) MASurvival of DC is mainly mediated by the PI3K pathway . On monoThe downstream signaling effects of CD40/CD4L are therefore diverse and differ between cell types.CD40 activation on DCs and monocytes upregulates the expression of other costimulatory molecules such as CD80 and CD86. Their effects on MHC molecules enhance antigen presentation, license DCs, and activate CD8+ T cells ,34,35,36CD40/CD40L binding, therefore, activates multiple innate immune cells, including DCs, macrophages, NK cells, and B cells, resulting in CD8+ and CD4+ T cell stimulation. Activation of cytotoxic and effector T cells leads to cell kill and apoptosis. n = 20,501) in samples from 534 clear cell renal cell carcinomas (ccRCC), 456 cutaneous melanomas, and 178 pancreatic adenocarcinomas, selecting tumor types that tend to be responsive to immunotherapy (melanoma and ccRCC) versus pancreatic adenocarcinoma which is resistant. Applying a cutoff Spearman correlation rho of \u2265 0.3 and an adjusted p-value of <0.001, we found that 198 genes in ccRCC, 361 genes in melanoma, and 935 genes in pancreatic adenocarcinoma were correlated with CD40 gene expression. The 50 genes with the highest correlation to CD40 in each cancer type are listed in The important role that CD40 signaling plays in anti-cancer immunity prompted us to explore genes that may be included as potential co-targets with a CD40 agonist-based therapy. We used RNA sequencing data from The Cancer Genome Atlas (TCGA) database to analyze the correlation of CD40 expression and that of other protein-coding genes  is a receptor for the cytokine IL-27. Although the role of IL-27RA in cancer is unknown, preclinical studies have shown that IL-27 inhibits tumor cell growth ,46. Tripn = 82) and include several C-X-C chemokine receptors including CXCR3, which is the ligand for CXCL10, as well as CXCR4 and CXCR6. We found several genes that are involved in the cytotoxic activity of T cells and NK cells including granzymes GZMM, GZMA, GZMH, and natural killer cell granule protein 7 (NKG7), that were similarly correlated with CD40 expression. NKG7 is a cytolytic-related protein expressed in NK cells and T cells, preferentially those polarized to Th2 direction ..106].Studies of the various CD40 agonistic antibodies remain in the early phase, and randomized data are not available yet. The activity has been seen as monotherapy, or in combination with other drugs, but optimal combinations and disease settings have yet to be established.Pharmacological activation of CD40 by antibodies activates signaling cascades in a variety of immune cells, particularly dendritic cells, B cells, and macrophages, resulting in increased inflammation and activation of cytotoxic T cells. Co-stimulation of innate and adaptive immunity might result in enhanced anti-tumor activity, and CD40 agonistic antibodies have therefore been the subject of intense preclinical and clinical research. Pre-clinical models assessing CD40 agonists alone or in combination with radiation, chemotherapy, immune-therapy, or angiogenic therapy have largely demonstrated superior activity to either modality alone. Additional pre-clinical studies are needed to further determine the mechanisms of action of these drug combinations and refine the dosing and sequencing of drugs and regimens. Early phase clinical trials of CD40 agonists in a variety of tumor settings have demonstrated activity, and additional clinical studies are needed to determine whether CD40 agonists can be added to the expanding repertoire of cancer drugs to either increase response (and preferably cure rates) in the frontline setting or to treat patients whose tumors have progressed on other frontline regimens.The importance of signaling of CD40/CD40L has been demonstrated in a variety of tumor models, and activation of CD40 affects multiple innate immune cells, providing the promise of harnessing CD40 as a therapeutic target in cancer. Early phase clinical trials have demonstrated that CD40 agonistic antibodies are generally safe, when administered alone and in combination with other anti-neoplastic modalities. Further studies are warranted to extensively evaluate CD40 agonists in cancer patients."}
{"text": "Nanoparticles generated during laser material processing are often seen as annoying side products, yet they might find useful application upon proper collection. We present a parametric study to identify the dominant factors in nanoparticle removal and collection with the goal of establishing an in situ removal method during femtosecond laser machining. Several target materials of different electrical resistivity, such as Cu, Ti, and Si were laser machined at a relatively high laser fluence. Machining was performed under three different charge conditions, i.e., machining without an externally applied charge ) was compared to machining with a floating potential and with an applied field. Thereby, we investigated the influence of three different charge conditions on the behavior of laser-generated nanoparticles, in particular considering plume deflection, nanoparticle accumulation on a collector plate and their redeposition onto the target. We found that both strategies, machining under a floating potential or under an applied field, were effective for collecting laser-generated nanoparticles. The applied field condition led to the strongest confinement of the nanoparticle plume and tightest resulting nanoparticle collection pattern. Raster-scanning direction was found to influence the nanoparticle collection pattern and ablation depth. However, the laser-processed target surface remained unaffected by the chosen nanoparticle collection strategy. We conclude that machining under a floating potential or an applied field is a promising setup for removing and collecting nanoparticles during the machining process, and thus provides an outlook to circular waste-free laser process design. Over the past decade, ultrafast laser ablation has emerged as a simple method for the direct generation of nanoparticles. The mechanism of femtosecond laser-assisted material removal is complex and has already been explored in detail ,2,3. ManThe effectiveness of copper as a broad antimicrobial  agent has been known since ancient times . RecentlNow, considering that nanoparticles are an essential resource for the above-mentioned applications but constitute a poorly managed waste stream in laser surface machining, the paradigm of a circular economy comes to mind . This leZheng et al., introduced a new technique to reduce the amount of Si nanoparticle redeposition onto a femtosecond laser machined target by using an external electric field . In thisIn this report, we build upon Zheng et al.\u2019s study and investigate nanoparticle collection and removal efficiency during femtosecond laser machining by applying either a high strength electric field or a varying electric field between the collection plate and target. While the collection of Cu nanoparticles is of primary interest, we compare two more target materials of different electrical resistivity. The effect of the polarity of the applied electric charge and the laser scanning direction on nanoparticle collection efficiency is also considered. The comparison and assessments are carried out on the basis of photography of the collection plate upon machining, high-speed videography of the nanoparticle plume deflection, scanning electron microscopy (SEM) and confocal microscopy of the resulting target surface topography.3 \u2126\u00b7m). Unpolished copper collection plates  with dimensions of 50 mm \u00d7 10 mm were used to collect the generated nanoparticles. All the target samples and the collection metals were ultrasonically cleaned in acetone for 5 min before laser experimentation. The scanning electron microscope images of each of the target material\u2019s surface and the collection plate surface are shown in Two metals and a semiconductor target were used in this study to generate laser-ablated nanoparticles: copper , titanium  and silicon . The target materials were selected according to their electrical resistivity ranging from low  to intermediate  and to high  of 1 kHz. The laser energy per pulse (E) was reduced to a desired lasing energy using a computer-controlled attenuator composed of a polarizing beam splitter and half-wave plate. The horizontally polarized Gaussian beam (TEM00) was then focused down to 40 \u00b5m spot diameter (thF) of the respective target materials which were raster scanned under the stationary beam. The ablation threshold of each material was experimentally determined by plotting the square of the laser-irradiated line width as a function of the logarithm of the incident laser fluence [2, respectively.Samples were machined in air using an amplified Ti: Sapphire solid-state laser system  operating at a central wavelength of 800 nm, a pulse duration of ~100 fs and a repetition rate , which is schematically represented in The nanoparticle plume was filmed using a high-speed camera  equipped with an 18\u2013108 mm macro zoom lens . The camera was temporally synchronized with the femtosecond laser system. The videos were gamma corrected, the region of interest was fixed, and each frame was converted into a .tiff image using PFV software . These processed images were then fed into a MATLAB code. An intensity threshold was set to distinguish between bright (plume) and dark (background) pixels. Upon identification of the bright pixels and their center points, the plume deflection angle of each frame was determined by fitting the center of bright pixels into a first order polynomial function.The machined surfaces were imaged with a scanning electron microscope (SEM)  both immediately after laser micromachining and once again after having been ultrasonically cleaned in acetone for 10 min. 3D confocal microscopy  was carried out to determine the ablation depth on the target. The depth of all the machined surfaces relative to the pristine surface were measured using the LEXT software , as shown in All the results presented in the following stem from experiments carried out with parallel laser scanning direction. From Finally, we note that collection seems to be directly correlated with the materials\u2019 electrical properties , which are a signature of the number of free electrons present on the material: the seemingly densest nanoparticle packing and maximum collection spread were observed for copper, which has the lowest electrical resistivity among the studied materials, whereas the smallest collection spread with seemingly looser packing was exhibited by Si, which has the highest electrical resistivity. We suggest that the appearance of the nanoparticle packing density and collection spread provide an indication of the absolute number of nanoparticles collected. A study by Irimiciuc et al., showed that an increase in electrical conductivity/decrease in resistivity on metals led to a release of more charge volume and a more intense electrostatic field, which again results in a greater ablation . The higWe acknowledge that photographs merely provide a 2D presentation of a 3D phenomenon, and thus only allow for qualitative comparisons. Ideally, quantitative measures substantiated these conclusions drawn from the \u201clook\u201d of the photographs. Unfortunately, our current experimental setup does not include a sufficiently precise force sensor to make significant in situ high-precision weight measurements of the nanoparticle agglomerates, and any attempts to carefully transfer collection plates from their holder to another instrument to conclusively assess and compare the collected nanoparticle weight proofed ineffective and error-prone. Furthermore, in situ microscopic imaging methods with possible 3D reconstruction would enable a direct analysis of the spatial distribution of the collected nanoparticles.Next, we studied the dependence of nanoparticle collection on the electric field intensity. The nanoparticles plume deflection was visualized using a high-speed camera synchronized with the femtosecond laser system.Conversely, a confinement in the nanoparticle plume was observed in the presence of a floating potential b and appWhen considering the electric field condition, the potential difference across the target and collection plates is definite at \u00b12000 V. Under such conditions, the electric charge accumulated during the laser ablation process is constant from pulse to pulse, resulting in a steady local electric field which controls the trajectory of the ionized species: the nanoparticles generated during the process of laser ablation. Such a strong and steady local electric field explains the high degree of confinement and the weak angular deflection of the nanoparticle plume c seen unIn contrast, neither the conventional PLD condition nor the floating potential conditions are electrically well defined, since the potential difference across the target and collection plates varies from pulse to pulse. The induced electric field will completely depend on the amount of charge left over from the previous pulse (effective field). Thus, this varying local field from pulse to pulse will cause diverse effects on the ionized particle; the behavior of the resulting nanoparticle plume will accordingly be correlated with the varying local electric field intensity. The local electric field for the conventional PLD machining conditions is expected to be weaker than the electric field resulting from the floating potential with an externally applied voltage of \u00b12000 V because of a relatively small charge accumulation during the laser ablation process. Thus, the differences in local field intensity of an externally applied floating potential compared to that observed under conventional PLD conditions explain the greater nanoparticle plume confinement and the weaker variation in the deflection of the nanoparticle plume. The electric charge accumulated during the laser ablation process is seemingly small; hence, the local electric field induced during the conventional PLD condition is less intense in comparison with the floating potential condition counterpart.That the applied field intensity influences the nanoparticle plume was further confirmed through plume videography, as seen in A confinement in the nanoparticle collection pattern is also observed under increasing floating potential c. The coHaving investigated the nanoparticle collection patterns on the collector plates, we now present the effects of the machining conditions on the appearance of the laser-machined target surfaces.Directly after laser machining, i.e., before ultrasonication, we observe that the actual topography of the machined surface shows some variation with the actual machining conditions a\u2013c. The While the experimental conditions under investigation here showed no impact of ultrasonication on the laser-induced surface structure after removal of the remaining nanoparticles by, the ablation depth\u2014as determined by confocal microscopy\u2014indicated considerable effects due to the actual experimental conditions. Detailed experimental results are presented in the The laser machining experiments were performed with two different laser scanning directions  as depicted in Conversely, for the perpendicular case, the distance between the nanoparticle generation sites along the long scan lines and the short edge of the charged collection plate varies. As a consequence, the force acting on the nanoparticles due to the charged collection plate varies throughout the machining of a single long scan line. When the laser incidence position on the target moves away from the short edge of the collector plate, the force decreases; when it approaches the collector plate again in the return scan, the force successively increases. As a result, initially ablated nanoparticles are attracted to a single spot on the collector plate, which is where the initial agglomeration starts and grows into a significant protrusion. The nanoparticles produced during later laterally translated scans will then drive towards this initially generated agglomerate protrusion rather than to the pristine collector plate which is at a slightly farther distance. As a consequence, the perpendicular scanning protocol results in a narrower collection pattern on the Cu collection plate.Plume videography showed no significant differences between the two scanning protocols compare .The comparison of SEM micrographs of the surfaces machined with parallel and perpendicular scanning direction for a given material after sonication revealed that the scanning direction does not affect the laser-induced structures compare . Yet, whIn this study, we carried out a parametric study to identify the dominant factors in nanoparticle collection and removal with the goal of establishing therefrom an in situ removal method during femtosecond laser machining. The influence of actual laser machining conditions  in removing the laser-produced nanoparticles, collecting them on a Cu collector, and the respective nanoparticle plume deflection behaviors were investigated. The connection between the different surface properties of the machined surface (topography before and after ultrasonication and the ablation depth) and the actual machining conditions were also explored.Machining in the presence of a floating potential results in a broader nanoparticle collection pattern for all tested targets  in contrast to machining in the presence of an applied field, where nanoparticle collection appears confined to a very small area on the collection plate. This observation correlates with a stronger confinement and a weaker angular deflection in the nanoparticle plume that was observed in proximity of an applied field in comparison with the floating potential condition. Under the conventional PLD condition in air, the nanoparticle plume was found to expand freely. The strong and steady local electric field induced under the applied field condition in contrast to the varying local field under the floating potential condition explains the high degree of confinement of the nanoparticle plume and the weak angular deflection of the nanoparticle plume. Furthermore, an increasing confinement of the nanoparticle plume and the resulting nanoparticle collection pattern was also observed with an increase in the applied field intensity and floating potential. Qualitatively judged from the collector plate photographs, the nanoparticle collection quantity directly correlates with the materials\u2019 electrical properties . Furthermore, the actual accumulation pattern on the Cu collector varies from one material to another, which is particularly evident for the collection patterns obtained upon machining with a floating potential. The topography of the machined surface was examined using SEM and the ablated depth was analyzed with the aid of a confocal microscope. Our results confirm that the surface topography of the machined surface before sonication and the ablation depth strongly depend on the actual charge conditions during machining. Yet, from our experiments, we found no notable impact of the charge condition during laser machining on the resulting laser induced surface textures, which were previously hidden under agglomerated and un-sintered nanoparticles. Whether an electric field or a floating potential is better suited to collect higher amounts of otherwise volatile nanoparticles will be an interesting subject of future studies.Such experimental efforts might benefit from a setup inside a closed system with a larger collection plate. Ideally, both target and collector are connected to high-precision force sensors. In addition, extremely valuable information would be gained if an alike experimental setup also allows for in situ imaging and sizing of the redeposited and collected nanoparticle clusters.Based on our findings, we conclude that machining metals and semi-conductors, in particular, under the floating potential or applied field condition with a parallel laser raster scanning protocol is a promising route for removing and collecting nanoparticles during the machining process. The current technique designed for collecting a substantial amount of such laser-generated nanoparticles in air directly during surface machining and patterning without affecting the target surface provides an outlook to circular waste-free process design."}
{"text": "The aim of the present study was to analyze the prevalence, epidemiology and relevance of shoulder injuries in polytraumatized patients in a large national trauma database.We hypothesize a high prevalence of shoulder injuries in traffic accidents and a high prevalence of concomitant injuries of the thorax leading to an aggravated clinical course and higher Injury Severity Score (ISS). Furthermore, we hypothesize an increased rate of surgical treatment with the severity of the injury.The retrospective analysis is based on the database (2002\u20132013) of the TraumaRegister DGU\u00ae and includes statistical data from 608 hospitals. The severity of injuries and trauma were scaled using the Abbreviated Injury Scale (AIS), and the Injury Severity Score (ISS), respectively. Patients with an ISS\u2009\u2265\u200916 were included in the study, and injuries were subdivided according to their anatomical involvement and analyzed with respect to the trauma mechanism and the resulting injuries.In this study, 54,076 cases of patients with an ISS\u2009\u2265\u200916 were analyzed. Shoulder injuries occurred in 15,115 patients (27.9%). Of these, 68.5% were caused by traffic accidents, especially in motorbike, bicycle, and pedestrian accidents. We found more shoulder injuries in blunt trauma mechanisms. Moreover, patients with shoulder injuries spent on average 1.7 more days on the intensive care unit (ICU), or intermediate care unit (IMCU), according to the severity of the injury, and had longer overall hospital stays (26.2 vs. 24.1\u00a0days) than patients without shoulder injuries. The overall ISS was increased in patients with shoulder injuries, whereas an increase of mortality could not be identified. Concomitant thoracic injuries occurred significantly more often in patients with shoulder injuries (82.9% vs. 69.6%). Injuries of the abdomen, pelvis, and lower extremity showed no correlation with shoulder injuries, whereas head and spine injuries showed a significant correlation.Shoulder injuries are very common in polytraumatized patients. Together with their distinctive concomitant injuries, they have an aggravating impact on the clinical progress. Our data confirm the correlation with thoracic injuries. Furthermore, we identified an increased risk of shoulder injuries in motorbike, bicycle, and pedestrian accidents. An increase in mortality could not be identified. Severe trauma is the sixth leading cause of death worldwide, and among young adults under 35\u00a0years of age, it is the leading cause of death and disability . This chIn major trauma, injuries to the shoulder, including severe nerve and vessel injuries, are frequently seen  and may In the present literature, several studies highlight the relevance of scapular fractures in a severe trauma or shoulder injuries in general \u201315. NeveThe purpose of this study was to investigate the prevalence and clinical features of shoulder injuries in polytraumatized patients. Furthermore, the impact on mortality was assessed, and concomitant injuries were identified.We presumed that, in the treatment of severely injured patients, the early identification of patients with a high risk for shoulder injuries might help reduce delayed diagnoses and decrease complications. We hypothesized a high prevalence of shoulder injuries in traffic accidents and a high prevalence of concomitant injuries of the thorax leading to an aggravated clinical course and higher ISS.The TraumaRegister DGU\u00ae (TR-DGU) of the German Trauma Society [Deutsche Gesellschaft f\u00fcr Unfallchirurgie (DGU)] was initiated in 1993. The aim of this multi-center database is to provide a pseudonymized and standardized documentation of severely injured patients. Data are collected prospectively in four consecutive time phases from the site of the accident until discharge from hospital: (a) Pre-hospital phase, (b) emergency room and initial surgery, (c) intensive care unit, and (d) discharge. The documentation includes detailed information on demographics, injury pattern, mechanism of injury, comorbidities, pre- and in-hospital management, a course on intensive care unit, relevant laboratory findings including data on transfusion, length of hospital stay, and outcome of each individual. The inclusion criterion is admission to hospital via an emergency room with subsequent ICU/IMCU care or reaching the hospital with vital signs but dying prior to admission to ICU/IMCU.The infrastructure for documentation, data management, and data analysis is provided by the Academy for Trauma Surgery , an affiliation of the German Trauma Society. The scientific leadership is provided by the Committee on Emergency Medicine, Intensive Care and Trauma Management (Section NIS) of the German Trauma Society. The participating hospitals submitted their data pseudonymized into a central database via a web-based application. Scientific data analysis is approved according to a peer-review procedure established by Section NIS.The participating hospitals are primarily located in Germany (90%), but an increasing number of hospitals from other countries contribute data as well. Currently, more than 33,000 cases from over 600 hospitals are entered into the database each year.Participation in TraumaRegister DGU\u00ae is voluntary. For hospitals associated with the trauma network of the DGU\u00ae, however, the entry of at least a basic data set is obligatory for reasons of quality assurance.The present study is in line with the publication guidelines of the TraumaRegister DGU\u00ae and was registered as TR-DGU project ID 2014\u2013029.In Germany, 608 participating hospitals contributed their data to the TR-DGU in the dataset from 2002\u20132013. All patients with an initial ISS\u2009\u2265\u200916 that were documented in the TR-DGU from 2002\u20132013 were included in the present study. Excluded were all patients with an ISS\u2009\u2265\u200916 due to a single craniocerebral injury (CCI). In sum, 54,076 patients were included in the study. Patients were divided into two subgroups: the \u201cshoulder\u201d group with patients presenting shoulder injuries , and the \u201cnon-shoulder\u201d group which included patients without the presence of any shoulder injuries . The severity of injuries was documented and coded according to the 2005 revised version of the AIS [Concerning the outcome and specification of injury, the average Revised Injury Severity Classification II (RISC II), mean ISS, mortality, trauma mechanisms, and further clinical and epidemiological data  transfusion, Glasgow Come Scale (GCS), and hospital stay including intensive care unit (ICU) and intermediate care unit (IMCU) were assessed. The concomitant injury pattern of polytraumatized patients demonstrates the prevalence of concomitant injuries (any injury of the specified anatomic region with an AIS score\u2009\u2265\u20092) that occurred in conjunction with a shoulder injury.t test for continuous data, whereas the Pearsons\u2019 Chi-Square test was used for categorical data. Continuous values are presented as mean\u2009\u00b1\u2009standard deviation (SD).Differences between the groups were evaluated with Student\u2019s p value\u2009\u2264\u20090.05 (two tailed) was considered to be statistically significant. The statistical analyses were performed with SPSS .A n\u2009=\u200915,115, 27.9%). Patients in the shoulder group had a significantly increased ISS  and were older . As men comprised the majority of all patients in severe trauma, at 72.5% of all cases, gender could not be identified as a risk factor , especially in contrast to the non-shoulder group . Focusing on traffic accidents, shoulder injuries were caused significantly more often by motorbike (20.6% vs. 13.4%), bicycle (10.5% vs. 6.8%), or pedestrian accidents (9.6% vs. 8.1%) . Accidents with cars and trucks (26.7% vs. 31.2%), falls\u2009<\u20093\u00a0m (9.1% vs. 11.9%) and falls\u2009>\u20093\u00a0m (18.8% vs. 20.5%) were less frequently associated with shoulder injuries  . Injuries of the shoulder are less common in patients with abdominal (26.6% vs. 28.4%), pelvic (25.4% vs. 26.6%), or lower limb (35.5% vs. 38.2%) trauma  , head (56.1% vs. 52.2%), and spine (40.5% vs. 36.8%) injuries   occurred significantly more often in combination with a proximal humerus fracture. Whereas lesions of the clavicle (11.0% vs. 12.4%) (p\u2009<\u20090.0001) occurred less often in combination with a proximal humerus fracture , the AC/SC-joint (1.3% vs. 1.2%), nerves (6.6% vs. 0.9%), vessels (1.3% vs. 0.2%), and other (4.2% vs. 2.1%) . In addition, a lower RISC II score was found in patients with shoulder injuries , highlighting the better prognosis of shoulder patients after multiple trauma. Furthermore, shoulder patients showed a more aggravated clinical course than non-shoulder patients, as they had a greater need for red blood cell concentrate (RBC) transfusions  and were more likely to be admitted to the ICU/IMCU . Patients in the shoulder group presented a GCS\u2009\u2264\u20098 less often than non-shoulder patients  , vessel injuries 77.4%), and prox. humeral fractures (74.3%) were most likely to undergo surgical treatment. Clavicle fractures, nerve lesions, scapula fractures, and injuries to the AC/SC-joint were more often treated conservatively Fig.