{"text": "In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age. A study of human aging in the kidney reveals similar changes in the transcriptional profile in cortex and medulla, suggesting that a common underlying aging process is taking place This indicates that aging is a complex process driven by diverse molecular pathways and biochemical events. As such, a powerful approach to study aging is to use systems biology, which allows a multitude of factors affecting aging to be analyzed in parallel. For example, DNA microarrays and gene expression chips have been used to perform a genome-wide analysis of changes in gene expression in old age. Extensive studies in ed genes . Severaled genes , 2000 aned genes , 2004. Tsir2, and oxidative damage have each been shown to affect lifespan in diverse model organisms  in the renal cortex. The primary function of the tubules within the medulla is to concentrate or dilute the urine so as to maintain fluid balance. The major age-related change in kidney function is a 25% decline in the glomerular filtration rate starting at age 40 . The abiIn this study, we present a molecular portrait of the aging process in the human kidney by analyzing gene expression as a function of age on a genome-wide scale. We show that age regulation is similar in the cortex and the medulla, and that age-regulated genes in the kidney are broadly expressed. We show that the expression profiles of age-regulated genes correlate well with the morphological and physiological state of the kidney in old age. Finally, we analyze the set of age-regulated genes to identify specific metabolic processes and cellular functions that change as a function of age, and discuss their possible roles in specifying the functional lifespan of the kidney.http://genome-www5.stanford.edu/) . Using our dataset, the expression level for every gene as a function of age could be plotted. For example, the expression of CDO1 (which encodes a cysteine dioxygenase type 1 protein) tended to increase with age. There was also variation between subjects and between the cortex and the medulla . We saw large differences in expression between tissue types and between the sexes. These differences were of similar magnitude for both young and old subjects, so that aging in one tissue (or sex) typically ran parallel to aging in the other , Tissuei is one if sample i was a medulla sample (and zero for cortex), and ij\u025b is a random error term. The coefficients kj\u03b2 for k = 0, 1, 2, and 3 are values to be estimated from data. Our primary interest is in \u03b2j1, which describes how quickly the expression of gene j changes with age, with\u03b2j1 = 0 for genes with no linear age relationship.In kjcan differ from zero, even when the true coefficient is zero. We judged statistical significance through p-values, where a value of pji near zero corresponds to a large absolute value |\u03b2^kj|unlikely to have arisen by chance. Such p-values do not distinguish genes that increase with age from those that decrease with age. We also use one-tailed p-values, written p\u02dckj, taking values near zero to be significantly decreasing trends and those near one to be significantly increasing trends  gave no reason to suspect that a curved relationship was needed. Similarly, a piecewise linear age relationship (with bends at ages 50 and 70) was not significantly better than a linear one. Large and statistically significant differences in expression were found for the two tissue types, and so the tissue type was included in p < 0.001 for both sex and age.To make p < 0.001), which is considerably greater than would be expected by chance . Of these, 742 genes increase expression with age and 243 decrease expression with age  were localized to an isolated region of the kidney. Our normal samples were obtained from the region of the kidney furthest from the carcinoma, were not directly contaminated with cancer cells, and appeared normal histologically see . This prp-value for whether renal cell carcinoma affected each of the 44,928 genes present on the Affymetrix DNA chip. The smallest p-value was 0.00013. We would expect to see almost six such p-values by chance alone. This result indicates that the presence of renal cell carcinoma does not significantly affect the expression of any gene in the normal tissue from the same kidney, compared to normal tissues taken from kidneys without renal cell carcinoma.Third, we used regression models to directly test whether our aging studies were affected by seven medical factors: renal cell carcinoma, transitional cell carcinoma, size of tumor, hypertension, systolic blood pressure, diastolic blood pressure, and diabetes mellitus. For renal cell carcinoma, we used a regression model predicting expression from age, sex, tissue type, and a zero/one variable indicating whether the sample came from a patient with renal cell carcinoma or not. The result gave a p < 0.001) age regulation using either of these models. Renal cell carcinoma does not significantly affect the age slopes for these genes . The regression slopes changed very little with and without these factors, indicating that these factors do not strongly affect our analysis of age regulation .Fourth, five of the samples were from kidneys that did not have tumors, and two of these were from donor kidneys used for transplantation that had no associated pathology at all. The expression profile from these five patients was similar to the profile from other samples used in our study. In summary, it is unlikely that these disease and medical factors have confounded our analysis of age-regulated changes in gene expression.Changes in the expression for some of the 985 age-regulated genes may directly reflect the aging process in the kidney; these genes would serve both as aging markers and provide clues about molecular mechanisms for aging in the kidney. Other changes may result from an age-related change in the relative proportion of cell types within the kidney, such as would result from increased infiltration of immune cells with age. Finally, the expression changes may reflect the downstream response of the kidney to an age-related process elsewhere, such as would result from age-related changes in blood pressure or vascular supply.Since the cortex and medulla contain different cell types and have distinct functions, it was of interest to test whether they age similarly. It is possible that the pattern of degeneration in a particular cell type reflects those metabolic pathways that are used most heavily by that cell. For example, there could be deterioration in cell adhesion in glomerular epithelial cells that form part of the filtration barrier in the cortex, while there could be an age-related decline in ion traffic or water flow across the apical or basolateral membranes of tubular epithelial cells in the medulla. Alternatively, distinct cell types could show a common pattern of age-related decline involving pathways common to all cells, such as protein synthesis and mitochondrial function. This degeneration of core cellular processes would affect every cell function, including filtration by glomerular epithelial cells and water and solute reabsorption by tubular epithelial cells.\u03b2j4 \u00d7 Tissue \u00d7 Age was added to the model in \u03b2j4. j4-values. Genes showing tissue-specific slopes would appear in peaks on the left and right. The figure shows neither of these peaks, indicating there is no statistically significant difference in aging between the two tissue types.To test whether age-related gene expression changes are different in cortex and medulla, we considered a model in which a term of the form To further investigate coordinate aging in the cortex and medulla, we searched for age-regulated genes in each of these tissues independently, and then tested whether age-regulated genes in one were also age-regulated in the other. Specifically, to find age-regulated genes in the cortex, we fit the modelusing the cortex samples only. To find age-regulated genes in the medulla, we fit the modelp < 0.001).using only the medulla samples. We found 634 genes in the cortex samples and 72 genes in the medulla samples that showed significant changes in expression with age (p < 0.001) in the cortex samples. If aging in the medulla were unrelated to aging in the cortex, we would expect to see a flat histogram. The actual histogram has a strong peak of genes on the right, indicating that significantly age-regulated genes in the cortex tend to also be significantly age-regulated in the medulla. Of the 634 genes that increased expression with age in the cortex, 22 also increased expression with age in the medulla, compared with the 0.6 genes expected at p = 0.001. We obtained similar results when we took the converse approach, first selecting the 72 age-regulated genes in the medulla, and then testing whether they were also age-regulated in the cortex (data not shown).Having identified age-regulated genes in the cortex, we next examined whether they were also age-regulated in the medulla. r = 0.487. Models 2 and 3 allow us to investigate whether the cortex and medulla age at the same rate as specified in model 1. For the 22 genes that are significantly age-related in both tissues, the age coefficients have a high correlation (r = 0.96), and the slopes themselves are numerically close (2 expression per year), corresponding to only a 6% divergence in expression over 50 y. Given the strong similarities in the aging profiles of these two tissue types, we are able to increase the statistical power of our analysis by pooling the cortex and medulla datasets (resulting in model 1).Next, we compared the slope of expression with respect to age in the cortex to that in the medulla C. The rely close D. We fouWe examined the list of 985 age-regulated genes, and immediately found evidence for increased expression of genes from immunocytes. Many of the 985 age-regulated genes are expressed specifically in B cells , T cells , or neutrophils  see . Nearly Immunohistochemical experiments using antibodies directed against markers specific for B cells, T cells, or neutrophils showed that the kidney samples contained a small proportion of immune cells (less than 1%) in sporadic clusters scattered throughout each section (data not shown). The number of immune cells varied greatly from section to section, and thus it was not possible to use immunohistochemistry to confirm a quantitative increase in the numbers of immune cells in the kidney with age.2 of the expression level for each gene, and then calculated an average expression level for the blood (five samples) and the kidney (134 samples). Of the 985 genes that change expression with age, 538 are expressed at higher levels in blood cells than in the kidney samples. Age-related changes in the RNA abundance of these genes may reflect either changes in the fraction of immune cells in the kidney or age-related changes in expression in kidney cells. The remaining 447 are expressed at higher levels in the kidney than in whole blood, and age regulation of these genes is likely to reflect expression changes in kidney cells themselves , indicating that regulation of expression due to differentiation (between the cortex and medulla) is much greater than that related to aging. This result is consistent with the idea that aging results from a slow degeneration of a core transcriptome in the cortex and the medulla of the kidney.Aging is thought to be caused by slow degeneration of the transcriptome (the entire set of genes expressed in a tissue), rather than a qualitative change in expression, as occurs during tissue specification. As such, changes in gene expression associated with aging should be less than expression differences between different types of tissues. To confirm this idea, we compared the magnitude of gene expression differences due to differentiation (cortex versus medulla) to those due to aging. We used the same approach as before to evaluate differences in expression in cortex versus medulla on a genome-wide scale. For every gene, we calculated the istogram . Genes cTo address whether different organs have distinct or common aging profiles, we analyzed whether the 447 age-regulated genes in the kidney were expressed specifically in the kidney or broadly in many tissues. If the kidney has its own specific pattern of aging, one might expect that the set of 447 aging-regulated genes would be enriched for those expressed specifically in the kidney, such as genes that have direct roles in forming the filtration barrier or in regulating ion or water reabsorption. If there is a common profile for aging shared among tissues, one might expect that most of the list of 447 aging-regulated genes would be expressed in many tissues.We determined the level of expression of the age-regulated genes in different tissues using data from a previous study reporting a genome-wide profile of gene expression in 26 different human tissues with Affymetrix gene arrays . Of the The expression levels of these 447 age-regulated genes constitute a molecular profile of aging, and we can examine the expression profile of individual patients to observe how they compare to the average for their age group. Older individuals tended to express age-induced genes at higher levels and age-repressed genes at lower levels than younger individuals. However, certain individuals had unusual expression profiles, in which genes were expressed at levels more typical of a different age group. For example, patient 81 was 78 y old but had an expression profile as though she were older see . Her kidDo the molecular gene expression profiles correlate with the physiological ages of the kidney samples? That is, does patient 81 have a kidney showing excessive age-related damage and does patient 95 have a kidney with unusually good health? To answer these questions, we determined the morphological and physiological states of the kidneys from each of the patients by examining histological stains. As people grow older, there is a general decline in the morphological appearance of the kidney: (1) the glomeruli lose their structure and their capillaries are replaced with fibrous tissue (glomerular sclerosis), (2) the tubules collapse and atrophy, and the interstitial space between them widens and scars , and (3) there is a thickening of the innermost layer of the arteriole wall due to the accumulation of hyaline material . We gave three scores to each kidney section corresponding to the appearance of the glomeruli, the tubules, and the arterioles. Scores ranged from zero for normal appearance for youthful patients to four for an advanced state of glomerular sclerosis, tubular atrophy/interstitial fibrosis, or arterial intimal hyalinosis see . We thenWe then compared the chronicity index to the gene expression profiles of the 447 age-regulated genes as a function of age see . In genemortalin-2 (which encodes Heat Shock Protein 70), which decreases expression in the kidney in old age. Heat shock proteins act as protein chaperones, and likely function to counteract cell senescence by alleviating the accumulation of damaged proteins in old cells. In human fibroblasts, overexpression of mortalin-2 extends lifespan in vitro  extends longevity, and several genes encoding heat shock proteins decrease expression in old age , all but one of which increase expression in old age see . Forty-nAnother functional group is a set of 11 genes encoding ribosomal proteins, all of which increase expression with age. Protein synthesis rates are known to decline as animals grow older, and increased expression of these ribosomal protein genes may serve to offset this.Changes in the expression of regulatory genes with age may have particularly strong effects on kidney metabolism and function, since these changes are likely to initiate cascades of changes in downstream genes. We examined our list of 447 age-regulated genes for those that are likely to function as regulatory genes. Of the 447 age-regulated genes, 15 encode transcription factors and 51 encode proteins that are part of signaling pathways.As filtration of the blood takes place in the glomerulus, age-regulated genes that are enriched in the glomerulus may be especially important for understanding how kidney function declines with age. We identified genes enriched in the glomerulus using data from a previous study, in which cDNA microarrays were used to compare expression levels in the glomeruli relative to the rest of the kidney . Of the Old age is associated with a functional decline in a myriad of molecular and cellular processes. To gain a global perspective of the diverse pathways that change with age, we performed a whole-genome analysis of gene expression as a function of age for kidney samples from 74 patients ranging in age from 27 to 92 y. Many factors affect gene expression in addition to age, including variability between individuals, between different tissues within the kidney, and between sexes. The large number of samples in our dataset provided good power for identifying age-regulated genes in noisy data despite small changes in expression, and allowed us to use a statistical linear regression model to identify 985 genes that change expression with age.The results from this work show that transcriptional differences between young and old individuals involve an accumulation of small changes in expression from many genes, rather than resulting from large expression changes in a small number of genes. This observation suggests that functional decline in old age is not the result of the complete failure of a small number of cellular processes. Rather, it is the slight weakening of many pathways that cumulatively causes a significant decrease in cell function. Studying aging by analyzing one pathway at a time is difficult, because any single pathway might show only a small change with respect to age and might contribute only a small amount to the overall functional decline in old age. By contrast, functional genomics is a powerful approach to study aging, because many genes can be simultaneously scanned in parallel for small changes in expression.(r) of the regression slopes for these 985 genes was only 0.085 between the kidney and muscle aging experiments and hence accounts for only 0.0072 of the variance between these two tissues  in which the pathology was localized and did not involve the part of the kidney sampled. Key factors from the medical record for each patient used in this study are listed in Frozen tissues were placed in cryomolds, embedded in Cryo Tissue Tek O.C.T. Compound  and cut into 4-\u03bcm sections . Sections were stained with hematoxylin and eosin, and then histologically evaluated to exclude samples showing abnormal histology. Histology slides were also marked into two main functional sections, the cortex and medulla, to help aid in accurate dissection of these two areas. We reviewed radiological findings for all tumors and histology for all slides. We excluded any cases in which radiological imaging, gross examination at the time of resection, or histological review of the removed tissue indicated that there might be tumor involvement of the normal areas. Cases with incomplete or unclear medical records were excluded from this study.Frozen kidney tissue samples were dissected into cortex and medulla sections. Portions were weighed (0.05\u20130.75 g), cut into small pieces on dry ice, and then placed in 1 ml of TRIzol Reagent  per 50\u2013100 mg of tissue. The tissue was homogenized using a PowerGen700 homogenizer , and the total RNA was isolated according to the TRIzol Reagent protocol.A standard protocol designed by Affymetrix  for their HG-U133A and HG-U133B high-density oligonucleotide arrays was slightly modified by the Stanford Genome Technology Center , and all samples were processed in their facility see . Eight mhttp://genome-www5.stanford.edu/) and at the Web site http://cmgm.stanford.edu/approximatelykimlab/aging_kidney/. The Affymetrix probe IDs and the locus link IDs for the genes discussed in the paper are in Using the dChip program , microarp-values we use are based on t-tests from standard linear regression theory. Under the hypothesis H0 that kj\u03b2 = 0, the estimated coefficient \u03b2^kjis a random variable. The least squares value is a particular number, \u03b2^LSkj. The p-value measures the extent to which the least squares value is surprisingly large assuming H0 holds. Specifically, the two-tailed p-value isThe p-value we use isand the one-tailed kjis employed to test H0 against an alternative hypothesis of kj\u03b2 < 0. We use it because it distinguishes between significant increasing and significant decreasing coefficients. Under H0, the distribution of p is U, and so is that of p\u02dc. Numerically, the equationSometimes p\u02dcholds.t-test is derived under an assumption of normally distributed errors. The data showed estimated errors with heavier than normal tails. The t-test is known to be robust against heavy-tailed errors.The A linear regression is more appropriate for these data than is an analysis of variance (ANOVA) on age groups, because the latter is aimed at piecewise constant expression patterns, and it is not plausible that expression should change sharply at a given age. A genome-wide ANOVA (data not shown) did, however, find a similar group of age-related genes. Unlike ANOVA, regression summarizes the age effect in one coefficient. This is advantageous for interpretation and for statistical power when there is little nonlinearity.p-values for all the genes. If the histogram of p\u02dcvalues differed sharply from uniform, and if the smallest p-values were small compared to 1/44,928, then the coefficient was included.The decision of whether to include a variable in model 1 was based on the collection of p-value of 0.001. Such a gene list can be expected to have about 44 genes in it by chance, even if all of the coefficients are really zero. Thus, of the 985 age-related genes, it is plausible that about 44 of them are false positives. We have chosen to work with a fixed significance level, instead of attempting to fix the false discovery rate, because our test statistics are strongly correlated.Gene lists were made using a threshold 2 expression in the cortex from that in the medulla, and fit a regression of the difference versus age and sex. Such an analysis removes intra-subject correlations. There was again no evidence of genes aging differently in the two tissue types (data not shown).We were concerned that intra-subject correlations might have affected our results. For each of 59 subjects with both cortex and medulla samples, we subtracted logFigure S1Each row shows the presence of a medical or related factor. Age of patients is shown on the y-axis. Only transitional cell carcinoma showed a strong age bias. We have identified over 20 different factors that might potentially confound our study on aging, such as race, blood pressure, diabetes, and type and size of tumor adjacent to the normal section see .(221 KB PDF).Click here for additional data file.Figure S2We used regression models to directly test whether our aging studies were affected by seven medical factors: renal cell carcinoma, transitional cell carcinoma, size of tumor, hypertension, systolic blood pressure, diastolic blood pressure, or diabetes mellitus. Scatterplots show age-related slopes using a regression model that includes a term for the medical factor compared to slopes from a regression model that does not include that medical factor.p < 0.001) age regulation using either a model with a renal cell carcinoma term or without a renal cell carcinoma term. The vertical and horizontal axes show the slope from a model with and without the renal cell carcinoma term, respectively. The slopes change very little with and without the renal cell carcinoma term. As one might expect, many of the genes that are significant at the 0.001 level are just barely so. There were 866 genes significant in both models, 119 significant only when renal cell carcinoma was not in the model, and 86 significant only when renal cell carcinoma was in the model. The overall picture of age relationship changes very little whether a term for renal cell carcinoma is included in the model or not.(A) Effect of renal cell carcinoma (RCC) on age-related expression. We selected genes that showed statistically significant . We have not used false discovery rate techniques for this problem, because the age coefficients for different genes are far from independent. The scatterplot shows that transitional cell carcinoma does not affect age-related slopes very much.(B) Effect of transitional cell carcinoma (TCC) on age-related expression. Scatterplot showing age-related slopes with and without a term for transitional cell carcinoma. Transitional cell carcinoma was present in 13 patients, all of whom were old. Thus if transitional cell carcinoma affected gene expression in adjacent normal tissue, then it might bias our results on aging. (B) shows data for presence or absence of transitional cell carcinoma in the model. The gene with the smallest (C) Tumor size does not affect age regulation.(D) Hypertension (HTN) does not affect age regulation.(E) Systolic blood pressure (SBP) does not affect age regulation.(F) Diastolic blood pressure (DPB) does not affect age regulation.(G) Diabetes mellitus (DM) does not affect age regulation.(307 KB JPG).Click here for additional data file.Figure S3p < 0.004). A Pearson correlation value of 0.085 implies that 0.72% of the variance in the muscle regression coefficients is due to variance in the associated kidney regression coefficients. We note that the muscle dataset had a small sample size (n = 16), which may not be large enough to sufficiently detect similarity in age regulation with the kidney.We obtained the muscle dataset from the GEO database . To comp(59 KB XLS).Click here for additional data file.Figure S4C. elegans (D. melanogaster (We compared patterns of gene expression in the aging time course data from  elegans  and D. mnogaster  to thoseD. melanogaster. Open triangles denote age-regulated genes in humans and their orthologs in flies. Open circles denote age-regulated genes in flies and their orthologs in humans. The scatterplot shows the regression slopes from the human kidney and the fly aging datasets (r = \u22120.05 (p = 0.27) for human and fly, while the age-regulated fly genes paired with human orthologs show a Pearson correlation r = \u22120.05 (p = 0.12).(A) Regression slopes of age-regulated genes from human kidney and datasets . SpecifiC. elegans. Open circles denote age-regulated genes in humans and their orthologs in worms. Open triangles denote age-regulated genes in worms and their orthologs in humans. The scatterplot shows the regression slopes from the human kidney and C. elegans aging datasets (r = 0.05 (p = 0.54). The age-regulated worm genes paired with human orthologs show a Pearson correlation r = \u22120.01 (p = 0.08).(B) Regression slopes of age-regulated genes from human kidney and datasets . The ageThese results show no evidence for overlap in the aging process between different species.(509 KB PDF).Click here for additional data file.Protocol S1(40 KB DOC).Click here for additional data file.Table S1(33 KB XLS).Click here for additional data file.Table S2(13 KB XLS).Click here for additional data file.Table S3(135 KB XLS).Click here for additional data file.Table S4(75 KB XLS).Click here for additional data file.Table S5(53 KB XLS).Click here for additional data file."}
{"text": "Science, Stuart Kim, a Stanford developmental biologist, made the case for laying down the broad strokes of a complex physiological process before defining its mechanisms. \u201cA powerful, top-down, holistic approach,\u201d he wrote, \u201cis to identify all of the components of a particular cellular process, so that one can define the global picture first and then use it as a framework to understand how the individual components of the process fit together.\u201d To get a broad view of gene expression in the aging nematode, Kim's lab turned to DNA microarrays and functional genomics. In a new study, Kim and colleagues apply this same approach to the decidedly more complex problem of human aging and \u201cpresent a molecular portrait\u201d of the aging kidney.Four years ago in Scientists have identified a wide range of molecular pathways and mechanisms associated with aging. Many have been found in evolutionarily distant organisms, suggesting they have been conserved and could shed light on human aging. Yet other studies suggest that since few animals reach old age in the wild, any aging-related physiological changes aren't likely to impact the fitness of a population and so aren't likely to be conserved. Consequently, aging pathways in worms, for example, would have little bearing on humans. To investigate the molecular pathways associated with human aging, the authors focused on human tissue\u2014in this case, the kidney.Kidneys came from 74 patients, ranging in age from 27 to 92. Samples were extracted from donated kidneys or \u201cmeticulously harvested\u201d from kidneys with localized disease to ensure only normal tissue was taken. Two structures that are critical to kidney function (removing toxins from blood) were removed from each sample: the renal cortex, which filters plasma, and the medulla, which alters urine composition to maintain fluid balance. Both deteriorate with age. An extensive clinical history was noted for each sample to account for any potentially confounding medical factors.Kim and colleagues then isolated RNA transcripts from the samples to determine the activity of every gene, broken down by age and kidney section, through microarray analysis. Looking for differences in gene expression across the genome, they identified genes that showed a statistically significant change in expression as a function of age. Of 33,000 known human genes on the microarray, 985 showed age-related changes, most showing increased activity. These changes are truly age-regulated, the authors conclude, since none of the medical factors impacted the observed changes in gene expression.Although cortex and medulla have different cell types and perform different functions, their genetic aging profile was very similar, suggesting a common aging mechanism operates in both structures. In fact, these mechanisms may function broadly, as most of the age-regulated kidney genes were also active in a wide range of human tissues. Other organisms appear to lack these changes, however, prompting the authors to argue that understanding aging in humans will require human subjects.Most importantly, the genetic profile of the tissue samples correlated with the physiological and morphological decline of an aging kidney. An 81-year-old patient with an unusually healthy kidney had a molecular profile typical of someone much younger, while a 78-year-old with a damaged kidney had the profile of a much older person. Using the power of functional genomics, this study has identified a set of genes that can serve as molecular markers for various stages of a deteriorating kidney and predict the relative health of a patient compared to their age group. These gene sets can also serve as probes to shed light on the molecular pathways at work in the aging kidney, and possibly on the process of aging itself."}