\u00a0. The rat7.4%, andExtremity injuries have a high prevalence in severe multiple trauma, with 58.6% of multiple trauma patients presenting significant extremity injury. Affection of the clavicle is the third most common fracture in polytraumatized patients (10.4%), only surpassed by femoral (16.5%) and tibial 12.6%) fractures .6% fract.In this context, our findings can be summarized as followed.Show a higher rate of head and thoracic injuries.Suffer significantly more often from pneumothorax and hemothorax.Stay longer on the ICU and in the acute care hospitals.Present a higher ISS than severely injured patients without shoulder injuries.Were likely to have been involved in a traffic accident.Polytraumatized patients (ISS\u2009\u2265\u200916) with shoulder injuries:The high prevalence of shoulder injuries 27.9%) in severely injured patients underlines the importance of understanding the main mechanisms and factors leading to shoulder injuries. Banerjee et al. already showed a 39.6% prevalence of upper extremity injury in polytraumatized patients 7.9% in s and fracAs initially hypothesized, the severity of the injury determines the rate of surgical treatment. Surgical therapy of proximal humerus fractures and injured vessels are very common. In contrast, scapula fractures and clavicle fractures underwent surgical therapy less often. Extraarticular scapula fractures represent approximately two-thirds of all scapula fractures  and are The associated concomitant injuries of shoulder injuries in the shoulder group aggravate the clinical course after severe multiple trauma. They received RBC transfusions, which are known to trigger complications and mortality dose dependence , signifiWe hypothesize that the high correlation of thoracic injuries with shoulder injuries is caused by the anatomical proximity of these structures and, therefore, the thorax is likely to be simultaneously injured in a shoulder injury, and vice versa. Additionally, in the shoulder group we observed numerous motorbike accidents, which are known to increase the risk of thoracic injuries . In seveDue to severe concomitant injuries, our findings underline the importance of an early computed tomography scan (CT-scan) of severely traumatized patients, particularly as suggested by guidelines . AdditioNevertheless, structured and repeated clinical examinations (body check) remain a key aspect in acute trauma care, especially to identify concomitant injuries that may have been missed initially. Further studies showed that brachial plexus injuries may be present in 1% of multiple trauma patients . With 4%Focusing on the ISS and RISC II, shoulder patients presented a significantly increased ISS. Unexpectedly, the RISC II score and the mortality of the study population are lower in the shoulder group, and a contemporaneous increase with the ISS could not be shown. Initially, we assumed a higher RISC II and higher mortality in the shoulder group due to the increased ISS. Previous studies already showed a lower mortality rate in severely injured patients with scapular fractures and aggravated concomitant injuries , 23, butWith regard to the higher ISS in the shoulder group, a previous study showed that a higher ISS could predict lower general health condition with respect to the long-term outcome after trauma . HoweverRegarding the mechanism of injury, traffic injuries have a huge impact on trauma cases worldwide and are the leading cause of death of young adults in Europe , 51. ThiThe present study has limitations, as the analyzed data is retrospective. As the participating hospitals have different levels of patient-centered care, the treatment after trauma could differ. As shoulder injuries could have been initially overlooked, there is a chance that shoulder injuries have been under reported. Furthermore, in polytraumatized patients, minor injuries may be omitted. The choice of diagnostics in the database leading to the diagnosis is not reported, so the accuracy of the data database is dependent on the individual examiner and submission of data in the register. Unfortunately, the exact specification of soft tissue injuries could not be classified.In summary, our findings show that shoulder injuries in severely injured patients are frequently associated with severe concomitant injuries, mainly of the chest, head, and spine. High-speed traffic accidents are the most common injury mechanisms, particularly in motorbike, bicycle, and pedestrian accidents. Previous studies on extremity injuries of the TraumaRegister DGU\u00ae confirmed the increase of the ISS, duration of stay, and aggravated clinical course of extremity injuries , 17.Shoulder injuries have an aggravating impact on the clinical course and prolong the clinical stay. As multiple trauma patients with shoulder injury present more diagnoses, the clinical management and treatment of the patient are aggravated. This is demonstrated by a higher initial ISS and longer stay on ICU/IMCU. Nevertheless, an increased rate of mortality in patients with shoulder injuries could not be identified. Our results correspond with empirical data in our daily clinical routine and other national and international findings in the literature. Furthermore, because of the large sample size of the analyzed study population and the high number of included trauma centers providing data, the clinical relevance of our findings seems significant. In the emergency room (ER) and in the tertiary survey, potential shoulder injuries and identified concomitant injuries should be closely considered."}
{"text": "Learning and memory are essential to organism survival and are conserved across various species, especially vertebrates. Cognitive studies involving learning and memory require using appropriate model organisms to translate relevant findings to humans. Zebrafish are becoming increasingly popular as one of the animal models for neurodegenerative diseases due to their low maintenance cost, prolific nature and amenability to genetic manipulation. More importantly, zebrafish exhibit a repertoire of neurobehaviors comparable to humans. In this review, we discuss the forms of learning and memory abilities in zebrafish and the tests used to evaluate the neurobehaviors in this species. In addition, the pharmacological studies that used zebrafish as models to screen for the effects of neuroprotective and neurotoxic compounds on cognitive performance will be summarized here. Lastly, we discuss the challenges and perspectives in establishing zebrafish as a robust model for cognitive research involving learning and memory. Zebrafish are becoming an indispensable model in learning and memory research for screening neuroprotective agents against cognitive impairment. Danio rerio) are tropical fish native to southern Asia and became a vertebrate model organism in developmental biology pioneered by George Streisinger in the 1970s at the University of Oregon. Since then, zebrafish have emerged as one of the animal models in preclinical studies for understanding various physiological processes and diseases. The growing interest is attributed to the favorable features offered by this species. These include ex vivo fertilization, transparent embryos and larvae (facilitate imaging), small size (2\u20135 cm for adult), easy maintenance, prolific nature (more than 100 eggs produced per fish), rapid development , their accessibility for genetic manipulations such as CRISPR and high homology (70%) to the human genome [Zebrafish  is the most prevalent form of dementia 60\u201370%) that affects more than 57 million people globally 0% that a,9,10. DeZebrafish show a high degree of conservation in the neuroanatomical organization and neurotransmitter signaling pathways with humans ,11. The Non-human primates and rodents are the classical animal models for behavioral studies involving cognition . Non-humThis review describes the cognitive behaviors related to learning and memory in zebrafish and the assessment tools available for these behaviors. We summarize pharmacological studies that used zebrafish models to screen for the effects of neuroprotective and neurotoxic compounds on cognitive performance. Challenges and perspectives in establishing zebrafish as a robust model to study cognitive impairment are also discussed.Cognition is the mental process of acquiring knowledge and understanding through thinking, learning, memorizing and sensing. The cognitive process includes sensation, perception, motor skills, attention, memory, executive function, language and processing speed . LearninZebrafish display different learning abilities, such as associative, non-associative, social  and motor learning. In social learning, a group of zebrafish learned faster than a single individual . In motoNon-associative learning is a general lasting change in response strength toward a stimulus due to repeated exposure. Non-associative learning is further categorized into habituation and sensitization. Habituation is the decrease while sensitization is the increase in the animal\u2019s response to a\u2009sensory stimulus upon continuous exposure. The changes in responses could be in the short- and long-term. Habituation has been regarded as an evolutionarily conserved behavior for optimal survival. Habituation allows the animal to disregard repeated stimuli while focusing on important stimuli such as potential predators and danger in the environment ,19. In tAssociative learning is the process of acquiring new information by linking two elements. This type of learning is more commonly assessed than non-associative learning in zebrafish studies. The two forms of associative learning are classical (Pavlovian) and operant  conditioning ,16,27. IIn operant conditioning, an animal learns to correlate its voluntary behavioral responses with their consequences . The terClassical conditioning can be further divided into appetitive and aversive conditioning based on the nature of the US. Appetitive conditioning utilizes favorable stimuli as the US. Sison and Gerlai demonstrated that zebrafish are capable of associative learning by using food as the reward and a red card as the visual cue . After tIn aversive conditioning, the CS is paired with a fear-inducing US that leads to a fear-related conditioned response when the CS is administered alone. The nature of the CS can be contextual (location or environment) or cued . The most commonly used aversive US in zebrafish studies is the application of electric shock (ES). Fear conditioning is the most common form of classical learning assessed in zebrafish. Valente and colleagues demonstrated that both larval and adult zebrafish could perform cued fear conditioning in a paradigm that associated a checkerboard pattern (CS) with the ES (US) . After tIn contextual fear conditioning, a novel environment is associated with the US. Kenney and colleagues showed that contextual fear conditioning in zebrafish lasted for at least 14 days and varied in the fear extinction rates among different strains . ExtinctFear-related responses include freezing (immobility), erratic movements (zigzagging), bottom-dwelling, a tighter shoal, leaping (jumping) and thigmotaxis . In the Operant conditioning can be classified as reinforcement or punishment, such that the paradigm increases or decreases the response to modify the strength of the response over the course of training. The reinforcement in operant conditioning can be positive (addition of reward as a reinforcer) or negative . Both positive and negative reinforcement paradigms are aimed at strengthening the behavioral response.Positive reinforcement administers the appetitive US after the animal performs a behavioral response to strengthen the response. Manabe and co-workers developed an automated operant device for positive reinforcement conditioning in zebrafish. The response key was equipped with a sensor that dispenses brine shrimp eggs when it was approached by zebrafish . In the Negative reinforcement removes the aversive US when the animal engages in a behavioral response. This type of conditioning can be further divided into escape learning and avoidance (active and passive). Escape learning refers to the animal engages a behavior to terminate the ongoing aversive stimulus. For example, the crossing response of rodents will be increased from a compartment with existing foot shock to the opposite compartment in a shuttle box. The active avoidance paradigm requires the presentation of a cue  prior to the administration of an aversive stimulus. Active avoidance was performed to prevent the happening of the aversive stimulus. Xu and colleagues demonstrated that zebrafish increased crossing response to a compartment without ES upon presenting a light signal previously paired with the administration of ES . In passSimilarly, punishment-based operant conditioning is divided into positive (impose punishment) and negative (remove appetitive stimulus as punishment). Both positive and negative punishment paradigms are aimed at weakening the behavioral response. For positive punishment, an aversive US is administered when a behavioral response is engaged, eventually leading to a decreased behavioral response. The difference between positive punishment, escape learning and avoidance is that the behavior response in positive punishment leads to the administration of an aversive stimulus, while the behavioral response in the latter two negative reinforcement results in the removal/prevention of an aversive stimulus. For example, in positive punishment conditioning, a rodent learns to refrain from pressing a lever that will lead to foot shock.For negative punishment, an appetitive US is removed when the animal performs a behavioral response. For instance, this conditioning is used in dog training to correct undesired behavior by taking away the reward (food or toy). To our knowledge, both positive and negative punishments have not been engaged in pharmacological studies using zebrafish as models to assess cognitive performance.In summary, zebrafish are capable of performing both non-associative and associative learning tasks. This allows the researcher to assess the zebrafish\u2019s cognitive performance using various tests. From a survival perspective, operant learning allows the animals to find a safe and rewarding outcome while avoiding danger in a complex environment. Unsurprisingly, operant conditioning is pervasive in zebrafish studies because task performance could be enhanced by adding an operant component . NeverthMemory can be classified mainly into sensory, short-term and long-term ,37. SensNot all forms of memory can be evaluated in zebrafish as model organisms. Similar to rodent models, the most common types of memory assessed in zebrafish for cognitive studies are spatial, recognition and associative memory. To score the memory retention index, a time delay between the training (familiarization) and test (probe) phases is required. Spatial memory in zebrafish is assessed based on the preference of zebrafish for new space/area. The cue to a novel place could be contextual or visual . Cognato et al. showed that spatial memory in zebrafish lasted up to 3 h after the familiarization session and diminished after 6 h of exposure . ObjectiThe main types of behaviors assessed in zebrafish as pharmacological models for learning and memory performance are locomotion, emotion (anxiety-like), cognition  and social interaction  . Table  There coThe locomotor activity test (LAT) is used to evaluate the spontaneous swimming behaviors of zebrafish . It is nLAT is generally performed first in a battery of behavioral tests before other more complicated paradigms. This can prevent the animals from becoming exhausted and less motivated to perform the subsequent paradigms. To our knowledge, there is no consensus on a standardized environment or environmental conditions for LAT. Most behavioral tests developed for zebrafish serve as a general guide rather than a standardized protocol. Considering that zebrafish are increasingly used as study models across different laboratories worldwide, standardization may be necessary to make the findings from different studies comparable. Recently, Maeda et al. developed a standardized method for assessing the behavioral response (including locomotor activity/response) in zebrafish larvae (6 to 7 dpf) using a light\u2013dark locomotion test . The chaA novel tank test is, similar to the LAT setup, used to assess the anxiety-like behavior of zebrafish . The tanThe inhibitory avoidance test (IAT) is one of the most commonly used cognitive tests to evaluate the learning and memory functions of zebrafish. The test can be established using a two-chamber tank A and a mSimilar to IAT, a rectangle tank or maze is used to establish the CS\u2013US pairing over repeated exposure. Chen et al. covered the left and right arms of a T-maze with green and red colored sleeves as visual cues . One of The Y-maze test is used to inspect the spatial memory of zebrafish based on their innate behavior that tends to explore the novel place . ZebrafiSimilar to the Y-maze that is based on the fish response to novelty, a novel object recognition test is used to examine the recognition memory. In the test conducted by Capatina et al., a zebrafish was placed in a tank with two familiar objects (red cubes) and allowed to explore the tank for 10 min . After aThe neurobehavioral tests for zebrafish have been reported for over two decades . The utiOriganum vulgare spp. hirtum (Lamiaceae)) essential oil on the scopolamine-induced amnesia zebrafish model [Juglans regia L.), on the scopolamine-induced cognitive deficit zebrafish model [Cholinergic signaling is involved in memory acquisition and consolidation. Reduction of acetylcholine (ACh) due to hydrolysis by acetylcholinesterase (AChE) could implicate the development of dementia and AD. Scopolamine is an alkaloid that acts as a nonselective muscarinic receptor antagonist. It blocks the receptors to induce cognitive impairment. Scopolamine-induced amnesia in zebrafish is a widely used model for screening drugs with neuroprotective effects . Capatinsh model . Scopolash model . The autsh model , lithiumsh model , sulforash model , adenosish model , flavonosh model , and physh model .Pediococcus acidilactici LAB4 and Lactobacillus plantarum LAB12 [Lactobacillus plantarum ST-III using the triclosan-induced neurodegeneration zebrafish model [Lim et al. used a high-cholesterol diet-induced cognitive impairment model of zebrafish to explore the effect of two lactic acid bacteria (LAB) strains: um LAB12 . The fissh model . They prEmbelia ribes), on acute and chronic PTZ-induced seizures and cognitive impairment. In the acute seizure model, pre-treatment of embelin (0.078\u20130.625 mg/kg) reduced seizures and ameliorated epilepsy-associated cognitive dysfunction induced by a single dose (170 mg/kg) of PTZ [ccl2, tlr-4, tnf-\u03b1, il-1, ifn-\u03b3).Kundap et al. developed a zebrafish model of epilepsy that induced seizure-like behaviors and cognitive dysfunction using pentylenetetrazole (PTZ) . The mod) of PTZ . Time sp) of PTZ . Daily pQuercetin has neuroprotective properties but is limited with lower bioavailability and permeability across the blood\u2013brain barrier. Rishitha and Muthiraman studied the efficacy of nanoparticle-formulated quercetin on PTZ-induced cognitive impairment in zebrafish . IntrapeN-Methyl-D-aspartate receptors are ionotropic glutamate receptors related to cognitive functions. MK-801, an N-Methyl-D-aspartate receptor antagonist, has been shown to induce amnesia in zebrafish ,82,83. OAlcohol consumption in higher doses can impair cognitive functions leading to amnesia or blackout. Rapid elevated ethanol levels impair memory consolidation, resulting in memory gaps for individuals during intoxication. Bertoncello et al. developed a novel ethanol-induced amnesia model in zebrafish to evaluate the effect of taurine on memory consolidation . The aniA receptors. Using the hypoxic zebrafish model, the same research group showed that magnesium sulfate prevented cognitive deficits and brain infarction via the up-regulation of EET4A glutamate receptor [Hypoxia results in ischemic injuries to neurons and cognitive deficits. It inhibits glutamate uptake into the neurons. Urinary trypsin inhibitor (UTI) is a urinary glycoprotein that inhibits proteolytic enzymes such as hyaluronidase plasmin, \u03b1-chymotrypsin and trypsin. Kim et al. demonstrated the neuroprotective effect of UTI on a cognitively impaired zebrafish model induced by hypoxia . UTI-trereceptor .c-fos and bdnf in the zebrafish brain. Pusceddu et al. utilized zebrafish to evaluate the chronic effects of Mediterranean natural extracts, namely licorice (Glycyrrhiza glabra) root extract and rosemary extract, on cognitive functions [Healthy and cognitively unimpaired zebrafish are models for screening compounds or conditions with memory enhancement (nootropic) properties ,88,89,90unctions . Both exunctions . Methyleunctions . Braida unctions . They foZebrafish are a popular model used to screen for compounds with neurotoxicity effects on learning and memory. These include metals/elements ,100,101,Shang et al. used zebrafish to validate the neurotoxic mechanism of occupational aluminum exposure in humans . Serum a2) reduced the fish locomotor activity and aggressive behaviors in a 3D LAT and mirror biting test, respectively. In the mirror biting test, a mirror was located next to the tank. The aggressive behavior was scored based on biting and fast swimming, as zebrafish tend to display boldness by biting the mirror and tracing their reflection by quick movement. The PbCl2 induced anxiety-like behaviors in a novel tank test and altered circadian rhythm locomotor activity. Fish exposed to PbCl2 had reduced latency to enter the dark compartment with ES in the IAT, indicating memory loss induced by PbCl2 poisoning. Elevation of cortisol and reduction of serotonin and melatonin in the brain could be related to the altered neurobehaviors observed in PbCl2-exposed zebrafish. Sarasamma et al. developed an AD model based on zinc chloride (ZnCl2)-induced cognitive impairment in zebrafish [2 decreased the latency to enter the dark chamber with ES in an IAT for trained fish, signifying lower memory retention. Elevation of reactive oxygen species level, lipid peroxidation markers  and stress hormones (catecholamine and cortisol) and reduction of melatonin and antioxidant enzyme activities  were observed in the brain following ZnCl2 exposure. The compound also affected neurotransmitters by increasing AChE activity, dopamine, glutamate and GABA levels, while decreasing glycine and histidine levels. More importantly, ZnCl2-exposed zebrafish displayed AD-like symptoms by elevating amyloid beta 42 and phosphorylated tau proteins in the brain.Bui Thi et al. utilized zebrafish to evaluate the effect of chronic lead exposure on neurobehaviors . Low levebrafish . ZnCl2 dAudira et al. investigated the adverse effect of chronic exposure to donepezil on cognitive functions and behaviors, using normal zebrafish as a model to represent healthy individuals without AD . Long-teZebrafish larvae can be used to perform toxicity screening on learning and memory. He et al. used zebrafish larvae to investigate the effect of prenatal exposure to propofol, a widely used general anesthetic drug, on learning and memory function . PropofoThere are several concerns when using zebrafish as models for drug discovery in cognitive research. First, aqueous immersion was the major route of drug administration used in many studies, which might be least applicable to humans. Aqueous immersion was chosen probably due to the convenient setup. However, the bioavailability of the drugs is difficult to assess in zebrafish via this method. Over the years, other routes of administration, such as intraperitoneal ,67,122, In this review, we have seen that zebrafish have emerged as one of the vertebrate models for cognitive research related to learning and memory functions over the past decade. The gaining popularity is supported by a repertoire of neurobehavioral displayed by the zebrafish and assessment tools available for these behaviors. Zebrafish have been proven useful as pharmacological models in drug screening for neuroprotective or neurotoxicity agents that affect learning and memory performance. Zebrafish emerged as a complementary model to rodents in preclinical studies before further testing on humans. One of the main advantages of the zebrafish model is lower maintenance costs for pharmacological screening. However, the clinically relevant routes of drug administration still require further exploration. In addition, innovative behavioral assessment apparatuses and software are needed to increase the assay throughput. By overcoming these limitations and combining the favorable features of zebrafish, this animal model will be indispensable in drug discovery research involving learning and memory."}