{"text": "We analyzed expression of 81 normal muscle samples from humans of varying ages, and have identified a molecular profile for aging consisting of 250 age-regulated genes. This molecular profile correlates not only with chronological age but also with a measure of physiological age. We compared the transcriptional profile of muscle aging to previous transcriptional profiles of aging in the kidney and the brain, and found a common signature for aging in these diverse human tissues. The common aging signature consists of six genetic pathways; four pathways increase expression with age , while two pathways decrease expression with age . We also compared transcriptional profiles of aging in humans to those of the mouse and fly, and found that the electron transport chain pathway decreases expression with age in all three organisms, suggesting that this may be a public marker for aging across species. Aging is a complex phenomenon characterized by the decay of biological function over time, eventually leading to death. High-throughput methods for examining changes in the expression of genes, such as DNA microarrays, have been successful in elucidating some of the genome-wide changes that occur with age in several human tissues. The authors profiled gene expression changes in the muscles of 81 individuals with ages spanning eight decades. They found 250 genes and 3 genetic pathways that displayed altered levels of expression in the elderly. The transcriptional profile of age-regulated genes was able to discern elderly patients with severe muscle aging from those that retained high levels of muscle function; that is, the gene expression profiles reflected physiological as well as chronological age. In order to find genetic changes that might affect most or all tissues during aging, the authors compared genome-wide profiles of aging in the muscle to those in the kidney and the brain, and found a common signature for aging shared among these three tissues consisting of six genetic pathways. One of these aging pathways (the electron transport chain pathway) is age regulated not only in humans but also in two model organisms (mice and flies), providing insights about shared age-related changes in animals with vastly different lifespans. Aging is marked by the gradual decline of a multitude of physiological functions leading to an increasing probability of death. Some aging-related changes affect one's appearance, such as wrinkled skin, whereas others affect organ function, such as decreased kidney filtration rate and decreased muscular strength. At the molecular level, we are just beginning to assemble protein and gene expression changes that can be used as markers for aging. Rather than search for molecular aging markers by focusing on only one gene or pathway at a time, an attractive approach is to screen all genetic pathways in parallel for age-related changes by using full-genome oligonucleotide chips to search for gene expression changes in the elderly. A genome-wide transcriptional profile of aging may identify molecular markers of the aging process, and would provide insight into the molecular mechanisms that ultimately limit human lifespan.INK4ap16 are molecular markers of aging, as they predict both the remaining lifespan and the physiological age of the mouse [Molecular markers of aging must reflect physiological function rather than simple chronological age because individuals age at different rates . In the he mouse \u20134. In thhe mouse . Whole-ghe mouse ,8, and dhe mouse , but chaIn this paper, we have performed a genome wide analysis of gene expression changes in the human skeletal muscle. As age increases, skeletal muscle degenerates, loses mass, loses total aerobic capacity, and becomes markedly weaker . One meaThe extent to which age regulation of genetic pathways is specific to a particular tissue or common across many tissues is unknown. Age regulation of gene expression between the cortex and medulla regions of the human kidney was found to be highly correlated . There wDrosophila melanogaster [Caenorhabditis elegans [Another key issue is whether there are genetic pathways that are commonly age regulated in different species with vastly different lifespans, such as human, mouse, fly, and worm. Transcriptional profiles of aging have been performed on both skeletal muscle and brains in the mouse ,15, in Dnogaster ,17, and  elegans . A compa elegans .In this work, we present a transcriptional expression profile of 81 human skeletal muscle samples as a function of age. The symporter activity, sialyltransferase activity, and chloride transport pathways all decrease expression with age in human muscle. The age-regulated genes were found to be markers of physiological age, not just chronological age. By comparing our results on aging in muscle to previous transcriptional profiles of aging in the kidney and the brain, we found a common signature for aging across different human tissues consisting of six genetic pathways that showed common patterns of age regulation in all three tissues. Finally, by comparing the signature for aging in humans to transcriptional profiles of aging in mice, flies, and worms, we found that expression of the electron transport chain decreases with age in humans, mice, and flies, constituting a public signature for aging across species with extremely different lifespans.http://www.ncbi.nlm.nih.gov/geo).In order to study the effects of aging in human muscle, we obtained 81 samples of human skeletal muscle from individuals spanning 16 to 89 y of age . Sixty-tWe used a multiple regression technique on each gene to determine how its expression changes with age, as had been done previously for age regulation in the kidney  . We analp < 0.001)  in addition to age, sex, and anatomy. We then compared the aging coefficient using this new model with the one from the original model that did not include the term. If any of the 250 genes were regulated by the medical factor and not by age per se, we would expect marked differences in the aging coefficients generated by the two multiple regression models. None of the fourteen medical factors, including hypothyroidism, had a significant effect on age regulation . Second,p < 0.001), and the large majority of these age-regulated genes change expression less than two-fold in 50 y. These results are consistent with a model in which age-related decline in cellular functions is caused by the accumulation of multiple, minute changes in the regulation of genes and pathways.In summary, we have generated a global profile of changes in gene expression during aging in human muscle . It is wCYP26B1 shows an average increase in expression of 90% in 50 y. CYP26B1 is a member of the cytochrome P450 family, which are monoxygenases used to metabolize toxic substances. Increased expression of CYP26B1 in old age could help eliminate toxins that accumulate with age.The genetic functions of many of the 250 genes shown in LASS5 decreases expression approximately 25% in 50 y. LASS5 is the human ortholog of the yeast lag1 longevity assurance gene. In yeast, lag1 expression decreases in older yeast cells [LASS5 is involved in the ceramide signaling pathway, which plays important roles on several lifespan-associated processes, such as stress resistance and apoptosis [LASS5 in old age could impair cell function by reducing ceramide signaling.st cells  similar poptosis . ReducedIn addition to searching for age regulation one gene at a time, we also screened known genetic pathways for those showing an overall change with age. With this approach, age regulation for every gene in a pathway is combined to determine whether there is an overall regulation of the entire pathway. Screening for coordinated age regulation of genetic pathways increases the sensitivity of our analysis, as the combined effects of small regulation of many genes in a pathway can be significant. For example, in a previous study of type 2 diabetes, screening genetic pathways for changes in expression provided key insights that were not possible from analyzing genes individually .We developed a variant of gene set enrichment analysis (GSEA) to determine whether a genetic pathway shows evidence for age regulation . We assap < 0.001) in this resampling. At this p value threshold, we would only expect to find 0.6 gene sets by chance alone. We also required the gene groups to show some practical significance by rejecting groups with a van der Waerden score smaller than 3.1 in absolute value. We found three pathways that passed both criteria: symporter genes, sialyltransferases, and chloride transporters showed decreasing expression with age  on average in response to increasing age. The increase is measured by a van der Waerden statistic. To judge whether a specific van der Waerden statistic is significant, we used bootstrap resampling. Each bootstrap sample was drawn by resampling the arrays and keeping the gene expression measurements linked with the age, sex, and anatomy variables. The 624 van der Waerden scores for the gene groups were recomputed for each of the 1,000 bootstrap samples. Six gene sets were found to have statistically significant van der Waerden scores (with age . Aging cSymporter genes (63 genes) and chloride transporters (35 genes) are necessary for transporting solutes during muscle contraction ; the decSome people age slowly and remain strong and fit in their 70s, whereas others age rapidly, becoming frail and susceptible to age-related disease. We wanted to determine whether the expression profile for the 250 aging-regulated genes correlated with physiological in addition to chronological aging. For example, patient V17 was 41 y old but expressed his age-regulated genes similarly to patients who were 10 to 20 y older, and we would like to determine whether this patient had poor muscle physiology for his age . ConversA simple correlation of gene expression with muscle type ratio would not be sufficient for our purposes. Such a correlation could arise simply because the gene expression and muscle type ratio are both correlated with age. Accordingly, we employed partial correlations of gene expression with muscle type ratios after adjusting for the effect of chronological age. To do this, we regressed type II/type I muscle fiber diameter ratio on age, regressed gene expression on age, and finally correlated residuals from both regressions to obtain partial correlation coefficients. The partial correlations for the 250 age-related genes are shown in p value below 0.0001 and concluded that the age-related genes are more likely than other genes to have some partial correlation with muscle diameter ratio. To illustrate this effect, we also sampled 250 genes from the genome 1,000 different times, each time counting how many had a partial correlation larger than 0.2 in absolute value. None of the samples had a count larger than 92  partial correlation was more than 0.2 . There w than 92 D.r > 0.2). We compared the distribution of partial correlations of the 250 age-regulated genes with type II/type I ratios to the distribution of partial correlations of the rest of the genes in the genome using nonparametric methods . This result indicates that the apparent physiological basis of our gene set is not a consequence of our having chosen 0.2 as a threshold.Our result indicating that the 250 age-regulated genes are enriched for genes regulated by type II/type I muscle fiber diameter ratio is valid even when we use other selection thresholds for muscle physiology . Aging coefficients for all genes in each of these pathways are listed in p < 0.05 in all three tissues, and hence a combined p < 1.25 \u00d7 10\u22124).Our initial attempt to compare transcriptional changes between tissues relied on a Venn analysis, in which we directly compared the overlap in the lists of the age-regulated genes from the three tissues. Next, we searched for a common aging signature by comparing the Pearson correlation of age regulation between two tissues. Both of these straightforward methods showed only borderline statistical evidence for similarities in aging between the three tissues , but neither is expected to be powerful. Ultimately, we compared tissues using a grouped gene analysis. Grouping genes can be more powerful if there are small but consistent effects in each of a number of genes. Furthermore, the specific biological processes associated with each genetic pathway provide insights into mechanisms of aging. We used the modified GSEA described above to analyze previously published data on age regulation in the kidney and the brain ,6. As be tissues . The cytTIMP1, which encodes tissue inhibitor of metalloproteinase 1, shows the largest increase in expression with age (average of 236% in 50 y).Increased overall expression of the extracellular matrix gene set (152 genes) with advancing age may contribute to widespread fibrosis in the elderly . FibrosiTGFB1 and FGFR1. Induction of genes in this gene set may reflect an attempt to repair tissue damage that accumulates over lifespan.The cell growth gene set (29 genes) includes genes coding for growth factors, such as Although complement activation genes (22 genes) are induced in muscle, the kidney, and the brain, they are expressed primarily in liver . TherefoCytosolic ribosomal genes include 85 genes that show a general increase in expression with age in all three tissues. This result is interesting because the rate of protein synthesis is known to decrease in old age , and yetThe chloride transport pathway is composed of 35 genes that show an overall decrease in expression with age in all three tissues. Ion transport of many types is important not only in the contraction of muscle , but als+-ATP synthase (complex V), and the uncoupling proteins. The finding that expression of genes involved in the electron transport chain decreases in old age supports the mitochondrial free-radical theory of aging [The mitochondrial electron transport chain was found to show an overall decrease in expression with age. This group contains 95 genes, including genes associated with the NADH dehydrogenase family (complex I), succinate-coenzyme Q reductase (complex II), ubiquinone-cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), Hof aging , as freeThe above results show that there is common age regulation for these six genetic pathways in the kidney, muscle, and the brain. Next, we determined that there was little statistical evidence for the correlation of age regulation of individual genes in a pathway in one tissue with their age regulation in another tissue . Thus, it is unclear whether or not the same genes or different genes within a pathway show age regulation between different tissues. For example, certain genes in the electron transport pathway might be age regulated in the kidney, whereas other electron transport genes might be age regulated in the muscle.Having identified genetic pathways that are commonly age regulated in different human tissues, we next determined whether their age regulation is specific for humans (private) or whether these groups are also age regulated in other species (public). Genetic pathways that are age regulated in different species would be of particular interest because they would identify mechanisms that are inextricably related to aging, even in animals that have vastly different lifespans.D. melanogaster [C. elegans [. To examine age regulation in aging mouse kidneys, we collected a kidney sample from ten C57BL/6 mice at 1, 6, 16, and 24 mo of age for a total of 40 mouse kidney samples. RNA from each kidney was extracted, labeled with P33-dCTP, and hybridized to cDNA filter membranes comprising 16,896 cDNA clones corresponding to 11,512 unique genes. We normalized expression values using the Z-score method [We compared age regulation in humans to previously published studies of age regulation in nogaster  and C. e elegans . To exame method , and anaWe first identified orthologs of human genes in each of the other three species. Next, we determined the change in expression with respect to age for each gene in each species, using multiple regression techniques similar to the ones used for our studies of aging in human muscle . We took the six gene sets shown to be aging-regulated in diverse human tissues, and then asked whether they also showed age regulation in any of the other three species. We analyzed the expression of each of the gene sets using modified GSEA to determine whether they showed an overall bias in expression with age in each species. Extracellular matrix genes, cell growth genes, complement activation genes, cytosolic ribosomal genes, and chloride transport genes did not show age regulation in other species.Drosophila, but did not show significant age regulation in C. elegans (p < 0.02) and flies (p < 0.001) but not C. elegans. The electron transport chain gene set also shows a large van der Waerden score in mice and flies (less than \u22123.7). In summary, humans, mice, and flies show decreased expression of the electron transport chain during aging, defining a public pathway for aging across species with very different lifespans. In C. elegans, it is unclear whether the lack of support for age regulation of the electron transport chain pathway is because the pathway is not age regulated or because the DNA microarray experiments lack statistical power to detect age regulation.The electron transport chain genes showed a consistent overall decrease in expression with age in humans, mice, and  elegans . To showIn this study, we have generated a high-resolution transcriptional profile of aging in the human muscle. Welle et al. have previously used DNA chips to profile expression changes during aging for the human muscle ,8, and tPeople age at different rates, especially with regard to muscular aging. Some remain fit and strong, whereas other become frail and weak when they are old. The transcriptional profile for aging in this study reflects the physiological age of the subjects, as measured by muscle diameter ratio, after making an adjustment for their chronological ages. Previous work on age regulation in the kidney also identified molecular markers that could predict the physiological age of the kidney .Our results provide the some of the first evidence for a common signature of changes of gene expression in different human tissues. Specifically, we found similar patterns of age regulation for six biological pathways in the muscle, the kidney, and the brain. Previous studies found similar patterns of aging between different parts of the same tissue, but not between entirely different organs  but also to degeneration of tissue-specific functions  that rely on housekeeping pathways. By identifying a common aging signature across tissues, we can now focus on aging pathways that are general instead of tissue-specific. The common aging signature reflects the age of diverse organs, whereas genes that are age regulated in just one tissue reflect the age of that tissue. Finally, treatments or therapies that alter expression of the four common age-regulated pathways might be expected to affect diverse tissues instead of a specific tissue, and may therefore have an overall effect on longevity.Although some patterns of aging are similar between different human tissues, much of aging is tissue-specific. Decreases in expression of the sialyltransferases and symporter genes are changes specific to muscle, and do not appear to occur in either the kidney or the brain.Nearly all of the age regulation that we found is specific to humans, and does not seem to occur in old mice, flies, or worms. Thus, much of age regulation in humans is species-specific (private) rather than universal for all animals (public). This result emphasizes the importance of studying aging in humans rather than model organisms with short lifespans in order to understand how people grow old.Nevertheless, we did find one pathway that was age regulated in humans, mice, and flies. The electron transport chain gene pathway decreases expression with age in all three species. Previous studies found little or no similarity in age regulation between humans and mice  or primaC. elegans, reducing the activity of eight genes involved in the electron transport chain using RNAi increased lifespan significantly [(NDUFA10) is one of the genes showing the largest decrease in expression with age in humans, and its ortholog in worms, K04G7.4, was also found to cause one of the largest increases in lifespan using RNAi in C. elegans [In mammals, direct genetic tests of the functional relevance of reduced expression of the electron transport chain pathway on lifespan have not yet been reported. However, in ficantly ,36. A ge elegans . The genWhat types of upstream events might cause a decrease in expression of the electron transport chain pathway with age? Other mitochondrial pathways, such as the mitochondrial ribosome, do not show age regulation similar to the electron transport chain pathway. One potential cause of decreased expression of the electron transport chain pathway is that metabolism may slow in old age, resulting in reduced expression of the energy producing machinery of the cell. Another possibility is that oxidative damage to the proteins in the electron transport chain in the mitochondria may lead to reduced expression of the corresponding genes in the nucleus. The electron transport chain creates free radicals in the process of generating energy that would preferentially damage protein components of the electron transport chain .It seems unlikely that common age regulation of the electron transport chain pathway is directly due to evolutionary conservation. Events in old age are unlikely to have a significant effect on fitness of a population because old animals (such as 3-y-old mice and 80-y-old people) are a small fraction of natural populations (except in recent human history). It could be that the electron transport chain is regulated during aging as an indirect consequence of regulation during development (antagonistic pleiotropy) . AlternaIt is interesting that the level of age regulation of the electron transport chain is nearly the same in each species, whereas lifespan varies greatly. Compared to humans, mice age 20- to 30-fold and flies age 400-fold more rapidly. Thus, the kinetics of the changes in gene expression for the electron transport chain genes precisely matches the difference in lifespan between species. This suggests that decreased expression of the electron transport chain pathway with age may be particularly informative as a marker of physiological aging.The muscle samples were obtained from patient biopsies collected either during surgery or in an outpatient procedure, and the medical conditions associated with each biopsy are listed in Frozen muscle samples were weighed (50\u2013100 mg), cut into small pieces on dry ice, and then placed in 1 ml of TRIzol Reagent . The tissue was homogenized using a PowerGen700 homogenizer , and the total RNA was isolated according to the TRIzol Reagent protocol.A standard protocol designed by Affymetrix  for their HG-U133 2.0 Plus high-density oligonucleotide arrays was slightly modified by the Stanford Genome Technology Center , and all samples were processed in their facility (see Protocol S1). Eight micrograms of total RNA was used to synthesize cRNA for each sample, and 15 \u03bcg of cRNA was hybridized to each DNA chip. The samples were processed in random order with respect to age.2-transformed expression values for all subsequent analyses.We used the DChip program  to normac oxidase preparations) and measured by hand. All of the diagnostic muscle biopsies were considered, and 32 of the 81 muscle samples were sufficiently intact for measurement, the remainder being inadequately oriented for cross-sections or too small for meaningful data. Digital analysis consisted of measuring the shortest width through the approximate center of the cell. After calibration with a known length, the diameters were measured and converted to microns using SigmaScan Pro 5.0 software . Diameters were tabulated by type I and type II cell types. The counts ranged from approximately 30 cells per type to more than 100 depending on the sample size. Print analysis was by similar methodology. Raw measurements in millimeters were used to calculate the ratio of type II to type I diameters without converting to microns.Cross-sections of muscle cryosections were photographed at 200\u00d7, and the pictures were either measured digitally  or printed , iAnatomy is the anatomic location from which the muscle sample was harvested , ij\u025b represents an error term, 1j\u03b2 is the change of expression with age, 2j\u03b2 is the change of expression with sex, 3j\u03b2 is the change of expression with anatomical origin of sample, and 0j\u03b2 is the regression intercept. For each gene j, we used least-squares to determine all of its coefficients, with our primary interest in the one with respect to age 1j)., indicating a significant overlap  .Here, ibution) . The 92 Second, we repeated our age analysis taking into consideration the effect of type II/type I muscle fiber diameter ratio on age regulation. To do this, we used a four-term multiple regression model that includes terms for both age and type II/type I ratio:p < 0.01) and 12,786 genes regulated by type II/type I ratio . We randomized the age variables of muscle samples 1,000 times while maintaining the sex and anatomy variables with the sample. p values in every randomization. Theory predicts, and our simulation verifies, that on average about 32 genes pass our threshold (p < 0.001) by chance. This result suggests that there are about 13% false positives in our set of 250 age-regulated genes in the muscle. In 95% of the permuted datasets, 107 or fewer genes were significant at the 0.001 level.We used a permutation analysis to simulate the number of genes that would pass our cutoff by chance . In our analysis, we have replaced the weighted Kolmogorov-Smirnov test by a weighted sum, the van der Waerden normal scores test.GSEA  uses a nG of genes to be age related than does the weighted Kolmogorov-Smirnov test. When N is large, then any small group that contains the single most age-related gene is significantly age related by the weighted Kolmogorov-Smirnov test. Such a group displays a genuine statistical significance and comprises strong evidence that F \u2260 G, but isn't necessarily biologically increasing or decreasing expression as a mechanistic unit with age. For example, a group of 30 genes with two of the most age-increasing genes and 2 of the most age-decreasing genes could be found to be both an age-increasing group and also an age-decreasing group with significance, even when the other 26 genes are not particularly age related. Here it is clear that F \u2260 G, but perhaps it is simply because G has higher variance than F.The van der Waerden test conforms more closely to our interpretation of what it means for a group G. This rank is the number of the original N genes with an age coefficient smaller than that of gene j. The raw van der Waerden score isTo compute the van der Waerden test, we first find the rank r(j) for every gene j \u2208 \u03a6 is the standard normal cumulative distribution function. When the N age coefficients are independent with a common continuous distribution F = G, then the distribution of Y is very nearly normally distributed with mean 0 and a variance V(Y) close to N-n. We replaced the GSEA enrichment score by the van der Waerden statistic, Z = Y/ under the null hypothesis. When distribution G is shifted left or right relative to F, then the value of Z tends to increase beyond what we would expect from the N distribution.where  null distribution to assess the significance of the enrichment score Z. The reason is that there are ordinarily correlations among the expression levels of the genes in G. When the expression levels of two genes in G are correlated, the age coefficients for those genes are correlated as well [. The value of Z can become large either because the genes are age related or because they are correlated with each other. Both may be biologically real, but the second is not an interesting finding, except possibly as confirmation that the group G is well constructed.It is better to use resampling methods instead of the N as well . It thenThe original GSEA  randomlyWe adopted instead a bootstrap approach. We resampled the data and recomputed enrichment scores, obtaining a histogram roughly centered over the observed enrichment score. If the null value (zero) is far outside the resampled histogram, then the enrichment score is statistically significant. The bootstrap approach also preserves correlations among genes as well as correlations between genes and covariates.The primary motivation for bootstrapping is the presence of covariates in our problems. Consider for example data with age, sex, and expression variables. If we permute the ages with respect to the expression data and repeat the regression, we have to decide whether the sex variable should be attached to the ages or to the expressions in the random permutation. Attaching sex to the age variables will leave us with simulated data sets in which females express Y chromosome genes as much as males. Because of such artifacts, this is not a suitable null distribution. Attaching a covariate to the expression variables is also problematic. Suppose that one of the covariates is somewhat correlated with age. The effect will be to increase the variance of the originally sampled age coefficient. In permutation samples where the covariate is attached to the expression data, it is resampled independently of age. Such independence reduces the variance of the age coefficient in the permutation data. The consequence is that the permutation-based histogram of age coefficients is then too narrow and false discoveries will result.In the bootstrap approach we generated 1,000 sample datasets. In each sample dataset we mimicked the sampling process that gave rise to the data by resampling 81 subjects from the population of 81 subjects. The resampling keeps age, expression, and all covariates of any given subject together. Bootstrap sampling mimics the random process that generated the data.We remark that both bootstrap and permutation sampling of the van der Waerden scores gave rise to Z scores that were nearly normally distributed, but not necessarily N (unpublished data). In permutation sampling, the histogram of enrichment scores tended to have means near zero, but several groups had variances larger than 1.0. In bootstrap sampling, the variances often differed from 1 and the means were usually between zero and the original enrichment score.p < 0.09, hypergeometric distribution), five aging-regulated genes in both muscle and the brain (p < 0.07), and 13 aging-regulated genes in common between the kidney and the brain (p < 0.29). There were no genes that were strongly age regulated in all three datasets. The Venn analysis approach is very interpretable but lacks power because it replaces actual measured correlations by a less informative notion of whether they are over a threshold.The most direct way to compare aging in muscle, the kidney, and the brain is via a Venn analysis: we find which genes attain a stringent significance level for each tissue and judge whether the overlap is statistically significant according to a hypergeometric distribution. We did a pairwise comparison between each tissue to find genes that are aging-regulated in both sets. There are six aging-regulated genes in both the muscle and the kidney ((r) of the resulting points (r = 0.219), and smaller but positive overlaps in aging between the muscle and the kidney (r = 0.103) or the muscle and the brain (r = 0.078).A more sensitive comparison can be based on correlating the age coefficients of genes in two tissues. We selected all genes that are age regulated in either of two tissues, plotted the age coefficient of each gene in one tissue versus that gene's coefficient in the other tissue, and computed the Pearson correlation g points . We founp value for a Pearson correlation between kidney and muscle, we used 1,000 sets of random genes. The number of genes in each set was the same as the number we used to compute the correlation in p value of 0.006 for kidney\u2013muscle. We similarly found a p value of 0.001 for kidney\u2013brain but only 0.058 for muscle\u2013brain. With the possible exception of the kidney\u2013brain pair, the age-related genes have more consistent age coefficients across tissues than randomly selected genes do.Because the genes are correlated we cannot use textbook formulas to judge the statistical significance of these Pearson scores. To get a p value of 0.078 for kidney\u2013muscle. To save computation, we used the same set of genes in each bootstrap sample instead of making the age-related gene set vary with the sample separately. The p value for muscle\u2013brain was 0.07 while that for kidney\u2013brain was 0.001. Based on these individual gene-level analyses, the age-related genes in the kidney and brain tended to be very similar. The muscle\u2013kidney and muscle\u2013brain comparisons were weaker.We also ran a bootstrap test of the tissue comparisons. In this test we resampled the microarray data with replacement 1,000 times. Each time we recomputed the correlations between age coefficients for genes in the kidney and muscle. In 1,000 trials we saw 39 in which the sample correlation was less than or equal to zero. After converting to a two-tailed test, this corresponds to a To test for the correlation of gene ranks between tissues within those gene sets found to be commonly age regulated in the human, we used a two-tailed Spearman correlation method to first calculate a correlation coefficient for every pairwise combination of tissues  for that age-regulated gene set . In order to test for the significance of the calculated correlations, we used a permutation-based Monte Carlo method, randomizing the ranks for each gene and tissue in the gene set and recalculating Spearman correlations 1,000 times. We found that most of the correlations between tissues were not significant .Figure S1x-axis. Sex, biopsy location, and 12 medical factors are shown in the legend. Only hypothyroidism shows any overt association with age.Each row denotes a medical or pharmaceutical factor. Age of patients is shown on the (253 KB TIF)Click here for additional data file.Figure S2y-axis) and without (x-axis) the coronary artery disease term was plotted. If coronary artery disease affected expression, we would expect a large deviation in age coefficient. No significant deviation was seen for any of the 250 age-regulated genes, indicating that coronary artery disease does not adversely affect our study of age regulation.(A) Coronary artery disease was included as an additional term in (B\u2013L) Similar to (A) for 11 other medical factors. (B) Coronary artery disease. (C) Colorectal cancer. (D) End-stage renal disease. (E) Hyperlipidemia. (F) Hypertension. (G) Hypothyroidism. (H) Pancreatic cancer. (I) Prostate cancer. (J) Radiotherapy. (K) Statins. (L) Villous adenoma.(319 KB TIF)Click here for additional data file.Figure S3Samples are clustered on the basis of 250 age-regulated genes in muscle, shown by the top dendrogram. Columns are individual muscle samples, marked by age of the patient. Top seven rows correspond to the expression of the first seven age-regulated genes. The diagram shows anatomical, medical, and pharmaceutical factors for each patient. Each row corresponds to one medical or pharmaceutical factor.(1.2 MB TIF)Click here for additional data file.Table S1(2.1 MB XLS)Click here for additional data file.Table S2(59 KB XLS)Click here for additional data file.Table S3(63 KB XLS)Click here for additional data file.Table S4(45 KB XLS)Click here for additional data file.Table S5(16 KB XLS)Click here for additional data file.Table S6(27 KB XLS)Click here for additional data file.Table S7(32 KB XLS)Click here for additional data file.Table S8(14 KB XLS)Click here for additional data file.Table S9(101 KB XLS)Click here for additional data file.Table S10(982 KB XLS)Click here for additional data file.Table S11(15 KB XLS)Click here for additional data file.Table S12(15 KB XLS)Click here for additional data file."}