{"text": "Serological testing for SARS-CoV-2 IgG antibodies is used to assess their presence in blood samples from exposed individuals and provides a measure of the magnitude of immune response to infection. The measurement of neutralizing antibodies (NAbs) in particular provides information about the severity of prior infection and level of protective immunity against re-infection. Much of the work investigating the association between prior infection severity and NAb levels has been conducted among clinical populations, and less is known about this relationship in the general population. Accordingly, we utilize data from a large (n\u2009=\u2009790) community-based cohort of unvaccinated, seropositive participants. We analyzed the association between NAb response, measured via surrogate virus neutralization assay, with patterns of symptoms and household exposure. Our results indicate no detectable NAb activity in 63.8% of the seropositive participants (n\u2009=\u2009504). Those with detectable NAb levels demonstrated a positive relationship between NAb activity and both self-reported previous symptom severity and household exposure. These findings are significant in light of recent concerns about degree of protective immunity conferred by prior infection or vaccination, and we highlight the value of community-based research for investigating variation in immune response. Response to infection with SARS-CoV-2 can be highly variable, including differential degrees of symptom severity, hospitalization, and immune response. Serological testing has been a vital tool in tracking SARS-CoV-2 infection across communities, as it is used to detect the presence of antibodies to SARS-CoV-2 in blood samples from exposed individuals, while also providing a measure of the magnitude of immune response to infection4.As of July 22, 2021, more than 190 million people worldwide have been infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19)6. NAbs are effective at inhibiting this initial interaction, as well as downstream events, and thereby preventing viral entry into cells5. Following infection, NAbs have been shown to persist for many months, albeit with decreasing levels detectable in the blood8.Serological testing facilitates assessment of exposure and immune response via laboratory measurement of antibodies that impede infection by SARS-CoV-2. Some, but not all, antibodies directed against the spike protein of SARS-CoV-2 can neutralize virus infectivity in laboratory assays. These neutralizing antibodies (NAbs) bind to the SARS-CoV-2 surface spike protein on the virion surface, the part of the virus that engages the human angiotensin-converting enzyme 2 (ACE2) receptor in order to gain entry into host cells9. Similarly, COVID-19 patients who were treated in an intensive care unit demonstrated significantly higher peak neutralizing antibody titers compared with those who were not11. Further, higher neutralizing antibody levels among vaccinated individuals have been shown to predict a lower likelihood of SARS-CoV-2 breakthrough infections11. These results point to a consistent relationship between disease severity and level of NAb activity in clinical populations.Because of their key role in hindering viral entry, it is expected that neutralizing antibody activity can provide information about severity of prior infection and the level of protective immunity against re-infection. This expectation is supported by results indicating that NAbs appeared earlier and at higher levels in patients who experienced severe or moderate infections, when compared with patients who experienced mild or asymptomatic illness12. We utilized data from a large community-based cohort in Chicago in order to investigate these dynamics in a non-clinical population that includes people who did require treatment or even know they had been infected with SARS-CoV-2. Because the majority of COVID-19 cases are asymptomatic or mild13, our community-based sample adds a clearer picture of the nature of the association between disease severity and the production of NAbs, which likely provides at least partial protection against re-infection in the broader population of all previously infected and unvaccinated persons.Less is known about this relationship in the general population. Increasing our understanding of variation in NAb levels across severity of infection in the community is particularly critical as many people remain hesitant to receive the SARS-CoV-2 vaccine, based on beliefs that any prior exposure will provide adequate protection against re-infection by the virus15. Previous work also indicated that household exposure to the virus, which is likely greater or more prolonged than exposure outside the home, is associated with both greater disease severity and higher antibody concentrations15. However, it is not yet known whether there is a similar association between the level of NAb activity and either COVID-19 symptom severity or household exposure history in a non-clinical population.Previous work by our team using data from a community-based observational study in Chicago has shown that those who reported more symptoms of infection in months prior to antibody quantitation had higher concentrations of immunoglobulin G (IgG) antibodies directed against SARS-CoV-2 spike receptor binding domain (RBD)Here, we analyze the association of magnitude of NAb response, using a surrogate virus neutralization assay, with patterns of symptoms and household exposure history among unvaccinated, seropositive participants. Our results indicate that the majority of individuals who tested seropositive for prior COVID-19 infection had no detectable neutralization activity. In individuals who had detectable levels of neutralizing antibodies, we report a positive association between COVID-19 symptoms and neutralization activity. We also report higher neutralization activity in individuals living with household members who reported symptoms or diagnosis of COVID-19.4. The study included a total of 4463 adults at this time point.A large community-based sample of adults living within the Chicago, IL metropolitan area was recruited to participate in a study called Screening for Coronavirus Antibodies in Neighborhoods (SCAN). Samples for this analysis were collected between June 24 and November 11, 2020, prior to availability of SARS-CoV-2 vaccination in the areaParticipation was facilitated by a web-based, \u201cno contact\u201d protocol, which allowed individuals to provide information and dried blood spot (DBS) samples outside of a clinical setting. Participants were recruited via advertisements in social media, emails, print flyers, newspapers, participant registries, participant referrals, community outreach, and local press. Recruitment was conducted in neighborhoods throughout the Chicago metropolitan area and at the Northwestern University Feinberg School of Medicine (FSM) in Chicago. To ensure racial and gender diversity and representation within the sample, we carried out stratified random sampling to adaptively match enrollment of white participants and women (groups that were more likely to complete the screener) to enrollment of non-white participants and men.17. All research activities were implemented under protocols approved by the institutional review board at Northwestern University (#STU00212457 and #STU00212472). Written informed consent was received from all individuals prior to participation in this study, and all methods were performed in accordance with relevant guidelines and regulations.Study data were collected and managed using REDCap electronic data capture tools hosted at Northwestern University2), smoking, number of individuals in their households, and whether any cohabitants had been diagnosed with COVID-19 or had symptoms of COVID-19.Participants provided information about COVID-19 testing, diagnoses, and symptoms experienced after March 1, 2020. Other variables included sex (based on assignment at birth), self-identified racial/ethnic identity, pre-existing chronic medical conditions  and the timing of blood collection (Supplementary Table 18. IgG antibodies were quantified using an enzyme-linked immunosorbent assay (ELISA) that has received emergency use authorization from the United States Food and Drug Administration . This assay was adapted and validated for use with DBS samples18.Analyses presented here focus on the 17.7% (n\u2009=\u2009790) of the 4463 total participants that tested seropositive for prior infection based on the presence of IgG antibodies against the RBD of SARS-CoV-215. The symptoms not included in the cluster of eight are itchy eyes; runny nose; and sore throat.Prior to DBS collection, participants indicated whether they had experienced any of the following eleven symptoms potentially associated with COVID-19: headache; fatigue or excessive sleepiness; sore throat; cough; muscle or body aches; runny nose; fever or chills; diarrhea, nausea, or vomiting; shortness of breath; loss of sense of smell or taste; and itchy eyes. In a previous study, we identified a cluster of eight of these symptoms that were associated with higher SARS-CoV-2 IgG concentrations: headache; fatigue or excessive sleepiness; cough; muscle or body aches; fever or chills; diarrhea, nausea, or vomiting; shortness of breath; loss of sense of smell or taste15). Briefly, each of the eight symptoms associated with higher SARS-CoV-2 IgG antibody levels was weighted by the regression coefficient that resulted from a bivariate model that included the symptom as the independent variable and log10-transformed SARS-CoV-2 IgG concentration as the dependent variable. For the symptom score, our goal was to establish the independent association between each individual symptom and SARS-CoV-2 IgG antibody levels\u2014which was facilitated by using bivariate models (that did not include other symptoms). Because participants reported distinct combinations of symptoms , we did not seek to create scores that included the correlation between symptoms or their mutual influence, as doing so might under-or over-estimate the significance of the individual symptom. The resulting symptom weights are: fever\u2009=\u20090.22, cough\u2009=\u20090.13, shortness of breath\u2009=\u20090.20, headache\u2009=\u20090.09, muscle or body aches\u2009=\u20090.19, fatigue or excessive sleepiness\u2009=\u20090.13, diarrhea/nausea/vomiting\u2009=\u20090.17, loss of taste/smell\u2009=\u20090.3215. This composite variable indicated how severely each participant experienced symptoms associated with COVID-19 infection.A \u201cCOVID-19 symptom severity score\u201d for each participant was created following methods described previously  protocol . This method, which can be implemented with immunoassay techniques, contrasts with other conventional methods for measuring neutralizing activity that require the presence of live virus and specialized laboratory containment facilities. Excellent concordance between results of these types of assays of NAb activity has been reported20. Based on these validation results, the threshold for determining the presence of surrogate neutralization activity was set at 13.2% or higher20.The sVNT method was adapted and validated to measure neutralizing antibodies in DBS samples described here21.Patterns of association between symptoms and the presence of NAb were established using summary statistics and multiple linear regression analyses. Due to the skewed distribution of NAb values, median values are presented as descriptive statistics and log10-transformed values were used for regression analyses. Covariates in the multiple linear regression analyses included age, race/ethnicity, sex assigned at birth, and chronic pre-existing conditions. All data analyses were conducted using R (version 4.0.4) in RStudio Version 1.4.1106Table Of the seropositive participants included in these analyses, 67.7% of participants reported experiencing one or more of eleven COVID-19 symptoms listed above since March 1, 2020. Notably 32.3% reported no prior symptoms.2\u2009=\u20090.12; p\u2009<\u20090.001)  (Table Seropositive participants (n\u2009=\u2009790) had a mean symptom severity score of 0.32 . COVID-19 symptom severity scores were higher among participants with detectable NAb activity than in participants with no neutralization activity . The median symptom severity score for participants with neutralization activity was 1.16, and median score for participants without detectable neutralization activity was 0.30. A linear regression model adjusted for the same covariates demonstrated a positive linear association between surrogate neutralization levels and COVID-19 symptom severity score . Similarly, participants with neutralization activity were also more likely to have a household member who reported one of the 11 symptoms potentially indicative of COVID-19 . A regression model adjusted for age, birth sex, race, and preexisting chronic medical conditions demonstrated that living with someone previously diagnosed with COVID-19 or having a household member with symptoms potentially indicative of COVID-19 was positively associated with levels of surrogate neutralization activity  . Symptom severity score of the study participant remained positively associated with surrogate neutralization levels in this model .Individuals with neutralization activity were more likely to report a household member who had been diagnosed with COVID-19 than participants without detectable levels of NAb activity (23.4% vs. 4.8%; \u03c7Our results indicate a positive relationship between neutralizing antibody activity and both self-reported previous symptom severity and household exposure to the virus, in a community-based sample of unvaccinated, seropositive persons. We also found no detectable surrogate neutralization activity in the majority of seropositive individuals, indicating that lightly symptomatic or asymptomatic SARS-CoV-2 infection does not elicit a strong NAb response.24. Our results are consistent with findings from clinical populations that indicate that more severe symptomatic cases of COVID-19 are associated with higher NAb levels, suggesting that these cases are more likely to provide increased and longer-lasting protective immunity following infection25.These results enhance our understanding of the variation in NAb levels across individuals who have different responses to exposure to SARS-CoV-2. These results are particularly significant in light of recent concerns about the persistence of both total antibodies and NAbs to SARS-CoV-2 following infection, particularly in those with mild or asymptomatic cases, and the degree of protective immunity conferred by prior natural infection26. Results such as these support previous findings indicating that prior SARS-CoV-2 infection, especially when mild or asymptomatic, may not be a reliable indicator of \u2018natural immunity\u2019 from reinfection, and consistent with the expectation that many previously infected persons are likely to have a relatively low level of protection from reinfection25. These results are consistent with our previous findings that two doses of the mRNA vaccine were required for individuals who had mild/asymptomatic seropositive cases to attain a level of surrogate neutralizing antibody response comparable to individuals who had previously been diagnosed with COVID-1927. Taken together, these findings suggest that natural infection\u2014particularly mild/asymptomatic seropositive cases\u2014are most likely to provide a level of immune protection comparable to one dose of mRNA vaccine29. Further, our results indicate that, to clinically assess the potential protection conferred from previous infection, it will be helpful to assess symptom severity and method of exposure to the virus, as a surrogate for NAb activity.However, our results also highlight that the majority of seropositive individuals in this community-based study did not exhibit neutralization activity. These findings are consistent with other studies conducted with non-clinical populations that have reported a low proportion of seropositive individuals with detectable NAbs30. In addition, completion of two mRNA vaccine doses was reported to elicit a broader NAb response better covering all variants of concern than did full vaccination of previously uninfected persons31. However, it is not yet known whether previously infected individuals with varying levels of disease severity differ in their antibody responses that may impact vaccine effectiveness, potential for onward transmission, including for more transmissible Delta variants and other variants of concern that may emerge in the future, after the full course of mRNA vaccinations. Because samples collection for this study predated wide-scale vaccination efforts and the emergence of the Delta variant, we are not able to test these questions directly. Nevertheless, the results of our study highlight the value of community-based research including the full spectrum of SARS-CoV-2 infection for investigating variation in immune response across infected individuals with differential symptomatology. Continuing to examine these dynamics with attention to symptom severity, as well as vaccination history and vaccination responses, can further inform public health strategies to control impacts of SARS-CoV-2 infection.Our findings are also relevant to questions concerning the degree of immune protection experienced by individuals who have received a COVID-19 vaccination following SARS-CoV-2 infection. Individuals with previous symptomatic or asymptomatic COVID-19 infection have been shown to have higher neutralizing antibody titer responses to a single dose of mRNA vaccine than those who were not previously infectedSupplementary Information."}
{"text": "Synedra, fungal microparasite Zygophlyctis, and co-growing bacteria), and even \u226517-fold in field-sampled populations . Additional data obtained using the Synedra\u2013Zygophlyctis model system reveals that fungal infections reduce the formation of aggregates. Moreover, carbon respiration is 2-fold higher and settling velocities are 11\u201348% lower for similar-sized fungal-infected vs. non-infected aggregates. Our data imply that parasites can effectively control the fate of phytoplankton-derived organic matter on a single-cell to single-aggregate scale, potentially enhancing remineralization and reducing sedimentation in freshwater and coastal systems.Phytoplankton forms the base of aquatic food webs and element cycling in diverse aquatic systems. The fate of phytoplankton-derived organic matter, however, often remains unresolved as it is controlled by complex, interlinked remineralization and sedimentation processes. We here investigate a rarely considered control mechanism on sinking organic matter fluxes: fungal parasites infecting phytoplankton. We demonstrate that bacterial colonization is promoted 3.5-fold on fungal-infected phytoplankton cells in comparison to non-infected cells in a cultured model pathosystem (diatom  Fungal parasites are found to effectively control the fate of phytoplankton-derived organic matter, potentially enhancing remineralization and reducing sedimentation in freshwater and coastal systems. Most of this decaying organic matter is remineralized within the euphotic zone, but a critical fraction gets exported to deeper layers as sinking particulate material in diverse systems, including freshwater4 and coastal environments6. Particulate organic matter is thereby transported through the water column towards sediments\u2014a mechanism that drives the bentho\u2013pelagic coupling, i.e., the exchange of energy, mass, and nutrients between pelagic and benthic habitats. Sinking organic matter thus sustains life below the euphotic zone7, while, on the other hand, it can also lead to expanding oxygen-deficient waters worldwide in areas where oxygen consumption through organic matter remineralization exceeds the available oxygen supply10. The fate of phytoplankton-derived organic matter is, therefore, key to aquatic food webs and biogeochemical cycles.Phytoplankton grows fast, renewing its biomass once per week and leaving behind several gigatons of decaying organic matter in the water column of the aquatic biosphere11. In this regard, sinking aggregates are primary vehicles of (in-)organic matter to depth12. The formation, remineralization, and sedimentation of aggregates are commonly explained by physical and biological processes, the latter being intimately linked to zooplankton grazing, bacterial degradation, and viral lysis16. Recent data, however, suggests that we are still missing some crucial links in the network of biological control mechanisms on vertical organic matter fluxes17. Aquatic fungi, for instance, are hardly considered in this context although they are abundant and active across various aquatic systems19, reaching from high-mountain lakes20 over coastal areas21 to the deep sea22. For instance, members of the fungal division Chytridiomycota, referred to as chytrids, can thrive as microparasites on phytoplankton cells. These chytrids are traditionally well documented in lakes23, especially during bloom events when host abundances are high25. More recently, they have also gained increasing attention in coastal systems after being observed via microscopy, for instance, in the highly productive upwelling region off Chile26, in the Arctic Ocean28, and during harmful algae blooms in the Mediterranean Sea29. Using high-throughput sequencing methods, Chytridiomycota have also been detected in other marine regions34, and DNA-based abundances of Chytridiomycota have been correlated to phytoplankton blooms or chlorophyll in coastal regions38. Chytrids thus occur frequently in freshwater and coastal regions where a strong benthic\u2013pelagic coupling is commonly assumed39, whereas in open ocean regions, their distribution is less evident with rare observations so far40.After blooming, phytoplankton cells together with fecal pellets and other detritus can coagulate as aggregates\u2014referred to as lake or marine snow\u2014which sink rapidly with velocities of up to several hundred meters per day24 or even 80\u2013100% of their specific phytoplankton host population43, including diatoms, dinoflagellates, and cyanobacteria. They thereby alter the dynamics of phytoplankton populations44 and are presumed to impact organic matter remineralization and sedimentation45. In a natural lake, chytrid-infected phytoplankton has been shown to mainly decompose in surface waters, whereas non-infected cells settled to depth41. It has further been estimated that 20\u201325% of the photosynthetically derived carbon from the infected host population is channeled to parasitic chytrids 46 and further to zooplankton (through the mycoloop)47. Chytrids have therefore been proposed to be an integral part of aquatic food webs and element cycling48. Yet, their impact on the fate of phytoplankton biomass in terms of aggregate formation and vertical organic matter fluxes remains unknown.Parasitic chytrids infect up to 1\u201344%50 and often mediate high proportions of the particle export52, are highly susceptible to fungal infections in freshwater and coastal systems44. We thus used one of the few available diatom\u2013parasite model pathosystems (freshwater diatom Synedra and chytrid Zygophlyctis)53 to investigate the impact of fungal infections on the formation and characteristics of sinking diatom aggregates and associated organic matter fluxes. We hereby considered cell aggregation, mass density, settling velocity, sticky polymers, bacterial colonization, and respiration\u2014parameters that are all intimately linked to organic matter remineralization and sedimentation processes. Moreover, we complemented these culture-based investigations with analyses of bacterial abundances and infection prevalence in aggregates that were formed from field-sampled phytoplankton communities. Our results demonstrate that epidemics of fungal microparasites need to be appreciated as an episodically impactful control mechanism on organic matter fluxes in the aquatic biosphere.Diatoms, which form fast-sinking aggregatesSynedra sp. and chytrid Zygophlyctis planktonica pathosystem are indicated as model system and data from the field-sampled populations as natural systems.Data obtained from the cultured diatom Synedra and co-growing bacteria. The designated infected treatment additionally included the parasitic chytrid Zygophlyctis, which developed Synedra-associated sporangia and free-living zoospores, both being part of the life cycle of this fungal microparasite53. One set of both treatments (each in triplicates) was grown and sub-sampled over a growth period of nine days, to monitor the culture development (referred to as culturing). An additional set was grown for six days and thereafter transferred into rotating cylinders, to analyze the formation and characteristics of aggregates (referred to as rotating cylinders). Symbols, terms, and abbreviations used in the following are summarized in Supplementary Box\u00a0We incubated non-infected and chytrid-infected diatom co-cultures\u2014hereafter non-infected and fungal-infected treatment, respectively. Both culture treatments included the freshwater pennate diatom Synedra cells  on maturely-infected cells. Post-infected Synedra cells displayed remains of chitinous cell walls as a sign of previous infections after zoospore discharge. Decaying cells displayed no chlorophyll autofluorescence and also no signs of previous infections, and thus, they had most likely not undergone any fungal infection46.For cell enumeration, we distinguished non-infected, early-infected, maturely-infected, post-infected, and decaying lls Fig.\u00a0. Non-infSynedra abundances increased from approximately 0.8\u2009\u00d7\u2009104 to 3\u2009\u00d7\u2009104 cells mL\u22121  reached 47% on day 6 (mostly maturely-infected cells) and remained at the same percentage until day 9 , with similar chlorophyll a contents in both treatments during day 0\u20132  but significantly lower chlorophyll a contents in the infected vs. non-infected cultures during day 4\u20139 .During the 9-day growth period, total L\u22121 Fig.\u00a0, with si56. Photometrically-analyzed TEP concentrations increased until day 6 and decreased thereafter in both treatments . TEP concentrations were significantly higher (P\u2009<\u20090.001) on day 2, but significantly lower on day 9  in the infected treatment in comparison to the non-infected treatment. On the other sampling days, no significant differences were detected between both treatments (P\u2009>\u20090.22). Similar to TEP concentrations, CSP concentrations increased until day 6 and decreased thereafter . Microscopy-derived numbers of TEP and CSP were highly variable, ranging from 119 to 3672 TEP mL\u22121  and from 966 to 21,196 CSP mL\u22121  in both culture treatments .Transparent Exopolymer Particles (TEP) and Coomassie Blue Stainable Particles (CSP) were determined microscopically and spectrophotometricallySynedra cells. On day 9, the abundance of diatom-associated bacteria was lowest in conjunction with non-infected Synedra cells (6.5\u2009\u00b1\u20094.2 bacteria diatom\u22121) and increased with increasing infection stage, from early-infected to maturely-infected, post-infected, and decaying Synedra cells . Consequently, diatom-associated bacteria were in total 3-times more abundant in the infected culture  and day 6 (P\u2009=\u20090.0001) in the infected culture. On day 9, however, the abundances of free-living bacteria were similar in both treatments  free-living bacteria as well as diatom-associated bacteria based on their association with (ii) non-infected, (iii) early-infected, (iv) maturely-infected, (v) post-infected, and (vi) decaying ure Fig.\u00a0. Abundanvs. non-infected Synedra treatments  were 38\u2009\u00b1\u200914-times less abundant  and comprised 46\u2009\u00b1\u200924-times less cumulative aggregate volume  in the fungal-infected Synedra treatment. Thereafter, also large aggregates (>1.3\u20135.6\u2009mm) became abundant, and both size classes (d\u2009=\u20090.4\u20131.3\u2009mm and >1.3\u20135.6\u2009mm) were 6\u2009\u00b1\u20094-times less abundant (range: 2\u201314-times) and comprised 9\u2009\u00b1\u20097-times less cumulative aggregate volume  during fungal infections. Aggregates within the smallest size bin (d\u2009=\u20090.35\u20130.46\u2009mm) were most abundant  accounted for most of the cumulative aggregate volume 57, was significantly higher for non-infected aggregates than for infected aggregates  was significantly different between non-infected and infected aggregates . This indication, however, was not confirmed by similar porosities of non-infected and infected aggregates , but carbon-specific respiration rates were on average 2-fold higher for fungal-infected aggregates as compared to non-infected aggregates 63. Due to the slower settling velocities and higher respiration rates, infected aggregates were estimated to respire 2.4\u20123.7-times more carbon per meter settled than non-infected aggregates  and diatoms . Chytrid infections were highest on Planktothrix, Synedra, and Fragilaria cells, and thus, we inspected these taxa in more detail 24. Bacterial abundances were \u226517-times higher on infected cells than on non-infected cells .We sampled a natural phytoplankton community in a temperate lake (Lake Stechlin), which comprised mostly filamentous cyanobacteria , but the stickiness, i.e., the probability that two cells coagulate upon encounter, was presumably reduced during fungal infections. The stickiness of cells and particles can be increased by the presence of exopolymers like TEP and CSP, which are broadly classified as acidic polysaccharides and alkaline amino acids, respectively89. TEP concentrations showed significantly different dynamics over time in our non-infected vs. infected Synedra culture  fewer freely-suspended polymers, reducing the overall aggregation, but (2) more adhesive polymers at the cell surface of infected Synedra cells, enhancing their cell-to-cell stickiness. Indeed, the observed faster growth of bacterial populations in the infected treatments  than the field-sampled cells (Lake Stechlin), and their morphologies were different . The different aggregation patterns between cultured and field-sampled Synedra may thus have resulted from genera-specific differences (involving diatom-specific bacteria interactions) or even strain-specific differences, as known from viral infections on phytoplankton95. Moreover, in culture, post-infected Synedra cells showed a higher aggregation potential than early-infected and maturely-infected cells  as compared to siliceous frustules (1.82\u2009g\u2009cm\u22123)97. The lower mass density of infected Synedra cells and aggregates in comparison to non-infected cells and aggregates may further be explained by more cell-associated polymers (such as TEP), fewer cells per aggregate, or thinner silica frustules as bacteria are known to dissolve silica98. Following Stokes\u2019 law (Eq.