{"text": "No matter how healthy a life one leads, no person has managed to live much longer than a century. Even though the advances of the modern age may have extended the average human life span, it is clear there are genetic limits to longevity. One prominent theory of aging lays the blame on the accumulation of damage done to DNA and proteins by \u201cfree radicals,\u201d highly reactive molecules produced by the metabolic activity of mitochondria. This damage is expected to reduce gene expression by damaging the DNA in which genes are encoded, and so the theory predicts that the most metabolically active tissues should show the greatest age-related reduction in gene expression. In this issue, Michael Eisen and colleagues show that the human brain follows this pattern. A similar pattern\u2014which, surprisingly, involves different genes\u2014is found in the brain of the aging chimpanzee.The authors compared results from three separate studies of age-related gene expression, each done on the same type of DNA microarray and each comparing brain regions in young versus old adult humans. In four different regions of the cortex (the brain region responsible for higher functions such as thinking), they found a similar pattern of age-related change, characterized by changes in expression of hundreds of genes. In contrast, expression in one non-cortical region, the cerebellum , was largely unchanged with age. In addition to confirming a prediction of the free-radical theory of aging , this is the first demonstration that age-related gene expression patterns can differ in different cells of a single organism.The authors found a similar difference in age-related patterns in the brain of the chimpanzee, with many genes down-regulated in the cortex that remained unchanged in the cerebellum. However, the set of affected cortical genes was entirely different between humans and chimps, whose lineages diverged about 5 million years ago. The explanation for this difference is unknown, but the finding highlights the fact that significant changes in gene expression patterns, and thus changes in many effects of the aging process, can accumulate over relatively short stretches of evolutionary time. These results raise a number of questions about age-related gene expression changes, including whether metabolically active non-brain tissues display similar patterns of changes, and whether the divergence between human and chimp patterns was the direct result of selection, or was an inevitable consequence of some other difference in brain evolution. The patterns seen in this study also provide a starting point for understanding the network of genetic changes in aging, and may even reveal targets for treatment of neurodegenerative diseases."}
{"text": "It is well established that gene expression levels in many organisms change during the aging process, and the advent of DNA microarrays has allowed genome-wide patterns of transcriptional changes associated with aging to be studied in both model organisms and various human tissues. Understanding the effects of aging on gene expression in the human brain is of particular interest, because of its relation to both normal and pathological neurodegeneration. Here we show that human cerebral cortex, human cerebellum, and chimpanzee cortex each undergo different patterns of age-related gene expression alterations. In humans, many more genes undergo consistent expression changes in the cortex than in the cerebellum; in chimpanzees, many genes change expression with age in cortex, but the pattern of changes in expression bears almost no resemblance to that of human cortex. These results demonstrate the diversity of aging patterns present within the human brain, as well as how rapidly genome-wide patterns of aging can evolve between species; they may also have implications for the oxidative free radical theory of aging, and help to improve our understanding of human neurodegenerative diseases. Transcriptional profiles in human and chimpanzee reveal a diversity of aging patterns present within the human brain, as well as how rapidly genome-wide patterns of aging can evolve between species. Despite its ubiquity and importance, aging remains a poorly understood process. This lack of understanding is due in part to the complexity of aging, which is characterized by the gradual and progressive decline of numerous physiological processes and homeostasis, eventually leading to death \u20136. HowevCaenorhabditis elegans belong to the insulin/IGF (insulin-like growth factor) signaling pathway, indicating possible connections to metabolism and a state of arrested development known as \u201cdauer\u201d [Both environmental and genetic alterations in model organisms have been found to have profound effects on aging and lifespan. In particular, dietary restriction has been found to dramatically increase the lifespan of organisms including yeast, flies, nematodes, and mammals ,5; the m \u201cdauer\u201d ,5. Anoth \u201cdauer\u201d ,3.2O2), are produced in large part by the mitochondrial electron transport chain. The free radical theory has garnered widespread support in recent years; in addition to the genetic evidence mentioned above, studies from a number of model organisms showing that decreasing ROS levels leads to an increase in lifespan indicate that ROS can strongly modulate the aging process [The free radical theory of aging was first introduced by Harman almost half a century ago . This th process \u20135.Exactly how macromolecules damaged by ROS may lead to aging has been studied in detail in recent years, and the human brain has been intensively examined in this regard because of its overall importance in human senescence. For example, up to one-third of the proteins in the brains of elderly individuals may be oxidatively damaged, and these damaged proteins have been shown to sometimes have diminished catalytic function ,6. One rComplementing studies of aging differences in various tissues within a single species, research into the evolution of aging has begun to shed light on the similarities and differences between species, although the expectation for how well conserved the effects of aging will be on a gene-by-gene basis is still unclear. One the one hand, if aging is largely caused by the deleterious effects of many alleles late in life\u2014as is often the interpretation of two widely held models for the evolution of aging, known as \u201cantagonistic pleiotropy\u201d and \u201cmutation accumulation\u201d ,5,13\u2014theOne promising approach to answering this question of evolutionary conservation lies at the level of gene expression: Do orthologous genes tend to undergo the same patterns of expression changes with age in diverse species, or can a common factor such as ROS lead to different gene expression patterns in different organisms? Using DNA microarrays, this question can now be addressed in a systematic, genome-wide manner. One such study found that a small but significant portion of aging-related gene expression changes are shared by the very distantly related nematode and fruit fly ; anotherThe human brain is of particular interest for studying the divergence in phenotypes that have changed rapidly during evolution (such as aging). Brain-specific genes have undergone accelerated evolution in the lineage leading to human since the split with chimpanzee at the levels of both protein sequence and gene expression ,17, poinIn order to test whether different regions of the human brain show similar patterns of change with age, we utilized three independently published microarray expression datasets. These were: Lu et al. , mentionTo achieve the most comprehensive picture of brain aging possible with these data, we first sought to study the patterns of aging in all six brain regions from Khaitovich et al. . Becausep < 0.01) Spearman rank correlation between age and expression level in frontal pole, irrespective of their fold change in expression; most of these were expected to be true positives, because only approximately 126 genes would be expected to pass this significance threshold by chance . Values of r close to one indicate good agreement between aging patterns, whereas those close to zero indicate a lack of agreement. To assess the significance of these correlations, we randomly permuted the ages of the samples, and calculated the probability of observing a random correlation as strong as that found in the real data . We note that the true similarity of aging patterns in these regions is likely to be even stronger than is indicated by the correlations because, as mentioned above, approximately 15% of our genes are expected to be false positives with no true aging-related changes. In sharp contrast to cortex, the cerebellum and caudate nucleus showed far less agreement with frontal pole (r| < 0.1 and p > 0.4 for each). These results have several implications. First, the agreement between frontal pole and four regions of cortex indicates that we were able to accurately measure the direction of gene expression changes with age for most genes, even with only three samples from each region; thus the age range, number of samples, etc., are all sufficient to reflect the pattern of gene expression changes previously reported in frontal pole [Strikingly, all four regions of cerebral cortex for which we had expression data  showed excellent agreement with the aging pattern in frontal pole . Although the cerebellum correlation is stronger here than in p = 0.17), even though the two cortex samples are both significant (p < 0.01 each). This finding supports our conclusion that cerebellum ages differently than cortex.In order to further test the similarity of aging patterns within the brain, we compared a third independent dataset to the data from Lu et al.  and Khair| < 0.5) in either dataset. This comparison showed the expected reproducibility of aging patterns across all four regions of the cortex: r > 0.76 for all four . In contrast, neither cerebellum nor caudate nucleus showed a significant correlation (r| < 0.04 and p > 0.4 for each), as expected from their lack of correlation with the frontal pole data shown in For a third test of aging patterns throughout the human brain, we determined the correlation of aging patterns between a single cortex region from Evans et al.  with allr = 0.952. The cerebellum data from Evans et al. [r = 0.964. Thus differing data quality could not explain the lack of correlation in cerebellum and caudate nucleus.The lack of correlation between the aging pattern in cerebral cortex with those in cerebellum and caudate nucleus might arise if the quality of data in the cerebellum and caudate nucleus samples was lower than that of the cortex samples from both Khaitovich et al.  and Evans et al.  was of sfewer genes changing expression with age than cortex does, and they thus lack a reproducible pattern of aging-associated gene expression changes altogether.There are two possible explanations for the difference in the aging patterns between cerebellum/caudate nucleus and cerebral cortex. One is that cerebellum and caudate nucleus have their own aging patterns distinct from that in cortex. The other possibility is that cerebellum and caudate nucleus are different from cortex because they each have far To distinguish between these possibilities, one could attempt to calculate exactly how many genes change expression with age in each region; if cerebellum and/or caudate nucleus have aging-related changes in as many (but a different set of) genes than cortex, then the number of genes identified as changing in cerebellum and/or caudate nucleus should be comparable to any region of cortex. Unfortunately, as mentioned above, there is not enough statistical power to pursue this approach, given only three samples per region .Another way to differentiate between the two possibilities listed above would be to compare two datasets of cerebellum and/or caudate nucleus aging patterns to one another. If these regions have a reproducible pattern of many genes changing expression with age , including the cerebellum\u2013cerebellum comparison. From these results, we conclude that cerebellum has a different pattern of aging than cortex because significantly fewer genes appear to change expression with age in cerebellum.We thus expected to see a strong positive correlation between cerebellum aging patterns in our two datasets if and only if a large number of genes change expression with age in cerebellum. Because this analysis was carried out on all informative genes  for the 841 genes changing strongly with age in frontal pole, in both young (45-y-old) and old (70-y-old) samples from Khaitovich et al. . As expeIn order to study the relationship between brain aging patterns in humans and chimpanzees, we required gene expression data from the chimpanzee brain. Although four studies have already produced such data, three of these ,22,23 exr| > 0.5) present on the microarray. Although we found no significant correlation when comparing cerebellum to either cortex region (r| < 0.07 and p > 0.3 for both comparisons), we found a very strong agreement when comparing aging patterns of prefrontal cortex to anterior cingulate cortex . This result is precisely analogous to our findings in human, where the entire cerebral cortex shares a single pattern of gene expression changes with age that is not found in the cerebellum . Similar results were found when comparing chimpanzee aging changes in any brain region to the patterns from either of our other two human expression datasets [We then tested whether brain aging in chimpanzee is similar to that of human. Using the 841 genes that change expression with age in human frontal pole , we testdatasets ,20 for eGiven this difference in aging patterns between humans and chimpanzees, we examined the expression levels of the chimpanzee orthologs of the 841 human genes that change expression with age in frontal pole in order to see if the expression levels of the chimpanzee orthologs of these genes resemble young humans, old humans, or neither. To test this, we first reanalyzed the expression data by masking all microarray probes with sequence differences between humans and chimpanzees . We thenr| > 0.5) are nearly all genuinely up- or down-regulated with age. Using this list of genes with a consistent aging pattern in the two cortex regions, there were 1,252 down-regulated and 700 up-regulated genes. Note that although the false-positive rate is likely to be low, we have no way to estimate the false-negative rate, so these numbers should not be interpreted as the total number of genes changing expression with age in chimpanzee cortex. Using this list of aging-associated genes, we tested for any significant enrichments of these genes in Gene Ontology annotation categories [The high correlation of gene expression aging patterns between the two regions of chimpanzee cortex implied that the genes for which both regions show the same direction of change  and the National Institute of Mental Health (NIMH) Silvio O. Conte Center, and from ArrayExpress and GEO databases. Only the seven \u201cType 1\u201d control individuals [Human microarray data was obtained from Pritzker Neuropsychiatric Disorders Research Consortium . Data were normalized using the Robust Multichip Average (RMA) method [Chimpanzee postmortem samples were obtained from Yerkes Regional Primate Center, Biomedical Primate Research Centre and Anthropologisches Institut und Museum, Universit\u00e4t Z\u00fcrich. All individuals suffered sudden death for reasons other than their participation in this study and without any relation to the tissues used; time between death and preservation of brain tissue  did not correlate with expression of genes that changes with age. Age and sex for all individuals are listed in ) method .Among the chimpanzees used for this work, one was of indeterminate age, having been caught in the wild 40 y before its death . HoweverAdditionally, one pair of chimpanzees used were full siblings, and another pair were half siblings , which cThere are several possible artifactual explanations for our results not addressed in the main text. First, there is the possibility that gene expression changes correlated with age were caused by an unknown factor unrelated to the normal aging process. For example, if in the study by Khaitovich et al.  the 70-ySimilarly, one possible explanation for the results in If gene expression changes in primates tend to be dependent on chronological age , then the different patterns of expression changes with age seen in humans and chimpanzees could be caused by the different age ranges of the human (18\u2013106 y) and chimpanzee (7 to approximately 45 y) samples. To control for this possibility, we truncated the data of Lu et al. ,20. TherAnother possible explanation for the difference between human and chimpanzee aging patterns B is thatOne caveat concerning our interpretation of fewer genes having aging-associated changes in expression in cerebellum than in cortex is that the two human cerebellum datasets, although both consisting of grey matter of the cerebellum, were from different regions of the cerebellum ; in these cases the test was two-sided. This randomization test is somewhat conservative; for example, analyzing the leftmost bar  of p-value of approximately 10\u221299, as opposed to approximately 0.015 from randomization. This large difference is due to the nonrandom structure of the expression data, which makes it more likely to observe strong correlations than would be expected in a set of random data. Finally, we note that our finding of significant agreement between aging patterns in datasets containing various numbers of microarrays does not imply that these numbers of microarrays will always yield sufficient power to find such a correlation if one exists.All correlation coefficients reported here were calculated by Spearman rank correlation, a nonparametric method that is robust to the presence of any outliers. The correlation coefficients from comparisons of aging profiles between two tissues or species as in  and 4 weTable S1p-value cutoff for each GO group is 0.005.\u201cAll detected\u201d is the number of genes on the microarray that had an annotation in each of the three Gene Ontology (GO) categories. \u201cDetected/group\u201d is the number of genes on the microarray that belong to the specific GO group listed. \u201cAll selected\u201d is the number of genes up-regulated with age in chimpanzee cortex that have annotations in each of the three GO categories. \u201cSelected/group\u201d is the number of genes up-regulated with age in chimpanzee cortex that belong to the specific GO group listed. The uncorrected (21 KB XLS).Click here for additional data file.Table S2(18 KB XLS).Click here for additional data file."}
{"text": "Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown.To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging . We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types .Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases. Aging is a complex biological process influenced by stochastic occurrence of molecular damage over time, but despite inherent randomness, the hallmarks of aging appear to be specific and uniform between individuals and even among species Despite a broad consensus, many changes in gene expression appear to be tissue- or even cell-type specific Age is a major risk factor for age-related macular degeneration (AMD), a leading cause of untreatable vision loss in the elderly Rod photoreceptors represent the predominant retinal cell type in most mammals and are uniquely vulnerable to the effects of age. In the human retina, about 30% of central rods are lost by the ninth decade, whereas cone density remains essentially unchanged http://research.jax.org/faculty/harrison/ger1vLifespan1.html and We had previously developed mice that express enhanced Green Fluorescent Protein (eGFP) under the control of the Neural Retina Leucine zipper promoter (Nrlp), which restricts eGFP expression in the retina to developing and mature rod photoreceptors max), sensitivity k, and a- (Vamax) and b-wave (Vbmax) amplitudes at maximum flash intensity of 1.09 log cd-s/m2. ERGs of Nrlp-eGFP mice showed a mild decline with age in rod function similar to that of age-matched C57Bl/6 controls . The dark-adapted b-wave, Vbmax, which reflects signaling downstream of the photoreceptors, declined with age in Nrlp-eGFP and C57Bl/6 mice . Rod-mediated scotopic amplitude Vmax, also declined with age but was not significantly different in Nrlp-eGFP as it was the case for C57Bl/6 mice (p<0.01). There was no change in sensitivity k with increased age in either group. For all ERG parameters, age-dependent changes in Nrlp-eGFP mice mirrored those observed in C57Bl/6 mice. Expression of eGFP in rod photoreceptors, therefore, does not appear to cause any significant difference compared to C57Bl/6 mice in retinal histology or function up to 12 months of age, though a slight decline in rod function by 12 months of age is detected in both strains.We first evaluated whether eGFP expression in rod photoreceptors had any effect on the physiology or cellular morphology by comparing Nrlp-eGFP mice to wild-type (C57Bl/6) mice at different ages. Nrlp-eGFP mice developed normally and showed no histological signs of retinal degeneration by 12 months of age . ONL thicontrols . There w6 and 9.54\u00d7105, respectively; n\u200a=\u200a4, p\u200a=\u200a0.846 by Student's two-tailed t test). Gene expression was compared among the three selected time points by one-way ANOVA that yielded 413 probe sets (corresponding to 375 transcribed sequences) showing >1.5-fold change at p<0.05 between at least two of the three time points ; immune system regulation - histocompatibility 2 K1, K region (H2-K1), interferon regulatory factor 6 (Irf6), and endoplasmic reticulum aminopeptidase 1 (Erap1); lipid and insulin metabolism - stearoyl coenzyme A desaturase (Scd1), oxysterol binding protein-like 1A (Osbpl1a), and insulin receptor substrate 1 (Irs1); and two genes that have not yet been characterized .Twenty-five genes with at least 2-fold change and/or potential biological relevance were selected for validation by quantitative RT-PCR (qRT-PCR) using independent rod photoreceptor RNA samples . Seventeen of the 25 genes exhibited changes in the predicted direction at both 5 months and 12 months of age relative to 1.5 months; of these, 16 showed increased expression with age, and one showed reduced expression compare . Of the Grem2, Rdh9, and Btnl9. Irf6 demonstrated a higher magnitude of change (over twofold) in retina samples than photoreceptor samples, but the remaining genes exhibited comparable fold changes with age. Expression of only one gene, C030034E14Rik, changed in an opposite direction with age in whole retinas compared to isolated rod photoreceptors.To evaluate whether gene expression changes in our analysis were characteristic of rod photoreceptors, we performed qRT-PCR analysis of the 17 validated genes using total RNA from whole retina from young (1.5 month old) and old (12 month old) Nrlp-eGFP mice . Whole rEye Morphogenesis, Electron transport chain, Motor Axon Guidance, and Antigen presentation among the top ten  enrichment analysis  top ten . Some ofRdh9) mostly increased. Both higher and lower expression changes were detected with age for genes in the tight-junction signaling and Cxcr4 signaling pathways.Ingenuity\u00ae Pathway Analysis (IPA) sorts genes into canonical pathways based on the scientific literature and indicates which are significantly overrepresented Ingenuity\u00ae network analysis revealed biological processes that were enriched among the 100 genes showing progressively higher or lower expression across the three time points see . We thenNr3c1), involved in stress response and inflammation; RhoA, an important signaling molecule associated with cytoskeletal remodeling; and Cdkn1b, which maintains cell cycle arrest. Additionally, several genes involved in overrepresented pathways (described above) were present in the direct interaction network. Overall, genes associated with stress response appeared to occupy prominent positions (hubs) in the network topology.Finally, we generated a network reconstruction using MetaCore\u2122 from GeneGo, Inc. C. elegans have identified a genetic basis for the modulation of aging phenotypes that extends to higher organisms in vivo as it is the most accessible part of the central nervous system. Furthermore, evidence suggests shared characteristics between age-related diseases of the retina and the brain, such as AMD and Alzheimer's disease www.sph.uth.tmc.edu/Retnet/) and is uniquely amenable to non-invasive evaluation, which facilitates the study of natural history and potential therapies. However, the direct impact of aging on molecular pathways that guide photoreceptor metabolism or function has yet to be elucidated. Here, we report adaptive changes in both conserved and cell-specific age-related pathways through temporal profiling of purified rod photoreceptors. We show that a majority of these expression changes begin in young adults and continue to progress with age. Furthermore, our study reveals that single cell-type profiling provides better resolution of subtle and progressive age-related expression changes (see below), yielding a more comprehensive depiction of cellular aging.Aging-associated changes occur on multiple levels and contribute to cellular dysfunction and disease. Landmark studies in yeast and Though rod photoreceptors are highly specialized neurons involved in photoreception, we identified changes in consensus pathways of aging, including oxidative phosphorylation and stress response affecting transcription and inflammation . Rods arregulation of transcription and nucleosome assembly, and expression of genes for histone clusters 1, 2, and 3  progressively increased over time (Dnmt3a) and Suppressor of variegation 4\u201320 homolog 1 (SUV420h1), which was also noted to change with age in human retinas Hr) and Nuclear co-repressor 1 (Ncor1) both decreased with age, suggesting a derepression of transcription that has been associated with epigenetic changes in other aging cells Patterns of histone acetylation and DNA methylation have been shown to change with age  from yeast to humans, influencing DNA stability and gene expression patterns ver time . DNA-modIrf6 and antigen presentation genes Erap1H2-K1 suggest a similar upregulation of inflammation signals in photoreceptors. Increased expression of H2-K1 and the human homolog, HLA-C, was also reported in previous studies of aging mouse and human retina and eye tissue Fos and Nr3c1 (glucocorticoid receptor) signaling that may contribute to systemic and cognitive aging Upregulation of inflammatory response pathways has been associated with neuronal aging of rodents and humans CD59a is particularly interesting as it protects against complement attack Stress response processes, including angiogenesis and retinoid/lipid metabolism, are especially relevant for the aging retina . PhotorePpara) appear to play a role in photoreceptor aging. Altered retinoic acid metabolism has been implicated in some forms of retinal degeneration Rdh9 and Rdh18) increased dramatically with age in rod photoreceptors, perhaps representing a stress response to lipid peroxidation, another source of ROS Scd1 and Osbpl1a, and a progressive decrease in Ppara, all of which influence lipid and insulin signaling Ppara has also been observed in aging heart, liver, and kidney Ppara may represent a response to age-related changes in RA signaling or lipid peroxidation, since LCPUFAs and retinoids are endogenous ligands for PPAR-alpha Changes in retinoid and lipid metabolism via the retinoic acid receptor (RAR) pathway and the peroxisome proliferator-activated receptor alpha , and further investigations are in progress to examine their physiological relevance in rod photoreceptor aging. In addition to examining individual genes, we adopted a systems biology approach to analyze the expression data as a whole . This type of analysis keeps the focus on groups of genes and the pathways/categories/functional groups they belong to, and it is best applied to larger unbiased data sets. The strength of the observation derives from the genes in similar pathways that behave in a coordinated fashion.Our study provides an aging-associated gene expression signature for a single neuronal cell type. Studying rod photoreceptors instead of the entire retina has allowed us to identify significant age-related changes with better resolution. Both consensus and photoreceptor-specific age-related pathways, identified here, have implications for understanding the pathogenesis of AMD and other retinopathies. Notably, many expression changes are evident even at a relatively young adult age, suggesting that aging is a continuous process set in motion long before phenotypic changes are detected. Our studies reveal contributions of rod photoreceptors to retinal aging as well as potential changes in the immune-privileged status of the neuroretina with age. We cannot determine whether expression changes associated with normal aging are causative, permissive, or protective with respect to aging-associated functional decline and disease. Nonetheless, we propose that changes in gene expression represent an adaptive response of rod photoreceptors to microenvironment (such as exposure to light and oxidative stress) during the aging process. Identification of aging-associated pathways thus provides a foundation for future investigations and offers potential targets to develop treatments and therapies for age-related diseases.All mice were used in accordance with the approved Institutional Animal Care and Use Committee (IACUC) protocol.To evaluate retinal function, full field ERGs were recorded in Nrlp-eGFP mice and C57Bl/6 control mice at 4 and 12 months of age. Mice were dark-adapted overnight and were prepared under dim red illumination as described previously 2 recording system  to brief xenon white flashes from \u22125.8 to +1.09 log cd-s/m2/flash in steps of 0.5 log units for dark-adapted responses and from \u22120.91 to +1.09 log cd-s/m2/flash over a 2 log unit range in steps of 0.3 log units for light-adapted responses. Responses were amplified at 1,000 gain at 1.25 to 1000 Hz, and digitized at a rate of 2000 Hz. A notch filter was used to remove 60 Hz line noise. Dark-adapted b-wave amplitudes were fitted with a Naka-Rushton function to determine estimates for the b-wave saturated amplitude,Vmax and the response sensitivity, k. Light-adapted responses were recorded after 10 minutes of adaptation to a white 32 cd/m2 rod-suppressing background.ERGs were recorded with a Ganzfeld configuration using the Espion eRetinas from 1\u20132 and 10\u201312 months old Nrlp-eGFP and C57Bl/6 mice (n\u200a=\u200a3 each) were dissected, fixed and processed to generate sagittal thin plastic sections encompassing the optic nerve head. The optic nerve was oriented in the section to indicate the ventral portion. ONL thickness was measured using Zeiss, AxioVision Documentation Rel.4.7 software on 40x-magnified images. Eight microscopy frames were counted ventrally and dorsally to the optic nerve head and measurements were taken in the middle of the frame. Values represent mean \u00b1 SEM.Rod photoreceptors were purified by flow-sorting, as previously described Five nanograms of total RNA from flow-sorted rod photoreceptors from Nrlp-eGFP mice at 1.5 month, 5 months and 1 year of age  were used for target generation. Ovation RNA Amplification System V2  was used for cDNA amplification according to the manufacturer's protocol. FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies) was used to fragment and biotin label the amplified cDNA and for hybridization of the labeled target onto a GeneChip Mouse Genome 430 2.0 Array  with 45,000 probe sets for 34,000 well-characterized mouse genes. Washing and staining of the GeneChips was performed using the EukGE-WS2v4_450 protocol on the GeneChip Fluidic Station 450 (Affymetrix). GeneChip scanning was performed on the GCS3000-7G Scanner (Affymetrix).www.ingenuity.com, Redwood City, CA), MetaCore(TM) from GeneGO , and GO Enrichment Analysis The Robust Multichip Average (RMA) method Hprt) and ribosomal protein S26 (S26) served as internal controls. Primer pairs tested are listed in Retinas were dissected from four independent biological replicates for each of the three timepoints , enzymatically dissociated as described above, and rod photoreceptors were flow-sorted into RNAprotect (Qiagen). Whole retinas were harvested from additional mice and snap-frozen. RNA was extracted using the RNeasy Mini kit (Qiagen) and quality was evaluated as described above. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix  on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Candidate genes were selected for analysis based on microarray predictions of fold-change >2, pattern of change, and gene function. Hypoxanthine guanine phosphoribosyl transferase Click here for additional data file.Table S225 genes evaluated by quantitative RT-PCR (photoreceptor data).(0.02 MB XLS)Click here for additional data file."}