\u00a0d\u2009=\u20091\u20135\u2009mm) sank 11\u201348% slower than non-infected aggregates. And secondly, the herein-measured lower mass densities (measured in a density gradient) and lower excess densities (Eq.\u00a0vs. non-infected aggregates would both result in ca. 30% slower settling velocities of infected aggregates (following Stokes\u2019 law), similar to the size~settling velocity regression. We thus conclude that host-associated sporangia increased the cell size of diatom cells but reduced their mass per volume (g cm\u22123), slowing down their settling. Reduced aggregate formation and hence, smaller aggregates during fungal epidemics may have an even stronger impact on sinking velocities. For example, maximum aggregate diameters were 1\u2009mm in the non-infected treatment but only 0.5\u2009mm in the infected treatment after 6\u2009h of aggregate formation. Such a 2-fold decrease in aggregate diameter led to 3-fold slower sinking velocities  would have lost 37% and non-infected aggregates only 12%  of their initial carbon content after settling 50\u2009m, indicating a substantial reduction in export efficiencies due to fungal infections.Aggregates that comprised 71% fungal-infected cells respired twice as much of their carbon content per day as compared to non-infected aggregates with similar size and POC content Fig.\u00a0. We presies Fig.\u00a0, we roug44, including productive upwelling regions26, commercial mass cultures101, and areas impacted by harmful algal blooms29, their epidemics thus have the potential to diminish the strength and efficiency of vertical organic matter fluxes in natural and engineered aquatic environments.Taken together, our incubations revealed that fungal infections decreased the chlorophyll content, cell mass density, cell aggregation, aggregate size, and settling velocity, but increased bacterial colonization and carbon respiration on a single-cell to single-aggregate scale Fig.\u00a0. As a reSynedra sp. Ehrenberg, 1830  and the parasitic chytrid Zygophlyctis planktonicum (strain SVdW-SYN-CHY1), including host-associated sporangia  and zoospores  isolated from lakes in Northern Germany53. Bacteria were co-isolated with the diatom and chytrid and maintained in co-culture in a nutrient-replete medium for several months, which likely selected for copiotrophic taxa46. Chytrid infections on the diatom host were initiated by free-swimming zoospores, which attached to a host cell, encysted, and developed into epibiotic sporangia while penetrating and digesting the host\u2019s interior through a rhizoidal system53. Within the (zoo-)sporangium, the next generation of zoospores was formed and finally released through the rupture of the sporangium. One infection cycle took 1\u20132 days. Each infection was lethal and prohibited further reproduction of the host.The model pathosystem comprised the pennate diatom host \u22121m\u22122 during the 16-h light phase. Synedra was grown as 12\u2009\u00d7\u2009650\u2009mL in 1\u2009L Erlenmeyer flasks until reaching ca. 7500 cells\u2009mL\u22121. Half of those flasks (6\u2009\u00d7\u2009650\u2009mL) were thereafter inoculated with a Synedra\u2013Zygophlyctis co-culture , including mature fungal sporangia, which were projected to discharge new zoospores within the next hours (infected treatment). The resulting infection prevalence was 9% in the infected treatment on day 0. The other 6\u2009\u00d7\u2009650\u2009mL flasks remained without Zygophlyctis (non-infected treatment). To monitor the culture development over time, 6\u2009\u00d7\u2009650\u2009mL (three per treatment) were grown and sub-sampled for nine days (see culture characteristics). The remaining 6\u2009\u00d7\u2009650\u2009mL (three per treatment) were grown for six days and subsequently transferred into rotating cylinders to follow the formation of aggregates (see formation and characterization of sinking aggregates). Erlenmeyer flasks were gently shaken by hand once per day for cell resuspension and water mixing.Batch cultures were grown in CHU-10 medium  after 0, 2, 4, 6, and 9 days. CHU-10 medium served as a blank for all analyses.a and nutrients analyses, 3\u2009mL were transferred into 15\u2009mL Falcon tubes and centrifuged at 3000\u2009rpm for 20\u2009min at 4\u2009\u00b0C. The supernatant was gently removed and the remaining pellet was extracted in 90% acetone, frozen at \u221220\u2009\u00b0C, and analyzed after one month using a fluorometer . Concentrations of chlorophyll a were calculated and corrected for phaeopigments, measured after acidification103. Calibration was done with a chlorophyll a standard . Concentrations of nitrate/nitrite and soluble reactive phosphorous were determined from 0.45\u2009 \u00b5m-filtered water by flow injection analysis (FIA) and spectrometric detection . Concentrations of dissolved organic carbon (DOC) were analyzed from GF/75-filtered water on a TOC-V CPH with a nondispersive infrared sensor  using combustion catalytic oxidation.For chlorophyll Synedra cells were counted in Uterm\u00f6hl plankton chambers  under an inverted epifluorescence microscope . The chitinous cell walls of sporangia were stained with Calcofluor White 104. Synedra cells were differentiated as (i) non-infected, (ii) early-infected, (iii) maturely-infected, (iv) post-infected, and (v) decaying cells  and Wheat Germ Agglutinin 104. Bacteria were counted under a fluorescence microscope (Leitz Leica DMRB) at x1000 magnification. For each group and replicate, 20 Synedra cells (for attached bacteria) or 20 counting grids (for free-living bacteria) were examined, to reach representative mean values (standard error \u22645%).For bacterial abundance analyses, 0.5\u20131\u2009mL were preserved with paraformaldehyde , filtered onto polycarbonate filters , and stored at \u221220\u2009\u00b0C. Prior analyses, filters were stained with 4\u2032,6-diamidino-2-phenylindole  onto PC filters . TEP filters were stained with Alcian Blue  for 5\u2009s, and CSP filters with Coomassie Brilliant Blue G  for 30\u2009s. After staining, filters were rinsed with MilliQ to remove excess dye and stored at \u221220\u2009\u00b0C. Sterile-filtered water (0.2\u2009\u00b5m) from the cultures was used as blank. For spectrophotometric analyses, AB was extracted in 80% H2SO4 and CBB-G in 3% SDS in 50% isopropyl alcohol, as specified in Supplementary Methods\u00a0\u22121) and CSP concentrations relative to bovine serum albumin .Transparent Exopolymer Particles (TEP) and Coomassie Blue Stainable Particles (CSP) were determined microscopically and spectrophotometrically105. Images were corrected for uneven light intensities in the background and split into separate RGB channels. The blue channel was subtracted from the red channel, to remove chlorophyll-related areas and outlines of diatoms cells. The obtained 8-bit greyscale images were reversed in color and a global threshold was applied. Automated particle analyses in ImageJ provided the number of particles per filter area  and the cross-sectional area A of each particle . Particles with ECD\u2009<\u20093\u2009\u00b5m were not included. Since the automatic TEP and CSP recognition was not perfect, the processed images were compared visually with the original images, and falsely outlined TEP or CSP were manually removed from the data set. Cell outlines and the infection stages were poorly visible on our images, and thus, we removed all cell-associated TEP and CSP outlines to not introduce any bias. Only freely-suspended TEP and CSP were therefore included in the microscopy analyses, whereas the spectrophotometric analyses included freely-suspended and cell-associated TEP and CSP.For microscopic analyses, the stained filters were placed onto CytoClear slides and inspected under a microscope . For each filter, 40 images were taken randomly along two transects across the entire filter area. Care was taken to use the same microscope and camera settings during the entire image acquisition. Image analyses were done in ImageJ 1.51p\u22123). The viscous layers were prepared with mixtures of Ludox TM colloidal silica , sucrose, and distilled water49. After 12\u2009h and an additional centrifugation run , cells had settled into the density layer that was equivalent to the mass density \u03c1s-cells of their particulate material when hydrated in liquid 57. The intact density layers (visible to the naked eye) were sub-sampled into 2\u2009mL tubes and stored at 4\u2009\u00b0C. Half of each sample was analyzed for its mass density \u03c1s-cells (g cm\u22123) using a density meter (Anton Paar DMA 38). The other half was used to enumerate non-infected and infected diatom cells (after CFW staining) under an inverted fluorescence microscope with combined bright-field and UV excitation .To analyze the mass density of diatom cells (on day 6), 10\u2009mL were transferred into 15\u2009mL Falcon tubes and centrifuged . The supernatant was gently removed, and cells were re-suspended in 1\u2009mL CHU-10 medium and transferred onto a density gradient, consisting of six viscous layers with increasing density from top to bottom (1.18\u20131.38\u2009g\u2009cmN\u2009=\u20093 flasks) in the infected treatment. Thereafter, the cultures were diluted with CHU-10 medium to approx. 5000 cells mL\u22121 (resembling cell abundances during bloom scenarios87) and transferred into rotating cylinders  to mimic the aggregation of cells due to their stickiness and differential settling behavior  but their physical properties such as density, porosity, and composition are comparable to natural aggregates108. Initial diatom abundances were similar in both treatments . Cylinders were rotated at 1\u20132\u2009rpm at 17\u2009\u00b0C in darkness on a roller table. The aggregate formation was recorded over time using a Mini Deep-focus Particle Imager . The MDPI is a \u2018shadowgraph imager\u2019 using a near-infrared LED light source  behind a pinhole and a set of identical plano-convex collimator lenses to create parallel light beams between the LED light pod and the camera pod . The sampling volume of two image sequences covered 2\u2009\u00d7\u20090.35\u2009L, equal to 30% of the entire cylinder volume.Triplicates of non-infected and fungal-infected cultures were grown for six days until the infection prevalence had reached 59\u2009\u00b1\u20093% . We counted the number of aggregates per size class, reported as aggregate number concentration N(d) (# L\u22121). To describe the number concentration as a function of size, the aggregate size spectrum n(d) (# L\u22121 mm\u22121) was calculated as\u2206d (mm) is the width of each size class 110. The volume spectrum nVd was calculated by normalizing the volume distribution to the aggregate sizeVagg (mm3) is the average volume of single aggregates in each size class111. Vagg and d  were calculated from A derived from the image analyses and assuming spherical geometry. After stopping the rotation, individual aggregates were picked with a wide-bore glass pipette for various analyses. To ensure that aggregate sizes were similar for non-infected and infected aggregates, aggregates were sampled after 30\u2009h from the non-infected treatment and after 45\u2009h from the non-infected treatment . Aggregates were counted, measured, and binned into size classes, in which the upper diameter was 1.3-times the lower size class. The resulting eleven size classes ranged from 0.4 to 5.6\u2009mm 57. We measured the settling velocity U of aggregates in a settling column . The column was filled with the same water as the rotating cylinders and kept at the same temperature (17\u2009\u00b0C). It was double-walled, with a 2\u2009cm interspace filled with water and a sealed top, except for a 2\u2009cm inlet to introduce the aggregates. This setup minimized convective currents in the column during settling experiments112. Each aggregate was allowed to freely settle from the wide-bore pipette into the water. The settling was timed along 15\u2009cm using a stopwatch. Excess densities \u2206\u03c1 of aggregates were calculated using Stokes\u2019 law, which applies to ballasted phytoplankton aggregates113\u03bd is the kinematic viscosity (1.085\u2009\u00d7\u200910\u22122\u2009cm2\u2009s\u22121 at 17\u2009\u00b0C) and g the gravitational acceleration (981\u2009cm\u2009s\u22122). The drag coefficient CD and Reynolds number Re derived fromSet I was used to determining the size, porosity, excess density, settling velocity, and fractal dimension. Aggregates were imaged directly after sampling using a compact camera . The cross-sectional area D3. D3 was derived from the slope of d and U after log\u2013log transformation  respiration rates by assuming a respiratory quotient of 1.2\u2009mol O2 to 1\u2009mol CO2115 and by normalization to the aggregate-specific POC contents. Shifts in oxygen concentrations due to, e.g., temperature changes, were tested in control vials with 0.2\u2009\u00b5m-filtered water but without aggregates. The observed shift in oxygen concentration was significantly lower in the control vials than in the vials with aggregates . After incubations, aggregates were filtered individually onto pre-combusted GF/F filters  and stored at \u221220\u2009\u00b0C for later POC analyses. After storage, filters were fumed over HCl and dried at 50\u2009\u00b0C overnight. POC was analyzed after combustion using a carbon analyzer . Carbon respiration rates are given in the units d\u22121 and % m\u22121  analyses. Single aggregates were transferred into 5.9\u2009mL gas-tight Exetainer\u00ae vials with 0.2\u2009\u00b5m-filtered water from the rotating cylinders. Oxygen concentrations were measured with a Clark-type oxygen microsensor  immediately after aggregates were added and after 24\u2009h-incubations at 17\u2009\u00b0C in darkness. During incubations, the Exetainer rotated to keep the aggregates freely suspended and to minimize diffusion-limited gas exchange at the aggregate surfacem\u22121 Fig.\u00a0, which dfit Fig.\u00a0 and meanfit Fig.\u00a0.Synedra populations was analyzed in Lugol-preserved samples (x400 magnification), whereas abundances of Synedra-associated bacteria were analyzed in PFA-preserved samples (x1000 magnification). Aggregates were disaggregated via gentle shaking in 2\u2009mL microcentrifuge tubes prior to filtration and/or counting, to be able to inspect individual Synedra cells under the microscope. The number of aggregate-associated bacteria (per aggregate) was calculated by multiplying the number of associated bacteria per Synedra-cell type with the abundance of each Synedra-cell type per aggregate. We assumed 20,000 Synedra cells per aggregate since we counted on average 22,843\u2009\u00b1\u200934,443 Synedra cells per aggregate with d\u2009=\u20092.1\u2009\u00b1\u20090.8\u2009mm (N\u2009=\u200918).Set III and IV served to determine the infection prevalence and bacterial abundance. Single aggregates and 1\u2009mL of the ambient water were sampled and inspected under the microscope, as described above (see section Culture characteristics). In short, the infection prevalence in \u03c1s-agg. Single aggregates were gently transferred on top of a density gradient. The further procedure resembled the protocol used for \u03c1s-cell, i.e., after centrifugation and settling, the layer containing the aggregate was sampled and analyzed for its mass density .Set IV was used to derive the mass density of single aggregates d\u2009=\u20092\u20134\u2009mm) were formed. Individual aggregates were sampled with a wide-bore glass pipette, and sub-volumes of the ambient water were sampled with a plastic syringe. Ten replicates (10x aggregates and 10\u2009\u00d7\u20092\u2009mL ambient water) were sampled from each cylinder and split in half. One half was preserved for determining the infection prevalence on single phytoplankton taxa, and the other half for counting cell-associated bacteria. Sample preservation and microscopy analyses were done as described above (see section Culture characteristics).A plankton community was sampled from Lake Stechlin (Northern Germany) during the spring bloom in April 2018. Cells were concentrated from 0\u201310\u2009m using a hand-held plankton net , resuspended in 10\u2009L of in situ surface water, and transferred into three rotating cylinders. Cylinders rotated at 7.5\u2009\u00b0C  for 12\u2009h in darkness until macroscopic aggregates . Normal distribution was tested using the Shapiro-test and data variance with the F-test. Statistical differences between multiple groups were determined with the Kruskal\u2013Wallis test . Statistically significant differences between time series data were calculated based on generalized linear mixed models . Data were log-transformed if the assumption of homogeneity of variance was rejected . Pair-wise comparisons in time series data were run with emmeans (v. 1.7.2 in R). Statistical tests and plotting were done in R 3.3.0116 and Origin2021. Statistical tests were run two-sided. The significance level was 0.05. P values at very highly significant levels are given as <0.001 or <0.0001. Uncertainties (\u00b1sd) that derived from combined uncertainties of single variables were calculated following the laws of error propagation. The number of replicates is indicated for each mean value in the results section.Pairwise comparisons between mean values were calculated with the Mann\u2013Whitney test for non-normally distributed data, the Welsh-test for normally distributed data with non-equal variance, and the Further information on research design is available in the\u00a0Peer Review FileSupplementary MaterialsDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary"}
{"text": "EM) population and restricting the expression of tumor suppressors in a preclinical model of spontaneous colitis-associated colorectal cancer (CAC). We show that IFN-\u03b3 expression is significantly increased both in the T cells and the colonic mucosal epithelia of mice with a T cell-restricted deletion of the TGF-\u03b2 intermediate, SMAD4 (Smad4TKO). The increase of IFN-\u03b3 expression correlates with the onset of spontaneous CAC in Smad4TKO mice by 6 months of age. This phenotype is greatly ameliorated by the introduction of a germline deletion of IFN-\u03b3 in Smad4TKO mice . DKO mice had a significantly reduced incidence and progression of CAC, and a decrease in the number of mucosal CD4+ TEM cells, when compared to those of Smad4TKO mice. Similarly, the colon epithelia of DKO mice exhibited a non-oncogenic signature with a decrease in the expression of iNOS and p-STAT1, and a restoration of the tumor suppressor gene, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In vitro, treatment of human colon cancer cells with IFN-\u03b3 decreased the expression of 15-PGDH. Our data suggest that Smad4-deficient T cells promote CAC through mechanisms that include an IFN-\u03b3-dependent suppression of the tumor suppressor 15-PGDH.Immune cells and the cytokines they produce are important mediators of the transition from colitis to colon cancer, but the mechanisms mediating this disease progression are poorly understood. Interferon gamma (IFN-\u03b3) is known to contribute to the pathogenesis of colitis through immune modulatory mechanisms, and through direct effects on endothelial and epithelial homeostasis. Here we explore whether IFN-\u03b3 influences tumor progression by expanding the effector memory T cells (T Colorectal cancer (CRC) is the third most commonly occurring cancer and is the second most common cause of cancer-related deaths in the world , 2. Glob+\u00a0T lymphocytes  develop chronic inflammation in the intestinal mucosa, a phenotype associated with spontaneous CAC (+ effector memory T (TEM) cells (Transforming growth factor-beta (TGF-\u03b2) is a pleiotropic cytokine with important functions for the maintenance of immune homeostasis and is one of the key molecules regulating epithelial cell biology and immunity in the gut . Inadequeous CAC , and theM) cells . These rTKO model (The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is an enzyme responsible for the degradation of PGE2 into an inactive metabolite . 15-PGDHKO model . The synKO model . While TKO model , 33, infKO model .In vitro IFN-\u03b3 treatment suppresses tumor suppressor 15-PGDH in cultured colon cancer cells. Smad4TKO mice exhibit mucosal epithelial hyperplasia that is accompanied by increased inflammation in colonic mucosa and a significant reduction in the expression of 15-PGDH in colon epithelial cells. Germline deletion of IFN-\u03b3 in Smad4TKO mice  significantly reduces the incidence and progression of CAC via a decrease in the population of CD4+ TEM cells and a restoration of 15-PGDH expression in colon epithelia, when compared to Smad4TKO mice. These data demonstrate the direct link between alterations of TGF-\u03b2 signaling in T cells and malignant transformation in epithelial cells in the gastrointestinal tract, through an IFN-\u03b3-dependent alteration in the expression of the tumor suppressor 15-PGDH.Here we report that in the absence of TGF-\u03b2 signaling in T cells, the production of proinflammatory cytokines is significantly increased and IFN-\u03b3 expression is down-regulated through a Smad4-dependent mechanism. Anti-phospho-Stat1 (Tyr701) (58D6), anti-phospho-Stat3 (Thyr705) (D3A7), anti-phospho-i\u03baB (14D4), anti-iNOS (D6B6S), and anti-PD-L1 (D5V3B) were purchased from Cell Signaling. Anti-CD3, anti-CD28, anti-CD25, anti-CD44, anti-CD62L, anti-IFN-\u03b3, IL-6, and anti-TNF-\u03b1 were purchased from BD Biosciences. Anti-Foxp3 antibody was purchased from eBioscience.SMAD4 gene  in mice has been described previously (KO) was purchased from The Jackson Laboratory. To generate mice deficient for both IFN-\u03b3 germ line and for Smad4 in the T cell lineage only, IFN-\u03b3KO mice were crossed with Smad4TKO mice. The resulting F1 heterozygotes were then bred to generate all genotypes, including Smad4TKO/IFN-\u03b3KO (DKO) mice. Mice were housed in a specific pathogen-free facility. All animal experiments were performed in accordance with institutional guidelines and with approval of the Institutional Animal Care and Use Committee at Case Western Reserve University.T cell-restricted deletion of the eviously , 32. TheThe colon was excised from the ileocecal junction to the anal verge, flushed with phosphate-buffered saline (Invitrogen) and opened longitudinally. Gross examination was performed to evaluate tumor size and number and to measure colon length and colon weight. The colon length to colon weight ratio was measured to assess thickening of the intestinal mucosa. The incidence , the mean tumor size \u00b1 standard deviation, and the mean number of tumors/mouse \u00b1 standard deviation were calculated for each group. Tumor size was determined by image analysis using imaging software (ImageJ). Images were taken with a scale bar and lengths were measured in pixels and correlated to the known distance in scale bars. Colon tumors as well as colonic tissues were processed for histopathological evaluation and further biochemical analyses.Serum Nitric oxide (NO) levels were measured by photometric analysis by using a nitrite/nitrate assay kit , according to manufacturer\u2019s instructions.-\u0394\u0394Ct, in which \u0394\u0394Ct equals \u0394Ct of the experimental sample  minus \u0394Ct of the control sample (WT mouse sample).Colon mucosa was obtained from scrapings of full-length colon and total\u00a0RNA was isolated using Trizol reagent (Invitrogen). For reverse transcription-PCR (RT-PCR), cDNA was synthesized using a High Capacity cDNA synthesis kit (Applied Biosystems). Quantitative RT-PCR was performed using BioRad CFX96 Real-Time System C1000 Thermal Cycler. The expression of target genes was normalized to expression of housekeeping gene \u03b2-actin. The relative gene level was expressed as 22.\u00a0FET cells were seeded in 12-well plates at 1 x 105\u00a0per well as triplicates and transiently transfected with 0.2\u00a0mg of SBE promoter vector or 2.5 kb 15-PGDH \u2013PGL promoter vector and\u00a020 ng of CMV-renilla using\u00a0LipofectAMINE Plus as transfection agent according to the manufacturer\u2019s instructions (Invitrogen). Approximately\u00a024 hrs\u00a0past\u00a0transfection, cells were treated with\u00a0IFN-\u03b3 (20 ng/ml) or IL-6 (20 ng/ml)\u00a0for 24 hrs in medium.\u00a0Luciferase activity was measured using Promega Dual Luciferase Assay Kit\u00a0\u00a0and a ML3000 Microtiter Plate Luminometer.