{"text": "P-value < 0.05). In general, gene expressions that increased with age were associated with increased mortality. Gene expressions that decreased with age were generally associated with reduced mortality. A multivariate estimate of biological age modeled from expression data was dominated by CDC42 expression, and was a significant predictor of survival after blood draw. A multivariate model of survival as a function of gene expression was dominated by CORO1A expression. This model accounted for approximately 23% of the variation in survival among the CEU grandparents. Some expression levels were negligibly associated with age in this cross-sectional dataset, but strongly associated with inter-individual differences in survival. These observations may lead to new insights regarding the genetic contribution to exceptional longevity.We investigated the hypothesis that gene expression profiles in cultured cell lines from adults, aged 57\u201397 years, contain information about the biological age and potential longevity of the donors. We studied 104 unrelated grandparents from 31 Utah CEU (Centre d\u2019Etude du Polymorphisme Humain \u2013 Utah) families, for whom lymphoblastoid cell lines were established in the 1980s. Combining publicly available gene expression data from these cell lines, and survival data from the Utah Population Database, we tested the relationship between expression of 2151 always-expressed genes, age, and survival of the donors. Approximately 16% of 2151 expression levels were associated with donor age: 10% decreased in expression with age, and 6% increased with age. Cell division cycle 42 (CDC42) and CORO1A exhibited strong associations both with age at draw and survival after draw (multiple comparisons-adjusted Monte Carlo  Caenorhabditis elegans , consists of Epstein\u2013Barr virus-immortalized lymphoblastoid cell lines and DNA extracted there from, for 46 three-generation CEU families, each consisting of 5\u201315 siblings, their two parents, and two to four grandparents who were still alive at the time of the family blood draws in the early 1980s (The CEPH/Utah (CEU) family resource, maintained at the Coriell Cell Repositories in Camden, NJ, USA , we used only the 2151 always-expressed genes , to examine the relationship between individual expression levels and age at draw in the CEU grandparent cell lines. We modeled expression as a simple linear function of age at draw. Expression levels are reported on a logA more complex analysis of changes in gene expression with age over all three generations of CEU families revealed a large number of highly significant associations. However, interpretation of age-related changes in expression across three generations, ages 5\u201397, is difficult because senescence can be confounded with sexual or physiological maturation, as well as secular trends occurring over such a long span of donor birth years in these families. Methods and results of this analysis are described in the Supporting Information.P< 0.05. Of these, 125 increased with age and 220 decreased with age. P-value below the Bonferroni 5% threshold of 2.3 \u00d7 10\u22125. The strongest association of expression level with age was CDC42 (cell division cycle 42), which exhibited strongly increased expression with age, with a sex-adjusted P-value of 3.1 \u00d7 10\u22125, and an unadjusted P-value of 1.3 \u00d7 10\u22125. Among the ten most strongly age-associated expression levels  expressions were associated with age at draw in the CEU grandparents at a nominal n levels , equal nH2) for each gene listed, and the correlation of expression between spouses. Most of the genes listed in Also shown in \u22125, although CORO1A (coronin) comes quite close. Nine of the ten strongest associations of age-adjusted expression with mortality are negative, meaning that relative overexpression of the gene is associated with reduced mortality. Interestingly, the one exception is TERF2IP , which is thought to protect telomeric DNA from nonhomologous end-joining. Note that only one gene (CORO1A) appears in both We also tested for gene expressions most strongly associated with survival, independently of their associations with age at blood draw. P< 0.05. Of these, 48 were associated with age at blood draw at a nominal P< 0.05. A total of 167 (7.8%) expression levels were associated with survival in the CEU grandparents at a nominal The results shown in Z-scores for age effects and survival effects on (age-adjusted) expression levels, for the CEU grandparents. Using Fisher\u2019s likelihood ratio approach (Z-scores (large red dots) to those generated by a 10% sample of 1000 random permutations of the phenotypic  data, while keeping the expression vectors constant . The dashed ellipse is drawn at the 50th largest chi-squared value observed for the 2151 always-expressed genes and 1000 permutations. The results are shown this way to approximate the fifth percentile of the null distribution, adjusted for 2151 comparisons. Three genes fall outside the threshold: CORO1A (0%), CDC42 (0.2%), and AURKB . Expression of CDC42 increases with age among the CEU grandparents, and higher age-adjusted expression is associated with higher mortality. CORO1A and AURKB represent the opposite extreme of this same pattern: expression decreases with age, and higher age-adjusted expression is associated with lower mortality.approach , we compr = 0.51; P< 2.2 \u00d7 10\u221216) between Z-scores for age-related expression change and age-adjusted survival in Overall there is a fairly strong positive correlation , SEPT2 (1.6), PBX3 (1.1), CIB1 (\u22120.91), SH3BGRL (0.77), UBE2A (\u22120.71), RNH1 (\u22120.60), PPP1R11 (0.55), QDPR (\u22120.48), DDX24 (0.36), GINS2 (\u22120.30), LPXN (0.24), and ACAA1 (0.16). Clearly, the positive association of CDC42 with age at draw dominates this model. This remains true at steps 20, 40, 60, and 80 (data not shown). Expression levels of CDC42, SH3BGRL, QDPR, and RNH1 were also among the ten most strongly associated with age at draw for CEU grandparents in the univariate analysis reported in We used the step 14 model from An alternative approach to modeling survival as a function of estimated biological age would be to model it as a function of the difference between biological and chronological age. This is equivalent to forcing both biological age and chronological age to have the same slope (with opposite signs), and is efficient only if both variables are scaled identically. We evaluated this approach by rescaling biological age to have 0 mean and unit variance, then multiplying by the standard deviation of chronological age (7.9) and adding the mean chronological age (71.5); that technique produced results (not shown here) essentially identical to our original method, reported above.The above analysis demonstrates that biological age, estimated from gene expression levels that change with age, is a significant predictor of remaining life span. An important potential weakness of this analysis is that it places too much emphasis on gene expressions that vary systematically with age in cross-sectional data. As noted above, the confounding of differential survival effects with age-related changes in expression could mask the effects of genes that regulate or contribute to survival.In an effort to circumvent this limitation, we also applied the LASSO approach to model survival as a direct multivariate function of expression levels. We computed deviance residuals from a baseline survival model adjusted for age at draw and sex, and used LASSO to identify expression levels associated with variation in the deviance residual [see Methods and reference ]. We useP-value = 4.0 \u00d7 10\u22128; inter-quartile relative risk (IQRR) = 2.35; median estimated survival difference = 5.5 years. Predicted mortality from the model accounts for 23% of the variation (R2 = 0.23) in survival among the CEU grandparents. Model coefficients for individual genes are converted into IQRR in P-value given in The nominal Although Inter-individual differences in gene expression profiles in the Utah CEU lymphoblastoid cell lines may reflect not only heritable genetic influences, but also environmental exposures experienced at any time prior to blood draw. Therefore, we considered the possibility that gene expressions associated with survival may simply reflect exposures (or nonexposure) to a common toxic agent, such as cigarette smoke. The data available to us do not contain information on environmental exposures; however, affiliation with the Church of Jesus Christ of Latter-day Saints (or LDS church), available from the UPDB, indirectly provides information about exposures that affect mortality risks. r2 across the entire dataset is 0.962 while the maximum r2 for 100 random pairs of grandparents was slightly smaller . Overall, then, spouse expression profiles are slightly more strongly correlated than expected by chance, although the level of correlation among all expression profiles is very high. Interestingly, however, the correlation of mortality risk between spouses  is only 0.075 , so correlations in expression are not likely to confound the survival analysis.It is apparent in It is not surprising to find that gene expression patterns vary with age, nor that variation in gene expression correlates with variation in longevity; many studies in model organisms have clearly demonstrated these associations. However, the observation that patterns of gene expression in lymphoblastoid cell lines contain information about both the age and subsequent survival of human donors is both biologically interesting and potentially of great clinical utility.et al.:While \u201cOur findings that Cdc42 activation is associated with natural aging and that Cdc42GAP deficiency causes a DNA damage repair defect to allow cells to accumulate genomic abnormalities that leads to early senescence further suggest a functional link between Cdc42 activity and mammalian aging (The results of this study support this conclusion.CORO1A) is an actin-binding protein with potentially important functions in both T-cell mediated immunity and mitochondrial apoptosis , several single nucleotide polymorphisms near the IQGAP1 gene are strongly associated with IQGAP1 expression .Thus, the region immediately surrounding IQGAP1 may harbor genetic variants associated with variation in human lifespan.Several other genes exhibiting associations with either aging or survival are worthy of note. AURKB is a key member of the chromosomal passenger complex which is critical in the regulation and conduct of mitosis . Goring H2 = 0.09; P= 0.10 in our data) or untransformed . This may be a consequence of the cell transformation process, which activates telomerase so that cell lines may grow indefinitely in culture; possibly variable TERF2IP expression is marking some variation in telomerase activity in transformed lymphocytes that is indirectly related to longevity. A recent report links longer telomeres to increased risk of breast cancer  in lymphoblastoid cell lines is associated with increased mortality, because increased expression of TERF2IP should lead to increased telomere length , which hBecause of the large number of comparisons we have generated for a relatively small number of samples, and because the potential for measurement error is inherent in microarray data, the associations we have observed require confirmation in other settings. Associations between gene expressions and survival may be due to causal roles in regulating aging; alternatively, the expression of a gene may not reflect a regulatory role in aging, but nevertheless may serve as a reliable indicator of overall health and remaining life expectancy. Strong evidence for a regulatory role in aging would be provided by experiments in model systems showing that modulating expression extended lifespan. Within the CEU family data, we are pursuing linkage and genetic association studies to identify loci responsible for regulating expression of the heritable expression phenotypes associated with longevity. The identification of germ line variants that affect the expression of longevity-associated genes will facilitate further high-throughput genetic epidemiologic studies of human aging. However, even the nonheritable gene expression patterns we have reported may have considerable relevance to mechanisms of aging and age-related disease processes. Finally, we note that expression data are available for approximately 6000 genes not included in the \u2018always-expressed\u2019 list to which we restricted this analysis. Approximately half of these genes are expressed in many, but not all samples. The other half are rarely, if ever, expressed above background. We plan to investigate the association between longevity and expression of these genes as well.We have described some striking patterns of association of gene expression with age and mortality, based on lymphoblastoid cell lines derived from ordinary blood samples, and stored for years as a replenishable source of DNA for genetic studies. If these results can be confirmed by others, frozen cell lines may also have considerable value as sources of phenotypic information on transcription, translation, and other cellular processes helpful in predicting the future health of the donors.The CEPH/Utah family resource originated from bloods drawn from 46 three-generation families, each consisting of 5\u201315 siblings, their two parents, and two to four grandparents who were still alive at the time of the family blood draws in the early 1980s (http://locus.umdnj.edu/nigms/ceph/ceph.html) for a total of 247 CEU family members , including 104 of the grandparents for whom we had survival data. Expression levels for 8793 probesets were measured using Affymetrix HG-Focus arrays. The resulting expression data were deposited in the NCBI Gene Expression Omnibus database, accession numbers GSE1485 and GSE2552. We combined both datasets to maximize the number of individuals available for study.Cheung and co-workers  or \u2018marginal\u2019 (P< 0.06) in each of the grandparents\u2019 samples. This step left us with 2151 always-expressed genes.We tested whether each gene was expressed beyond baseline in each sample using the Wilcoxon signed rank test as described in the Affymetrix Microarray Suite version 5 (gcrma) method to normalize all expression levels.We used Wu and Irizarry\u2019s GeneChip robust multiarray averaging . For these individuals, we averaged gcrma-corrected expression levels for each probeset prior to analysis.We performed two separate analyses of expression as a function of age at draw. First, we considered only the grandparents, who were effectively unrelated to one another, although careful analysis of the records of the UPDB  has reveStrong genetic correlations in expression level should be present within each three-generation CEU family. For this reason, standard linear regression approaches are not appropriate for the study of age-related variation in expression patterns. Instead, we used linear mixed-effects models to adjust for the kinship among family members . The subr software package (http://www.r-project.org). Although some nonproportionality was detected with P-values below 0.0001, none of the genes strongly associated with survival had a P-value for nonproportionality lower than 0.28 (EMP3).Grandparents ranged in age from 57 to 97 years of age at the time of the blood draw. Median follow-up age was 84.7 years (range 65.7\u2013100.8). Survival was measured from age at draw to age at death or follow-up. We used a proportional hazards model adjusting for sex, year of birth, and age at draw to test the association of each expression level with survival. Each proportional hazards model was tested for nonproportionality using the cox.zph function in the We computed Adjustments for multiple testing are clearly appropriate in this context, but many methods mask hidden assumptions about the dependency structure of the data or the true proportion of false null hypotheses . We therCp or by cross validation.To assess the relationship between age at draw and multiple expression levels, we employed the LASSO algorithm of Tibshirani  in our sample of 104 grandparents. We compared the resulting LOOCV curve to curves generated from 100 random permutations of the data, performed as described above. Comparing the cross-validation curve to a null distribution of cross-validation curves not only gives information on the optimal setting of the tuning parameter, but also on the probability that the result is due to chance.We set For each value of the tuning parameter, corresponding to a step in which a predictor variable can be added or dropped, the model selected was used to predict each subject\u2019s age at draw. These \u2018biological age\u2019 estimates, together with the subjects\u2019 actual age and sex, were used to predict age at death in a proportional hazards model.We followed the approach of"}
{"text": "CYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as sex hormone metabolism. Because differences in the activity of the enzyme have been correlated with variant alleles of single nucleotide polymorphisms (SNPs), it represents an attractive candidate gene for studies into colorectal cancer susceptibility.We genotyped 597 cancer patients and 597controls for three CYP1B1 SNPs, which have previously been shown to be associated with altered enzymatic activity. Using the three SNPs, eight different haplotypes were constructed. The haplotype frequencies were estimated in cases and controls and then compared. The odds ratio for each tumour type, associated with each haplotype was estimated, with reference to the most common haplotype observed in the controls.The three SNPs rs10012, rs1056827 and rs1056836 alone did not provide any significant evidence of association with colorectal cancer risk. Haplotypes of rs1056827 and rs10012 or rs1056827 and rs1056836 revealed an association with colorectal cancer which was significantly stronger in the homozygous carriers. One haplotype was under represented in the colorectal cancer patient group compared to the control population suggesting a protective effect.Genetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations. Adverse interactions between DNA and environmental toxins may be mitigated through a range of biologic defense mechanisms, including DNA repair, cell cycle checkpoint control and xenobiotic clearance enzymes. Xenobiotic clearance is important for the removal of carcinogens and is primarily accomplished by hydroxyl conjugation, involving enzymes in the cytochrome P450 pathway . CancersAs part of a major initiative to identify major cancer susceptibility genes for colorectal cancer, we investigated three common variants in the CYP1B1 gene that individually confer a difference in the activity of the encoded protein. All three variants, codon 48G > C (rs10012), codon 119G > T rs1056827) and 432 G > C (rs1056836) have been shown to result in an increased metabolism of 16a-OH-estradiol [6827 and The Polish population is extremely homogeneous and lends itself to population based studies as there are no significant ethnic differences to take into consideration. 597 colorectal cancer patients were enrolled in the study. Enrollment occurred between 1998 and 2006 from 3 cities in Poland  and 114 bp, respectively . The resultant products were size separated on 4% agarose gels. The uncut product was 335 bp in size and the cut product was predicted to yield 4 fragments of 230,105, 91 and 14 bp in size. Only the 230,105 and 91 base pair fragments were visualized, since the 14 base pair product was too small to be readily detected using this approach  and Fisher's exact test, where appropriate. Bonferroni correction was applied to take into account multiple testing.http:// cran.r-project.org/src/contrib/Descriptions/haplo.stats.html as reported by Schaid et al. [Eight haplotypes could be generated from the three SNPs. The frequencies of each of these were estimated in the cases and controls using the genetic statistical package haplo.stats for R d et al. . The CGGEach SNP of the CYP1B1 gene generated three genotypes, including two homozygote states and one heterozygote state. For each SNP, for each site, the odds ratio and 95% confidence intervals were constructed. For these comparisons, the most common allele was considered to be the normal allele and odds ratios were constructed with reference to individuals with two common alleles. The control population for this study was used to determine the relative frequency of the CYP1B1 alleles in the underlying Polish population.For each individual SNP genotyped no significant association between it and colorectal cancer was observed (see Table When rs1056827 was investigated with rs1056836 a similar trend was forthcoming Table , where hIn order to determine if there were groups of individuals who may be greater risk by being homozygous for all three SNPs we investigated the combination of homozygote carriers against each other, where the wild type haplotypes were considered to represent population risk (see Table This study examines the potential influence of CYP1B1 genetic variants alone, and in combination, on colorectal cancer risk. Each of the three SNPs when considered individually appeared not to be associated with disease risk for colorectal cancer. When considered together, the magnitude of the colorectal cancer risk appeared to be consistent yet modest. One previous report of CYP1B1 and CRC risk provided some evidence for an association with disease but this disappeared once a correction was applied for multiple testing . The repSeveral studies have examined the relationship between individual CYP1B1 polymorphisms and cancer risk -17. The The variants in codons 119 and 432 are associated with different substrate specificities and consequently the catalytic activity of the enzyme ,17. TherFinally, in studies similar to the one reported herein it would have been improved by including the effects of environmental factors on disease risk. The current study was not intended to address environmental influences on disease risk rather to determine whether or not there is an over-representation of genetic markers in a case compared to a control population thereby providing information on the relative involvement of the genetic marker on disease risk.In summary, the CYP1B1 variants examined in this study suggest that they contribute to inter-individual differences in cancer risk and are potentially valuable in genetic risk assessment. These findings have potentially important implications for genetic risk assessment and for prevention studies, but it is important that they be confirmed in other colorectal cancer populations prior to introducing them in the clinical setting.Genetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations.The authors declare that they have no competing interests.JT participated in the design and coordination of the study and carried out the molecular genetic studies, performed the statistical analysis and the manuscript. EGK participated in the collection of samples and carried out the molecular genetic studies. JS participated in the collection of samples. BM performed the statistical analysis. PSF participated in the design of the study and performed the statistical analysis. GK participated in the collection of samples. CC participated in the design of the study. BG participated in the design of the study. TH participated in the collection of samples. TB participated in the collection of samples. JG participated in the collection of samples. EZ carried out the molecular genetic studies. JK participated in the collection of samples. ZB participated in the collection of samples. RW participated in the collection of samples. EK coordinated the preparation of material for the study. JL conceived of the study and participated in its design and coordination. RJS conceived of the study and participated in its design, prepared the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/420/prepub"}
{"text": "Pteridium aquilinum is triggered by herbivory and if so - whether it is regulated by the octadecanoid signaling pathway. Interestingly, feeding of both generalist  and specialist (Strongylogaster multifasciata) herbivores as well as application of singular and continuous mechanical wounding of fronds induced only very low levels of VOC emission. In contrast, treatment with jasmonic acid (JA) led to the emission of a blend of VOCs that was mainly comprised of terpenoids. Likewise, treatment with the JA precursor 12-oxo-phytodienoic acid (OPDA) and \u03b1-linolenic acid also induced VOC emission, albeit to a lower intesity than the JA treatment. Accumulation of endogenous JA was low in mechanically wounded fronds and these levels were unaffected by the application of oral secretions from both generalist or specialist herbivores. The emission of terpenoids upon JA treatment could be blocked with fosmidomycin and mevinolin, which are inhibitors of the MEP- and MVA pathways, respectively. These results indicate that similar to higher plants, terpenoid VOCs are produced via these pathways in bracken fern and that these pathways are JA-responsive. However, the very low amounts of terpenoids released after herbivory or mechanical damage are in stark contrast to what is known from higher plants. We speculate that S. multifasciata and S. littoralis feeding apparently did not induce the threshold levels of JA required for activating the MEP and MVA pathways and the subsequent volatile emission in bracken fern.Jasmonate-mediated regulation of VOC emission has been extensively investigated in higher plants, however, only little is known about VOC production and its regulation in ferns. Here, we investigate whether the emission of VOCs from bracken fern  Apart from this report, there is virtually no data available regarding indirect defence responses in lower plants such as ferns. In the present study, we investigate the regulatory events underlying VOC emission in the bracken fern P. aquilinum. Studying this trait, which functions in all higher plants studied so far as an indirect defence, in a phylogenetically more ancient plant like bracken might shed light on the evolutionary origin as well as the ancestral function of VOC emission.Bracken fern ?Spodoptera littoralis) and a specialist herbivore (Strongylogaster multifasciata)?Does bracken release a similar VOC blend after simple or continuous wounding and upon feeding of a generalist  involved that are also used by higher plants?Pteridium aquilinum (L.) Kuhn, Dennstaediaceae were collected from a forest about 15 km from Jena  and whole plants were brought to the greenhouse for further propagation. Experiments were done on plants vegetatively propagated from these in the greenhouse and grown at a temperature of 27\u201330\u00b0C and 45\u201350% humidity, under 16 h photoperiod in Klasmann clay substrate .Spodoptera littoralis Boisd.  was reared on artificial diet   were collected between May\u2013June 2009 at the same field site as the plants, identified to species level P. aquilinum.The generalist herbivore, Plant volatile response is known to be regulated by members of the octadecanoid signaling pathway. We used \u03b1-linolenic acid, JA, COR and the pore-forming peptide alamethicin (ALA) to test for their capacity to induce VOC emission in bracken fern. Since ion fluxes and membrane depolarisation are elements of the early responses to herbivory \u22121 water . ALA was initially dissolved in methanol at 10 mg ml\u22121 and this stock solution was diluted in tap water to obtain the final concentration. Fronds were damaged mechanically by puncturing 2\u20133 rows of holes with a pattern wheel. Continuous mechanical damage was inflicted using the MecWorm system S. littoralis or S. multifasciata (both reared on fern diet) were placed on each frond, allowed to feed for a day and VOCs were collected for 24 h. S. littoralis larvae were allowed to feed on fern fronds for 1\u20132 days before the onset of the experiments.For elicitor treatments, JA (1 mM), OPDA (1 mM), \u03b1-linolenic acid (2 mM) and COR (100 \u00b5M) were sprayed as aqueous solution on the fern fronds of intact plants. These concentrations were chosen based on previous literature reports, in which these compounds have been shown to induce VOC emission in other plant species Spodoptera littoralis grown on P. aquilinum diet, or from field-collected Strongylogaster multifasciata larvae. To reproducibly mimic feeding of an herbivore, 20 \u00b5l of the OS was diluted 1\u22361 with de-ionized water and applied to mechanically damaged (pattern wheel) intact fronds. Mechanically wounded fronds treated with water served as a control in all experiments.Oral secretions (OS) were collected from third instar larvae of JA was prepared by saponification from commercially available MeJA . COR, OPDA and the deuterated standards for phytohormone analysis were synthesized according to literature procedures Terpenoids are the main constituents of the VOC blend emitted from herbivore-induced plants. In higher plants, these VOCs are synthesized via either the MVA  or the MEP (methylerythritol phosphate) pathways. In order to understand the origin of the terpenoids produced by bracken fern, each of these pathways was specifically inhibited using certain inhibitors. Fosmidomycin was used to inhibit the DXP-reductoisomerase of the MEP pathway and mevinolin to block the HMGR-CoA reductase, the main enzyme of the MVA pathway 2H2]JA and 40 ng [13C6]JA-Ile was added to the samples and after vortexing the samples were centrifuged for 15 min at 13,200 g (4\u00b0C). The upper organic phase was transferred into a fresh tube and the leaf material was re-extracted with 0.5 ml ethylacetate. The organic phases were pooled and evaporated to dryness. The dry residue was reconstituted in 0.4 mL of 70/30 (v/v) methanol/water for analysis with an LC-ESI-MS/MS instrument . Ten \u00b5l of the sample were injected in a ProntoSIL\u00ae column  connected to a pre-column . As mobile phases 0.05% formic acid in water (solvent A) and methanol (solvent B) were used in a gradient mode with the following conditions: time/concentration (min/%) for B: 0.0/15; 2.5/15; 4.5/98; 10.5/98; 12.0/15; 15.0/15; time/flow (min/mL): 0.0/0.4; 1.5/0.2; 1.5/0.2; 10.5/0.4; 15.0/0.4. Compounds were detected in the ESI negative mode and multiple reaction monitoring (MRM) mode 2H2]JAIle.For analysis of the octadecanoids JA, JA-Ile and OPDA, 0.2 g of frozen leaf tissues were homogenized to a fine powder in liquid nitrogen. 1.0 mL of ethylacetate spiked with 200 ng [S-(OOH)-18:3 and 9(R/S)-OOH-18:3 were used as standards .As a next step, the precursors of JA biosynthesis namely the hydroperoxy and free fatty acids were analyzed in order to understand precursor availability. Hydroperoxy-fatty acids were extracted with the chloroform-methanol method as follows: 1 g of frozen leaf material was homogenized in 10 ml glass tubes containing 3.75 ml of ice-cold 2/1 (v/v) chloroform/methanol spiked with 5 ng of 15-hydroperoxy-eicosadienoic acid . After adding of 1.25 ml chloroform and 1 ml water, samples were vortexed and the phases were separated. The organic phase was collected and the water phase re-extracted with 3 ml hexane. The hexane and chloroform fractions were pooled and the solvent was evaporated under a stream of nitrogen. The samples were reconstituted in 70% (v/v) methanol/water and analyzed by liquid chromatography-(ESI)-tandem mass spectrometry (LC-MS/MS) as previously described 2SO4/water were added and after centrifugation, the organic phase was collected and the aqueous phase re-extracted with 3 ml of 3/2 (v/v) 2-propanol/hexane. The organic phases were combined, the solvent evaporated under a stream of nitrogen and the samples reconstituted in 1 ml of 1% (v/v) H2SO4/methanol. The samples were heated for 1 h at 75\u00b0C and the fatty acid methyl esters (FAMES) were extracted with hexane. FAMES were analyzed in a Varian CP-3800 GC coupled with a Varian Saturn 3800 ion trap MS in electron ionisation  mode . 1 \u00b5l of the sample was injected in splitless mode on a DB-WAX column  with helium at a constant flow of 1.0 ml min\u22121 as the carrier gas. The injector was at 230\u00b0C. The oven temperature program was: 130\u00b0C for 5 min, 220\u00b0C at 3.0\u00b0C/min, 5\u00b0C/min ramp to 240\u00b0C and hold for 1 min. EI spectra were recorded on Scan mode from 40 to 400 amu. Quantification was performed in the linear range of detection and based on calibration curves generated with increasing concentrations of commercial FAMES (fatty acid methyl esters) mixes  and the IS (17\u22360).For fatty acid analysis, 0.5 g of leaf material was ground in liquid nitrogen, transferred into 10 ml glass tubes containing 2 ml of 2-propanol and quenched by heating for 10 min at 80\u00b0C. On cooling, 3 ml of hexane and 2 ml of 6.7% (w/v) NaBratschlauch\u2019  that does not emit detectable volatiles by itself . The traps were eluted with 2\u00d720 \u00b5l dichloromethane containing 200 ng \u00b5l\u22121 of 1-bromodecane as an internal standard. Leaves were dried for dry weight determination. VOCs samples were analysed on a Thermo Finnigan Trace GC-MS  equipped with a fused silica Alltech EC5 column  using 1.5 ml min\u22121 helium as carrier gas. Separation was achieved under programmed conditions . MS analysis was performed in electron impact full-scan mode at 70 eV with source temperature at 200\u00b0C and GC interface temperature at 250\u00b0C. Compounds were identified tentatively by comparison to the NIST database and subsequently collated with spectra from reference compounds . Individual compounds were quantified with respect to the peak area of the internal standard and related to the dry weight of the frond.Treated fronds were bagged individually in a PET foil \u2018see e.g.  and VOCsDifferences between treatments were evaluated as a \u2018general linear model\u2019 command with \u2018treatment\u2019 as fixed and \u2018plant individual\u2019 as random factor. For multiple comparisons, LSD post hoc test tests were used in case variances were homogeneously distributed and Tamhane's T2 post hoc test if this assumption was violated. All statistical analyses were done using SPSS 17.0 .Strongylogaster multifasciata nor the generalist herbivore Spodoptera littoralis on intact fronds increased VOC emission rates in bracken . Damaging the fern fronds using a pattern wheel (singular damaging event) induced low VOC emission levels that were statistically indisting-uishable from undamaged controls. To verify whether this result was merely the consequence of the damage levels, we employed a mechanical device .Interestingly, neither feeding of the specialist herbivore  bracken . Even inP<0.02, n\u22653\u20136 per treatment). Both coronalon (COR) and jasmonic acid (JA) induced the highest production levels of VOCs of all treatments.Next, The total amount of VOCs released by bracken upon treatment with various elicitors  was analyzed and compared to the effect of mechanical wounding and herbivore damage . The totE)-(\u03b2)-farnesene, aromadendrene epoxide, as well as a methylester of palmitic acid. Qualitative composition of the emitted VOC blends also differed strongly between different treatments (E)-\u03b2-farnesene was the most dominant compound emitted after JA or COR treatment (E)-\u03b2-farnesene was only detected after ALA-, but not after OPDA treatment. Limonene was emitted after all elicitor treatments, except after \u03b1-linolenic acid- and damage treatments. Interestingly, herbivory inflicted by a generalist herbivore  induced the emission of limonene, yet in very small amounts -\u03b2-freatment . 1-Octenreatment . ALA ind amounts .P<0.03, n\u200a=\u200a4).To verify to which extent the observed VOC emission pattern was dose-dependent, the JA concentration applied to each intact frond was varied and the resulting emission rate of VOCs quantified. Fronds, which have been treated with different JA concentrations ranging between 0.05 mM and 1 mM, indicated that a concentration of 1 mM JA was required to significantly increase total VOC emission levels over control levels  accounted for 34% and unsaturated fatty acids  constituted 65% of the total amount of these molecules , the initial precursor for JA biosynthesis  upon wounding as a function of time . Endogenolecules . Mechaniynthesis .Spodoptera littoralis; reared on fern diet; Strongylogaster multifasciata; reared on fern diet; P>0.05, n\u200a=\u200a5).To analyze the effect of herbivory on endogenous JA levels, oral secretions (OS) from generalist herbivores , compared to JA-treated plants, whereas the amount of monoterpenes emitted after inhibition with fosmidomycin was significantly lower compared to JA-treated plants . The observation that fosmidomycin was more effective in blocking monoterpene production than mevinolin, indicated that the MEP pathway accounted for the formation of these compounds (E)-\u03b2-farnesene was suppressed by both inhibitors by almost 90% as compared to JA treatment, which suggests that the plastid-derived MEP pathway supports the formation of both mono- and sesquiterpene synthesis in bracken fern.Since bracken produced both mono- and sesquiterpenes in response to JA treatment, we investigated which metabolic pathways were involved in the production of these compounds using specific inhibitors (fosmidomycin and mevinolin). Treatment with these inhibitors did not affect the emission rates of Compounds . On the Several fascinating functions for the emission of plant VOCs have been discovered. However, the basic questions why there is such a structural diversity of VOCs as well as how these evolved to function as an indirect defence mechanism in plants remain largely unanswered. VOC emission could have originated as a direct defence strategy against herbivores or pathogens or as a means to attract pollinators P. aquilinum is also regulated by jasmonates, ii) the same elicitors  that are known from higher plants trigger VOC emission in bracken. The impact of mechanical damage as a component of herbivore feeding was studied using a computer-controlled device (i.e. \u2018Mecworm\u2019).For many higher plants, it is well known that insect feeding induces the emission of a VOC blend that strongly resembles the one released upon exogenous application of JA Phaseolus lunatus, treatment with ALA mainly induced the emission of homoterpenes; the emitted VOC blend resembled OPDA-treated, rather than JA-treated plants. In the present study, ALA treatment elicited a VOC profile that was more similar to OPDA-treated than JA-treated plants E)-\u03b2-farnesene (E)-\u03b2-farnesene was also the most dominant VOC emitted from the fern Pteris vittataArabidopsis thalianaBotrytis cinerea, suggesting that 1-octen-3-ol may be recognized by the plant as a signal for presence of fungal pathogens When ferns were treated with COR, the VOC blend emitted resembled the one emitted from JA treated plants both qualitatively and quantitatively  and 2a. arnesene , a sesquPteridium aquilinum), elicitors like JA, COR OPDA, \u03b1-linolenic acid, and ALA induced the emission of certain VOCs, which is in line with previous studies using higher plants. Interestingly, continuous damage by the \u2018Mecworm\u2019 did not result in significantly increased VOC emission levels in the fern . Larval feeding of this herbivore, however, resulted in much lower emission levels of VOCs than were released from elicitor-treated ferns \u22121  and 6. TGinkgo biloba has been shown to produce increased amounts of VOCs upon JA treatment, but failed to emit any volatiles after tissue damage A. thaliana, tobacco, tomato, soybean, lima bean and maize Pteris vittata responded with an emission of terpenoids after herbivory Interestingly,"}
{"text": "Cancer/testis (CT) antigens represent promising targets for immunotherapy. We investigated the composite expression of 13 CT antigens by RT-PCR in 79 lung cancer tissues and by immunohistochemistry in 22 lung cancer tissues. In the 79 lung cancer tissues, MAGE-3 (42%) was expressed most frequently and followed by NY-SAR-35 (33%), NY-ESO-1 (30%), MAGE-1 (27%), CT-7 (20%), MAGE-4 (19%), LAGE-1 (16%), and MAGE-10 (14%). Twenty-one tissues did not express any of the CT antigens tested, 58 (73%) expressed at least one, 36 (46%) co-expressed two, 24 (30%) co-expressed three, 17 (22%) co-expressed four, 14 (18%) co-expressed five, 8 (10%) co-expressed six, 4 (6%) co-expressed seven and 2 tissues expressed 9 of the 13 examined CT antigens. Expression of CT antigens was significantly associated with age (P<0.001), smoking history (P=0.009), and gender (P=0.001) of patients, whereas no correlation was found between the expression of CT antigens and other clinical factors, such as pT status, pN status, tumor stage, and histology history. The present results show that CT antigens are potential candidates in lung cancer patients for polyvalent immunotherapy. Lung cancer is the major cause of cancer-related mortality worldwide and is also one of the most frequent causes of death in Korea . The higAn essential condition for the development of effective immunotherapeutic strategies is the existence and identification of tumor specific antigens that are either exclusively or preferentially expressed in malignant compared to normal tissues . Human tCT antigens are immunogenic proteins expressed in normal testis and in different types of tumors ,9. CT anThe present study analyzes the frequency of expression of 13 CT antigens in 79 lung cancer tissues. The correlation between CT antigen expression patterns and pathological characteristics of lung cancer tissues was also studied.Human tumor tissues were obtained from lung cancer operations performed at the Pusan National University Hospital, Busan, Korea. Analyses were performed on tumor tissue samples of 79 patients with lung cancer confirmed pathologically. The tumor samples consisted of 33 cases of adenocarcinoma, 24 of squamous cell carcinoma, 6 of neuroendocrine carcinoma, 4 of pleomorphic carcinoma, and 12 of others . Among the 79 lung cancer patients from whom tissue samples were obtained, 68 had available records, but 11 did not. Data for the 68 tissues including gender, age, classification of TNM status, and stages were obtained from the clinical and pathological records. Tumor stage and progression were classified according to the International Staging System .Total cellular RNA was extracted from frozen tissue specimens of 79 lung cancer samples, using TRI Reagent . RNA extraction using TRIzol , a common protocol, was used. Tumor tissues removed at surgery were snap-frozen and stored at \u221270\u00b0C. Total-RNA was isolated from ~100 mg of each tissue sample using 1 ml TRI Reagent , extracted with chloroform, precipitated with isopropyl alcohol, washed with ethanol and re-dissolved in RNase-free water. The amount of isolated RNA was measured by a spectrophotometer  at 260 nm.The cDNA preparations used as templates in the RT-PCR reactions were prepared by using 500 ng of total-RNA in conjunction with the SuperScript First Strand Synthesis kit (Invitrogen Life Technologies).The 13 primer sets for the 13 CT antigens and the lengths of each PCR product are shown in The M3H67 monclonal antibody (to MAGE-3) was obtained from the Ludwig Institute for Cancer Research, New York Branch at the Memorial-Kettering Cancer. Immunohistochemical staining was performed according to the previously report . Briefly2 test was used to compare the correlation between disease stage, grade, and CT antigen expression. Statistical significance was accepted at P<0.05.Statistical analysis was performed with the SPSS program . Pearson \u03c7Expression of the 13 CT antigens  was assessed by RT-PCR in 79 lung cancer tissue samples. Representative RT-PCR results from lung cancer tissues are shown in The percentages of co-expressed CT antigens in lung cancer tissues are shown in The characteristics of the patients are summarized in Among the 79 lung cancer tissue specimens, 22 lung carcinoma samples from which sufficient material was available were investigated for the expression of the frequently expressed MAGE-3 protein by immunohistochemistry. Representative immunohistochemistry results of lung cancer tissues with the mAb MAGE-3 are shown in CT antigens are immunogenic proteins expressed in normal testis and in different types of tumors. Because of their tissue-restricted expression, CT antigens are ideal candidates for antigen-specific cancer immunotherapy. In general, CT antigens are expressed in 20\u201340% of specimens from a given tumor type . The preIn addition, 58 of 79 lung cancer tissue specimens (73%) were found to express at least one of the CT antigens. According to our results, 73% of our lung cancer patients would be eligible for specific immunotherapeutic approaches with at least one CT antigen. A possibility for the high observation frequency of CT antigen expression in lung cancer tissues could be due to the characteristics of the patients. These results indicate that expression of CT antigens was significantly associated with the age and gender of the patients, whereas no significant correlation was detected between CT antigen expression and other clinical factors such as pT status, pN status, and tumor stages . In thisThe highly homologous NY-ESO-1 and LAGE-1  are highly immunogenic, and a CTL response to an immunodominant, HLA-A2-restricted NY-ESO-1/LAGE-1 epitope can commonly be detected in cancer patients ,30. We cOf particular interest was NY-SAR-35, which represents a CT antigen as it appears to be a rare example of a cell surface antigen , which wTo confirm the existence of MAGE-3 protein expression, 22 lung carcinoma samples from which sufficient material was available were immunohistochemically stained by a specific MAGE-3 monoclonal antibody. Similarly to our previous study , we founFrom the data of our study, a high proportion  of lung cancer tissues was positive for at least one of these 13 CT antigens as targets will greatly increase the number of candidates for CT antigen-based lung cancer immunotherapy. However, our results showed no expression of CT antigens in 27% (22/79) of lung cancer tissues. For these patients, it is necessary to screen other CT antigens or tumor-specific antigens serving as immune targets for lung cancer tissue immunotherapy. In conclusion, this study demonstrated that lung cancer tissues frequently express CT antigens and a high percent express more than one CT antigen, suggesting that CT antigens are potential candidates for polyvalent immunotherapy."}
{"text": "Lung cancer is the most common cause of cancer-related mortality worldwide. Survival rates after non-small cell lung cancer (NSCLC) diagnosis still remain poor, despite standardization of surgery and adjuvant treatment. Cancer testis (CT) antigens as targets for immunotherapy in cancer patients have been heavily investigated. We aim to evaluate the expression of CT antigens in non-small cell lung cancer and the correlation with the clinical characteristics.The expression of MAGE-A3, MAGE-A4, MAGE-C2 and NY-ESO-1 in fresh cancer tissues from 113 patients with non-small cell lung cancer were analyzed by RT-PCR. The expression of HLA-A2 was detected by flow cytometry.49.6% non-small cell lung cancer samples were HLA-A2 positive. CT antigens were frequently expressed in non-small cell lung cancer. The expression percentage of each gene in cancer tissues samples were as follows: MAGE-A3, 70.8%; MAGE-A4, 46.9%; MAGE-C2, 61.1%; and NY-ESO-1, 15.0%. In all, Seventeen tissues did not express any of the CT antigens tested, 85.0% expressed at least one, 66.4% co-expressed two, 34.5% co-expressed three, 6.2% co-expressed four examined CT antigens. The percentage of samples co-expression HLA-A2 and CT genes were: MAGE-A3, 51.6%; MAGE-A4, 36.3%; MAGE-C2, 44.0%; and NY-ESO-1, 12.1%. MAGE-A3 was associated with male, smoking history, tumor stage, tumor differentiation and lymph node metastasis (P<0.05); MAGE-C2 was associated with tumor stage (P<0.05). There was no correlation MAGE-A4 or NY-ESO-1 expression with clinical characteristics such as gender, age, HLA-A2 positive, clinical stage, grade of differentiation and lymph node metastasis (P>0.05).CT antigens might be prognostic markers and factors related to the progress of NSCLC, and also be served as the immunotherapeutic targets of NSCLC, especially in multi-antigen vaccine preparations and tumor antigen specific lymphocytes infusion."}
{"text": "Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers  and 189 breast cancers, while MAGE-A proteins were present in 15% and 7\u201316% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer. Modulation of the immune system in cancer patients has shown to efficiently generate anti-tumor immune responses, but selection of targets for effective and specific intervention remains challenging. The unique expression pattern and immunogenic properties of cancer/testis (CT) antigens make them ideal targets for different types of cancer immunotherapy, such as vaccination and adoptive transfer with recombinant T-cell receptor-transduced T cells. CT antigens are male germ cell proteins ectopically expressed in various malignancies \u20133. Male http://www.cta.lncc.br), but only a small number of these have been investigated for expression profiles. Although some CT antigens tend to be co-expressed in a subset of tumors, others have distinct and cancer-subtype specific expression profiles [More than 200 different CT antigens have been identified  were collected as diagnostic specimens from patients treated at the University Hospital of Odense. The ethical committee of Funen and Vejle County (VF20050069) approved the use of these tissues, without informed consent from participants. The lung (LC1502) and breast (BRC1502) carcinoma tissue microarrays were purchased from BioCat GmbH, Heidelberg, Germany. The lung carcinoma tissue microarray LC1502 contained 23 cases of lung squamous cell carcinoma, 21 lung adenocarcinoma, 5 each of lung adenosquamous carcinoma and bronchioalveolar carcinoma, 7 small cell undifferentiated lung carcinoma, 1 each undifferentiated lung carcinoma and malignant mesothelioma, 2 each of large cell lung carcinoma and carcinosarcoma, 3 neuroendocrine lung carcinoma, and 1 each of lung chronic bronchitis, lobar pneumonia and pulmonary tuberculosis, 2 normal lung tissue, duplicate cores per case (duplicated cores from the same patient were put onto upper and lower rows in the same position). The breast carcinoma tissue microarray BRC1502 contained 62 cases of ductal carcinoma, 2 lobular carcinoma and 1 each of papillary carcinoma, sarcoma, mucinous adenocarcinoma and tubular carcinoma. The estrogen receptor and HER2 status of this first cohort of breast cancers were not available. Tissue microarrays of the second cohort of breast cancers with information on receptor status were subsequently analyzed. The generation and characterization of this second cohort has previously been reported . The exp202 in Tris-buffered saline (pH 7.5) for 10 min to block endogenous peroxidase activity. Thereafter, they were rinsed in distilled H2O, demasked, processed for antigen retrieval and washed in TNT buffer . To optimize conditions for staining with antibodies raised against amino acids 122\u2013215 of ADAM2 , amino acids 195\u2013294 of CALR3  and amino acids 497\u2013641 of SAGE1 , different concentrations were tested in combination with different antigen retrieval protocols using sections of human testis. The methods of antigen retrieval included microwave boiling for 15 min in 1) T-EG buffer , 2) 10 mM citate buffer, pH 6.0 or 3) Dako Target retrieval solution (Dako S1699), or subjected to proteolytic treatment using 4) 0.05% protease type XIV  in TBS, pH 7.0 for 15 min at 37\u00b0C, or 5) 0.4% pepsin  in 0.01M HCl for 20 min at 37\u00b0C. A protocol for immunohistochemical staining with mouse anti-MAGE-A  has previously been described [2O and counterstaining of sections with Mayers hematoxylin before mounting in AquaTex .Paraffin-embedded, formalin-fixed tissues were cut in 6 \u03bcm sections, deparaffinized, treated with 1.5% HTissue sections were evaluated by immunohistochemistry and considered positive if staining was observed in more than 5 cells, regardless of intensity.Breast  and lung cancer cell lines  were cultured in RPMI or DMEM  supplemented with 10% fetal bovine serum (Sigma Aldrich), penicillin (100 U/ml) and streptomycin (100 mg/ml). For MCF7 and T47D, 6\u20138 ng/ml insulin (Sigma Aldrich) was added to the media. When indicated, cells were grown with 5\u03bcM 5-Aza-2\u2019-deoxycytidine (Sigma-Aldrich) for 48 hours. Cell lines were obtained from ATCC, Wesel, Germany.Total RNA was purified from cultured cells using Isol-RNA lysis reagent  and RevertAid Premium Reverse Transcriptase kit (Fermentas/Life Technologies) was used for cDNA synthesis with random hexamer primers. Relative quantification of gene expression was performed with SYBR green PCR Mastermix . PCR primers were: ADAM2 , CALR3 , SAGE1  and PUM1 .http://www.cta.lncc.br), in contrast to MAGE-A , which constitute about 85% of lung cancers ; Fig 2. We also investigated the expression of ADAM2, CALR3 and SAGE1 in 13 breast cancer cell lines  and in bThe absence of ADAM2, CALR3 and SAGE1 expression in lung and breast cancer prompted us to test their expression in a panel of melanoma cell lines ; Fig 3, Infrequent or heterogeneous expression of tumor antigens in tumors may be an obstacle to development of efficient and widely applicable cancer vaccines. Importantly, agents that inhibit the function of DNA methyltransferases have been demonstrated to induce the expression of CT antigen genes. For instance, 5-aza-2\u2019-deoxycytidine activates MAGE-A, GAGE, CT45 and many other CT antigen genes , 26\u201329. We investigated the potential of three previously uncharacterized CT antigens, ADAM2, CALR3 and SAGE1, as targets for treatment of lung and breast cancer. Although we demonstrated that these CT antigens exhibit a germ cell-specific expression pattern in normal tissues, which is an important feature of antigens for cancer immunotherapy, none of these antigens were expressed in lung and breast cancer. Furthermore, the expression of these antigens could not be induced by DNMT-inhibitor treatment, in contrast to many other CT antigens. In conclusion, ADAM2, CALR3 and SAGE1 should not be considered targets for treatment of lung and breast cancer. However, our study also provides the reagents and conditions for assessing expression of these antigens in other cancer types. Furthermore, SAGE1 was detected in about 40% of melanoma cell lines, suggesting that this protein may be an interesting target. Our study represents an important step in the tedious evaluation of the long list of potential tumor antigens to identify novel therapeutic targets for different types of cancer.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
{"text": "A robust and transferable algorithm is presented to objectively describe and rank robotically captured images of crystallization droplets according to their likelihood of crystalline behaviour for the efficient and accurate identification of successful crystallization. The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallo\u00adgraphy. Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes; here, a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals, thereby prioritizing for visual inspection those images that are most likely to contain useful information. The use of textons is introduced to describe crystallization droplets objectively, allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence or absence of crystals or microcrystals. Unlike multi-class classification, this two-class system lends itself naturally to unidirectional ranking, which is most useful for assisting sequential viewing because images can be arranged simply by using these scores: this places droplets with probable crystalline behaviour early in the viewing order. Using this approach, the top ten wells included at least one human-annotated crystal or microcrystal for 94% of the plates in a data set of 196 plates imaged with a Minstrel HT system. The algorithm is robustly transferable to at least one other imaging system: when the parameters trained from Minstrel HT images are applied to a data set imaged by the Rock Imager system, human-annotated crystals ranked in the top ten wells for 90% of the plates. Because rearranging images is fundamental to the approach, a custom viewer was written to seamlessly support such ranked viewing, along with another important output of the algorithm, namely the shape of the curve of scores, which is itself a useful overview of the behaviour of the plate; additional features with known usefulness were adopted from existing viewers. Evidence is presented that such ranked viewing of images allows faster but more accurate evaluation of drops, in particular for the identification of microcrystals. Approaches to the problem include edge detection . An image is filtered with all 38 filters in the filter bank, but only the maximum filter response for the edge and bar filters at each scale is recorded, resulting in a vector with eight  rotation-invariant filter responses per pixel. An example of the final filter response vector is shown in Fig. 2b). We chose this filter bank as it was shown by Varma and Zisserman to be the best filter bank when compared with the filter bank proposed by Leung & Malik  building a texton dictionary containing unique texture prototypes and (ii) comparing the filter response of each pixel in a new image to this dictionary to find the closest texton and label the pixel with the corresponding texton label. The conventional way of building this dictionary is by taking a fixed number of textons from each class of textures and combining them to form the final dictionary. However, precipitation behaviour yields a continuum of patterns which are difficult to classify objectively by eye, and since no labelled data set is available in the community, we took a different approach to generate this dictionary, as outlined in the following section.2.1.2.To ensure that crystal-related textons are well represented, we used separate sets of 52 arbitrarily selected crystal-containing images and 100 precipitation images to cover a wide range of patterns  was calculated using the \u03c72 distance between their histogram,ix and iy are the normalized bin counts of the ith feature of droplets x and y, respectively, n = 300 in this case and the distance is weighted inversely by the sum of the bin counts. The resulting 50 clusters were manually inspected and found to have visual consistency; examples of these clusters are shown in Fig. 5The precipitation patterns of 1400 images randomly selected from 28 crystallization plates were clustered to examine whether the method is able to discriminate droplets based on their patterns. Hierarchical clustering with Ward\u2019s minimum variance method  is masked and the remaining pixels are intensity-normalized to have a mean intensity of 0 and a standard deviation of 1. The following statistical descriptors are calculated from the gradient image of the normalized image: mean, standard deviation, skewness, kurtosis and the distribution of the absolute gradients at fixed 50 bin centres ranging from 0 to 50. These 54 features are used with the area, centroid, eccentricity (ratio of difference between foci and the major axis length), major and minor axis length of the segmented droplet (described next) as inputs to a random forest classifier which predicts whether a droplet image is good or faulty. The classifier used here was trained with 11\u2005326 images (5225 faulty and 6101 non-faulty images) and has an accuracy of over 94%. The majority of inaccurate classification occurs in experiments set up with detergent leading to the much lower contrast of the droplet boundary in such droplets .Errors owing to inaccuracies of the droplet-dispensing robots may result in empty subwells, unusually small droplets (where either the protein or reservoir solution was not added) or droplets sitting at the edge of the subwell. Errors in the imaging system may also produce images where the subwells are partially out of the field of view of the camera. To identify these failed droplets, background images were obtained for each of the subwells by taking the average of that subwell for all wells from an empty plate. This should be performed for every type of plate since it is plate-specific; in this case, we generated background images for the 3-Well Crystallization Microplate (SWISSCI), which is most commonly used at the SGC Oxford. This background image is rigidly registered to a new image by searching for the ts Fig. 7d.2.2.2.DroplIT  of the original segmented droplet. For each column (1\u2013360\u00b0) in the polar image the droplet boundary is extended by replacing 13 pixels  outside the radius of the droplet with the median of intensities of the ten pixels closest to the droplet boundary. The median is used instead of the mean to avoid outliers from possible imperfect segmentation. The new extended polar image is then converted back into rectangular space, resulting in a padded droplet. The ring of added pixels is copied to the original droplet image. This provides a sufficient number of neighbouring pixels for the calculation of filter responses for pixels in the original edge; the filter responses for pixels in the extended region are ignored in the final histogram count. Figs. 9b), 9c) and 9d) show a comparison of texton labels with and without droplet extension.Filter responses at the edge of droplets will be dominated by the droplet boundary. To avoid the strong boundary effect, droplets are artificially extended radially and the filter response of this extended region is ignored in the final frequency count. Fig. 92.2.5.The gamma-corrected and extended image is intensity-normalized to have \u03bc = 0 and \u03b4 = 1 for intensity invariance and is scaled by a factor of 0.25 to match the textons in the dictionary. The texton distribution is then calculated as described in \u00a72.3.2.3.1.At the SGC, crystallization experiment droplet images are automatically captured with a Minstrel HT system (Rigaku). Experimenters label images with scores between 1 and 10, where labels 1 and 2 are for precipitates, 3\u20135 for microcrystals and 6\u201310 for mountable crystals. It should be noted that labelling is optional, and it is usually only the interesting droplets that are labelled. Two sets of training images were selected: (i) \u2018interesting\u2019 images, a random subset of 2501 images of droplets that were given labels of 3 and above captured between April 2013 and July 2013, and (ii) \u2018uninteresting\u2019 images, a random subset of 3553 images from the same period with scores <3 or with no scores recorded.2.3.2.TreeBagger function of MATLAB. The random forest algorithm was chosen for its speed and its ability to deal with the large numbers of both instances and features, unlike competing algorithms. Given a set of features from new test images, the random forest produces the posterior probability for a particular class, which is used directly as scores for the images. In this case, for the \u2018interesting\u2019 class a score of 1 indicates that the droplet is likely to contain crystals or crystalline behaviour, while a score of 0 indicates that it is \u2018uninteresting\u2019. In a typical classification exercise, a threshold is set to determine which class the data point belongs to. Tenfold cross-validation of the classifier at a cutoff of 0.5 gave an average area under the ROC curve of 0.9418 \u00b1 0.0027, indicating good separation of scores for both classes, and an average accuracy of 0.8930 \u00b1 0.0044. However, as the intention is to rank and not to classify droplets, no threshold was selected; instead, the scores were used to rank droplets directly.The set of 2501 \u2018interesting\u2019 droplets and 3553 \u2018uninteresting\u2019 droplets were used to train a two-class random forest classifier  with at least one recorded crystal . Of these, 101 plates were sparse-matrix screens, while the remaining 95 were optimization experiments. Each plate contained 96 wells with three subwells each, where the subwells share the same protein solution and reservoir solution at different mixing ratios . Droplets in these plates were ranked by the algorithm and the highest rank of crystals marked by crystallo\u00adgraphers was determined either by subwell or by well, where the maximum score of the three subwells was used.c). When DroplIT failed on the original image, it was instead applied to a gamma-corrected image, as described in \u00a7For validation of the robustness of the algorithm across imaging platforms, we analysed images acquired by the Structural Biophysics group of the Novartis Institute for Biomedical Research (NIBR), Basel using a Rock Imager (Formulatrix). Images of 134 plates with at least one recorded crystal were selected. A comparison of this data set with that from the SGC is summarized in Table 12.5.A and B). Each set consisted of three sparse-matrix screens and two optimization screens of different protein targets. Two groups of five crystallographers from the SGC with varying experience were asked to evaluate each plate in a standardized time interval of 2\u2005min, which was insufficient to view all 288 droplets in a plate and thereby simulates the end goal of the program of minimizing the time required to identify all crystals.  The first group viewed set A in a ranked order and set B in an unranked order, while the second group viewed set B in a ranked order and set A in an unranked order. The majority vote  of each group of crystallo\u00adgraphers was used to compare against the other group for missed (micro)crystal annotations.To show that such ranked viewing of images results in drops with microcrystals being more carefully evaluated, ten plates containing (micro)crystals were randomly selected from the SGC and divided into two sets  of their probability of being \u2018interesting\u2019: this means that crystals or microcrystals are likely to be viewed first, while the \u2018uninteresting\u2019 droplets  are viewed later. In the real-world environment where time is limited and attention level decreases with time, the ranking system focuses resources on what is most likely to matter, namely the likely presence of crystallinity.The ranking score is obtained by repurposing a two-class classifier trained on images assigned manually into only two categories, namely containing crystallinity (\u2018interesting\u2019) or not (\u2018not interesting\u2019), and therefore containing the respective textons. When applied to new images, the classifier generates a score (0 to 1), which ordinarily would be compared against a threshold in order to assign images to either category; instead, here the score is directly employed for sorting a given set of images .e.g. \u2018crystal\u2019 over \u2018microcrystal\u2019 over \u2018spherulite\u2019 and so on); however, this ranking is algorithmically arbitrary , and moreover it is non-obvious how to rank images within classes, since the classifier scores are no longer uni\u00addirectional. On the other hand, Buchala & Wilson , which shows that for most plates the first human-annotated crystal is very high in the ranking order. We show both the results for viewing by well and subwell in Table 2Our approach appears to rank images effectively, if judged by criteria that are reasonable in routine laboratory usage: for plates where wells were viewed with our new ranking order, the first well was a crystal-containing image for 128 out of a total of 196 plates (65.31%). The number of such \u2018successful\u2019 plates increases if the criterion is relaxed to expect at least one crystal in the top 10 or top 32 ranked wells Table 2, as alsob) and Supplementary Fig. S3 illustrate how the top of the viewing order is generally enriched in images with crystals and microcrystals. Moreover, where the ranking failed to move the human-annotated crystal to the top, it was usually questionable whether the images had been correctly marked  or else the images themselves were problematic  to approximately match the resolution at the filtering stage. Table 3This result indicates that the texton approach does not need a very high level of detail in the images: SGC and Novartis images have similar field of view . A common method in vendor software is to label the images with tags and different colour schemes ; on the contrary, annotation appears to become more stringent if larger (micro)crystals have already been observed.We show here that enriching the number of crystal-containing images to inspect first does indeed result in closer attention apparently being given to interesting droplets. We tested this by comparing how effectively crystallographers could evaluate crystallization plates under extreme time pressure  both with or without the ranking system. All plates were viewed by two groups of five crystallographers, with plates viewed in rank order by one group being viewed unranked by the other, but with both groups performing both ranked and unranked viewing; the consensus score within each group was taken to reduce human error ; however, full integration with existing database and imager systems and deployment of the viewer will invariably require bespoke effort. The executable for image processing and generating ranking scores requires little effort to run once the reference background images have been generated, but it is the viewer that must be set up to fit into the infrastructure of a given laboratory; an application programming interface (API) is under development to simplify this process. We have shown that integration is possible for both the Rigaku and the Formulatrix systems, and for these particular versions of the systems it could take as little as 3\u2005days to integrate fully for someone familiar with either system and with general database knowledge. The code and executables can be downloaded from https://github.com/thesgc/TeXRank.The software is available as a standalone package, 4.We show that textons are effective at describing crystallization experiments objectively with fast computation times, making them suitable for everyday use. These descriptors are effective for ranking droplets by their probability of containing crystals or microcrystals, and the algorithm was transferable between two widely used commercial imaging systems for sitting-drop vapour-diffusion experiments. The collection of scores across a plate forms a useful profile for a quick overview of the suitability of the protein for structural studies; the algorithm and the custom viewer can be integrated into the existing infrastructure and are freely available. We have also shown that with the system, crystallographers have been able to identify crystals and microcrystals more efficiently and accurately by prioritizing which images to view and spend more time on.10.1107/S1399004714017581/nj5198sup1.pdfSupporting Information.. DOI:"}