\u00a0Data shown represent the mean of three independent experiments. For IFN-\u03b3 promoter luciferase activity, pCMV5 plasmid or pCMV5-Smad4 plasmid were transfected one day before transfection of 3.6 Kb IFN-\u03b3 promoter in 293T, Jurkat or primary murine T cells and 24 hrs after IFN-\u03b3-promoter transfection, luciferase activity was measured. For T cell activation, T cells were plated on the plate coated with anti-CD3/CD28.The\u00a0FET\u00a0human colon carcinoma cells were cultured in MEM (Invitrogen) with 10% fetal bovine serum (Germini) and glutamine (2 mg/ml) at 37 \u00b0C in 5% COFor Western blot, colon mucosa was obtained from scrapings of full-length colon and lysed by incubation in lysis buffer  on ice for 30\u00a0min. 20 \u03bcg aliquots of proteins were separated by electrophoresis in 10% SDS/PAGE minigels and transferred to nitrocellulose membrane (Invitrogen). Following blocking, membranes were incubated in buffer containing the primary antibody, followed by washing and incubation for 1hr at room temperature with horseradish peroxidase-conjugated secondary antibodies. Immunostaining was visualized by ECL.For hematoxylin and eosin staining (H&E), excised colons were washed with PBS and fixed in 10% formalin. Samples were embedded in paraffin wax, sectioned, stained with H&E, and examined by light microscopy. For immunohistochemistry (IHC), slides were deparaffinized and rehydrated and heat-induced epitope retrieval was performed prior to blocking with Peroxidazed 1  and Rodent Block M . Slides were incubated with primary antibodies, CD3 and PD-L1 for one hour at room temperature. Antibodies were detected using Rabbit-on-Rodent HRP polymer  and visualized using Betazoid DAB chromogen kit . The percentage of CD3 positive cells were assessed on six randomly selected field using digital eyepiece.LP immune cells were isolated using Mouse Lamina Propria Dissociation Kit (Miltenyi Biotec). Colon sections were washed clean, cut into small pieces, and incubated with 5 mM EDTA, 1 mM DTT (MilliporeSigma), and 5% FBS in HBSS buffer (Thermo Fisher Scientific) for 20 minutes. Then tissue was collected into gentle MACS C tube (Miltenyi Biotec) and dissociated to single cells using gentleMACS Dissociator (Miltenyi Biotec).Cell suspensions were prepared from spleens or colon lamina propria by filtering through nylon mesh (40-\u00b5m diameter). Erythrocytes were lysed using ACK lysis buffer (BioWhittaker) and cells were washed twice in RPMI 1640 supplemented with 10% heat-inactivated FBS, 50 \u00b5M 2-ME, penicillin, and streptomycin (Invitrogen). Viable cells were counted using trypan blue exclusion on a hemocytometer. All Antibodies used in FACS analyses were purchased from BD Pharmingen. Pan T cells were purified from spleen and lymph node using a Pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer\u2019s instructions (purity greater than 95%).in vitro by plate-bound anti-CD3 and anti-CD28 antibodies in 24-well plates in the absence or presence of TGF-\u03b2 for 72 hrs. The cells were harvested, washed with PBS, stained with anti-CD4 and CD8 antibodies, and stained for intracellular Foxp3 using a Foxp3 staining Kit (eBioscience) according to the manufacturer\u2019s instructions.For intracellular staining for cytokines, lymphocytes from spleen and colon lamina propria were activated with plate-bound anti-CD3 and anti-CD28 antibodies for 48 hrs and re-stimulated with PMA and ionomycin for the last 5 hrs in the presence of Golgi stop solution prior to washing and staining with antibodies for CD4 and CD8. Intracellular staining for IFN-\u03b3, IL-6 and TNF-\u03b1 was performed using an intracellular staining kit (BD Biosciences) according to the manufacturer\u2019s instructions. For inducible regulatory T cell assay, splenocytes from each genotype were activated p-value less than or equal to 0.05, with *p<0.05, **p<0.01, ***p<0.001.Data are expressed as means \u00b1 SE. Statistical significance was determined by 1-way ANOVA with Tukey\u2013Kramer Multiple Comparisons Test. The Fisher\u2019s Exact Probability test was used for comparison of the incidence of lesions between the two groups. Statistical significance was accepted to be a co/co;Lck-cre, Smad4TKO) leads to spontaneous mucosal inflammation, and epithelial cancers throughout the gastrointestinal tract. Smad4TKO mice develop CAC, with the majority of tumors arising after 6 months of age  cells (CD44HighCD62LLow) was significantly increased (78.74%) compared to wild type mice (35.29%)  cells in the colon of Smad4TKO was decreased (17.74%) compared with that of WT (27.71%) while there were no significant changes in the splenic Treg population  through a T cell lineage-restricted deletion of the Smad4 gene in mice . In this35.29%) , 35, 36,.29% , 35(35.29%) . In a T EM cells and the malignant transformation of colonic epithelium in Smad4TKO mice, we examined the expression levels of tumor suppressors in the colon of Smad4TKO mice. The expression level of the tumor suppressor 15-PGDH was significantly reduced in the colons of Smad4TKO mice, implying that soluble factors from the pathogenic TEM may be responsible  onto a genetic background with a germ line IFN-\u03b3 deletion (IFN-\u03b3KO) to generate a \u2018double knockout\u2019  model. DKO mice harboring the T cell-restricted deletion of the tumor suppressor SMAD4 and a germ line deletion of proinflammatory cytokine IFN-\u03b3 ameliorated CAC and inflammatory infiltration of the mucosa as early as 8 months of age, at which point the mortality rate of DKO mice began to decrease compared with the Smad4TKO mice. The survival rate of DKO was 75% at the age of 10 months, compared to 38% for Smad4TKO mice  in DKO was significantly smaller than that (2.3\u00a0mm) in Smad4TKO mice. Furthermore, tumor multiplicity was less than 1 tumor/mouse in DKO mice, whereas more than 4 tumor/mouse were found in Smad4TKO mice. DKO mice showed a similar phenotype as WT or IFN-\u03b3KO, with a delayed disease presentation (12 months) as compared with the Smad4TKO (8 months). These data clearly demonstrate that germ line IFN-\u03b3 deletion decelerates CAC development and tumorigenesis in the Smad4TKO mice. Histological analysis of intestinal sections from Smad4TKO mice revealed disrupted villus architecture with regions of epithelial atypia, as well as adenomas and invasive carcinomas as compared to the colon histology of the WT, IFN-\u03b3KO and DKO mice  cells in cancer is complex, and is often dysregulated within the TME  were validated by introducing the germ line deletion of the IFN-\u03b3 gene into Smad4TKO mice . Utilizing the DKO mouse model, we discovered that the CAC phenotype in Smad4TKO mice is linked to gain of IFN-\u03b3 expression in lymphocytes, with significant skewing of the mucosal CD4+ T cell repertoire toward an activated, effector memory phenotype. However, other types of cells, such as CD8 T cells, NK cells, and NKT cells cannot be ruled out as a potential important source of IFN-\u03b3 expression in this model system. Invariably, the colonic epithelium of Smad4TKO mice exhibited an inflammation-driven oncogenic signature that includes a significant suppression in the expression of 15-PGDH and an elevation in the expression of iNOS, p-Stat1 and p-Stat3.In this study we have demonstrated a role for IFN-\u03b3 production by Smad4-deficient pathogenic T cells in the pathogenesis of inflammation-related CRC in mice. Our data support the finding that IFN-\u03b3 abundance accelerates gastrointestinal epithelial malignancy by promoting epithelial cell transformation through suppressed production of the tumor suppressor 15-PGDH in the colonic epithelial cells as well as enhanced production of pro-inflammatory mediators by tissue resident CD4In vitro IFN-\u03b3 treatment of cultured colon carcinoma cells suppressed 15-PGDH expression  and concomitant suppression of 15-PGDH expression in epithelial cells. Importantly, strategies designed to modulate the expression and activity of soluble factors in both the epithelial and stromal compartment in the TME may serve to concomitantly support the maintenance of mucosal epithelial homeostasis and suppress the expansion of pathogenic, tissue-resident effector T cells producing inflammatory cytokines, thereby forming a unique and effective approach to cancer immunotherapy.To our knowledge, this is the first report describing dual, tandem mechanisms for tumor promotion by IFN-\u03b3, that include the expansion of pathogenic effector CD4The original contributions presented in the study are included in the article/The animal study was reviewed and approved by The Institutional Animal Care and Use Committee at Case Western Reserve University.SC, JL, and B-GK designed the studies and developed the methodology; SC and B-GK performed experiments; SC, AH, JL, and B-GK interpreted the data; SC and B-GK wrote the initial draft of the manuscript; SC, AH, JL, and B-GK edited the manuscript and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health grants (R01CA168586 and 1R03CA259901-01A1).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 SFthose 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": "Pathologies of the blood\u2013brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood\u2013brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic \u201ccheckpoint\u201d of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation. Changes in the extracellular milieu of the brain are reported during neurological disorders, including low pH and high extracellular potassium ,2. For eThe BBB is comprised of cerebral capillary endothelial cells, locked together by tight junction  and adherens junction  proteins to create a dynamic, highly selective permeable barrier between the blood and CNS ,25,26. D2 standard flasks  with Dulbecco\u2019s modified eagle media (DMEM) . DMEM was supplemented with 2 \u03bcM L-glutamine , 10% fetal bovine serum (FBS) , and 1% penicillin-streptomycin ). C8-D1A murine astrocytes  were cultured in 75 mm2 standard flasks with DMEM supplemented with 10% (FBS)  and 1% penicillin-streptomycin . Both cell lines were split upon reaching 80% confluence to prevent overgrowth. Cell culture flasks were then incubated in a 37 \u00b0C humidified incubator with 5% CO2:95% air atmospheric conditions. b.End3 murine immortalized endothelial cells  were cultured under sterile conditions in 75 mm2O, 148.19 mM NaCl, 3 mM KCl, 1.85 mM CaCl2, 1.71 mM MgCl2 1.80 mM NaHPO4, 229.20 \u00b5M NaH2PO4) in ACM at equivolume to 60 mM KCl was used as a control (vehicle); (2) ACM buffered to a pH of 6.8 to model release of H+ ions into the extracellular space at the CSD wave front, prepared by titrating ACM down to a pH of 6.8 \u00b1 0.05 with 12 M HCl ; (3) glutamate  dissolved in ACM at concentrations of 10 \u03bcM, 30 \u03bcM, and 100 uM; and (4) 60 mM KCl  dissolved in ACM, serving as a positive control. A total of 60 mM KCl treatments at relevant physiological levels were utilized as it is a typical condition used to evoke K+-ion-triggered spreading depolarization in live brain slices [b.End3 endothelial cells utilized in these studies were cultured in astrocyte conditioned media (ACM) for at least 24 h prior to any type of treatment, collection, or fixing. The usage of ACM for endothelial co-culture was integral to the functional culture of an endothelial barrier by supplying the critical growth factors and modeling the in vivo critical role of astrocytes in the maintenance of proper endothelial barrier function. ACM was produced in house by culturing fresh DMEM (Gibco 11995-065) cell culture medium supplemented with FBS  and penicillin 100 UI/mL-streptomycin 100 \u03bcg/mL  for 24 h with a confluent growth of C8-D1A mouse astrocytes. This media was aliquoted and frozen at \u221220 \u00b0C for use when needed. Culturing endothelial cells in ACM allows for the formation of a functional cell monolayer and tight junctions in vitro. To model a CSD event, cells were treated with a 5 min pulse of one of the following: (1) artificial cerebrospinal fluid (aCSF)  uptake assays [2:95% air atmospheric conditions. Upon the formation of a luminal monolayer on the Transwell insert, co-cultures were then used for downstream analyses.In vitro modeling of the BBB was performed on a Transwell monoculture system, utilized for TEER, e assays . b.End3 n = 3.The TEER technique utilizes measured changes in electrical resistance between two chambers filled with an aqueous solution and separated by a cultured cell barrier, with increased electrical resistance indicative of increased barrier integrity, as the free flow of ions between the chambers is prevented by the cellular barrier, manifesting as an increased electrical resistivity due to loss of electrical conductance between the chambers. a. b.End3TM fluorescent secondary antibodies  in 70% ethanol for one hour and air-dried them for 30 min under a UV lamp in a fume hood. Dried coverslips were placed in a 12-well plate and treated with a 20% collagen solution for two hours. Collagen solution was then removed by vacuum suction, and b.End3 cells were aliquoted in 80 \u03bcL volumes of DMEM media and incubated at 37 \u00b0C until the formation of a monolayer, after which a 24 h ACM incubation was initiated. Cells were then washed in 1\u00d7 PBS and treated with one of the following preparations: (1) vehicle (aCSF in ACM), (2) ACM buffered to pH = 6.8, or (3) 60 mM KCl in ACM. After treatment cells were washed with 1\u00d7 phosphatate buffered saline , then fixed with a 1% paraformaldehyde solution, permeabilized with 0.2% Triton X-100  in 1\u00d7 PBS for 10 min at room temperature, and blocked in a 10% bovine serum albumin (BSA) solution  with 0.1% Triton X100 for 1 h. Primary antibodies  prepared2:95% air to allow the formation of a cell monolayer, upon which abluminal media was replaced with ACM and another 24 h incubation initiated. After incubation treatment, the pulses consisted of the following: (1) vehicle (aCSF in ACM), (2) ACM buffered to a pH = 6.8, and (3) 60 mM KCl in ACM. Pulse treatments were aliquoted in triplicate into a new 24-well plate, with inserts transferred from the original culture plate to the treatment plate for 5 min of treatment. A 30 min time course was initiated, and abluminal media was collected at 5 and 30 min timepoints in new collection plates. Scintillation vials for radiolabel quantification were prepared with Optiphase Supermax cocktail  to act as a suspension agent. Radiolabeled 14C-sucrose  was prepared by suspending 100 \u00b5L stock 14C-sucrose in 10 mL of DMEM, and 50 \u00b5L of this preparation was assayed for the working range of a radioactive emission of 50,000 counts per minute (CPM) \u00b1 15,000. Once prepared, 14C-sucrose suspension was added to the luminal side of each insert, which was then immediately subjected to an abluminal treatment pulse, then transferred to a collection plate containing abluminal ACM. Abluminal media from the 5- and 30 min collection plates was then collected and aliquoted into a 5 mL scintillation vial , placed on a scintillation counter, and allowed to run overnight to capture CPM values. Samples were run in triplicate for each condition for n = 4. All radioactive material was disposed of according to University of Arizona regulations (RAM Protocol #698).b.End3 cells were seeded on the luminal side of Transwell inserts pretreated with 20% calf collagen and incubated at 37 \u00b0C with 5% CO2 for 72 h until the formation of an endothelial cell monolayer on the insert. Upon the formation of the monolayer, 1000 \u00b5g/mL of 4 or 70 kDa fluorescein isothiocyanate-dextran1  solution was prepared in DMEM. Once prepared, the FITC preparation was added to the luminal side of Transwell inserts and pulsed abluminally for 5 min with the following treatments: (1) vehicle (aCSF in ACM), (2) ACM buffered to pH = 6.8, and (3) 60 mM KCl in ACM. All abluminal treatment media was then removed and replaced with fresh ACM, and a 180 min time course for the experiment was initiated. Aliquots of 10 \u00b5L were then removed from abluminal wells of each insert at timepoints of 10, 20, 30, 60, 120, and 180 min, and diluted into 90 \u03bcL DMEM to allow for a working volume and placed into a black clear-bottom microplate for fluorescence reading. Fluorescence readings were obtained with a ClarioStar plate reader (BMG Labtech) at an excitation wavelength of \u03bb = 483 nm and emission wavelength of \u03bb = 530 nm. The experiment was repeated 4 times in triplicate for an overall n = 4.b.End3 cells were seeded onto the luminal side of Transwell inserts pretreated with 20% calf collagen. Inserts were cocultured with abluminal ACM and incubated at 37 \u00b0C in 5%:95% air CO2:95% air and grown to confluence. Growth media was then removed and replaced with ACM, and cells were incubated for an additional 24 h. Cells were removed, washed with 1\u00d7 PBS, and treated with a 5 min pulse of either (1) vehicle (aCSF in ACM), (2) ACM buffered to a pH = 6.8, and (3) 60 mM KCl in ACM. Treatments were removed and cells were then washed in 1\u00d7 PBS, and 200 \u03bcL of cell lysis buffer  containing 1% by volume of protease and phosphatase inhibitor cocktail  was added to each well and harvested via cell scraping. Lysed cell material was then added to a new 1.7 mL microfuge tube and centrifuged for 10 min at 13,000\u00d7 g at 4 \u00b0C. Supernatant was then transferred to a new 1.7 mL microfuge tube for protein quantification.b.End3 cells were seeded on 6-well culture plates and incubated at 37 \u00b0C with 5% COProtein quantification was performed using a Pierce BCA protein assay kit . Quantification standards were prepared from a 2 mg/mL Pierce BSA Standard  serially diluted for the generation of quantification curve. Albumin standards and cell lysate samples were pipetted into a microplate in duplicate, and working reagent was added to each sample, covered, and incubated at 37 \u00b0C for 30 min. Subsequently, an incubation plate was assayed for optical density (OD) on a BMG ClarioStar plate reader at \u03bb = 562 nm. OD values for BSA standards were used to generate a quantification curve, which was used to calculate the total protein concentration in each sample. Samples were then diluted to a concentration of 1 \u03bcg/\u03bcL in lysis buffer with 5\u00d7 Lane Marker Reducing Sample Buffer  and Dithiothreitol (DTT) . Samples were then frozen at \u221280 \u00b0C.\u00ae 20 Detergent  for 5 min, then blocked in 5% milk in 1\u00d7 tris-buffered saline  for 30 min. Primary antibodies were then added to the membrane (prepared in 5% BSA in 1\u00d7 TBS), and the membrane was incubated at 4 \u00b0C for 48 h on a rocker. Following incubation, primary antibodies  were loaded into a criterion gel electrophoresis cell, then filled with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-Page) running buffer . Wells were then loaded with 30 \u03bcg of previously prepared cell lysate samples in triplicate by treatment group. An electrophoresis cell was run at 150 V for 10 min, then 190 V for 40 min. Following electrophoresis, gels were removed from their plastic cassettes and placed into a membrane transfer apparatus with a nitrocellulose membrane  and filled with SDS transfer buffer . The voltage was set at 20 V and transfer was run for 120 min. Following the transfer, apparatus was disassembled and the nitrocellulose membrane was removed and washed 3 times in 1\u00d7 tris-buffered saline with 0.1% Tweentibodies  were remtibodies  at room 2O), and (3) one 3 min wash with 500 \u00b5L no-salt buffer (10 mM tris-HCl pH = 7.5 in H2O). Pellets were then eluted into 60 \u00b5L 2\u00d7 Laemmli dye  with 10% DTT, and heated to 95 \u00b0C for 10 min, cooled, and loaded onto a 10% SDS-PAGE gel for electrophoresis, then placed into a transfer apparatus with a nitrocellulose membrane for the transfer of proteins to the membrane. Following the transfer, the membrane was blocked in 5% milk, and incubated with anti CL-5 antibody at 1:500  vehicle (aCSF in ACM), (2) ACM buffered to pH = 6.8, and (3) 60 mM KCl in ACM. Treatments were removed, and cells were washed in 1\u00d7 PBS at pH 8.0 and chilled to 4 \u00b0C. NHS-SS (Succinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate) biotin linking reagent buffer from a Thermo Cell Surface Isolation Kit  was then added to cells and incubated at 4 \u00b0C for 25 min. A fresh aliquot of biotin buffer was added, and cells were incubated at 4 \u00b0C for another 25 min. Following incubation, the biotin buffer was removed, and cells were washed with pH = 8.0 buffered 1\u00d7 PBS. Cell lysis buffer was prepared in house with protease and phosphatase inhibitors added at a ratio 1:100 and added to cells. Cell lysate was then scraped off the culture plates and transferred to a 1.7 mL microfuge tube and incubated on ice for 1 h. Subsequently, incubation tubes were centrifuged at 14,000 RPM for 10 min at 4 \u00b0C and supernatant was pooled by treatment group into a new 1.7 mL centrifuge tube. Neutravidin beads from the Cell Surface Isolation Kit were then equilibrated in lysis buffer and added to the supernatant previously collected. Tubes containing supernatant and beads were then sealed with parafilm and incubated overnight at 4 \u00b0C on a rocker to allow for mixing. The following day, each tube with was re-aliquoted into a new 1.7 mL microfuge tube and centrifuged at 14,000 RPM for 5 s to pellet Neutravidin beads. After removing and freezing supernatant, Neutravidin beads were washed following the following protocol at 4 \u00b0C: (1) three 3 min washes with 500 \u03bcL of lysis buffer, (2) two 3 min washes with a high-salt buffer . The search variables used were as follows: 10 ppm mass tolerance for precursor ion masses, and 0.5 Da for product ion masses, trypsin digestion, maxima of two missed tryptic cleavages, variable modifications of phosphorylation of threonine, tyrosine, and serine, and oxidation of methionine. Scaffold software  was used to cross-correlate Mascot search results with X! Tandem software. Significance value was set at p \u2265 0.05. Ion intensity-based label-free quantification was performed using Progenesis QI for proteomics software . Raw files were imported and converted into two-dimensional maps with y axis defined as time and x axis defined as m/z, which was then followed by the selection of a reference run for alignment. The aligned runs were then used to create an aggregate data set containing all peak information from all samples, after which the data pool was narrowed down to only +2, +3, and +4 charged ions for further analyses. The top 8 most intense precursors of a given feature were grouped into a peak list of fragment ion spectra and exported in a Mascot generic file (.mgf) and searched against the Mus musculus SwissProt_2018_01 database utilizing Mascot software. The following search variables were used: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses, trypsin digestion, maxima of two missed tryptic cleavages, variable modifications of oxidation of methionine, and phosphorylation of serine, tyrosine, and threonine, 13C = 1. The data were collected into a Mascot .xml file and imported into Progenesis allowing for the assignment of peptides and proteins. Peptides with a Mascot ion score < 25 were not used for further analyses. Non-conflicting peptides and precursor ion abundance values were normalized using a reference run to perform protein quantification. A heat map of principal component analyses (PCAs) and unbiased hierarchal clustering analyses were performed in Perseus [b.End3 murine endothelial cells were treated with either a 24 h hormone treatment or pulsed with one of the CSD constituent substances, as previously described. b.End3 cells were then lysed post treatment and loaded into SDS-PAGE for separation by electrophoresis. A total of 200 \u03bcg of harvested cell lysate supernatant was separated on a 10% SDS-PAGE gel and stained for total protein presence with Bio-Safe Coomassie G-250 Stain. Lanes from the gel were separated and cut into six slices, which then underwent trypsin digestion, and resulting peptides were purified by C18 desalting performed as described by Kruse et al. . High-pe Perseus ,34.n = 4) underwent tryptic digestion and enrichment of phosphopeptides with sequential enrichment from metal oxide affinity chromatography, as per the manufacturer\u2019s instruction (Thermo Scientific). A Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer fitted with an EASY Spray source was used to perform HPLC-ESI-MS/MS in positive-ion mode, according to the manufacturer\u2019s protocol. NanoLC was performed using a Thermo Scientific UltiMate 3000 RSLCnano System with an EASY Spray C18 liquid chromatography column ; loading phase for 15 min at 0.300 mL/min; mobile phase, linear gradient of 1% to 34% buffer B in 119 min at 0.220 mL/min, followed by a step to 95% buffer B over 4 min at 0.220 mL/min, hold 5 min at 0.250 mL/min, and then a step to 1% buffer B over 5 min at 0.250 mL/min and a final hold for 10 min ; buffer A 5 0.1% formic acid/H2O; and buffer B 5 0.1% formic acid in 80% acetonitrile. All solvents utilized were liquid chromatography mass spectrometry grade. Xcalibur software  was used to acquire Spectra, and Progenesis QI  was used to perform ion-free intensity-based label-free quantification. .raw files were imported and converted into two-dimensional maps with y axis defined as time and x axis defined as m/z, which was then followed by a selection of a reference run for alignment. The aligned runs were then used to create an aggregate set containing all peak information from all samples, after which the data pool was narrowed down to only +2, +3, and +4 charged ions for further analyses, which were then grouped by treatment. A peak list of fragment ion spectra was generated and exported in Mascot generic file (.mgf) and searched against the Mus musculus SwissProt_2018_01 database, utilizing Mascot software with the following search variables: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses, trypsin digestion, maxima of two missed tryptic cleavages, variable modifications of oxidation of methionine, and phosphorylation of serine, tyrosine, and threonine, 13C = 1. Protein or peptide assignment was performed by importing the resultant Mascot .xml file into Progenesis, while peptides with a <25 Mascot ion score were not used further. Precursor ion abundance values for peptide ions were normalized to all proteins. Differences were assessed as significant if a difference between vehicle and treatment groups was p < 0.05 assessed with one-way analysis of variance (ANOVA). Consensus phosphorylation sequences were determined using iceLogo [b.End3 cells were cultured and treated as previously described. A total of 5 mg of protein lysate per sample  policy (NOT-OD-15-102), so that differences of 20% were detected with 80% power at a significance level of 0.05. Post-experimental data analyses were performed in GraphPad Prizm 7.0 (GraphPad Software). Unless otherwise noted, data were analyzed with either a two-way paired or unpaired Pronociceptive substances released at the wavefront of CSD events were screened to qualitatively assess the significant induction of a paracellular leak in the BEB. Substances found significant were then assayed for their functional impact on BEB with quantitative transport assays to assess the magnitude of the BEB breach and functional outcomes on BEB.p = 0.0010) and acidified media (** p = 0.0011), (p = 0.2941) and 100 \u03bcM ATP (** p = 0.0058) demonstrated no significance, and a gradual drop in TEER became significant 20 min following treatment, respectively. The rapid reduction in TEER values following KCl and acidified pH delayed the effect of ATP, and no effect from glutamate suggests a fast-acting mechanism regulating BEB functionality.The integrity of the BEB was assayed via TEER a,b to as0.0011), b, mainta14C-sucrose, 4 and 70 kDa FITC uptake assays were performed over a 30 and 180 min time course, respectively, to assess the functional alterations to the BEB as well as quantifying the magnitude of the BEB breach. Both 4 and 70 kDa FITC uptakes demonstrated no significant differences between treatments  and acidified pH treatment (**** p < 0.0001)  c, which  0.0001) d. This oOur previous in vivo studies indicated that the total detection of expressed TJ proteins CL-5 and OCC was unchanged in isolated microvessels following CSD induction . To detep = 0.0351) reducing and acidic pH (* p = 0.521) increasing CL-5 CTCF. These treatments had no significant effect on both ZO-1 and VE-CAD fluorescence. These observations suggest the rapid onset of apical protein relocalization in vitro.Alterations to the localization of the proteins CL-5, ZO-1, and VE-CAD were assayed to observe changes at the TJ (CL-5), AJ (VE-CAD), and intracellular compartment (ZO-1) of the endothelial cells following KCl and acidified pH insult a. Confocp = 0.0415), indicative of potential homeostatic stress response following KCl insult.The structural integrity of the endothelial cell TJ complex is dependent on intracellular actin cytoskeletal linkage to transmembrane TJ proteins, such as CL-5 and OCC via ZO-1, 2, and 3. Cells undergoing structural insult can be identified by the increased detection of filamentous actin (F-actin) vs. normal globular (G-actin) actin. FITC-conjugated phalloidin was used to stain f-actin filaments following KCl and acidified pH treatment a,b. FollCL-5 has recently been validated as a clinical biomarker for several CNS disorders; therefore, focus was placed on this protein to further characterize its utility as a tool for investigating alterations to TJ integrity under experimental conditions, and to further characterize the potential of clinical diagnostic use. Biotynylation cell surface protein isolation assays were utilized to quantify the changes in surface localization of CL-5 following KCl and acidified pH insult. p = 0.0231) was shown to increase the surface detection of CL-5 vs. vehicle  to the global proteome. p < 0.0544). Acidified pH downregulated 68 proteins and upregulated 160 proteins (p = 0.2286) and ZO-1 upregulated following KCl exposure (* p = 0.0207) (p = 0.0034) while downregulating Caza2 (p = 0.0006) and Caz2b (p = 0.0508). KCl exposure significantly downregulated Caza2 (p < 0.0001). Taken together, these data indicate that the loss of BEB integrity is due to a synergistic convergence of unrelated individual deleterious processes induced by KCl and acidified pH exposure to TJ proteins CL-5, ZO-1, and actin maintenance proteins AFAP1, Caza2, and Cap2b. Global proteome analysis of b.End3 cells post treatment detected a total of 7113 proteins, 6279 of which were identified as being statistically significant  D. Degrad 0.0207) D. Actin p-values to visualize the functional effect on pathways and processes related to TJ and cytoskeletal homeostasis  and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatics databases to assess the functional impact of protein expression changes detected in our analysis following KCl and acidified pH exposure. Separated by treatment group, significant proteins (\u03b1 < 0.05) were queried through the following three GO bioinformatic databases: biological processes (BPs), cell compartment (CC), and molecular function (MF). Additionally, the KEGG pathway database was screened, and positive hits were scored by fold enrichment (FE), with the top ten from each database listed, separated by treatment group, and graphed by FE score against log transformed -Log10 eostasis a,d. Dataeostasis D. These Phosphorylation of CL-5 is associated with increased permeability of the BBB, but it is unknown if CSD-like conditions induce these post-translational modifications (PTMs). Following treatment with vehicle, KCl, and acidified pH, cells were quantified for total phosphorylation enrichment and amino acid residue enrichment of phosphorylation sites by individual protein. A total of 25,546 total phosphorylation adducts were detected, of which 11,371 were deemed significant by 3-way ANOVA (\u03b1 = 0.05). When analyzed by treatment group, 237 proteins experienced phosphorylation following vehicle treatment vs. KCl and acidic pH A. A totap-values  as opposed to Evans Blue and the exclusive use of 70 kDa FITC. These differences in methodology may have contributed to the opposing findings contained herein. The variable detection of F-actin stress fibers following KCl and acidified pH insult was confirmed in the global proteomic analysis. The expression of actin-associated proteins AFAP1 and Caza2 and Cap2b were significantly altered following acidic pH exposure, and KCl heavily downregulated Caza2. Mirroring the treatment-dependent functional changes observed at the TJ, differential molecular mechanisms may underlie these changes. The findings from total proteome analyses following modeled CSD insult indicate a potential major homeostatic disruption to several key structural proteins in endothelial cells, evidenced by a significant alteration to the enrichment of actin-associated proteins AFAP1, Caza2, and Capzb, in addition to CL-5 and ZO-1 changes. The discrete manner by which each specific CSD constituent plays a role in these observations warrants further investigation into a potential PTM mechanism.These proteomic data seem to contrast with our previous report : the preData from this study demonstrate that acidic pH dynamically regulates more proteins and phosphorylation PTMs than KCl in murine brain endothelial cells centered on the downregulation of actin reorganization and capping, while upregulating responses to exocyst organization and negative regulation of membrane tubulation. Phosphorylation of proteins related to adheren junction\u2019s reorganization and localization was also downregulated by acidified pH. KCl globally facilitated microtubule formation by downregulating depolymerization at the signaling level by upregulating actin formation pathways. This treatment-dependent effect was further validated by the absence of overlap in total protein enrichment between treatments, suggesting changes in brain pH and K+ concentration increase BEB permeability via distinct mechanisms. Phosho-proteomic analysis indicated PTMs as a likely mechanism for divergent treatment response, as phosphorylation is a reversable and highly dynamic mechanism of protein regulation. ZO-1, a major linking protein between the cytoskeleton and TJ was significantly enriched in phosphorylated residues specific to each treatment resulting in similar functional outcomes. This functional convergence on ZO-1 from both treatments may also indicate a central regulatory role for ZO-1 in response to CSD-like insult; specifically when considering the critical function, it performs maintaining structural homeostasis. Therefore, similar functional outcomes due to divergent KCl and acidified pH phosphorylation sites on ZO-1 demonstrate a potential mechanism driving protein relocalization and the paracellular leak of the BEB.Low brain pH and high extracellular K+ during neurological disorders promote BEB/BBB paracellular leak by inducing TJ and actin cytoskeletal reorganization and functional alteration through independent mechanisms. The elucidation of these molecular mechanisms may be utilized as a \u201cdiagnostic fingerprint\u201d specific to a unique pathology potentially useful as a biomarker."}