{"text": "The aim of this survey was to evaluate attitudes and preferences for the clinical management of hypertension and hypertension-related cerebrovascular diseases (CVD) in Italy.A predefined 16-item survey questionnaire was anonymously administered to a large community sample of general practitioners (GPs), trained by specialized physicians (SPs), who have been included in an educational program between January and November 2015. A total of 591 physicians, among whom 48 (8%) training SPs and 543 (92%) trained GPs, provided 12,258 valid answers to the survey questionnaire. Left ventricular hypertrophy was considered the most frequent marker of hypertension-related organ damage, whereas atrial fibrillation and carotid atherosclerosis were considered relatively not frequent (10\u201320%). The most appropriate blood pressure (BP) targets to be achieved in hypertensive patients with CVD were <140/90\u00a0mmHg for SPs and <135/85\u00a0mmHg for GPs. To achieve these goals, ACE inhibitors were considered the most effective strategies by GPs, whereas SPs expressed a preference for ARBs, both in monotherapies and in combination therapies with beta-blockers.This survey demonstrates that Italian physicians considered left ventricular hypertrophy frequently associated to CVD and that drugs inhibiting the renin-angiotensin system the most appropriate therapy to manage hypertension and hypertension-related CVD.The online version of this article (doi:10.1186/s40885-017-0066-0) contains supplementary material, which is available to authorized users. Effective control of high blood pressure (BP) reduces incidence of major cardiovascular complications, and improves event-free survival from cerebrovascular diseases (CVD), mostly stroke . DespiteIn order to improve BP control, several interventions have been proposed and applied in various countries, including Italy. Among these, a closer attention to global cardiovascular risk stratification and a proper selection of antihypertensive drug therapies on the basis of individual global CV risk profile have emerged as the best way to ameliorate hypertension management and control \u20136. HowevMore recently, an extensive use of epidemiological surveys and observational studies has emerged as a valuable option to evaluate physicians\u2019 workflow, particularly when managing hypertensive outpatients at different CV risk \u201315. In tIn the present survey, we evaluated the clinical attitudes and preferences for the management of patients with hypertension and hypertension-related CVD, expressed by a large sample of physicians in Italy.The primary aim of this survey was to evaluate the clinical attitudes and preferences of both general practitioners (GPs) and specialized physicians (SPs), who were included in an educational program performed in Italy in 2015.Secondary aims of the survey were to analyse pharmacological preferences  in patients with hypertension and CVD.The methodology of the study has been previously described . BrieflyThe study conformed to the Declaration of Helsinki and its subsequent modifications. Confidentiality on demographic and personal data of each physician included in the present survey was carefully preserved and strictly protected during each phase of the study. No access was made to individual data of neither physicians\u2019 own patients nor their medical databases. Written consent to participate to the educational program was obtained by all involved physicians.The survey questionnaire included a total of 16 questions addressing the following items: 1) estimated concomitant prevalence of hypertension and CVD and prevalence of hypertension-related markers of organ damage and comorbidities in patients with hypertension and CVD (questions num. 01\u201306); 2) diagnostic options to assess the presence of CVD in hypertensive patients (questions num. 07\u201308); 3) BP targets and the most appropriate therapeutic targets to be achieved in a setting of clinical practice, when managing hypertensive patients with CVD (question num. 09\u201310 and 13\u201314); 4) preferences for antihypertensive drug classes in hypertensive patients with CVD to be used as first line therapy (monotherapy) or combination therapy (questions num. 11\u201312 and 15\u201316). The full survey questionnaire is reported in Additional file Physicians\u2019 engagement was carried out during the first 6\u00a0months of 2015. As per study protocol , particiPhysicians were invited to participate to an educational program, aimed at improving knowledge on hypertension-related cardiovascular diseases and implementing strategies for achieving better BP control in their practice. Educational program was structured into two distinct sections: one start-up meeting, held in January 2015 and pointed to SPs, and local meetings, distributed throughout the whole Italian territory, during which SPs involved in the start-up meeting trained GPs. Before starting each educational meeting, involved physicians were asked to fill the survey questionnaire anonymously. The entire survey questionnaire was completed by participants on-site and then delivered to the data collection centre. Physicians who completed the survey did not receive any compensation for their participation.All data derived from the survey questionnaires were reported into a computerised sheet . Then, analyses were made to generate proportions of individual answers to each question of the survey questionnaire. A separate sheet, containing graphs, was produced for the 16 questions of the survey questionnaire. Data were presented as a percentage of the total answers to each question.Overall, 591 physicians  were included in the survey. Among these, 48 (8.1%) were training SPs and 543 (91.9%) were trained GPs. Involved physicians provided 12,599 answers to the survey questionnaire, among which 312 (2.5%) were considered inappropriate or incorrect and 29 (0.2%) were missing or not reported. Thus, a total of 12,258 valid answers were included in the present analysis, which represents the 97.3% of the overall results generated by the survey questionnaire.As shown in Table\u00a0Renal organ damage was considered to be frequent by 54% of SPs and 45% of GPs, although about one third of both groups of physicians considered this marker relatively not frequent in their clinical practice. At the same time, vascular organ damage  was considered relatively not frequent in hypertensive outpatients by the vast majority of involved physicians. Of note, estimated prevalence of CVD, including transient ischemic attack and stroke, was reported to be relatively low mostly in those hypertensive outpatients followed by SPs compared to those followed by GPs.As shown in Table\u00a0On the other hand, the majority of GPs considered the carotid vascular ultrasound the most appropriate diagnostic tool to be used in patients with hypertension for excluding the presence of CVD in their clinical practice, whereas, the majority of SPs expressed a clear preference for brain imaging techniques, including CT or MR.As shown in Table\u00a0Also in the clinical management of hypertensive outpatients with stroke, the achievement of the recommended BP targets was considered the most important therapeutic target by 47% of GPs, followed by absolute BP reductions (38.4%) and protection from hypertension-related organ damage (10.9%), whereas SPs gave similar clinical relevance to protection from organ damage (40.4%) and achievement of the recommended BP targets (38.3%). Also in this case, adherence to prescribed medications was relatively partially considered by SPs (10.6%) and GPs (3.5%). Of note, reduction of drug-related side effects and adverse reactions was basically not considered of clinical relevance by both groups of physicians.Differences between two groups of physicians were observed with regard to BP goals in hypertensive outpatients with TIA Fig.\u00a0. Indeed,In hypertensive outpatients with TIA Fig.\u00a0, angioteIn hypertensive outpatients with stroke Fig.\u00a0, about 6Combination therapies based on ACE inhibitors with beta-blockers, diuretics or calcium-channel blockers represented the preferred options for treating patients with hypertension and TIA by GPs Fig.\u00a0. On the Similar proportions were observed for physicians\u2019 preferences with regard to different combination therapies used for treating hypertensive patients with stroke Fig.\u00a0. In partIt is well known that hypertension management and control have been not achieved for many years, and that this relative failure has largely contributed to a persistently high burden of hypertension-related CVD, mostly including TIA and stroke, worldwide. It has been also shown that many factors can be advocated to try to explain the reported poor rates of BP control observed in various Western Countries, including Italy. Among these factors, patients\u2019 clinical characteristics and behaviours , as well as poor effectiveness of antihypertensive drug strategies  have been acknowledged.As a matter of fact, all these items are mostly focused on hypertensive outpatients rather than on treating physicians. Indeed, minor data are available to evaluate physicians\u2019 preferences and behaviours in the clinical management of hypertension in real practice. In this view, we recently analysed the preferred options for the clinical management of outpatients with hypertension and hypertension-related diseases expressed by Italian physicians with different medical skills \u201319. ThesFirst of all, concomitant presence of hypertension and CVD was considered to be relatively not frequent in a setting of clinical practice. The vast majority of both groups of Italian physicians reported an estimated prevalence of hypertension and CVD between 10 and 20%. On the other hand, physicians reported a relatively high prevalence of cardiac organ damage, namely left ventricular hypertrophy, which has demonstrated high predictive value on the risk of hypertension-related CVD. This highlights the need for having specific diagnostic and therapeutic indications, in order to improve the clinical management of patients with hypertension and cardiac organ damage and reduce the potential risk of CVD.In addition, Italian physicians considered the presence of cardiac organ damage, namely left ventricular hypertrophy, as the most common marker of organ damage. Also, the estimated prevalence of left ventricular hypertrophy was considered to be substantially higher than those reported for other hypertension-related markers of organ damage, including renal abnormalities and carotid atherosclerosis, thus highlighting the clinical relevance given by both groups of physicians to hypertension-related cardiac organ damage. However, the existence of left ventricular hypertrophy can be easily detected by simple ECG, whereas tests for other markers of organ damage except for cardiac one, may result in additional costs. Given this consideration, lower prevalence of some types of organ damage may be due to incomplete evaluation in a setting of real clinical practice.Preferred options expressed by involved physicians for BP targets to be achieved in treated hypertensive patients with CVD resulted of particularly relevance, because of Italian GPs aimed to achieve more ambitious targets than those expressed by specialized physicians and recommended by current guidelines. In the most recent guidelines , 20, it As applied for previous analyses , 17, somAlthough limited by the descriptive nature of the survey, this study provides some relevant information on attitudes and preferences, as well as on different diagnostic and therapeutic approaches applied by physicians when managing hypertensive outpatients with CVD in Italy. The main findings of our analysis, in fact, demonstrated that the concomitant presence of hypertension and cardiac markers of organ damage is reported to be high, whereas that of hypertension and CVD is considered to be relatively low in a setting of clinical practice by both groups of involved physicians. Even in the absence of specific indications from international guidelines, GPs tended to attain more ambitious BP targets in hypertensive outpatients with CVD. To achieve these BP targets, pharmacological therapies based on ACE inhibitors, either in monotherapy or combination therapy (mostly with beta-blockers), represented the preferred options."}
{"text": "Primary hyperparathyroidism (PHPT) is uncommon among children with an incidence of 1:300,000. This diagnosis is often missed in children in contrast to adults where it is detected at a pre symptomatic stage due to routine blood investigations. Etiology of PHPT can be due to adenoma, hyperplasia or rarely carcinoma.A 12\u00a0year old Sri Lankan girl presented with progressive difficulty in walking since 1\u00a0year. On examination she had bilateral genu valgum. Skeletal survey revealed valgus deformity of knee joints, bilateral subluxation of upper femoral epiphysis(SUFE), epiphyseal displacement of bilateral humeri, rugger jersey spine and subperiosteal bone resorptions in lateral aspects of 2nd and 3rd middle phalanges. There were no radiological manifestations of rickets. Metabolic profile revealed hypercalcemia with hypophosphatemia. Intact parathyroid hormone levels were elevated at 790\u00a0pg/ml. Vitamin D levels were deficient. She had low bone mineral density with Z score of \u22123.4. Vitamin D supplementation resulted in worsening of hypercalcemia without reduction in PTH levels. Tc 99 Sestamibi uptake scan showed abnormal tracer retention in left inferior pole of thyroid. A large parathyroid gland was removed with histology favoring parathyroid adenoma. Post operatively she developed hypocalcemia. Bilateral osteotomy was done for SUFE and further surgeries for correction of limb deformities planned.PHPT in children is usually diagnosed late when irreversible organ damage has occurred. Children can present with non specific symptoms involving gastrointestinal, musculoskeletal, renal and neurological systems. PHPT can cause disarray in bone and epiphysis in children during pubertal growth spurt. Genu valgum and SUFE are rare skeletal manifestations in PHPT and only 10 cases of genu valgum and 9 cases of SUFE have been reported up to now. So far no cases have been reported on epiphyseal displacement of humeri. Awareness regarding the occurrence of these rare skeletal manifestations especially during puberty is important for early diagnosis to prevent irreversible outcomes. Primary hyperparathyroidism (PHPT) is a common condition among adults. However it\u2019s an uncommon disorder in children and adolescents associated invariably with delay in diagnosis. This is mainly attributed to incidental detection of hypercalcemia in routine blood investigations done in adults thus allowing diagnosis at an asymptomatic stage. In contrast diagnosis in children is often when they are symptomatic with target organ involvement. It is postulated that puberty may result in unmasking of certain skeletal manifestations which result due to PHPT.We present a case of an adolescent with PHPT who presented with rare skeletal manifestations of genu valgum, bilateral SUFE and bilateral epiphyseal displacement of humeri.A 12\u00a0year old Sri Lankan girl presented with crooked legs and progressive difficulty in walking for 1\u00a0year duration. She did not complain of any leg pain, joint pain or proximal muscle weakness. There was no history of trauma, fractures, abdominal pain, vomiting constipation, neck swelling or renal calculi. She had been relatively healthy up to now taking part in athletic events in school until the development of these symptoms. Her menses were regular following attaining menarche at the age of 11\u00a0years. She was a product of a non consanguineous marriage and both her siblings were healthy. Family history was nil of note for any renal disease or multiple endocrine neoplasia (MEN). Her diet was deficient in milk and meat products. Bilateral stapling of knees done 6 months back did not lead to any symptomatic resolution. On examination she had bilateral valgus deformities of the knees without any other clinical features of Rickets or dysmorphism. No other joint deformities were noted. Height and weight were age appropriate and she was in Tanner 2 in pubertal development. The rest of the systemic examination was normal.X ray imaging of the lower limbs revealed bilateral slipped upper femoral epiphysis (SUFE) and bilateral genu valgus deformity of the knees. As SUFE is associated with a wide range of endocrine and metabolic disorders she was screened for these diseases. Ionized calcium levels were 1.5\u00a0mmol/l (1\u20131.3\u00a0mmol/l) which was repeated and confirmed. Serum phosphate levels were low at 2.3\u00a0mg/dl (2.5\u20134.5\u00a0mg/dl). Serum Alkaline phosphate levels (ALP) were elevated at 3210\u00a0U/L (age matched reference range 182\u2013587\u00a0U/L). Serum intact Parathyroid Hormone (PTH) levels were 796\u00a0pg/ml (8\u201376\u00a0pg/ml) indicating hyperparathyroidism. Calcium creatinine ratio was 0.06 (>0.02 suggestive if primary hyperparathyroidism). Vitamin D levels were 25\u00a0nmol/l (<30\u00a0nmol/l suggestive of deficiency). Anterior pituitary hormones inclusive of Thyroid function tests, 9\u00a0am Cortisol, Follicular Stimulating Hormone, Luteinizing Hormone, Prolactin and baseline hematological indices, serum electrolytes, renal and liver profile were all normal. A skeletal survey revealed evidence of epiphyseal displacement of bilateral humeral heads, rugger jersey spine and subperiosteal bone resorptions involving lateral aspects of 2nd and 3rd phalanges. There were no radiological features of rickets such as widening, cupping or fraying of the metaphysis. An ultrasound scan performed to detect a possible parathyroid adenoma failed to detect any masses. Ultrasound scan of abdomen did not show any evidence of nephrocalcinosis or renal calculi. Bone densitometry of spine revealed low bone mineral density with a Z score of \u22123.4. At this stage a differential diagnosis of Primary hyperparathyroidism (PHPT) with vitamin D deficiency or Tertiary hyperparathyroidism due to vitamin D deficiency was considered. It was decided to reassess her following adequate supplementation with vitamin D. She was treated with 50,000\u00a0IU of vitamin D2 weekly for 8\u00a0weeks followed by maintenance therapy of vitamin D3 1000\u00a0IU daily Table As adequate vitamin D supplementation failed to reduce the PTH levels a diagnosis of primary hyperparathyroidism was made. A Tc99 Sestamibi parathyroid scan revealed focal abnormal and persistent tracer retention in right inferior thyroid pole. As the diagnosis of PHPT was most likely due to parathyroid adenoma she underwent surgery where a large 2\u00a0\u00d7\u00a03\u00a0cm parathyroid gland was removed from the right inferior pole of the thyroid gland. Ipsilateral parathyroid gland was atrophied. Histology favoured a parathyroid adenoma. Post operatively she developed perioral numbness and paresthesia in fingers. Serum ionized calcium levels were 0.8\u00a0mmol/l, serum intake PTH levels were 61\u00a0pg/ml with normal levels of magnesium 0.82\u00a0mmol/l (0.75\u20130.95\u00a0mmol/l) and phosphate 1.0\u00a0mmol/l (0.8\u20131.5\u00a0mmol/l). A probable diagnosis of hungry bone disease was made and she was treated with a single dose of intravenous calcium followed by oral calcium carbonate 3\u00a0g/day and calcitriol 2\u00a0\u03bcg/day with monitoring of serum calcium levels. She required calcium supplementation for 3\u00a0months following surgery. Bilateral osteotomy was performed for SUFE even prior to completion of metabolic evaluation as delay in treatment can have detrimental effects such as progression of the slippage and avascular necrosis. Series of surgeries for correction of limb deformities were planned following the improvement of quality of bone after treatment of PHPT Figs. , 5 and 6PHPT is common disease among adults. However it\u2019s occurrence is very rare among children and adolescents with about 200 cases reported worldwide up to now . It\u2019s inThe characteristic feature in older children is delayed presentation. Almost 80% of children are symptomatic and have end organ damage at the time of presentation , 4. ThisPHPT in our 12\u00a0year old patient was unmasked due to these skeletal deformities as she progressed through puberty. Apart from these she did not have any other clinical features suggestive of PHPT. Following excision of the parathyroid adenoma she developed biochemical resolution. However further surgeries will be required to correct the limb deformities.PHPT is a rare disease in children which is often diagnosed late once target organ involvement had developed. In contrast, adults with PHPT are incidentally detected in the asymptomatic phase during routine blood investigations. Apart from the well known skeletal manifestations, PHPT can affect the bone and epiphysis resulting in rare skeletal deformities such as genu valgum, SUFE and epiphyseal displacement especially during the pubertal growth spurt in adolescents. Having a high degree of suspicion when children and especially adolescents present with skeletal symptoms may aid in speedy diagnosis and prevention of detrimental long standing deformities."}
{"text": "LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8."}
{"text": "Remarkably, when B-\u03b3-CsSnI3 perovskite is deposited from a dimethylformamide solution onto a 180\u2013190\u2009nm thick CuSCN film supported on an indium-tin oxide (ITO) electrode, the CuSCN layer is completely displaced leaving a perovskite layer with high uniformity and coverage of the underlying ITO electrode. This finding is confirmed by detailed analysis of the thickness and composition of the film that remains after perovskite deposition, together with photovoltaic device studies. The results of this study show that, whilst CuSCN has proved to be an excellent hole-extraction layer for high performance lead-perovskite and organic photovoltaics, it is unsuitable as a hole-transport layer in inverted B-\u03b3-CsSnI3 perovskite photovoltaics processed from solution.We report the findings of a study into the suitability of copper (I) thiocyanate (CuSCN) as a hole-transport layer in inverted photovoltaic (PV) devices based on the black gamma phase (B-\u03b3) of CsSnI However, the possibility of lead contamination of the environment due to failure of the device encapsulants or improper disposal at the end of life is a serious concern for commercial exploitation6. The latter is because lead is a highly toxic element that accumulates in the food chain7 and lead perovskites decompose upon exposure to moisture and water to form lead compounds with significant solubility in water9. Consequently, there is a need for lead-free alternative matched to the needs of photovoltaic (PV) applications. Recent research has shown that tin (Sn) is a potential replacement for Pb in halide perovskites, an element with much lower toxicity than Pb10. PPVs based on wholly inorganic tin perovskite PVs are however at a very early stage of development and so to date the highest reported power conversion efficiency is only 6.4%11. Much of the dramatic improvement in the power conversion efficiency of lead PPVs has resulted from identification and optimisation of the electron transport layer (ETL) and hole transport layer (HTL)12 which interface the light harvesting perovskite layer with the electrodes. These interfacial layers are critically important because they: (i) enable the optimization of the light distribution in the device; (ii) facilitate efficient and selective charge extraction, by minimizing the barrier to the extraction of one carrier type, whilst presenting a large barrier to extraction of the charge carriers with opposite polarity carrier; (iii) physically separate the metal electrode form the perovskite layer, blocking the adverse reactions between them.Perovskite photovoltaic (PPV) devices using lead (Pb) halides as the light harvesting semiconductor have shown an unprecedented evolution over the span of less than a decade, with the power conversion efficiency increasing from 3.8% in 200913. It is however recognized that the high cost and instability of this material will negatively impact the prospects of commercialization and so there is a need to identify lower cost alternative HTLs. In addition to offering long term stability, the charge transport layer at the transparent electrode must also offer high optical transparency, and so there is interest in using wide band gap wholly inorganic HTLs14 including NiO15, CuI16, Cs2SnI617 and copper (I) thiocyanate (CuSCN)22. Among these CuSCN has proved to be a particularly effective HTL for high performance Pb-PPVs with both a conventional12 and inverted device architecture18, and also for high performance organic PVs22. The success of this material as a HTL is due to its wide band gap (\u22653.50\u2009eV), excellent solution processability at room temperature and high hole-mobility combined with an ionization potential suitable for the efficient extraction of holes22. Herein, we report the findings of an investigation into the suitably of CuSCN as a HTL in inverted PV devices based on B-\u03b3-CsSnI3, a semiconductor that is attracting considerable attention because it\u00a0offers near ideal optoelectronic properties for a single junction PV devices and can be processed at room temperature from dimethylformamide (DMF) solutions of CsI and SnI223.In the context of Pb-PPVs the doped organic semiconductor spiro-OMeTAD  has proved to be the best HTL to date in terms of the device power conversion efficiency22. A solution concentration of 50\u2009mg/ml deposited at room temperature yielded a film thickness of 180\u2013190\u2009nm as measured by step height analysis using atomic force microscopy (AFM): Supporting Information Figure\u00a03\u2009+\u2009SnCl2 (10\u2009mol% excess) |phenyl-C60-butyric acid methyl ester (PC61BM)|bathocuproine\u00a0(BCP)|Al. The B-\u03b3-CsSnI3 layer was deposited from DMF solutions at room temperature, with 10\u2009mol% excess SnCl2 added as a source of excess Sn, according to our previously reported optimized procedure23.\u00a0In a dry nitrogen-filled glovebox CsI, SnI2 and SnCl2 were mixed together in 1:1:0.1 molar ratio. To this mixture DMF was added to make an 8\u2009wt% solution , which was stirred overnight before use. To deposit films, two drops of solution were cast onto a substrate spinning at 4,000\u2009rpm for 60\u2009s. To confirm that DMF is an orthogonal solvent for CuSCN a 180\u2013190\u2009nm thick film of CuSCN was washed with DMF using the same spin-coating procedure as used for perovskite film deposition. The AFM images in Fig.\u00a0q), measured over an area of 10\u2009\u03bcm\u2009\u00d7\u200910\u2009\u03bcm is reduced from \u223c30\u2009nm before spin casting DMF to \u223c10\u2009nm after washing with DMF by spin casting. This reduction in surface roughness is consistent with partial dissolution of the CuSCN layer during DMF washing and its possible  removal. However, it is clear from Fig.\u00a019.In this study diethylsulfide was the solvent of choice for CuSCN deposition because it has been shown to be an excellent solvent for the formation of CuSCN films for electronic applications3 perovskite films deposited from 8\u2009wt% DMF solutions by spin coating directly onto CuSCN supported on ITO glass have a high surface coverage with improved uniformity as compared to perovskite films deposited directly onto ITO glass , open-circuit voltage (Voc), fill factor (FF) or shape of the external quantum efficiency (EQE) spectrum for PV devices with and without a CuSCN HTL. Given that the CuSCN layer increases the total semiconductor thickness in the device by up to a factor of 5, it is surprising that the Jsc remains unchanged (8.1\u2009mA\u2009cm\u22122vs 8.2\u2009mA\u2009cm\u22122) and the shape of the EQE spectrum remains unchanged, since the light distribution in the device would be significantly changed by the inclusion of such a thick additional layer24. It is also unexpected that Voc for the devices with and without CuSCN; 0.31\u2009V and 0.30\u2009V respectively, is essentially unchanged since the CuSCN layer would be expected to reduce the reverse saturation current by blocking the unwanted extraction of electrons by the ITO electrode, giving rise to an increase in Voc23.It is evident from Fig.\u00a03 based PV devices without a HTL23, and so it is unexpected that PV devices incorporating a CuSCN HTL should exhibit the same evolution in performance with storage.Additionally, devices with and without a CuSCN layer improve significantly with storage under nitrogen: Fig.