{"text": "Restriction on hospital visits for COVID\u201019 infection control continues to have a significant negative impact on patients and their families. For a patient receiving palliative care, this social isolation may deteriorate their mental health. In such situations, home care could be a viable solution to this problem. In the COVID\u201019 pandemic, providing end\u2010of\u2010life care at home may allow patients to spend more time with their families, improve communication between them and their health professionals and reduce the possibility of hospital\u2010acquired infections. In an attempt to limit the spread of COVID\u201019 infection, hospitals and nursing homes have imposed visiting restrictions, which prevent patients from spending precious time with their loved ones in their final days of life. This separation might further worsen the mental health of both vulnerable patients and their family members.Home care could be a preferable solution to this problem. Home care is a form of care in which patients stay at home with the help of their families, nurses, care workers, doctors and other health care professionals. This form of care is actively practiced in some countries, including Japan, where reimbursement for home care is available, including for patients with cancer and palliative care needs.2A man in his late 80s living in Tokyo with his wife and two daughters experienced sudden abdominal pain in July 2020 and went to seek help in a nearby tertiary hospital. That same day, he underwent an emergency laparotomy and appendicectomy after being diagnosed with generalized peritonitis associated with the perforation of an appendiceal tumor. Once the diagnostic tests were conducted, the patient was confirmed to have Stage IV appendiceal cancer. Postoperative contrast\u2010enhanced computed tomography (CT) scan did not show any abnormal findings such as local recurrence, lymphadenopathy, or distant metastasis associated with cancer. However, histopathological tests revealed scattered lymphatic invasion in the sub\u2010serous layer. Accordingly, the patient was offered palliative care instead of invasive procedures such as surgery.Family members were informed about the severity of the disease highlighting the need for end\u2010of\u2010life care. However, due to COVID\u201019, the hospital imposed certain restrictions, which limited the number of visitors it can allow per patient. Only one family member was allowed to visit an inpatient, and only with approval from the hospital or physician. Accordingly, the family started seeking updates on the patient's condition through phone calls with the medical staff. However, family members were unable to comprehend all information clearly, leading to misunderstandings and even conflicts between healthcare staff and family members. For example, there was an incident where the family members thought that the intravenous drip was administered through the peripheral intravenous catheter, but later they found it was administered through the peripherally inserted central venous catheter. So, family members were having difficulty adjusting to such incidents. They were particularly concerned when hospital staff asked them not to call too often and were told that medical staff would contact them directly if anything happened to the patient.acute respiratory distress syndrome (ARDS) secondary to heart failure. The patient received well\u2010organized, proficient home\u2010based health care, and spent the final days of his life with his family, friends, and other loved ones.The patient's family was worried about home care initially because the hospital told them it would be difficult and expensive. However, considering the patient's desire to spend his final period at home with his family, along with the restriction on hospitals visits due to the pandemic, the patient and his family chose to receive professional home care services in November 2020. After his discharge, he started receiving home care where home care nurses visited twice a day and the home doctor visited once every 2\u00a0weeks. Moreover, two daughters assisted his wife as family caregivers, and professional caregivers provided clinical care through a publicly funded nursing care insurance scheme. The patient was able to spend ample time with their loved ones, which was not possible in the hospital. On weekends, his grandchildren and great\u2010grandchildren also visited him, and the whole family was able to spend quality time with the terminally ill patient. Moreover, communication between the healthcare professionals and the family was also enhanced. Nurses communicated on a daily basis, and the doctor in charge made house calls as needed. In April 2021, the patient passed away peacefully after experiencing 3This case illustrates the importance of home\u2010based end\u2010of\u2010life care for cancer patients, particularly during crises such as the COVID\u201019 pandemic, as it allows patients to spend quality time with their families along with optimal care if managed effectively. Typically, end\u2010of\u2010life care is provided around the globe in hospice or hospital, aiming to provide holistic, personalized care to patients and to support families and caregivers.In our case, the terminally ill patient was able to enjoy the comfort of his own home, cherish his lifetime memories, and spend quality time with his loved ones. Besides the psychosocial implications, we believe that there are several other reasons to choose home\u2010based end\u2010of\u2010life care during a COVID\u201019 pandemic. First, this would prevent communication problems between healthcare professionals and family members. Restrictions imposed due to COVID\u201019 have reportedly reduced the quality of communication between hospitalized patients and their family members, resulting in severe distress that may affect the quality of death and bereavement.However, home care also has its drawbacks. Previous studies have reported challenges related to delivering urgent care when necessary4The findings of this case study highlight that in crises situations such as COVID\u201019, home care could be considered as a viable option for terminally ill cancer patients.Sakamoto R, Bhandari D, Ozaki A, Yoshida M, and Tanimoto T wrote the manuscript. All authors conceptualized and designed the study and contributed to making critical revisions for improving the intellectual content of the manuscript.AO reports personal fees from Medical Network Systems, MNES Inc. TT reports personal fees from Medical Network Systems, MNES Inc. and Bionics co. Itd. Other authors declare no competing interests.Written informed consent was obtained from the patient's child.The patient is deceased and we obtained a consent form from the patient's child."}
{"text": "Endothelial cell density variation was 5.51%, 3.06% and 2.82% in groups A, B and C, respectively (p = 0.19). Total Endothelial Cell Loss (ECL) was 4.37%, 5.32% and 7.84% in groups A, B and C, respectively (p = 0.39). Endothelial cell morphology was comparable in all three groups. Conclusions: In the DMEK RAPID Mini, low temperatures (<2 \u00b0C) may affect the quality of pre-loaded grafts, inducing a higher ECL after 72 h of preservation, although no significant differences among groups could be proved. Our data would suggest maintaining grafts loaded in the DMEK RAPID Mini at temperatures between 2\u20138 \u00b0C for appropriate preservation.The aim of the study was to assess different temperature ranges for the preservation of pre-loaded Descemet Membrane Endothelial Keratoplasty (DMEK) grafts in the DMEK RAPID Mini device. Methods: Three groups of 15 DMEK grafts (five per group) were pre-loaded in the DMEK RAPID Mini and preserved in Optisol-GS for 72 h at different temperatures: group A at >8 \u00b0C, group B between 2\u20138 \u00b0C and group C at <2 \u00b0C. After stripping and preservation, the viability of the endothelium, cell loss and morphology were assessed through light microscopy following trypan blue and alizarin red staining. Results: Overall mortality was 4.07%, 3.97% and 7.66%, in groups A, B and C, respectively, with percentages of uncovered areas of 0.31%, 1.36% and 0.20% (all  Eye banks are playing a crucial role in the development of more standardized and surgeon-friendly techniques for preparing, loading, transporting and delivering grafts for Descemet Membrane Endothelial Keratoplasty (DMEK) ,2,3,4. IIn the eye bank, the DM can be loaded inside the injector either with the corneal endothelium folded outwards (ENDO-OUT configuration) or inwards (ENDO-IN configuration). Once separated completely from the posterior stroma, the DM naturally adopts a scrolled, ENDO-OUT configuration, an effect likely due to the different degrees of elasticity of the anterior and posterior surfaces of the DM . InsteadEye banks have been collaborating with surgeons to provide pre-trephined and pre-loaded membranes preserved either with ENDO-IN or ENDO-OUT configurations. To improve such preparations, efforts are being made to minimize Endothelial Cell Loss (ECL) associated with preparation ,3,12,13,While no dedicated device is so far available for membranes folded inwards, the DMEK RAPID Mini system  has recently received CE marking for the transportation of pre-loaded, ENDO-OUT grafts . This syThe aim of this study was, therefore, to evaluate the effects of preservation at different low-temperature ranges on corneal endothelial cells viability, simulating the transport of pre-loaded DMEK grafts in the DMEK RAPID Mini device.Donor corneal tissues unsuitable for transplantation due to poor endothelial cell counts, procured by Fondazione Banca degli Occhi del Veneto Onlus  were used for the research purposes and the validation studies described in this manuscript, according to the realms of the law 91/99 and after an informed consent form was signed by the donor\u2019s next of kin.n = 15) retrieved from the morgue and preserved in Cold X Medium (homemade medium) at 4 \u00b0C for up to 3 days. The membranes were prepared following a standard stripping protocol as previously reported [In this study, we used fifteen corneas ;Group A: >8 \u00b0C storage (-n = 5);Group B: 2\u20138 \u00b0C storage in a \u201ctransport simulation\u201d condition (-n = 5).Group C: <2 \u00b0C storage  and stored at different temperatures, as described below:Transport simulation was performed by leaving the pre-loaded tissues on a laboratory shaker  placed inside a temperature-monitored refrigerator for the entire duration of the experiment. All the membranes were preserved for 72 h.2, a mortality rate lower than 2.5% and percentages of uncovered areas lower than 1% at T0.The tissues were analyzed after the stripping step (T0). The membranes were stained with trypan blue dye and evaluated by means of light microscopy  using a hypotonic analysis solution (HAS) to highlight the intercellular borders and to assess Endothelial Cell Density (ECD), cell mortality rate, acellular areas . To be included in this study, tissues had to have an ECD > 1700 cells/mmAfter 72 h of preservation (T72), the membranes were carefully extracted from the injector, gently placed and unfolded on a glass slide, stained with trypan blue dye and immersed in hypotonic analysis solution to perform the second light microscopy evaluation.In general, trypan blue positive cells appear within the corneal endothelium as a result of different possible discomfort events or situations, such as poor initial quality of the tissue, long post-mortem time, long storage time, stressful storage conditions, iatrogenic trauma, etc. All these conditions lead to changes in the cell membrane permeability, allowing the dye to stain the cellular nuclei. These cells are considered dead and ready to detach from the Descemet membrane. Uncovered (or acellular) areas are a natural consequence of cellular deaths or are caused by severe mechanical traumas leading to cell detachment .2 squares of a 10 \u00d7 10 mm reticule inserted in the eyepiece of an inverted microscope were counted manually at 100\u00d7 magnification. The number of trypan blue positive cells allowed us to determine the percentage of cell death and uncovered areas (2) and 8.25 mm (53.43 mm2). For the confirmation of uncovered areas, Alizarin red  was topically applied, and the tissues were incubated for 5 min at RT away from bright light. The tissues were then re-evaluated under the light microscope.To estimate the ECD and the extent of damaged areas, the cells in five 1 mmed areas  in relatThe overall mortality was obtained by calculating the difference between the mortality detected at T72 (post-preservation) and the mortality found at T0 (post-stripping). The overall uncovered areas were calculated using the same method. By doing so, the damage that occurred during the stripping phase could be excluded from the final data evaluation, which will be only referred to in the conservation phase. Total ECL was obtained by combining these two values .Descriptive data were summarized using the mean (standard deviation) and percentages where appropriate.The normal distribution of outcomes was checked by means of Shapiro\u2013Wilk tests and visual inspection of Q-Q plots.One-way ANOVA was used to compare the effect of the 3 temperature groups on ECD, mortality rate and uncovered areas at T0 and at T72, as well as ECD variation, overall mortality, overall uncovered area and total ECL between T0 and T72. Homogeneity of variance and covariance was confirmed by means of Levene\u2019s test and Box\u2019s M-test, respectively.t-tests with Bonferroni correction were used for comparisons between groups in case of a significant ANOVA result.Post hoc pairwise p-value of 0.05 was considered statistically significant. All analyses were conducted using R software version 4.2.2 .A Membranes with similar biological features were used for all the groups .2, the mortality rate was 0.26 \u00b1 0.31% and the percentage of uncovered areas was of 0.16 \u00b1 0.27%. In Group B, the average ECD was 2110 \u00b1 260 cells/mm2, the mortality rate was 1.02 \u00b1 0.21% and the percentage of uncovered areas was of 0.07 \u00b1 0.16%. In Group C, the average ECD was 2060 \u00b1 134 cells/mm2, the mortality rate was 0.54 \u00b1 0.47% and the percentage of uncovered areas was of 0.14 \u00b1 0.28% (After stripping (T0), in Group A the mean \u00b1 SD ECD was 2160 \u00b1 134 cells/mm \u00b1 0.28% .p = 0.01). The post hoc pairwise comparison showed that the mortality rate was significantly greater in group B compared to group A (p = 0.01).At T0, only the mortality rate was significantly different among the three groups , in Group A the mean \u00b1 SD ECD we found was 2040 \u00b1 114 cells/mmp > 0.05).At T72, no significant difference in ECD, mortality rate or uncovered areas could be identified . Likewise, no difference was found in Overall Uncovered Areas (p = 0.1). In group C, Overall Mortality (7.66 \u00b1 4.95%) and Total ECL (7.84 \u00b1 4.94%) were higher than in group A (4.07 \u00b1 3.76% and 4.37 \u00b1 3.86%) and group B (3.97 \u00b1 1.97% and 5.32 \u00b1 2.90%). However, these differences were not statistically significant (p = 0.25 and p = 0.39).The mean \u00b1 SD ECD variation between T0 and T72 was 5.51 \u00b1 1.79%, 3.06 \u00b1 2.80% and 2.82 \u00b1 2.58% in Group A, B and C, respectively  compared to that seen in Group B (2\u20138 \u00b0C) and A (>8 \u00b0C), although these differences did not reach statistical significance.Along with preservation time, the temperature is a crucial parameter for appropriate corneal tissue preservation, especially when the isolated Descemet Membrane, and its endothelium, are preserved . The finTo date, two main storage systems of corneal tissue have been developed: cold storage, mainly used in America and Asia, maintains the cornea between 2 \u00b0C and 8 \u00b0C for a maximum of 14 days, while Organ Culture preservation, which uses room temperature up to 31\u201337 \u00b0C for storage of corneas, can preserve the tissues for up to 28 days ,23. The In this study, the preservation of corneal tissue was performed in Optisol-GS, the most widely employed medium for CS . Other s2 has been associated with a significantly lower 5-year graft survival rate in a study by Vasiliauskait\u0117 and colleagues [As DMEK continues to evolve, optimization of the preparatory steps leading to surgery will represent more and more a key area for development. The efforts to minimize ECL in the preparation, storing and shipping of preloaded grafts are paramount given its prominent role in early postoperative ECL. In fact, preoperative ECD has been directly correlated with 6-month post-DMEK ECD, which in turn was found to be a key predictor of late endothelial failure. Specifically, a 6-month ECD count of less than ~830 cells/mmlleagues . Given tlleagues ,3, the alleagues .Limitations of this study could be attributed to damage to the endothelium not related to hypothermic storage, which could have confounded our results. In the pre-loaded, ENDO-OUT configuration, the membrane is left free to roll up and then vacuumed through a tubing system connected to a syringe and can be accommodated in a larger space as compared to the devices for ENDO-IN membranes. Since the graft spontaneously rolls up with the endothelium folded outwards, corneal endothelial cells may be exposed to a certain degree of friction against the internal walls of the injector, especially during the injection phase when the diameter of the funnel is reduced . FurtherFinally, given the small sample size available, we might have been underpowered to detect significant differences among the three groups (Post Hoc Power range = 0.1\u20130.85).In conclusion, to preserve the quality of isolated DMEK grafts folded in the ENDO-OUT configuration in the DMEK RAPID Mini device in Optisol-GS, it is recommended to keep the grafts at temperatures between 2 to 8 \u00b0C. Lower temperatures can alter the morphological characteristics of the endothelium and possibly increase cell mortality. Conversely, no major issue has been observed with higher temperatures as long as these are kept below room temperature to avoid fungal growth ."}
{"text": "A conductive, stretchable, adaptable, and self-healing, GaInSn/Ni--based composite hydrogel by incorporating a magnetic liquid metal into the hydrogel framework through crosslinking polyvinyl alcohol with sodium tetraborate.The multifunctional composite hydrogels showed outstanding performance for magnetic repair movement sensing, and EMI shielding.The online version contains supplementary material available at 10.1007/s40820-023-01043-3. Hydrogels exhibit potential applications in smart wearable devices because of their exceptional sensitivity to various external stimuli. However, their applications are limited by challenges in terms of issues in biocompatibility, custom shape, and self-healing. Herein, a conductive, stretchable, adaptable, self-healing, and biocompatible liquid metal GaInSn/Ni-based composite hydrogel is developed by incorporating a magnetic liquid metal into the hydrogel framework through crosslinking polyvinyl alcohol (PVA) with sodium tetraborate. The excellent stretchability and fast self-healing capability of the PVA/liquid metal hydrogel are derived from its abundant hydrogen binding sites and liquid metal fusion. Significantly, owing to the magnetic constituent, the PVA/liquid metal hydrogel can be guided remotely using an external magnetic field to a specific position to repair the broken wires with no need for manual operation. The composite hydrogel also exhibits sensitive deformation responses and can be used as a strain sensor to monitor various body motions. Additionally, the multifunctional hydrogel displays absorption-dominated electromagnetic interference (EMI) shielding properties. The total shielding performance of the composite hydrogel increases to\u2009~\u200962.5\u00a0dB from\u2009~\u200931.8\u00a0dB of the pure PVA hydrogel at the thickness of 3.0\u00a0mm. The proposed bioinspired multifunctional magnetic hydrogel demonstrates substantial application potential in the field of intelligent wearable devices.The online version contains supplementary material available at 10.1007/s40820-023-01043-3. Liquid metals of Ga-based alloys have attracted intensive interest because of their deformability, nontoxicity, self-healing capability, high electric/thermal conductivity, and unique surface chemistry \u20133. In paCurrently, miniaturized, integrated, and high-power electronic devices are being rapidly developed for wireless communication, and significantly large electromagnetic interference (EMI) is produced as an inevitable by-product , 17. EMI3C2-MXene-functionalized poly:polystyrene sulfonate (PEDOT:PSS) hydrogels [However, the EMI shielding properties of liquid metal-based composites are mainly achieved by reflecting electromagnetic waves, which will cause secondary pollution. Moreover, their limited self-healing ability hampers their applications in intelligent, flexible devices. In contrast to metal liquid-filled elastomers, hydrogels are a type of engineering material composed of a crosslinked network of hydrophilic blocks surrounded by water , demonstydrogels , ionic lydrogels , polyacrydrogels , MXene oydrogels , and mulydrogels . HoweverHerein, we present a simple ultrasonic method to fabricate PVA/GaInSn\u2013Ni composite hydrogels with rapid self-healing capability and excellent stretchability and shape adaptability. The multifunctional composite hydrogels demonstrated high performance for magnetic repair and prototyping, body movement sensing, and EMI shielding. The abundant reversible hydrogen bonds between PVA and borate ions and the fluidity of liquid metals render the PVA/liquid metal hydrogel self-healing features in the absence of any external stimulus. In the presence of a magnetic field, broken wires can be repaired remotely by placing them in a sealed space with one end warped by the magnetic liquid metal hydrogel. The composite hydrogel could be used as a strain sensor to detect body motions and as a signature sensor that sensitively and rapidly responds to various external stimuli. The water-rich hydrogel with moderate conductivity provides absorption-dominated EMI shielding performance to the composite. Importantly, the composite hydrogel exhibited long-term stability for EMI shielding even after storage for 1\u00a0year.PVA  was purchased from Meryer Co., Ltd. Sodium tetraborate , gallium (99.9%), indium (99.9%), and tin  were obtained from Shanghai Macklin Biochemical Co., Ltd. Nickel microparticles (99.9%) were obtained from Shengshida Metal Materials. Co. Ltd., China. All reagents were used as received without further modification.The EGaInSn liquid metal alloy was fabricated by melting a mixture of Ga (68\u00a0wt%), In (22\u00a0wt%), and Sn (10\u00a0wt%) in an oil-bath pan at 150\u00a0\u00b0C for 1\u00a0h.First, PVA white powder (0.4\u00a0g) was added to 5\u00a0mL of deionized (DI) water and magnetically stirred at 80\u00a0\u00b0C for 7\u00a0h until all powders were dissolved to obtain a PVA solution (8\u00a0wt%). Thereafter, EGaInSn and Ni were added to the solutions, and then, EGaInSn\u2013Ni was evenly dispersed in the solution by ultrasonic treatment for 2\u00a0h. Meanwhile, sodium tetraborate was dissolved in hot DI water (60\u00a0\u00b0C) and shaken well until the particles were completely dissolved to produce a solution of 4\u00a0wt%. After cooling to room temperature, the sodium tetraborate solution was slowly blended with the PVA mixture to prepare composite hydrogels. PVA-based hydrogels with different mass ratios of EGaInSn and Ni, namely 1:0.5, 1:1, 1:2, 1:4, and 1:8, are denoted as PVA/EGaInSn\u20130.5Ni, PVA/EGaInSn\u20131Ni, PVA/EGaInSn\u20132Ni, PVA/EGaInSn\u20134Ni, and PVA/EGaInSn\u20138Ni, respectively.A powder X-ray diffractometer  was used to record the XRD patterns. X-ray photoelectron spectroscopy (XPS) analyses were performed on a Kratos AXIS Ultra spectrometer equipped with a monochromatized Al K\u03b1 X-ray source. Fourier transform infrared (FTIR) spectra were acquired using a spectrometer . The functional groups of the PVA-based hydrogels were examined using an FTIR spectrometer (Thermo Nicolet iS10). The morphology and elemental distribution of the PVA\u2013LM hydrogels were characterized using Scanning electron microscopy  coupled with energy-dispersive X-ray spectroscopy . The rheological behavior of the hydrogels was investigated using a Thermo HAAKE MARS 60 machine with a 20\u00a0mm parallel plate. A dynamic frequency sweep of 0.1\u201310\u00a0Hz was conducted at 25\u00a0\u00b0C with a fixed oscillation strain of 0.2% in the oscillation mode. The electrical resistivities of the PVA/EGaInSn\u2013Ni and pure PVA hydrogel samples were tested using an RTS-8 four-probe resistivity meter. An LCR instrument (TH2830) operated using a LabView software was used to collect all relevant data. In the EMI shielding measurement, the composite hydrogel is cut into rectangle shape with dimensions of 22.86\u00a0mm\u2009\u00d7\u200910.16\u00a0mm\u2009\u00d7\u20093.0\u00a0mm. The EMI shielding properties of the PVA/liquid metal composite hydrogels were measured using a vector network analyzer  in the X band (8.2\u201312.4\u00a0GHz), and more details can be found in the Supporting Information.Figure\u00a0d , the peak at 18.5\u00a0eV is associated with metallic gallium (Ga0) [1+ in Ga2O and Ga3+ in Ga2O3 [s peak at 23.8\u00a0eV is also observed in the Ga 3d spectrum, confirming the oxidation of Ga [d core-level spectrum, two distinct peaks at 443.1 (In 3d5/2) and 450.7\u00a0eV (In 3d3/2) are attributed to In0 . Similarly, in the Sn 3d XPS spectrum , the peaks centered at 484.8 and 493.2\u00a0eV are related to the metallic Sn0. These results suggest that the surface of the EGaInSn particles a mainly composed of Ga oxides and a small amount of the mixture of In and Sn oxides. The O 1s XPS spectrum  is deconvoluted into two peaks at 531.7 and 533.3\u00a0eV, suggesting the presence of intact stoichiometric oxides and oxygen vacancies, respectively [The EGaInSn particles were synthesized through an isothermal sonication process at 150 \u2103 for 60\u00a0min. The long sonication process yields highly dispersed liquid metal droplets with uniform elemental distribution . A more detailed study on the surface composition of EGaInSn was performed using XPS . In the XPS spectra of Ga 3um (Ga0) , while ton of Ga . The funectively .\u22121 due to the absorption characteristic of water and stretching vibration of the \u2013OH group, which is a typical indication of hydrogen bonding [\u22121. The carbonyl  and hydroxyl groups  can produce a high density of hydrogen bonds [\u22121.Figure\u00a0 bonding . The peaen bonds , which ip core-level spectrum , the metallic indium exhibits two peaks centered at 443.7 (In 3d5/2) and 451.4\u00a0eV (In 3d3/2), with a separation of spin\u2013orbit components d XPS spectrum , the peaks centered at 484.9 and 493.5\u00a0eV correspond to the metallic tin [s core-level spectrum has three-peak components, i.e., C\u2013C/C=C (284.9\u00a0eV) and C\u2013O (286.5\u00a0eV). The strong C signal indicates the presence of abundant hydrophilic groups on the PVA chains, which offer abundant crosslinking sites in the hydrogel [s peaks are deconvoluted to metal oxide, hydroxide, and lattice oxide component peaks , demonstrating the formation of mixed liquid metal oxides and the Ni oxidation states on the shell [The chemical bonding states of the PVA/EGaInSn\u20138Ni composite hydrogels were confirmed using XPS  values are smaller than the storage modulus (G) in the frequency range of 1\u201310\u00a0Hz, indicating their solid-like states. Significantly, the G\u2033 value of PVA/EGaInSn\u20138Ni exceeds G\u2032, exhibiting a liquid-like state . Therefore, Ni in the hydrogels facilitated the generation of a more liquid-like condition without damaging the complex network structure. Furthermore, for the liquid metal hydrogels with different Ni contents , the G\u02b9 of all composite hydrogels is less than G\u2033, confirming their liquid-like behaviors. Therefore, for the subsequent experiments, we chose PVA/EGaInSn\u20138Ni as the smart sensor hydrogel.The rheological properties of the liquid metal hydrogels were studied using oscillatory rheology . For pure PVA and PVA/EgaInSn hydrogels, their loss modulus (Ms) value of 12.2\u00a0emu\u00a0g\u22121 . The disconnected wire, in which one end is wrapped in the hydrogel, is placed in a plastic container. A permanent magnet is placed outside the plastic container. When the magnet is moved closer to the hydrogel, the entire hydrogel shifts toward the magnet. The strong adhesion between the liquid metal and PVA prevents the liquid metal droplets from leaving the hydrogels during magnetic field-driven movement. The magnet can drag the hydrogel to repair the wire conduction and finally lit the diodes up (Movie S1). In addition, the liquid metal hydrogel can be guided using the magnetic field to fill the custom-designed heart-shaped pattern  indicate the good reliability of the magnetic PVA/liquid metal hydrogel monitoring. When the PVA/liquid metal hydrogel is attached to the wrist, the current increases when bending the wrist, and the current\u2013time curve exhibits significant stability and reproducibility  in the band (8.2\u201312.4\u00a0GHz) using a vector network analyzer. In everyday applications, an EMI shielding effectiveness (SE) of 20\u00a0dB can block approximately 99% of incident EM wave energy [T) of 36\u00a0dB is obtained in PVA, which shields 99.975% of EM waves  and 65.8\u00a0dB (blocking 99.9999747% EM waves) for PVA/EGaInSn and PVA/EGaInSn\u20138Ni hydrogels, respectively; the increase percentage is 28.3% and 83.0%, respectively. These results indicate that introducing liquid metals into the PVA hydrogel can prompt EMI shielding. In comparison with EMI shielding properties and sensor ability of state-of-the-art hydrogels (Table S1), our PVA\u2013liquid metal composite hydrogels demonstrate the competitive performance for EMI shielding, magnetic repair and prototyping. The contributions from absorption loss (SEA) and reflection loss (SER) were analyzed to assess the EMI shielding mechanism of the hydrogels , and 62.5\u00a0dB (increase percentage of 96.8%), respectively, while the SER values decrease from 4.2 to 3.9 (decrease percentage of 7.1%) and 3.3\u00a0dB (decrease percentage of 22.0%), respectively. This demonstrates that adding liquid metals can effectively increase the absorption and decrease the reflection, and it seems that the EMI shielding mechanism mainly results from absorption. However, the calculated SER was based on the total power of EM waves, whereas SEA was based on the power of incident waves [A) and reflection (R) for the PVA, PVA/EGaInSn, and PVA/EGaInSn\u20138Ni composite hydrogels are shown in Fig. S12. The R values are higher than A values over the entire frequency range for the PVA and PVA/EGaInSn hydrogels, suggesting that the EMI shielding is dominated by reflection. However, in the case of PVA/EGaInSn\u20138Ni, the A value is higher than the R value in one-third of the X-band, indicating that introducing magnetic Ni is favorable for absorption. Although reflection dominates the EMI shielding mechanism, absorption plays an important role in the shielding contribution.EMI between electronic devices often results in equipment failure. Self-healed conductive hydrogels with good EMI properties are ideal for soft robotic applications that integrate many electronic components. The EMI shielding capabilities of PVA\u2013liquid metal composite hydrogels (1.0\u00a0mm in thickness) were calculated by testing the scattering parameters , the R is higher than A in the frequency range of 8.2\u201310.7\u00a0GHz; however, the A is higher than R in the frequency range of 10.7\u201312.4\u00a0GHz. This illustrates that the reflection dominates in most measured frequency ranges. Most conventional EMI shielding materials show deteriorated shielding performance when placed in an air atmosphere for a long time [SET values increased from 65.8 to 75.2\u00a0dB, and SEA values increased from 62.5 to 72.9\u00a0dB. The increase percentage of SET and SEA reached 14.3% and 16.6%, respectively. However, SER values decreased from 3.3 to 2.3\u00a0dB, and the decrease percent was 28.5%. By analyzing power coefficients of A and R , PVA/EGaInInSn\u20138Ni presents an absorption-dominated EMI shielding mechanism after storage for one year. In an air atmosphere, hydrogels could absorb environmental water molecules to generate more free ions and dipoles, which was beneficial for EM energy dissipation and enhancing SET and SEA values.The EMI shielding performance of PVA/EGaInSn\u20138Ni before and after self-healing was verified Fig.\u00a0g, h. Allong time , 41. NotSE values significantly increased to 65.8\u00a0dB (blocking 99.99997% of EM waves) for PVA/EGaInInSn\u20138Ni hydrogels, and the increase percentage reached as high as 83.0% in comparison with those of the pure PVA hydrogel. Introducing liquid metals to PVA could effectively increase the absorption loss and decrease the reflection loss, and absorption might dominate the EMI shielding mechanism. Significantly, the excellent EMI shielding performance was maintained after storage for one year, showing long-term stability. Moreover, liquid metal hydrogels could conform to objects of arbitrary geometry and recover rapidly from damage, demonstrating significant application potential in flexible electronics and artificial skin. The hydrogel served as a strain sensor to detect various body movements and a signature sensor for sensitive and rapid responses to external stimuli. Based on the above functions, the present study offers a novel strategy to develop intelligent hydrogels for multifunctional applications as well as a versatile method for fabricating liquid metal composites with extended performance.In summary, we developed a multifunctional PVA/liquid metal composite hydrogel with rapid self-healing ability, excellent stretchability, shape adaptability, magnetic prototyping, sensing capability, and good EMI shielding properties. The fluidity of liquid metal and reversible hydrogen bonds between PVA chains and borate ions enabled the PVA/liquid metal hydrogel to complete self-healing rapidly without external stimuli. The proposed PVA/liquid metal hydrogel could effectively repair broken wire joints when they were placed in a precision-sealed space under an applied magnetic field. The synergy between the moderate conductivity and inherent moisture-rich environment endowed the composite hydrogel with high-efficiency EMI shielding. The total Supplementary file1 (PDF 1523 kb)Supplementary file2 (MP4 831 kb)Supplementary file3 (MP4 284 kb)Below is the link to the electronic supplementary material."}
{"text": "A 46-year-old female patient was diagnosed with a rare and benign intrapulmonary schwannoma, a neurogenic tumor that represents approximately 20% of adult mediastinal tumors, with schwannomas being the most common subtype. The patient was initially asymptomatic; however, after a period of four years, the patient presented with bilateral extremity edema, chronic venous stasis, elevated right ventricular systolic pressure, and a slightly enlarged inferior vena cava. These symptoms were caused by the lung tumor compressing intrathoracic structures.This case highlights the need for early evaluation and proper management of neurogenic tumors to avoid serious symptoms and complications. It also emphasizes the importance of vigilant monitoring and prompt surgery to achieve the best outcome for patients with neurogenic tumors. Approximately 20% of adult mediastinal tumors are neurogenic tumors . These nA 46-year-old Hispanic female with a history of a benign lung tumor, diagnosed four years prior, rheumatoid arthritis, and tobacco dependence presented to the hospital complaining of progressively worsening bilateral lower extremity edema, accompanied by an ulceration on the right medial malleolus. The patient was diagnosed with a primary intra-pulmonary schwannoma via biopsy of the lung mass approximately four years prior and referred to University of California, Los Angeles (UCLA) for surgical resection. However, she could not travel for the procedure due to financial constraints. At the time of her initial diagnosis four years ago, the patient was asymptomatic and reported no lower extremity edema.On this admission, the patient reported the onset of bilateral lower extremity edema approximately six to seven months prior, which worsened over time. She first noted the appearance of a right medial malleolus ulcer about two months ago and was prescribed antibiotics by her primary care provider for possible cellulitis. However, the ulcer failed to heal despite antibiotic treatment. The patient reported experiencing mild dyspnea with exertion but denied any chest pain, syncope, shortness of breath at rest, hemoptysis, changes in vision, fever, or chills. Her current medications include dicyclomine, meloxicam, and ondansetron.Upon admission, the patient was afebrile; vitals were remarkable, with a heart rate (HR) ranging in the 105-110s and blood pressure ranging around 150/80s mmHg. There were no signs of apparent respiratory distress, with normal oxygen saturation at mid-95%. On physical examination, there were diminished breath sounds in the anterior and posterior right upper lobes, with no crackles appreciated on lung auscultation. Jugular venous distension and prominent distension of neck veins on the right side were also noted. On peripheral vascular examination, bilateral lower extremity edema was observed, extending up to the knees, with grade 2 pitting edema and clean, non-purulent ulceration on the right medial malleolus.The complete blood count (CBC) was remarkable for normocytic anemia, with a hemoglobin level of 9.6 g/dL. Otherwise, no abnormalities were noted. The comprehensive metabolic panel (CMP) results were within normal limits. The blood and wound cultures taken from the right medial malleolus ulcer were negative for growth. The chest X-ray (CXR) taken upon admission, as shown in Figure A computed tomography (CT) angiography of the chest, shown in Figure At the initial presentation four years prior, a fluorodeoxyglucose-18 positron emission tomography (FDG-18 PET) scan was obtained, as shown in Figure A CT-guided core needle biopsy performed four years prior showed that the mass tested positive for S-100, cytokeratin (CK), and AE1/AE and was negative for CD34, alpha-smooth muscle actin (SMA), and desmin on immunohistochemistry. The microscopic histology of the mass demonstrated wavy nuclei in hypercellular regions known as Antoni and hypocellular regions known as Antoni B, shown in Figure The echocardiogram during this hospitalization revealed a left ventricular ejection fraction (LVEF) of 60-65%\u00a0with a mild increase in left ventricular wall thickness. The right ventricle size was normal; however, the RSVP was mildly elevated at 38 mmHg . Additionally, a mild dilation of the IVC was observed, measuring 2.8 cm . The bilateral lower extremity venous Doppler study did not reveal the presence of deep vein thrombosis. A right ankle X-ray showed evidence of soft tissue swelling at the medial malleolus.The cardiothoracic vascular surgery (CVTS) team was consulted to evaluate this lung tumor, which has increased in size and possibly compressed intrathoracic neurovascular structures. Upon thorough evaluation and complexity of the tumor, CVTS referred the patient to a tertiary care center for requiring complex surgical intervention. The patient was discharged with a recommendation to follow up with the UCLA\u00a0medical team for ongoing management and monitoring. In addition, the patient was advised to use Unna boots to treat a right medial malleolus ulcer and to wear compression stockings to manage bilateral leg edema.Schwannomas, also referred to as neurilemmomas, are the most frequently occurring nerve sheath tumors. They typically originate from the sensory root of an intercostal nerve or, in less frequent cases, from the phrenic or vagus nerve -4. A retIntrapulmonary Schwannomas are a rare subtype of lung tumors that are typically benign and solitary. They can be located either centrally or peripherally within the lung tissue. These tumors' recurrence and malignant transformation rates are low -8. The mThe metabolic characteristics of schwannomas on FDG-18 PET are poorly understood and cannot be used to differentiate benign from malignant forms . ReportsThe primary treatment for intrapulmonary schwannomas is surgical resection, which is highly effective in achieving complete removal and has a low recurrence rate. Video thoracoscopy is a minimally invasive and safe approach for removing intrathoracic neurogenic tumors -16. HoweComputed tomography angiography (CTA) is a widely adopted protocol for the preoperative evaluation of patients diagnosed with neurogenic tumors in the paravertebral sulci between T5 and T12 . CTA aimOur patient, initially diagnosed with benign intrapulmonary Schwannoma based on immunochemistry and microscopic histology of the mass Figure , was asyVon Recklinghausen's disease (neurofibromatosis type 1), resulting from a Schwann cell development issue, is characterized by numerous neurofibromas on the skin and within internal organs. In a study of four cases of MPNST, 75% exhibited clinical signs of Von Recklinghausen's disease. The presence of widespread neurofibromas raises the likelihood of malignancy or future malignancy in posterior mediastinal tumors . Our patOver the four years following the initial diagnosis, the size of the patient's intrapulmonary Schwannoma grew, possibly causing symptoms such as bilateral lower extremity edema and a moderately elevated RSVP of 38 mmHg, which may be a result of compression of intrathoracic neurovascular structures. Surgical resection is the preferred method of treating intrapulmonary schwannomas, which offers a high success rate for complete removal and minimal chance of relapse ,16. ConsIntrapulmonary Schwannomas are a rare type of lung tumor that is benign. The most common symptoms include thoracic pain, respiratory symptoms, and Horner's syndrome. CT scans of the thorax are crucial in evaluating mediastinal neurogenic tumors. Schwannomas, which are benign tumors, often exhibit high FDG-18 uptake, making it difficult to differentiate them from malignant neurogenic tumors. Hence, a biopsy is necessary to establish a definite diagnosis. Surgical resection remains the primary treatment modality for intrapulmonary schwannomas. Multidisciplinary collaboration between specialties such as neurosurgery and cardiothoracic surgery is essential to optimize surgical outcomes and minimize postoperative complications."}
{"text": "Infectious bursal disease (IBD) is an infectious immunosuppressive disease that affects young chickens. Instead of strict biosecurity practices, vaccination is used to control IBD. However, the disease has not been effectively managed. Variations in the observed clinical symptoms lead to confounding diagnoses. The study aimed to obtain pathological lesion data from chickens suspected of IBD virus (IBDV) infection by gross pathology, confirm IBDV infection through molecular diagnostics, and genotype the VP1 gene fragments of circulating IBDV in the field.The bursa of Fabricius, thymus, spleen, proventricular\u2013ventricular junction, thigh muscles, and kidneys samples were collected from chickens suspected of IBDV infection from four commercial broiler farms in Central Java and The Yogyakarta Special Region Province between 2021 and 2022. The collected samples were examined histopathologically. Infectious bursal disease virus RNA was extracted from the bursa of Fabricius and VP1 gene was identified by reverse-transcriptase polimerase chain reaction (RT-PCR). The RT-PCR positive sample were sequenced and analyzed in Mega X for homology search and phylogenetic tree analysis.Macroscopic pathological lesions in the bursa of Fabricius were demonstrated by enlarged edema and thickened plica, presence of gelatinous exudate, hemorrhage, atrophy, and caseous exudate in the lumen. Moreover, the thymus had atrophy and small gray foci were observed in the spleen. Petechiae or hemorrhage was detected on the thigh muscle, and the kidney was dull and pale. Hemorrhage in the proventricular\u2013ventricular junction was distinct. The histopathological examination of the bursa of Fabricius showed follicular vacuolization, edema, heterophilic infiltration, follicular atrophy, congestion, and hemorrhage. The thymus and spleen showed the presence of multifocal necrosis. Hemorrhage was observed in thigh muscle and mucosal part of proventricular\u2013ventricular junction. Vacuolization was seen in renal tubules (nephrosis). Reverse transcriptase-PCR of 26 bursa of Fabricius samples from chickens suspected of IBDV infection showed four negative and 22 positive samples. Phylogenetic analysis of the VP1 gene fragment has indicated very virulent IBD (vvIBD) and belonged to B2 genotype.Infectious bursal diseases virus infection in broiler chicken generated macroscopic and microscopic primary lesions in the bursa of Fabricius and thigh muscle. Other organs such as the spleen, thymus, proventricular\u2013ventricular junction, and kidney, were also involved. Molecular analysis of the VP1 gene confirmed the causative agent and grouped the virus into vvIBD and B2 genotype. All samples were collected from vaccinated birds therefore, the efficacy of available vaccine is required for urgent evaluation. Since most studies only focused on VP1, further exploration on VP2 gene is suggested notably for new-generation vaccines. Monitoring clinical signs\u2019 transformation over time could assist field diagnostics. Birnaviridae family. The virus has an icosahedral symmetrical shape, is 55\u201365 nm in diameter, and is non-clustered. The viral genome is dsRNA with two segments: A and B. Segment A  encodes VP5 and a precursor polyprotein (VP2-VP3-VP4). This protein is autocatalytically cleaved by the viral protease VP4 into the main structural proteins of the virion VP2 and VP3. Segment A also encodes a non-structural protein VP5. [et al. [Infectious bursal disease (IBD) or Gumboro, is an immunosuppressive disease that affects young chickens. The virus is an RNA virus of the genus Avibirnavirus of the nt A 3,26 bp encodnt A 3,26 bp encodein VP5. . In B-lyein VP5. . Allan e [et al.  reported [et al. . The ver [et al. . Lesionsnt A 3,26 bp encod [et al. , 13. Chi [et al. .Infectious bursal disease virus is classified into pathogenic serotype I and non-pathogenic serotype II. Based on pathogenicity, IBDV belonging to serotype I is classified into mild, intermediate, intermediate plus, classical, variant, and very virulent strains . Due to In Indonesia, subclinical IBD cases in 1991 caused low mortality. The first outbreak of IBD reported an acute increase and expansion of IBD cases, with high mortality reaching 25% in broilers and 60% in laying hens . Based oThus, this study aimed to obtain pathological lesion data and VP1 gene characterization of the latest cases of IBD in commercial broiler farms in Indonesia. This is the first genotype analysis of IBDV VP1 gene from Indonesia.The samples were obtained from IBDV case in commercial broiler farms. There were no challenge or live animal experiment involved. Thus, ethical clearance is not required. The method has been reviewed and obtained approval from Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia .The study was conducted from December 2021 to October 2022. The samples were collected from commercial broiler farms in the Special Region of Yogyakarta (DIY) and Central Java. The histopathology slides were processed at the Wates Disease Investigation Center, Yogyakarta. The RT-PCR test was carried out at the Department of Microbiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada University.Samples were obtained from chickens suspected of IBDV by gross pathology in four commercial broiler farms: Sragen, Wonogiri, Batang District of Central Java Province, and Sleman District of Yogyakarta Special Region Province. A total of five chickens from each farm with indicative clinical symptoms of IBD were selected. The clinical signs were recorded, and necropsies were performed to observe lesions on the bursa of Fabricius, thymus, spleen, proventricular\u2013ventricular junction, thigh muscles, and kidneys. The collected organs were preserved in 10% formalin prior to histology slides processing. A part of the bursa of Fabricius was stored at \u221220\u00b0C for molecular analysis.Histology slides were processed at the Wates Disease Investigation Centre, Yogyakarta Special Region Province, according to the standard protocols of the institution. Histopathology examination was conducted at the Department of Pathology, Faculty of Veterinary Medicine, UGM.All samples were tested for the VP1 gene by RT-PCR. The genetic material of IBDV was extracted from the bursa of Fabricius using Geneaid Viral Nucleic Acid Extraction Kit II  according to the manufacturer. The VP1 gene was amplified using specific primers: Forward 5\u2019-cta cgg gag tgg gac cta ca-3\u2019 and reverse 5\u2019-acc acg tgt tgg agt gaa ca-3\u2019, which yielded a 749-bp amplicon . The polOrgan samples were collected from 26 chickens with signs of depression, trembling, whitish diarrhea, feather loss, paralysis, and dirty cloaca, with mortality rates between 3.82%\u201320.41%. The organs evaluated included the bursa of Fabricius, thymus, spleen, proventricular\u2013ventricular junction, thigh muscles, and kidneys. All observed changes were recorded and are summarized in Macroscopic lesions of involved organs in IBD cases were collected from commercial broiler farms are shown in Obtained histopathological changes involving organs are grouped according to lesion type . ObserveThe RT-PCR analysis revealed 22 out of 26 bursal samples positive for the VP1 gene. The sequences were analyzed against the VP1 gene sequence available in GenBank. Homology analysis indicated variations in amino acid residues 166, 167, 360, 399, 401\u2013404, 410, and 412 . The phyet al. [et al. [The clinical symptoms observed in this study are consistent with those reported elsewhere. Chickens infected with IBDV exhibit general weakness, whitish diarrhea, dirty cloaca, and tremors \u201328. Theset al. , or expeet al. . Furtheret al. . The mor [et al.  reported [et al. , 51% [25 [et al. .et al. [et al. [The bursa of Fabricius is the primary organ affected by IBDV infection and generally presents with inflammation, edema, hyperemia, hemorrhage, and atrophy . Bursal et al.  consiste [et al.  reported [et al. . Liver l [et al. . In an e [et al. .et al [et al. [Histopathological lesions from bursal samples  exhibiteet al . Furtheret al , 43. Widet al . Challenet al . Howeveret al . Other det al . Multifo [et al. . A previ [et al. . The ren [et al. .The molecular analysis could only confirm 22 samples by RT-PCR, possibly the viral load was too low because samples were collected after peak infection . This evBased on sequence analysis, some variations were detected in amino acid residues D242E, T287A, L360M, and E393D of all isolates in this study. Mutations in residues D242E, T287A, L360M, and E393D were reported to alter pathogenicity from classic to very virulent strain , 52, 53.et al. [et al. [et al. [The phylogram of the VP1 gene included IBDVs from serotype I  and serotype II (BII). Based on the VP1 gene sequence, IBDV is divided into genogroups B1 (non-vvIBD) and B2 (vvIBD) . By contet al.  divided et al. , the VP1et al. ; Wibowo  [et al.  that the [et al.  suggeste [et al. .Our study has limitations. The genetic properties of identified vvIBD strains must be further explored. Moreover, this study should include a larger area of samples and include cases from layer farms.Broiler chickens infected with IBDV showed specific macroscopic and microscopic lesions in the bursa of Fabricius and thigh muscle. Moreover, other organs such as the spleen, thymus, proventricular\u2013ventricular junction, and kidney were affected. Molecular analysis of the VP1 gene fragment has grouped the virus into vvIBD of B2 genotype. This study has provided evidence of the latest IBDV genotype circulating in broilers in Indonesia. Most studies on IBDV have focused on the VP1 gene; however, some studies have suggested using VP2 for generating more effective IBDV vaccines. Periodic monitoring of the latest isolate for the probability of genome mutation and clinical signs update of IBD field cases. Because all samples were obtained from vaccinated chickens, our findings provide a new outlook regarding vaccine efficacy.BAD: Collected samples, performed laboratory work, analyzed the data, and drafted the manuscript. KP: Supervised the molecular work and reviewed the manuscript. MHW: Designed the study, supervised all the laboratory work and the analysis, and reviewed the manuscript. All authors have read, reviewed, and approved the final manuscript."}