\u00a02, and the result was the same  analysis  of the films of CuSCN and B-\u03b3-CsSnIs Figure\u00a0 which pr7 Figure\u00a0 confirmi7 Figure\u00a0 is consi7 Figure\u00a0 and all 3 perovskite does not form directly from DMSO solutions at room temperature, but there is rapid crystallization to form a solvated intermediate29 which must be annealed for conversion to the perovskite. Figure\u00a030 environment  as the solvent for perovskite film deposition. Notably, unlike for DMF, B-\u03b3-CsSnIt Figure\u00a0 shows tht Figure\u00a0. Taken t3 perovskite or a solvated intermediate29. Tin perovskites are known to crystallize much more rapidly than their lead analogues due to the high Lewis acidity of Sn2+\u200930, and the enthalpy of formation of the B-\u03b3-CsSnI3 perovskite is very low as compared to that of CuSCN (\u223c0.06 eV31vs 1.14 eV32), so the process of complete ejection of the CuSCN under layer is evidently a strongly kinetically driven process.The complete displacement of the 180\u2013190\u2009nm thick CuSCN film by the perovskite film  is remarkable, not least because of the high speed of film formation during spin-casting. This finding is attributed to a combination of the increased solvation power of DMF and DMSO for CuSCN, due to the high ionic strength associated with halide and metal ions, together with the tendency for very rapid crystallization either directly to the B-\u03b3-CsSnI3 based PPV with an inverted device architecture. Unencapsulated PPV based on B-\u03b3-CsSnI3 with an inverted architecture have been shown to exhibit promising device stability23, although the power conversion efficiency has remained stubbornly low primarily due to a low Voc. The ionization potential of B-\u03b3-CsSnI3 is particularly small amongst tin and lead halide perovskites33 and so in order to achieve a significantly increased Voc, energetic losses that occur during hole-extraction must be minimized. The identification of a suitable HTL layer, which has hitherto proved elusive31, is therefore essential to improve the efficiency of B-\u03b3-CsSnI3 based PPVs, and so represents a fertile area of research.In conclusion, whilst CsSCN has proved to be a low cost HTL for Pb-PPV and high performance organic PVs, here we have shown that is unsuitable as a HTL in solution processed B-\u03b3-CsSnIA solution concentration of 50\u2009mg/ml CuSCN in diethyl sulfide was drop cast onto a freshly cleaned ITO glass substrate spinning at 3000\u2009rpm for 60\u2009seconds.2 and tin(II) halide were mixed together in 1:1:0.1 molar ratio. To this mixture N,N-dimethylformamide (DMF) was added to make an 8\u2009wt% solution , which was stirred overnight before use. To deposit films, two drops of solution were cast onto the substrate (sufficient to cover the whole substrate), followed by spinning at 4,000 r.p.m. for 60\u2009s. The B-\u03b3-CsSnI3 phase forms immediately upon solvent evaporation.In a dry nitrogen-filled glovebox CsI, SnI\u22121.) were held in vertical slide holders and ultrasonically agitated in an acetone bath, followed by a high purity water bath with a few drops of surfactant, followed by high purity deionized water only bath, acetone and finally an isopropanol bath. Slides were then UV/O3 treated for 15\u2009minutes. Immediately after UV/O3 treatment the slides were transferred into a dry nitrogen filled glovebox for CuSCN film deposition followed by deposition of the perovskite and finally the PC61BM layer from 13\u2009mg\u2009ml\u22121 chlorobenzene solution using a spin speed of 1500\u2009rpm. This was followed by thermal evaporation of 6\u2009nm bathocuproine (BCP) deposited at 0.5\u2009\u00c5\u2009s\u22121 and then 60\u2009nm of Al deposited at 1\u2009\u00c5\u2009s\u22121. Thermal evaporation was performed at a pressure of 1\u2009\u00d7\u200910\u22125 mbar with substrate rotation. The Al electrode was deposited through a shadow mask to make six devices per slide, each with an area of 6 mm2.Indium tin oxide (ITO) coated glass slides (Thin Films Devices Inc. 15\u2009\u00b1\u20093 \u03a9 sqJ\u2013V) curves were measured using a Keithley 2400 source-meter under AM1.5\u2009G solar illumination at 100\u2009mW\u2009cm2 (1 sun), scanned from 1\u2009V to\u2009+\u20091\u2009V at 0.1 Vs-1. External quantum efficiency (EQE) measurements were carried out using a Sciencetech SF150 xenon arc lamp and a PTI monochromator, with the monochromatic light intensity calibrated using a Si photodiode (Newport 818-UV). The incoming monochromatic light was chopped at 180\u2009Hz. For signal measurement a Stanford Research Systems SR 830 lock-in amplifier was used. J\u2013V and EQE measurements were made using custom LabVIEW programs.Device testing was performed in the same glove box as used for device fabrication using a solar simulator inside the glove box. Current density\u2013voltage , with the photoelectrons being detected at a 90\u00b0 take-off angle. The sputtering was carried out at room temperature using a Minibeam I ion gun . A beam of 4\u2009keV Ar+ ions was incidenton a 3\u2009\u00d7\u20093\u2009mm area of the sample surface. Curve fitting was performed using the CasaXPS package, incorporating Voigt (mixed Gaussian Lorentzian) line shapes and a Shirley background.XPS analysis was performed using a Kratos AXIS Ultra DLD. Samples were unavoidably exposed to air for approximately 1\u2009min during transfer from an air-tight box to the vacuum chamber of the instrument. XPS measurements were carried out in an ultrahigh vacuum system with a base pressure of 5\u2009\u00d7\u2009103 prepared from 8\u2009wt%  DMF solution deposited onto a glass substrate (13\u2009\u00d7\u200913 mm2) spinning at 4000\u2009rpm for 60\u2009seconds. Measurements were made on a Panalytical X\u2019Pert Pro MRD equipped with an Anton Paar DHS 1100 domed stage under a flow of N2. Simulated diffraction patterns were calculated using the program Mercury 3.122 using CIFs from the Inorganic Crystal Structure Database (ICSD).XRD was performed on thin films of CuSCN prepared from diethyl sulphide solution and a bilayer of CuSCN and B-\u03b3-CsSnISupporting Information"}
{"text": "Effects on Fatty Acid Metabolism of a New Powdered Human Milk Fortifier Containing Medium-Chain Triacylglycerols and Docosahexaenoic Acid in Preterm Infants [We thank Bernard and colleagues for their careful reading and interest in our article  Infants . Bernard Infants , our rep Infants ,4,5,6,7. Infants , the levA second point raised by Bernard et al.  is that Finally, we agree with the remaining two points raised by Bernard and colleagues . First, We fully share the perspective of Bernard and colleagues that improving preterm infants\u2019 lipid nutrition is an urgent task, and we are appreciative of the opportunity to provide a corrected"}
{"text": "Designing, changing and adapting organizations to secure viability is challenging for manufacturing companies. Researchers often fail to holistically design or transform production systems. Reasons are often the conflict of interests between production and maintenance, the temporal divergence of their activities and their organizational structure. Thus, the aim of this study is to propose a holistic approach of how production and maintenance can be designed, changed or managed. Hereby, the Viable System Model was applied. This structure can be applied to any kind of structured organization and for its management with goals to be achieved in modern society; however, focus of the research is the coordination of production and maintenance management. The goal of the developed model is to be able to react to some potential production environments by taking coordinated decisions correctly and in the right moment based on the needed information. To ensure this, standardized communication channels were defined. In conclusion, this proposed approach enables production systems to have internal mechanisms to secure viability depending on all potential environment scenarios. The global logistics flows have increased dramatically in recent years due to a globalized economy that introduces inherent challenges to the establishment of international business  (p. 10).The success of the actors involved in a cooperative supply chain depends in an essential way on the extent to which they are able to deal with the dynamic market requirements  (p. 101)Information in real time and its processing for production planning and control can give the correct answer how to face increasing dynamic requirements. However, the current planning and control logics do not provide the necessary support for this, which causes unexpected deviations in planning which cannot be compensated in the short term  (p. 160)According to a survey carried out by the VDMA  organization of 240 European companies of all sizes and of different industries, in 2006, 78.5% of managers claimed that the importance of maintenance has increased significantly in recent years and 67.1% that it will continue to increase in the future  (p. 38).Many approaches have been considered to make production and maintenance planning as efficient and flexible as possible. All pursue and many theoretically achieve the partial or total optimization of global production systems by making them efficient. However, when applying these theoretical internal logistics management models to practice, information systems should be used as a vehicle to communicate the necessary information to be managed within the organization. The reasons why the majority of applications of such concepts in information systems, as in ERP (Enterprise Resource Planning) systems, have failed are: the delay or lack of information, the misuse of production planning tools and maintenance, lack of coordination or conflict of interest between the production and maintenance departments, the non-consideration of the environment and the requirements of the final customer in a dynamic process of continuous improvement, leading all of them to take strategic, tactical or operational decisions at an optimal time or in an optimal way.Research focus is the design and simulation of a coordination model for production and maintenance management using as a basis the current state of the art and pursuing high adaptability to environment changes. To achieve this, the Viable System Model (VSM) is used as a methodological structured approach and system dynamics is used to describe interrelationships between parameters and control loops in production and maintenance. The goal of the research is to generate an approach of how to design and develop production systems to be successful in all potential future environment scenarios considering developments in other areas of the company and in external fields of a company such as customer and supplier needs and conditions, politics, economy, technology, environment and energy regulations and society. In this context, simulation serves as tool to prove the approach in some potential future scenarios for production and maintenance coordination.The initial hypothesis is that a production system built using the structure of the VSM will be able to react faster to environment changes and therefore to improve short-, medium- and long-term goals of every producing company.This chapter contains a literature review of the viable system model, system dynamics as well as a description of the simulation technique and software employed.The Viable System Model (VSM), a cybernetic management model, was developed by Stafford Beer throughout his life  (p. 57).To validate the research methodology, research and practical applications using the VSM were searched. Many authors have used the VSM as basis to describe and develop models how to deal with complex challenges in social and industry fields. Some of the topics worked and that give an indication of the scientific value of the approach are: organizational models for companies  .System dynamics is a computer-guided approach for studying, managing and solving complex feedback problems with focus on policy analysis and design  (p. 342)SD has been applied to a great set of systems from corporate strategy to the dynamics of diabetes as well as for the cold war arms race between the USA and USSR. System dynamics can be applied to any dynamic system, with any time and spatial scale  .This chapter provides the basic terminology needed to successfully perform this work.Production is the foundation of human activity. Natural resources are transformed into useful products through production processes to meet the needs of society  p. 319)931]  model, which is a reference model for its analysis, evaluation and design, is used  (p. 29).All tasks are distinguished vertically in Industrial maintenance is defined according to DIN (German Institute for Standardization) 31051 as the \u201ccombination of all technical, administrative and management measures during the life cycle of an observation unit in order to maintain the functional status or restoring it so that it can fulfill the required function\u201d  (p. 26).The \u201cideal maintenance organization\u201d does not exist  (p. 65) According to experts, German companies spend around 140,000 million euros annually on maintenance of machines and installations. This, together with the fact that between 75% and 80% of the execution time of a maintenance order are activities without added value, defines a great potential for savings in production companies. Many of the time losses are in the interfaces between the different areas. The management of maintenance processes must ensure the change from a functional orientation to an orientation towards processes  (p. 60).Maintenance management of production units affects not only the maintenance personnel but also all the employees of the whole company  (p. 104)Total Productive Maintenance (TPM),Lean Maintenance,Total Lifecycle Cost Strategy (TLC),Reliability Centered Maintenance (RCM),Knowledge Based Maintenance.Logistics can be described as an interdisciplinary strategy to optimize production. Availability is, therefore, a key factor for logistics and is also the link between logistics and industrial maintenance  .This chapter describes the components of an integrated planning model for production and maintenance management. In a first step, the planning tasks are presented according to its planning level . Later, the recursion levels of the VSM are described. Then, the planning tasks are associated with the different recursion levels and the information flows are defined between recursion levels and systems of the VSM.Production systems are considered important in relation to aspects of quality, time and costs  (p. 1). The highest level, company (n \u2212 1),\u25cbThe recursion level of plant or production workshop, for example production management activities in an automotive production or assembly shop (n + 1),\u25cbThe level of machine group, machine or installation with the associated activities for the different production activities such as preparation of the machine, change of tools, operation, control of production, etc. (n + 2),The recursion level of production (n). In the same recursion level it can be found finance, human resources, IT, research and development, etc.\u25cbThe recursion level of plant or production workshop\u2014for example, maintenance management activities in an automotive production or assembly shop (n + 1),\u25cbThe recursion level of installation or machine with the associated activities for the different maintenance activities: preventive, planned repairs, corrective, etc. (n + 2).The recursion level of maintenance (n). In the same recursion level can be found finance, human resources, production, etc.A company is assumed as a viable system that is the first level of recursion in which the five systems neededto ensure viability are found. Therefore, in this research work, four levels of recursion can be differentiated as it is shown in The activities in the recursion level n + 2 are no longer viable systems in contrast to the higher recursion levels because they do not contain a structure like that of the VSM, since they are the elements of production or maintenance execution.Within this first level of recursion, company, the different functions of a company can be found, such as production, maintenance, commercial, finance, research and development, information systems, etc. In this research project, production and maintenance tasks will be analyzed in detail, recursion level n, but also considering the function of system 2 at the company level, n \u2212 1, whose function is to coordinate the different functional areas of a company.System 5 establishes the production and maintenance objectives and communicates them to the other management systems, systems 3 and 4.System 4 observes and collects essential information from the external environment of maintenance and production. Production environment is mainly represented by the demands of customers, but also by other factors such as information systems offered by the market for the management, planning and control of production, new manufacturing technologies and, in general, all factors affecting the production system such us market standards, delivery times, production strategies, delays, production costs in external companies for example to help in making decisions about outsourcing or to not manufacture certain parts or about the assembly of certain sets, etc. Maintenance environment could be described by the costs in the market for maintenance activities, average reaction times in the market as well as service level standards, information systems for maintenance, technology development, environmental and work safety regulations and customer delays. With these and other information from the external environment and information from system 5, system 4 creates a vision of what the production and maintenance areas have to be, and which should be the measures to be followed to reach that state. This vision is validated internally with system 3 so that system 4 makes the decision and system 3 makes the changes internally.System 3 is responsible for maintaining the internal stability of the model by optimizing the use of internal resources using the information received by system 4 about the clients as well as the information on the different divisions of system 1 through system 2. It would be related to functions such as operative production and maintenance management and control, information management, quality management, operative logistics planning and control, etc. Moreover, system 3* allows a quick response to possible emergencies in the manufacturing process or in the production control and monitoring by acting before information flows through system 2. It is capable of performing actions in real time if something happens outside of normal limits such as making changes in sequencing and production scheduling to avoid stopping production flow.System 2 is represented by the functions of coordination between the different production locations in daily activities. This system receives all the information of the different production plants and acts as a filter so that only the necessary information reaches system 3. The difference between both is in the time horizons of action. While system 2 performs functions in daily activities, the tactical system optimizes the performance of the internal system over a longer time horizon.System 1: each plant or workshop within the production system is an operational unit that includes the management of the unit and the division that performs the operational activities. An example could be an assembly workshop that contains the planning and production control departments responsible for the components of the workshop with their team leaders together with the operators that finally perform the production tasks.Environment: represents all the external factors that influence the production and maintenance management in a company. System 2 at company level plays the role of coordinator between the functional areas of the company trying to solve conflicts between them. Moreover, systems 1 at company level are all functional areas of every company such as production or maintenance. At the recursion level of production and maintenance (n), it is assumed that the different production plants or workshops will be the respective system 1, which also contains a viable system in each of these locations. The VSM of production and maintenance management within a company is described in the next paragraphs by the tasks performed by its five necessary systems:Production and maintenance management tasks were assigned to the VSM systems at recursion levels n and n + 1 defined before. In Between the company environment and system 4 at the production/maintenance recursion level,Between systems 5 of company and production/maintenance,Between systems 4 of company and production/maintenance,Between systems 3 of company and production/maintenance,Between systems 2 of company and production/maintenance,Between the operating units, systems 1, of company and production / maintenance,Between the alarm/monitoring filter (System 3*) of the company\u2019s recursion level and system 4 of production/maintenance.Current technical literature agrees that the connection interfaces between recursion levels is extremely important  (p. 59).Between the two normative systems of company and production/maintenance, there is a flow of information that defines the degree of freedom of decision-making in which production/maintenance recursion level can act. Specifically, it means that the decisions taken by the management of the company are communicated to production and maintenance management defining its guidelines for autonomous decision-making within the respective areas. These guidelines can be financial, on personnel, on affectation to other areas, etc. In the same way, the objective levels such as production in term, production quality and production costs and adaptation capacity are influenced by decisions from the management, defining the priorities and the limits for the coordination among production areas. An example could be: the direction of the company in its strategic plan establishes the target production volume for the following years as well as the required flexibility in percentage on the production as well as the decrease in target costs. Of course, these decisions would influence the decision-making framework for production and maintenance that should adapt their methods and tools to be able to optimize costs, times and quality based on that flexibility also securing the required availability and adapting its maintenance strategy.As explained during the research work, basic communication flows were defined. In total, 88 information connections were defined for the production recursion level specifying if the communication goes from company\u2019s recursion level to production recursion level or between systems in production recursion level. Moreover, for the recursion level of maintenance, a total number of 77 information connections were defined specifying if the communication goes from company\u2019s recursion level to maintenance recursion level or between systems in production recursion level. An extract is shown in Press shopBodywork shopPaint shopEngines shopAssembly shopsFinal assembly shopsThe automotive supply chain is composed of suppliers at three levels (Tier 1\u20133), OEM (car manufacturers), distributors. For the work, we are going to focus on the production process of an OEM   are growing. The complexity of customized models and variants is on the rise, especially as regards the way individual vehicles are equipped. The key trend in automotive production is the standardization of construction modules in common platforms. The modules refer to groups of components and related systems. The diversity of models is an important sales argument and delivery time is the key factor for the automotive market and in the manufacturing process. These requirements involve a change in assembly operations that need to be more flexible and agile  In this step, the conceptual model for a manufacturing process is specified. To do this, the interrelations between indicators are defined by Causal Loop Diagrams (CLD). Later, the concepts are modeled in the VENSIM commercial simulation program. The simulation models present a simplified depiction of a real production and maintenance process to quantify the effect of decisions in a production system. Moreover, it contains the validation of the models and the presentation of the results obtained. Finally, the main conclusions are summarizedas well as a critical reflection of the work including a perspective for future research activities.The possibility of developing simulation models for production and maintenance management starts from the knowledge of the interrelations between its elements. Using casual loop diagrams (CLD), the effects of the changes of certain factors in their dependent parameters are shown. Five CLDs are developed from production management to maintenance planning. Definition of the objective, hypothesis and methodologyNumber of simulation modelsSimplification of the complexity of the conceptual model through assumptionsCriteria that make possible a comparison between modelsDefinition of quantitative parameters to obtain results and compare modelsDefinition of the production flowCreation of the model based on the CLD Validation of the behavior of the simulation modelsDetermination of scenarios, simulation and extraction of resultsEvaluation of the results and derivation of conclusionsThe aim of the simulation is to observe the impact of the delay in decision-making in a chain of manufacturing processes from the steel stamping process to the end of assembly in the automotive industry. The hypothesis is that a simulation model applying the VSM will present better results in terms of study parameters compared to the one that does not apply the VSM or that has delays in internal decision-making. The methodology for the simulation design is:Production times for car models are not variableTimes for material transport and employee movements are not variableDistribution of finished products as givenProcurement of raw material as givenDemand does not change if customer service is better or worseEach order has a production unitBill of materials are not consideredFirst, assumptions are defined to simplify the model with focus on the simulation goal:Same demand, same patterns of demand, demand replicationSame production capacities per production stepSame breakdowns per production stepSame reaction times depending on the locationSame task execution timesSame workforce capacityThe same maintenance capacity is considered for the same class of workerSome elements are equal in all models to make possible a comparison between the models under the same conditions:Production on time (% per week)Total production (# thous. cars)Total downtime Availability of final assembly workshop (%)Capacity utilization of final assembly workshop (%)Cumulated stocks (# Mill. cars)MAD (Mean Absolute Deviation) (# thous. cars)Cumulated demand (# thous. cars)The objectives can be qualitative or quantitative. The research goal is to study the behavior of the different models in different situations of demand and configuration of maintenance and production areas. The results are quantified to evaluate the response according to the following key performance indicators:The production process consists of a process from steel stamping to the final revision shop. The plants in the process are shown in Press shopBodywork shopPaint shopPre-assembly shop\u2014assembly 1Mechanical assembly shop\u2014assembly 2Final assembly shop\u2014assembly 3Final inspection shopAll of them are in different shops and have production and maintenance units associated to each one of them.Time restrictions: first, the modeler must define a time horizon and units of time. It is easy to fulfill that step by asking to what extent the simulation should be considered. In the case of the study, it has been decided to simulate four working years to evaluate influences in the medium and long term.Production strategy: the model offers the possibility to manufacture according to the push or pull principle. A mixed strategy can also be chosen. It assumes the existence of a single product or model in the production flow.Maintenance strategy: the simulation allows defining a preventive maintenance ratio so that it reduces the corrective according to a factor that is assumed.Capacity and production methods: in the model there are two methods: CONWIP (CONstant Work in Progress) in which the quantity of products in production process is always the same and BOA (Workload-dependent order release) that initiates production of an order based on the current and expected workload for the plants.Organizational structure and tasks distribution between production and maintenance: the simulation allows personnel changes in the areas of maintenance and production as well as between them. In addition, assumptions about the influence of the changes are made, for example, if there are more maintenance personnel, it will take less time to perform preventive maintenance tasks.Demand characteristics and forecast: the model makes different forecasts using two different methods: moving average and linear regression.As shown in For a lower number of employees, production on time (%) must be lower and the total stock should be higher because the production facility provides more products than employees can process. Moreover, utilization of shop capacities should be lower and therefore also the production output.The validation of the simulation models can be done using different methods. In this process, some simulation variables will be used to observe their behavior and to evaluate if the models will be validated. Sterman defined 12 possible methods to validate system dynamics models  (p. 6). As shown in For higher initial stocks in WIP (work in progress) at initial time, production on time (%) must be higher and total stock should be higher in the first weeks because the production facility has more products within production process at the beginning. Moreover, utilization of shop capacities should be higher and therefore also the production output in the first weeks. Afterwards, due to the CONWIP method, the production WIP converges to a CONWIP goal that is equal to one week of demand and therefore all other indicators also converge.In For a make-to-order production, it is expected to have less stock during the production process, less capacity utilization and production output as well as less production on time than with a make-to-stock production.As shown in The model is validated because logical expected values are obtained for three different input parameters influencing multiple key performance indicators of the simulation.Case study 1\u2014reorganization of production and maintenance employees: the production area transfers workers to the maintenance area when there is a low customer demand. With this scenario, how the utilization of production workforce for maintenance activities could increase global production performance, if the decision is made in an early moment when demand starts to be lower than production capacities, is studied.Case study 2\u2014reorganization of production and maintenance employees: the maintenance area transfers workers to production when there is a high customer demand. With this scenario, how the utilization of maintenance employees in production activities could increase global production performance, if the decision is made in an early moment when demand starts to be almost at the maximum level of production capacities, is analyzed.Case study 3\u2014comparison between the CONWIP and BOA methods: for the same customer demand, the order initiation method varies. CONWIP (CONstant Work in Progress) initiates production of new orders maintaining the same WIP cars while BOA initiates production of new orders depending on current and future equipment workload. BOA tries to optimize utilization and CONWIP in the model tries to maintain a week of production demand always as WIP.Case study 4\u2014comparison between preventive and corrective maintenance: for the same demand, a low level of preventive maintenance activities and a high level of maintenance activities is simulated. With this scenario, the effect of the preventive level of maintenance activities can be simulated. In the models, a parametrization is done considering that preventive activities require a certain employee capacity but reduced the unexpected downtimes of the production shops.Four case studies are proposed in which two different model configurations are simulated, trying to reflect the importance of decision-making. The VSM model takes earlier these decisions and therefore can benefit earlier from the new production system configuration:Case study 1\u2014reorganization of production and maintenance employees with a low demand:Case study 2\u2014reorganization of production & maintenance employees with a high demand:Case study 3\u2014comparison between the CONWIP and BOA methods:Case study 4\u2014comparison between preventive and corrective maintenance:For the four scenarios, the following results are obtained:Case study 1: the production area transfers workers to the maintenance area when there is a low demand for production. This decision is only taken in the VSM simulation model and moves three employees of a total of 20 employees from each production area to its respective maintenance area. The logic reason for this change is, since production employees cannot be fully utilized, they can help the maintenance units to perform maintenance activities in order to improve the production system performance. As it can be seen in Case study 2: the maintenance area transfers workers to production when there is a high production demand. In this case, the Non-VSM model presents in one case 20 maintenance and production workers per plant or workshop while the VSM model changes to 23 workers for production and 17 for maintenance. Therefore, maintenance activities have less capacity while production capacity is increased. As it can be seen in Case study 3: it can be observed in Case study 4: in the simulation model, there is a parameter that indicates the level of intensity of preventive maintenance. As it can be seen in Based on the results presented, it can be said that the VSM simulation model improves relevant KPIs when making the decision to move employees and can help production systems when deciding which production methods should be used as well as deciding the optimal percentage of preventive maintenance to be performed.Identification of the objectives, key performance indicators and functions of production and maintenance management as well as basic methods and toolsClear evidence of the need for new approaches in the coordination of production and maintenance management after reviewing the state of the artConfirmation of the need for new communication and coordination systems as well as integral management of internal logistics as one of the biggest potentials to deal with current and future challenges in the manufacturing sectorThe application of the structure of the Viable System Model (VSM) for the design of the conceptual model for production and maintenance management provided a structure that has allowed the definition of information flows, planning levels as well as mechanisms of autonomy and escalation between levels within a company and within the areas of production and maintenance.System Dynamics has provided the notation and functions necessary for the design of simulation models using VENSIM as commercial software.The comparison of the different models for the four cases presented has shown the relevance of the organizational structure decisions and methods for production and maintenance management. In addition, it was proved how production and maintenance performance can be optimized by making decisions at the right moment. Key performance indicators such as total production, service level, downtime, etc. improved significantly when moving employees, changing production method or the maintenance strategy.After completion of the research work, the importance of the design and management of a productive system that integrates the planning tasks of production and maintenance of a company has been proved. In addition, the following points can be successfully concluded:According to the results of the comparison, it has been demonstrated that correct and early decision-making through the application of the VSM gives a better response of all the key performance indicators. A similar response of the models without application of the VSM can occur when there are no external or internal changes that affect the productive system, that is, if the production system would be in a static situation. As environments and companies are always dynamic, the usefulness of the VSM as a principle for the definition of responsibilities, interfaces, escalation methods and decision-making are confirmed.Improve the conceptual model with all the different methods and tools by level of recursion and task as well as its inclusion in the simulation modelExtend the study for all internal logistics processes to generate a tool to help management, since, as we have seen, the markets require flexibility and right now the companies do not have mechanisms to make the right decisions in most of the cases. Many of them centralize maintenance units and a few years later they decentralize it again, which suggests that there is no strategic or tactical direction.Apply the simulation model based on the VSM for a real productive systemIn conclusion, this proposed approach can increase the efficiency of internal logistics models such as the productive system in its interaction with industrial maintenance. Finally, it is important to point out new ways of research or new ways to continue improving the project carried out:"}