{"text": "Breast cancer (BC) remains a major challenge for oncology today, impacting the lives of countless individuals worldwide. Yet, in the journey toward improved outcomes and personalized care, a beacon of hope has emerged in the form of neoadjuvant systemic therapy (NST). NST is used in early stage BC to predict outcomes and increase eligibility for breast conserving surgery, recommended for patients with triple negative (TN) or human epidermal growth receptor 2 positive (HER2+) diseases. Magnetic resonance imaging (MRI) is an essential tool for assessing breast tumors, axillary lymph node status and response to NST, aiding in surgical decision-making. The goal of this Special Issue is to analyze closely related cutting-edge diagnostic and therapeutic approaches in early stage BC receiving NST, based on a collection of review and research articles with the latest evidence.As we said, neoadjuvant systemic therapy has revolutionized the approach to early stage BC in HER2+ and TN patients. Giffoni et al.  providedTN BC is known for its aggressive nature, poor prognosis, and early relapse and historically treatment has heavily relied on chemotherapy. However, recent advancements in biologic and targeted therapies are reshaping early stage TN BC treatment strategies as showed by Garufi et al. . The aut\u00ae, a classification system that incorporates the number, type, and distribution of CD3+ and CD8+ immune cells. The score was initially developed for the prognosis of colon cancer, but it is now widely used in clinical research for prognostic and predictive evaluation in many types of solid tumors. As a first clinical validation of the prognostic potential of the Immunoscore\u00ae in patients with BC, the authors assessed this test in BC in the neoadjuvant setting. The presence of high CD3+, CD8+ TILs, and a high Immunoscore\u00ae is predictive of better treatment responses. Patients with these characteristics are more likely to achieve pCR, which is a favorable prognostic index. These findings have significant clinical implications, as they suggest that assessing TILs and Immunoscore\u00ae could help tailor treatment strategies for BC patients.In their research article, Rapoport et al.  highlighMRI is an essential diagnostic tool for evaluating NST response and can influence the surgical management. In their reviews, Panico et al. and Conti et al. ,5 emphasThe use of MRI can also be useful for assessing the response to NST in specific biological tumor subtypes as demonstrated in the research article of Panthi et al. . They asIn parallel with the necessity of an adequate \u201cT\u201d staging after NST, it is also important to define the \u201cN\u201d stage, indeed it represents the main prognostic factor affecting the rate of recurrence and the therapeutic management so that a correct staging of the axillary lymph node status is fundamental. Di Paola et al.  in theirIn a prospective research article, Rella et al.  explore Finally, an important clinical issue to consider in selecting NST in BC is the likelihood of cancer recurrence. Rabinovici-Cohen et al. , in theiThe combination of these research and review articles collected in this Special Issue give an overview that allows us to better comprehend the wide and heterogeneous field of neoadjuvant treatment in early stage breast cancer. They provide a detailed and up-to-date view of the main advances in the field of pharmacological neoadjuvant treatment in early stage BC, and also in relation to tumor molecular subtypes, tumor staging (T) and lymph node assessment (N).The possibility of predicting pathological complete response and recurrence rate using clinical and radiologic information was also discussed promising new frontiers in personalized care and outcome."}
{"text": "The Great Debate session at the 2022 Melanoma Bridge congress (December 1\u20133) featured counterpoint views from leading experts on five contemporary topics of debate in the management of melanoma. The debates considered the choice of anti-lymphocyte-activation gene (LAG)-3 therapy or ipilimumab in combination with anti-programmed death (PD)-1 therapy, whether anti-PD-1 monotherapy is still acceptable as a comparator arm in clinical trials, whether adjuvant treatment of melanoma is still a useful treatment option, the role of adjuvant therapy in stage II melanoma, what role surgery will continue to have in the treatment of melanoma. As is customary in the Melanoma Bridge Great Debates, the speakers are invited by the meeting Chairs to express one side of the assigned debate and the opinions given may not fully reflect personal views. Audiences voted in favour of either side of the argument both before and after each debate. The Great Debate session at the 2022 Melanoma Bridge congress (December 2\u20133) featured counterpoint views from leading experts on five contemporary topics of debate in the management of melanoma. The debates considered the choice of anti-lymphocyte-activation gene (LAG)-3 therapy or ipilimumab in combination with anti-programmed death (PD)-1 therapy, whether anti-PD-1 monotherapy is still acceptable as a comparator arm in clinical trials, whether adjuvant treatment of melanoma is still a useful treatment option, the role of adjuvant therapy in stage II melanoma, what role surgery will continue to have in the treatment of melanoma. As is customary in the Melanoma Bridge Great Debates, the speakers are invited by the meeting Chairs to express one side of the assigned debate and the opinions given may not fully reflect personal views. Audiences voted in favour of either side of the argument both before and after each debate.The combination of nivolumab and ipilimumab was assessed in the phase III CheckMate 067 trial of 945 patients with previously untreated unresectable stage III/IV melanoma. After a follow-up 6.5\u00a0years, combination treatment with nivolumab 1\u00a0mg/kg plus ipilimumab 3\u00a0mg/kg or nivolumab alone was associated with significantly better overall response rate (ORR) and median overall survival (OS) than ipilimumab alone . This miLAG-3 is a marker of T cell exhaustion, with exhausted CD8+\u2009and CD4+\u2009T cells progressively co-expressing multiple inhibitory checkpoints during chronic antigen stimulation . ResponsStrong T cell receptor (TCR) signalling results in higher LAG-3 expression, meaning that the effect of LAG-3 blockade is amplified in conditions of strong immunogenic signalling . ConversGiven this, relatlimab in combination with nivolumab may be a better treatment option in patients who would otherwise be candidates for single-agent nivolumab or nivolumab plus low-dose ipilimumab. In addition, patients with or without BRAF-mutated melanoma, as well as those with acral and mucosal melanoma, a high tumour burden, or M1a/b baseline metastases, all benefit from nivolumab plus relatlimab versus nivolumab alone . The effNivolumab plus relatlimab is superior to single-agent nivolumab across patient populations, with the added toxicity consistent with single-agent therapy and largely manageable. This combination should replace nivolumab plus low dose ipilimumab. Nivolumab plus higher-dose ipilimumab still has a role in special populations, e.g., patients with brain metastases, but even then only until data indicating otherwise become available. The use of nivolumab plus ipilimumab should be limited to second-line or salvage therapy, as in the SWOG 1616 trial which reported improved PFS and a higher ORR with nivolumab plus ipilimumab versus ipilimumab alone in patients with advanced melanoma refractory to anti-PD-1/PD-L1 treatment . HoweverMultiple trials across multiple years in various melanoma patient populations have proven the benefit of nivolumab plus ipilimumab over nivolumab alone. This includes in patients with liver metastases , uveal mIn a comparison of ipilimumab 3\u00a0mg/kg compared with ipilimumab 10\u00a0mg/kg in patients with advanced melanoma, the higher dose was associated with significantly improved OS but with more treatment-related adverse events . HoweverIn patients with metastatic melanoma, choice of first-line therapy is critical given that the response to second-line therapy is never as good. For patients who do not respond to nivolumab and relatlimab, options for second-line treatment need to be considered. Most patients today have received adjuvant PD-1 treatment and so need a different option to nivolumab plus relatlimab. In the future, the best first-line option may be triplet or quadruplet therapy. For example, the interleukin-6 receptor blocking antibody, sarilumab, is being investigated in combination with ipilimumab, nivolumab and relatlimab in a phase II study of patients with unresectable stage III/IV melanoma. The discussion now is how to optimise checkpoint blockade. Second-line therapy will not be another checkpoint inhibitor but will involve an alternative approach, such as tumour-infiltrating lymphocyte (TIL)-based therapy or bispecific antibodies. These may be developed to have affinity to both cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and PD-1 receptors, allowing higher dosing with less toxicity. Other bispecific antibodies include XmAb104, which simultaneously targets PD-1 and the immune co-stimulatory receptor, ICOS, and bavunalimab (formerly XmAb 22841), that simultaneously targets CTLA-4 and LAG-3. These two agents will be assessed in combination in a phase Ib/II study in patients with metastatic melanoma refractory to prior immune checkpoint inhibitor therapy and with or without central nervous system (CNS) involvement. Anti-LAG-3 has good tolerability, so will have a role as second-line therapy in combination with newer agents. Examples include ImmTAC\u00ae molecules, which are engineered to recognise intracellular cancer antigens with ultra-high affinity and selectively target these cancer cells via an anti-CD3 immune-activating effector function, and could be combined with LAG-3 inhibitors. Off-the-shelf therapeutics, e.g., allogeneic natural killer (NK) cells with a bispecific innate cell engager, may also be a well-tolerated option which could be combined with LAG-3 or PD-1 blockade , high LDH or high tumour burden.More data is needed in relation to the long-term benefit of LAG-3 based therapy.In many centers, combined anti-PD-1 based immunotherapy is the most frequently used treatment regimen for patients with unresectable metastatic melanoma. This is most typically nivolumab plus ipilimumab in both Europe and the US, with nivolumab plus relatlimab also available in the US. However, anti-PD-1 monotherapy also remains a well-established regimen of choice.Future clinical trials in metastatic melanoma are likely to have several major aims. These include increased efficacy of first-line treatments, identifying new immune checkpoint inhibitors with increased efficacy or reduced toxicity, and defining the therapeutic role of specific vaccine-based and cellular products. Choice of therapy in the control arm of any clinical trial must be an established standard of care treatment in metastatic melanoma, with acceptable toxicity and proven survival benefit  as compared with previous treatments.The two pivotal trials used to support the use of anti-PD-1 based combination therapy over monotherapy are the CheckMate 067 trial of nivolumab plus ipilimumab and RELATIVITY 047 trial of nivolumab plus relatlimab. In the CheckMate 067 trial, PFS rates at 6.5\u00a0years were 34% with nivolumab plus ipilimumab in combination versus 29% with nivolumab alone and 7% with ipilimumab alone [In the RELATIVITY 047 study, median PFS was significantly improved with nivolumab plus relatlimab compared to nivolumab monotherapy  [In a post-hoc descriptive analysis of CheckMate 067, 6.5-year MSS rates were 56% with the combination, 48% with nivolumab and 27% with ipilimumab . The comIn conclusion, anti-PD-1 monotherapy remains an established standard of care in metastatic melanoma with a proven survival benefit over previous treatments, whereas any OS and MSS benefit of combination immune-checkpoint blockade over monotherapy is still unproven. Moreover, combination therapy is associated with a significant additional toxicity burden compared with monotherapy. As a result, single-agent anti-PD-1 treatment should still be considered an acceptable comparator arm in clinical trials.The time has now arrived that PD-1 blockade with nivolumab or pembrolizumab alone can no longer be considered a standard of care or an appropriate control arm for clinical studies in metastatic melanoma. The combination of nivolumab plus relatlimab resulted in significantly better PFS than nivolumab alone in the RELATIVITY 047 study and this was maintained from 1-year to 2-years of follow-up . In addiIn the neoadjuvant setting, nivolumab plus relatlimab resulted in a pCR rate of 57% and overall pathologic response rate of 70% among 30 patients with resectable stage III/IV melanoma [In a cross-trial comparison of nivolumab plus either relatlimab (RELATIVITY 047) or ipilimumab (CheckMate 067), the benefit:risk ratio, presented as the ratio of PFS improvement to toxicity\u2009\u00d7\u2009100, favoured nivolumab plus relatlimab . In anotFor future trials in melanoma, there should be no single agent anti-PD-1 arm as the single control arm. In both the metastatic and neoadjuvant settings, nivolumab plus relatlimab combination appears most active and less toxic than nivolumab plus ipilimumab, although the two combination approaches should be directly compared to make a strong definitive statement. A triplet combination of nivolumab/ipilimumab/relatlimab may become a future experimental arm in clinical trials. However, ultimately a better understanding of biomarkers is needed to help guide treatment choices among all these regimens in combination or as single agents Fig.\u00a0.Fig. 2IsKey points:Choice of therapy in the control arm of a clinical trial must be an established standard of care, with acceptable toxicity and proven survival benefit.Anti-PD-1 monotherapy has a proven survival benefit over previous treatments, without the additional toxicity burden of combination therapy.In both the metastatic and neoadjuvant settings, nivolumab plus relatlimab appears most active and less toxic than nivolumab monotherapy, which may no longer be the gold standard of care.Five-year survival rates for stage III melanoma in different cohorts range from 80% in stage IIIA to 30% in stage IIID, which justifies the use of adjuvant treatment. Studies of adjuvant therapy in high-risk melanoma (dabrafenib and trametinib in COMBI-AD and pembrolizumab in KEYNOTE 054) have shown HRs of 0.51\u20130.61 for reduced risk of relapse and 0.56\u20130.62 for distant metastases in stage III disease , 18. ThePrevention of recurrence is considered to be more important than concerns of adverse events or quality of life by patient who are candidates for adjuvant melanoma treatment. In a real-world survey of over 900 patients, approximately 25% with stage III disease declined adjuvant therapy, with the most cited reasons in descending order of frequency being age, presence of comorbidities, fear of relapse, fear of adverse events and loss of quality of life . However\u201cperioperative\u201d, since patients received treatment with pembrolizumab both before and after surgery, so it is not a pure neoadjuvant approach. These may be practice-changing data, with meaningful reductions in recurrence seen with just three cycles of pre-surgery pembrolizumab and no additional toxicity compared with conventional adjuvant therapy. It is unclear whether this may become a replacement for or an alternative to ipilimumab plus nivolumab.Alternative options to the current approach include improved selection of high-risk patients who will benefit from adjuvant treatment, e.g., through the use of nomograms, risk prediction scores and gene expression profiling (GEP). Another option is neoadjuvant therapy with or without adjuvant treatment. In the OpACIN-neo study, two cycles of neoadjuvant nivolumab plus ipilimumab had a pathologic response rate (pRR) of 77% . In the Only around 20% of all patients with stage III melanoma have palpable lymph nodes or skin metastases and are therefore candidates for neoadjuvant treatment. In the future, there may be even fewer candidates due to more effective adjuvant stage II treatment. To date, there are no neoadjuvant trials in stage IIB/C melanoma. Adjuvant treatment remains an important option, although a neoadjuvant approach represents a valuable alternative for selected patients.The goal of adjuvant therapy is to improve the OS of curatively treated patients. However, even after 5\u00a0years of follow-up we have not seen a statistically significant OS benefit, according to prespecified boundaries, with adjuvant immunotherapy or targeted therapy in melanoma. This does not mean we should stop adjuvant therapy completely, but better selection of patients that will truly benefit is needed.Adjuvant targeted or immunotherapy benefits less than 20% of patients with stage III melanoma, i.e., 80% of patents are treated for no clinical benefit but at a significant financial cost and exposure to adverse events. In COMBI-AD, the absolute difference in RFS rate at 4\u00a0years was 16% (4-year RFS of 54% with dabrafenib plus trametinib and 38% with placebo) . SimilarAn OS difference is also unlikely to be observed after even longer-term follow-up. Among patients with distant recurrence, approximately 20% will have distant metastases that can be salvaged with local therapy (radiotherapy or surgery) . ResponsThe same applies in stage II melanoma, where even less absolute benefit is of only around 10% of patients seeing a RFS or DMFS benefit with adjuvant immunotherapy . WithoutFurthermore, the neoadjuvant arm involved 15 cycles of pembrolizumab after surgery, i.e., neoadjuvant plus adjuvant, and it is not known whether this is needed for all patients. In the PRADO trial, patients with major pathologic response (MPR) had 2-year RFS and DMFS rates of 93% and 98%, without therapeutic lymph node dissection (TLND) and adjuvant therapy .Vice versa, adjuvant systemic therapy improved the RFS and DMFS rates of patients with no response to neoadjuvant checkpoint inhibition. In PRADO, 2-year RFS rate was 71% in non-responders, which compares with a 2-year RFS of 37% in non-responders, who did not receive adjuvant therapy in the OpACIN-neo study . This abIn conclusion, adjuvant therapy for all patients with resectable stage II/III melanoma is not a valid option, because for these whole unselected cohorts there will be no OS benefit, and this at high financial and adverse event costs of patients treated for no benefit. A neoadjuvant approach is the way forward to solve this dilemma. Identifying non-responders after neoadjuvant therapy might become a strong biomarker for identifying patients with stage III disease who need adjuvant therapy. In stage II melanoma, a neoadjuvant approach still needs to be developed Fig.\u00a0.Fig. 3IsKey points:Data suggest adjuvant treatment has a survival benefit with good tolerability in high-risk melanoma.Even after 5\u00a0years of follow-up we have not seen a statistically significant OS benefit with adjuvant immunotherapy or targeted therapy in melanoma so better selection of patients that will truly benefit is needed.Adjuvant treatment remains an important option, although a neoadjuvant approach represents a valuable alternative for selected patients.Stage II melanoma represents around 15% of patients with melanoma, with around one\u2010half of these patients having stage IIB or IIC disease and so being at higher risk of recurrence . The numAnti-PD-1 therapy reduces recurrence and distant metastasis. In the KEYNOTE 716 trial, adjuvant pembrolizumab significantly improved RFS and DMFS (both HR 0.64) versus placebo after 27\u00a0months of follow-up in patients with stage IIB or IIC melanoma . SimilarHowever, even without a proven OS benefit, adjuvant therapy may still be important. Although OS was rated the most important factor by patients when surveyed about attitudes to treatment decision-making, reduction of relapse was prioritized over risk of toxicity . As suchIn conclusion, resectable stage IIB/C melanoma has a significant risk of recurrence. Anti-PD-1 therapy improves RFS and DMFS and most likely also PRFS2. No OS benefit has been reported in any peri-surgical (neoadjuvant or adjuvant) anti-PD-1 trial but reduction in relapse is in itself considered a significant benefit by many patients.The proportion of patients with stage II melanoma who benefit from adjuvant therapy is limited and the vast majority with stage IIA/B disease do well without treatment; overtreatment is an issue given that only 12% with stage IIA disease, 18% with stage IIB and 25% with stage IIC benefit from adjuvant treatment .Adjuvant therapy in stage II disease is a controversial topic and this can in part be attributed to the unpredictable nature of relapse, which may or may not be salvageable. Patients with stage IIB and IIC melanoma have a high risk of recurrence after 24\u00a0months with poor outcomes . PembrolIn KEYNOTE 716, 16% of pembrolizumab-treated patients had grade 3\u20134 treatment-related adverse events, which is not a trivial proportion. Similarly, grade 3\u20134 treatment-related toxicity occurred in 10% of nivolumab-treated patients in the CheckMate 76\u00a0K study . AlthougTo date, we do not have data showing an OS benefit of adjuvant therapy in stage II disease. We do not know whether treating later will be equal in benefit to treating early. Those patients most likely to experience side effects cannot be predicted. Similarly, we do not have biomarkers for disease recurrence. More data on patterns of recurrence are required.Alternatives to adjuvant therapy include careful observation or enrolment in clinical trials, although opportunities for study enrolment are limited. Targeted therapy may still be an option, as previously shown by a benefit with vemurafenib versus placebo in patients with stage IIC/IIIB melanoma . ProspecImmunotherapy can offer the \u2018gold star\u2019 of a potential cure, but more data and rigorous biomarkers are needed to avoid overtreatment. It is essential that treatment goals and outcomes, including potential side effects and risks of recurrence with and without adjuvant therapy, are discussed with each individual patient Fig.\u00a0.Fig. 4StKey points:Anti-PD-1 therapy improves RFS and DMFS in resectable stage IIB/C melanoma.Although no OS benefit has been reported in any peri-surgical (neoadjuvant or adjuvant) anti-PD-1 trial, reduction in relapse is in itself a significant benefit for many patients.However, the vast majority patients with stage IIA/B disease do well without treatment and more data and rigorous biomarkers are needed to avoid overtreatment.The majority of patients diagnosed with early-\u00afstage melanoma are at low likelihood of relapse and will never require treatment beyond the primary site. A wide excision is recommended with margins based on the primary tumour thickness \u201340. OngoThe second Multicenter Selective Lymphadenectomy Trial (MSLT-II) reported that CLND did not result in a MSS benefit versus observation in patients with melanoma and sentinel-node metastases . This reIn long-term follow-up of MSLT-II, 80.2% of basins were free of nodal recurrence at 10\u00a0years in the observation arm, supporting that there is a therapeutic value of SLNB in patients with melanoma . Risk fahttps://melanomarisk.org.au/SNLForm) [Risk prediction tools which use conventional biomarkers, such as the Melanoma Institute Australia Prediction Tool for Sentinel Node Metastasis Risk (SNLForm) , or combSNLForm) . Low-risIn a retrospective study of 1377 patients with pT1\u2013pT4b primary cutaneous melanoma, the optimal maximum tumour deposit size cut-point was 0.7\u00a0mm for the pT1b-pT4a SLN-positive subgroups, but there was no cut-point for SLN-positive patients with pT4b melanoma . Nodal rAdjuvant therapy reduces risk of recurrence and improves survival following initial treatment and in whom there is no evidence of disease. However, clinical benefit is only possible if residual sub-clinical disease exists. It is important to recognize that absolute benefit is a function of hazard ratio and overall risk of recurrence. Moreover, since toxicity may occur in any patient who receives adjuvant therapy, with the potential for lifelong sequalae, it is critically important to weigh the potential benefits, risks, and alternatives of any potential treatment strategy. SLNB is an important staging technique that improves regional control, may have a therapeutic benefit, and helps clinicians and patients make more informed treatment decisions.In the metastatic setting, surgery may be selectively deployed, with long-term survival following metastasectomy in highly selected patients having a limited but important role, while the consolidation or resection of resistant clones in the setting of systemic therapy can help mitigate/palliate symptoms, such as pain, bleeding, or obstruction, and facilitate considerations for additional systemic and/or other approaches.To conclude, despite exciting times in this era of effective systemic therapy, there is still a significant role for surgery across the continuum of melanoma that continues to evolve and is unlikely to completely disappear.Clearly surgery is required for resection of primary melanoma. However, beyond this, the number of surgical interventions in melanoma could be reduced by up to 80% in Europe over the next 5\u00a0years. Importantly, lymphocytes are primed and \u2018educated\u2019 in the lymph nodes so their removal should be avoided where possible.Wide excision for primary cutaneous melanoma is recommended with margins based on the primary tumour thickness . HoweverPositive sentinel node staging used to provide an indication for CLND, although this is no longer the case since the MSLT-II and DECOG trials showed no OS benefit , 48. A pWith regard to TLND, this has largely been replaced by neoadjuvant immunotherapy, as shown by the PRADO trial in which patients with MPR in their index lymph node after neoadjuvant nivolumab and ipilimumab did not receive TLND or adjuvant therapy; 2-year RFS was 93% in these patients . NeoadjuFurther surgical uses include resection of In-transit metastases, which can mostly be treated with immunotherapy  although there is a still an occasional need for excision of oligometastatic disease. Distant metastases can be treated with neoadjuvant checkpoint inhibition, with only around half needing resection. Escape lesions are very rare and will be treated by organ specialist surgeons. Palliative interventions for local control may be sometimes needed but are also rare; neoadjuvant immunotherapy and radiation therapy may also have a role in these patients.To conclude, melanoma surgery will be reduced by\u2009>\u200960% in the short term and by\u2009>\u200980% in the next 5\u00a0years, being replaced by (neo)adjuvant therapies and the use of SLNB replacement technologies. Neoadjuvant immunotherapy will replace the standard adjuvant therapy model and reduce TLND, metastasis resections, and SLNB , 50 adjuvant therapies and the use of SLN biopsy replacement technologies.Neoadjuvant immunotherapy will replace the standard adjuvant therapy model and reduce TLND, metastasis resections, and SLN biopsy.The Melanoma Great Debate included the presentation of counterpoint views from leading experts on five contemporary clinical issues in the management of melanoma. Given the format and nature of the debates, presentations were not intended as a rigorous and/or systematic assessment of the field but instead allowed the opportunity to highlight some important questions and current controversies. These debates are obviously more nuanced than the simple for or against/yes or no format encourages; however, it is hoped that these discussions can help focus attention on these issues, stimulating further research needed to improve our understanding of different therapeutic approaches."}