{"text": "Plasma IL-18 levels were significantly decreased in patients with AN compared with controls. Plasma IL-18 levels correlated to BMI in controls, but not in patients with AN. These results suggest that a decline in plasma IL-18 levels in patients with AN is not only due to malnourishment, but other pathophysiologic changes as well. IL-18 has a role in the brain\u2019s reaction to sadness and chronic stress. Therefore, decreased levels of IL-18 may commonly occur in patients with chronic AN.Multiple studies on the dynamics of inflammatory cytokines in patients with anorexia nervosa (AN) have been published, although results are not consistent among reports. Thus the pathophysiologic roles of these cytokines are not clear. We performed an exploratory analysis that included (1) comparisons of plasma interleukin-18 (IL-18) concentrations between patients with AN ( Anorexia nervosa (AN) is sometimes fatal due to severe malnutrition. Leukocytopenia frequently accompanies AN , althougInterleukin-18 (IL-18) is an inflammatory cytokine first identified by Okamura et al. in 1995. It was formerly referred to as an interferon gamma-inducing factor and is known to activate natural killer cells in the spleen ,11. IL-1In this exploratory analysis, we compared (1) plasma concentrations of IL-18 between patients with AN and healthy controls, and (2) correlations between body mass index (BMI) and IL-18 concentrations in both groups to examine the relationship between malnourishment and IL-18 levels.The ethics review committees at Nagoya University Graduate School of Medicine and Nagoya University Hospital approved the study protocol, and written informed consent was obtained from all participants.2) based on the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5) [2 to 14.54 kg/m2. Patients\u2019 medical records were reviewed retrospectively for psychiatric comorbidities; two cases had bipolar I disorder, one had bipolar II disorder, one had major depressive disorder, and one had a history of sedative, hypnotic, or anxiolytic use. No other psychiatric comorbidities were identified. Patients were treated in the psychiatric ward of Nagoya University Hospital and received medical nutritional therapy aimed to increase weight by 1 kg per week along with supportive psychotherapy by psychiatrists. They also received psychotropics or plasma electrolyte replenishers, as needed. The control group consisted of 39 healthy participants recruited from medical and co-medical students and hospital staff. A structured interview confirmed the absence of any psychiatric history in the control group. BMI in the control group ranged from 17.12 kg/m2 to 33.23 kg/m2. Patients included 21 Japanese women living in Japan who met the criteria of extremely severe AN (BMI < 15 kg/m (DSM-5) , includiBlood samples from patients were drawn around 8 a.m., before breakfast. Body height and weight were measured under surveillance in the early morning, before the blood was drawn, every Monday and Thursday, and the data obtained closest to the date of blood draw within the same week were used for analysis. In the control group, blood samples, body height, and weight were acquired during a visit to our institute, so the time of day for sampling was not fixed.Plasma IL-18 concentrations of all subjects were measured by enzyme-linked immunosorbent assay (ELISA) using a human bioactive IL-18 ELISA kit from Medical Biological Laboratories .p-values were calculated between plasma IL-18 concentrations and BMI in each group. P values < 0.05 were considered statistically significant.All data were analyzed with Excel 2013  and JMP pro 13 . The Wilcoxon rank sum test was used to compare plasma IL-18 concentrations between groups. The coefficients (Pearson\u2019s r) and p = 0.014) lower concentrations  than controls .p = 0.01). However, no such correlations were observed in patients   .The lack of correlation between plasma IL-18 levels and BMI in patients with AN suggests that plasma IL-18 concentrations are affected not only by nutritional status but by an unknown pathophysiologic factor in these patients. Increased levels of IL-18 in plasma have been observed in patients with schizophrenia and moderate to severe depression , so a psWhen exposing mice to chronic stress, IL-18 expression is increased in the basolateral amygdala (BLA). IL-18 knock-out mice show tolerance to chronic stress, through a decline of vasopressin and oxytocin levels in the BLA, caused by suppression of phosphorylated nuclear factor-\u03baB induced from IL-18 ,16. PlasPatients with AN often show chronic depression ,21; howeOur research had a small sample size, so a replication study and subtype-sensitive research, with larger samples, are needed. Pharmacologic and nutritional effects on plasma IL-18 levels cannot be excluded. The time of day for blood sampling from control participants was not fixed, so diurnal effects of plasma IL-18 cannot be disregarded. Because the linear association of IL-18 levels in plasma and in cerebrospinal fluid (CSF) has not been proven, levels of IL-18 in CSF should be measured. However, lumbar punctures may be unsafe in patients with AN, who tend to have hemorrhagic issues. Thus we did not determine CSF levels. It is unclear why plasma IL-18 levels in patients with AN did not correlate with BMI levels. Pathophysiologic factors in patients with AN other than low body weight or malnutrition should be examined. Moreover, future prospective and longitudinal studies focused on disease duration, measuring IL-18 at different stages of AN including recovered patients, psychosocial factors and determining IL-18 and endogenous opioid levels in CSF may be needed. Our current results lead us to hypothesize that the decline of plasma IL-18 levels in patients with AN may affect disease chronicity."}
{"text": "Glutathione (GSH) is a critical endogenous antioxidant found in all eukaryotic cells. Higher GSH concentrations protect against cellular damage, tissue degeneration, and disease progression in various models, so there is considerable interest in developing interventions that augment GSH biosynthesis. Oral GSH supplementation is not the most efficient option due to the enzymatic degradation of ingested GSH within the intestine by \u03b3-glutamyltransferase, but supplementation of its component amino acids\u2014cysteine, glycine, and glutamate\u2014enhances tissue GSH synthesis. Furthermore, supplementation with some non-precursor amino acids and micronutrients appears to influence the redox status of GSH and related antioxidants, such as vitamins C and E, lowering systemic oxidative stress and slowing the rate of tissue deterioration. In this review, the effects of oral supplementation of amino acids and micronutrients on GSH metabolism are evaluated. And since specific dietary patterns and diets are being prescribed as first-line therapeutics for conditions such as hypertension and diabetes, the impact of overall diets on GSH homeostasis is also assessed. Glutathione (GSH) is a low molecular weight, water-soluble thiol that is present in all eukaryotic cells . It is sDue to the strong cytoprotective effects of GSH, it could be a therapeutic option to maintain cellular integrity and slow tissue degeneration in various diseases. Researchers have tested the impact of administering GSH through several routes, including intravenously , intranaThis review presents the effect of oral supplementation of individual amino acids on GSH homeostasis in mammals with emphasis on clinical studies. Then, we highlight particular micronutrient supplements that have been found to improve GSH status by generally enhancing cellular antioxidant systems. Lastly, since specific dietary patterns are commonly being prescribed as first-line therapeutics for certain chronic diseases, this review will also briefly assess the impact of these overall diets on GSH homeostasis.L-Glutamine is the most abundant free amino acid in the body . ThroughWhen healthy human volunteers were supplemented with oral glutamine at 0.3 g/kg/day for 10 days, both glutamine and glutamate concentrations increased in plasma, but blood GSH concentrations unexpectedly decreased . This ef+ patients were supplemented with glutamine (20 g/day) for 7 days, plasma GSH, glutamine, and glycine levels were normalized [It has been proposed that glutamine most effectively increases GSH levels during times of GSH depletion . Thereformalized . Howeverrmalized . Glutamine supplementation may not be as effective in patients in the intensive care unit (ICU) ,40 or wiRodent studies support the notion that GSH levels increase with glutamine supplementation ,43,44,45+ individuals, but not in ICU patients or those with sickle-cell disease. It is crucial that future research efforts expand beyond RBC, as RBC lack mitochondria, producing a unique biochemical milieu that may not fully represent other cell types [It must be noted that long-term glutamine supplementation should not exceed >40 g/day due to its negative effects on biochemical pathways . Glutamill types .Glycine is one of three amino acids used to form GSH , so it hGlycine seems to improve GSH concentrations in animals with higher levels of oxidative stress. When supplemented in the diet, glycine may exert a beneficial effect via promotion of GSH biosynthesis and attenuation of oxidation, which in turn protects against endothelial and vascular dysfunction. There is evidence that glycine supplementation could be even more effective when orally supplemented with cysteine in older HIV-infected individuals . The rolL-cysteine is the rate limiting amino acid in de novo synthesis of GSH [s of GSH , so natus of GSH , signifis of GSH ,60,61,62s of GSH ,64. BecaN-acetylcysteine (NAC), S-Adenosyl-L-methionine (AdoMet), and L-2-oxothiazolidine-4-carboxylate (OTC) supplementation on GSH levels are evaluated in the next sections. Methionine is also a cysteine prodrug that increases RBC GSH levels when supplemented orally [The efficacy of cysteine precursors d orally , but supd orally ,67, so iNAC is a cysteine precursor that, when administered orally, increases GSH concentrations in individuals with GSH deficiency caused by infections, genetic defects, or metabolic disorders . Orally + patients were orally supplemented with NAC (1 g/day), plasma GSH, cysteine, homocysteine, taurine, methionine, and glutamine concentrations increased significantly [In a study by De Rosa et al., HIV-infected individuals were orally supplemented with 8000 mg NAC daily for 8 months, and whole blood GSH levels significantly increased with no major adverse effects . An addificantly . In a seficantly . In addificantly , and mayficantly .Overall, orally administered NAC is an effective way to replenish GSH in HIV-infected patients, but more research is needed to better understand the effect of NAC supplementation on GSH levels in healthy individuals. Despite its potential benefits, NAC is limited by potentially harmful side effects, such as nausea or gastric distress, but investigators hypothesize that the intestinal side effects found in the trial may have been due to the ingestion of the excipient, which contained lactose .AdoMet is a precursor to the transsulfuration pathway, the metabolic pathway responsible for condensing homocysteine to form cystathionine, which is then converted to cysteine . AdoMet In baboons, supplementation of oral AdoMet resulted in significant hepatic uptake of the molecule, and eventually, restored GSH levels . These rAdoMet supplementation does not appear to have a significant effect on homocysteine concentrations in healthy individuals , so suppOTC is converted to cysteine through 5-oxoprolinase in cells  and has Studies suggest that OTC may be a safe and viable option for elevating GSH concentrations, but some limitations remain unaddressed. A study by Nishina, Ohta, and Obuka (1987) found that intraperitoneal delivery of OTC increased hepatic GSH concentrations in guinea pigs, but decreased renal GSH levels . It was 6, condenses homocysteine and serine to ultimately form cysteine [The transsulfuration pathway is a major source of sulfur for GSH formation . In the cysteine . PresentSirt1 [In mice fed a HFD, supplementation of serine in drinking water significantly decreased hepatic ROS compared to the control group . Serine Sirt1 .Sirt1 and other age-related pathways.In experimental models of oxidative stress, serine appears to conserve tissue GSH levels and attenuate oxidative damage. But more research is needed to better define mechanisms by which serine protects GSH pools and acts on Cysteine is a necessary metabolic precursor for taurine synthesis, so it has been postulated that taurine supplementation would spare cysteine for eventual incorporation into GSH . In reseEffects of taurine on the enzymes GPx and GR have been evaluated in both rodent and human studies. Taurine supplementation recovered hepatic GR activity in diabetic rats , whereasOverall, these results point to a limited impact of taurine on GSH, implying that other amino acid or micronutrient supplements may be a more effective option at targeting and raising GSH concentrations.Fat-soluble vitamins  have been evaluated for their potential effects on GSH homeostasis. These vitamins exhibit antioxidant properties, and there is evidence that status of vitamin A ,117, vitVitamin E is a lipid-soluble antioxidant that protects the cell membrane against oxidation and supports other antioxidant reactions, including those involving GSH . Studies1c levels decreased [The beneficial impact of vitamin E on GSH translates to clinical studies as well ,127,128.ecreased ; the resecreased . In addiecreased  and suppecreased .Based on these rodent and clinical studies, it appears that vitamin E, when taken in safe doses, has the potential to increase RBC and brain GSH concentrations.6, is a coenzyme in the conversion of homocysteine to cysteine, which supports GSH biosynthesis [Pyridoxal 5\u2032-phosphate (PLP), the biologically active form of vitamin Bynthesis . PLP is ynthesis ,135.6 is especially critical for maintaining GSH pools [6 supplementation increased renal and hepatic GSH levels in rats with chromium-induced toxicity [6 supplementation had no effect on hepatic GSH levels in control rats, with or without supplementation [6 supplementation increased blood GPx levels of control rats, indicating that the vitamin may directly stimulate the GSH redox system under normal conditions [Homocysteinemia and chromium toxicity are high stress environments wherein vitamin BSH pools ,136,137.toxicity . Conversentation , nor didentation . Unexpecnditions .6 also affects GSH status of human patients. Hepatocellular carcinoma (HCC) patients exhibit high levels of oxidative stress, and when 33 HCC patients\u2014who had recently undergone a tumor resection\u2014were supplemented with vitamin B6 (50 mg/day) for 3 months, plasma GSH/GSSG ratios and GR activity unexpectedly decreased while GSSG and GPx levels did not change [Vitamin Bt change . The autt change .6 supplementation with other vitamins has been also evaluated [6 (200 mg/day) and folic acid (15 mg/day) supplementation for 1 month increased RBC and whole blood GSH levels in both hemodialysis patients and healthy patients [12 and B6 had improved GSH status [The impact of vitamin Bvaluated ,141. ComH status .6 supplementation cannot be made due to the limited clinical research and conflicting results on the effect of vitamin B6 supplementation on GSH concentrations. Future research on vitamin B6 supplementation is warranted given the possibility of beneficial effects on GSH status.At this time, recommendations on BAscorbate, a potent free radical scavenger, and GSH are biochemically intertwined ,143. GSHRodent models have clarified the role of ascorbate in GSH metabolism. Ascorbate normalized hepatic GSH levels in homocysteinemic rats, despite the fact that serum homocysteine levels remained high . When agHealthy adults were supplemented with ascorbate to better understand the vitamin\u2019s effects on GSH homeostasis. Subjects were supplemented with a placebo for 1 week, 500 mg/kg/day for 2 weeks, 2000 mg/day for 2 weeks, then placed on a placebo-controlled withdraw week . RBC GSHAscorbate has also been combined with vitamin E since the two vitamins have an additive effect against oxidative stress . When asBased on this evidence, ascorbate, if used in safe and tolerable doses, appears to attenuate GSH depletion under stress conditions and promote an increased antioxidant status.In the enzymatic reduction of hydrogen peroxide to water, GSH is utilized by GPx, a selenoprotein that requires Se for proper functioning ,154. ResFurther research is needed to better define the impact of Se on GSH concentrations, given its proven ability to increase GPx protein levels. Although Se could be a viable option to increase GPx activity in individuals with high oxidative stress, Se supplementation may be most effective in combination with another therapeutic known to directly increase GSH. After all, without GSH, an increase in GPx may not induce significant health effects.Mg is an important mineral whose status has been linked to oxidative damage . When diBased on these clinical trials, it is possible that Mg supplementation may provide antioxidant protection. Importantly, more research is needed to identify the optimal dose of Mg supplementation to boost GSH metabolism.The Mediterranean diet (MedDiet) emphasizes high intakes of extra virgin olive oil, vegetables, fruits, cereals, nuts, pulses and legumes, and moderate intakes of fish, dairy, and red wine, while limiting intakes of eggs and sweet foods . There iEfforts to understand the relationship between the MedDiet and GSH levels should now be expanded. For instance, new efforts may be conducted in pediatric populations to identify any protective antioxidant mechanisms that may be enhanced when beginning the diet early in life.The DASH diet was developed in the 1990s and rose in popularity once the National Institutes of Health began supporting research efforts to identify dietary interventions that were efficacious against hypertension . The DASChronic disease patients often have impaired GSH statuses and heightened GSSG levels . It has Naturally, vegetarian diets exclude meat, a significant source of amino acids needed for GSH biosynthesis . Yet veg6 [A Western-type diet is characterized by high amounts of meats, saturated fat, refined grains, and sugar, with low amounts of fruits and vegetables . The Wes6 , methion6 , and cys6  status, 6 , suggest6 , so an aWhey protein is a rich source of cysteine, so research is being conducted on its relationship with GSH status. Evidence suggests that the consumption of whey protein can raise intracellular GSH levels in healthy individuals . In addiOral GSH supplementation is not the most effective means of increasing GSH status, so researchers anticipate that alternate forms of oral GSH may be more promising. Notably, food-grade sublingual and liposomal GSH supplements have been designed. Sublingual forms of GSH have received significant attention because sublingual compounds are able to by-pass hepatic first-pass metabolism and degradation, maintaining supplement bioavailability . A 3-weeTo supplement with lipoceutical GSH, GSH is first packaged into liposomes, small droplets that fuse with cellular membranes, directly releasing their contents into target cells. Recent studies have underscored the beneficial effects of liposomal GSH on intracellular GSH concentrations ,205. ForTo date, a limited number of animal and clinical interventions have evaluated the efficacy of liposomal GSH ,205,206.GSH is an exciting and promising therapeutic target against oxidative stress. Given the low bioavailability of oral GSH, researchers have supplemented with compounds that directly or indirectly affect the GSH system. The list of supplements discussed in this review is not exhaustive, and other amino acids and micronutrients, or their combinations, may have significant effects on GSH homeostasis in specific populations. It is important to ensure that interventions are tested in various age groups, in particular because pediatric populations have been consistently understudied. And it is important to consider the health status of each population under investigation, as dysfunction of the liver and other essential organs can impede the body\u2019s ability to synthesize GSH and thus, nutraceutical supplementation may not be as effective as it would be in healthy individuals. Investigators must also consider the analytical method used to quantify GSH and GSSG in each study. GSH and GSSG are often quantified via colorimetric assays , high pe"}
{"text": "Swimming in fecally contaminated recreational water may lead to gastrointestinal illness. A recreational water-associated outbreak of norovirus (NoV) infections affecting at least 100 people in The Netherlands occurred in August 2012. Questionnaire responses from patients indicated swimming in recreational lake Zeumeren as the most likely cause of illness. Most patients visited the lake during the weekend of 18\u201319 August, during which the weather was exceptionally warm (maximum temperatures 32\u201333 \u00b0C), and visitor numbers elevated. Patients, mostly children, became ill with gastroenteritis 1\u20136 days (median 2 days) after exposure. Four stool samples from patients were NoV GI positive. Subsurface sandy soil from one of the beaches where most patients swam was NoV GI positive; the water sample was negative. The epidemiological curve and the timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the source in recreational water outbreaks. A NoV outbreak in a recreational lake that is not subjected to external fecal contamination sources shows the need for active communication about human shedding of viruses during and after diarrheal episodes and the advice to refrain from swimming, even a few weeks after the symptoms have resolved. The virarticles ,3. Symptarticles ,4, but tn = 25) of the outbreaks [n = 14). In a recently published study of bathing water related outbreaks in Finland that all occurred in 2014, epidemiological and microbiological data showed that NoV was the main causative agent [NoV have been genetically classified into five genogroups (G) . GI and utbreaks . The majve agent .This paper describes the occurrence and investigation of a recreational water associated outbreak of NoV infections affecting at least 100 people in The Netherlands in August 2012.www.knmi.nl). Because of the warm weather, several thousands of people swam at Zeumeren during the weekend. Most patients became ill one to one-and-a-half day after exposure to the lake water, and had swum at two of the ten possible beaches surrounding the lake . All patients, who were mainly children, had visited the lake during the previous weekend of 18 and 19 August. During that weekend, the weather in The Netherlands was exceptionally warm with maximum temperatures of 32\u201333 \u00b0C   [Lake Zeumeren is located in a rural area (community Barneveld) in the center of The Netherlands. It is a 32-hectare isolated fresh water lake with stagnant water, with groundwater and rainwater as sources. The maximum water depth is 17 m and it has a sandy soil. Lake Zeumeren is an official bathing site, where the water quality is monitored for fecal indicator parameters (EU BWD)  with a fn = 36). The patients were asked to answer the questions and to forward the questionnaire to anyone they knew who had swum at the same site during the same period and had become ill with the same symptoms. This approach was chosen in an attempt to rapidly identify the source of the outbreak. Since there was no aim at performing a full case-control study, no matching controls were sought. By allowing questionnaires to be forwarded, no records were (or could be) kept of the number of questionnaires distributed. Apart from demographic questions about age and gender, questionnaires included information requests about having visited lake Zeumeren (date between 15 August and 24 August 2012), having entered the water (including specification of beaches visited), having swum (including duration of swimming), health conditions , using the on-site toilets, and having bought and consumed food and beverages on-site. Analysis of questionnaire responses was done by using SPSS .The province of Gelderland and the Public Health Service VGGM sent questionnaires by email on 24 August 2012 to all patients with whom they had primary contact and for whom they had kept records  and Yersinia, and NoV at the medical microbiological laboratory of the Rijnstate Hospital in Arnhem, The Netherlands, by using routine PCR. Later, the four samples were sent to the National Institute for Public Health and the Environment (RIVM) for further testing. At RIVM, the samples were analyzed for the presence of NoV by using a real-time RT-PCR assay, with primers, probes and PCR conditions as previously described [Five patients were asked to send in stool samples; samples from four patients were received and analyzed for the bacterial enteropathogens escribed .E. coli and intestinal enterococci, by using the most probable number methods specified in the EU BWD [In order to narrow down the outbreak and to identify the source, several leads were followed. In response to the reported cases of gastroenteritis, water samples were taken from lake Zeumeren at the two official beaches  on 20 Aue EU BWD . Although lake Zeumeren does not have a history of problems with cyanobacteria, the presence of a faint greenish colouring of the water led to visual inspection for the presence of cyanobacteria and sampling of the water at beach 4  for deteCampylobacter jejuni infection that was treated with antibiotics. Although the woman did not meet the case definition, it was decided to follow this lead because of the known nuisance caused by gulls at lake Zeumeren that take over the beaches after the bathers have left. Water samples were taken at the two official beaches on 23 August and analyzed for the presence of Campylobacter spp. by using a membrane filtration method with incubation on Karmali Agar at 42 \u00b1 0.5 \u00b0C for 48 \u00b1 2 h . On Wednesday, 22 August, a woman reported to the province of Gelderland having fallen ill after swimming in lake Zeumeren during the weekend prior to the outbreak weekend (11\u201312 August). She attended a physician and had blood, urine and feces examined, which revealed a When the four analyzed stool samples appeared to be NoV positive, RIVM was contacted on 30 August and asked to take environmental samples for NoV analysis at lake Zeumeren. Sampling was done on 3 September 2012. Water and subsurface sandy soil samples were only taken from the beaches where most of the patients had stayed or swum during the weekend of 18 and 19 August as was pointed out by the questionnaire results. One water sample was taken at sampling point A, and one subsurface sandy soil sample was taken at each of the sampling points A\u2013C .The water sample (600 L) was taken by using an on-site filtration adsorption-elution procedure for concentration of the sample ,21. The Subsurface sandy soil samples of approximately 50 g were taken by using a tubular soil sampler. Subsequently, 10 mL 0.05 M glycine buffer pH 9.0 was added to 5.0 g of each sandy soil sample and mixed at 500 rpm for 35 min at 4\u00b0C, after which it was allowed to settle for 10 min (in house method RIVM). RNA extraction was done according to Rutjes et al. , by addiA real time reverse transcriptase PCR (RT-PCR) assay as described by Verhaelen et al.  was perfn = 25) were female. The age of the respondents ranged between 0 and 59 years of age, with more than half being children of 0\u201312 years of age , while the others were adults of 18\u201359 years of age . All respondents visited lake Zeumeren between 15 and 19 August, but the majority were at the site on 18 August  and/or 19 August . In total, 27 cases were exposed on more than one day during the specified period. All cases went into the water for swimming and became ill afterwards, with health complaints starting 1\u20136 days (average and median 2 days) after exposure to the bathing water and beaches at Lake Zeumeren and lasting for 0.5\u20136 days (average and median 2 days). Reported health conditions were : diarrhea , vomiting , fatigue , and fever . None of the cases reported skin conditions, and eight cases reported other complaints than those indicated above, of which seven were related to gastro-intestinal discomfort. One case reported a headache.Public Health Service VGGM received 45 filled-in questionnaires that met the case definition. Of the respondents, 56% (n = 31) and/or beach 2 . Most cases  were in the water for 30 min to over an hour.Forty-four of the cases indicated at which beach they had swum , showing that most cases swam at beach 1  of the cases used the permanent on-site toilets, the others did not. Buying and consuming on-site sold food and beverages were not very common: 87% (n = 39) did not buy or consume food and 98% (n = 44) did not buy or consume beverages.Fifty-eight percent , and 275 overlapping with the typing region of ORF2. Genotype assignment by phylogenetic analysis was supported by 100% and 99% bootstrap analysis, for ORF1 and ORF2, respectively. Typing from RT-PCR products is reliable if the fragment has >100 nucleotides overlapping with both of the typing regions of ORF1 and ORF2.E. coli at the official beaches of 16 most probable number (mpn)/100 mL at beach 1\u20132 and 6 mpn/100 mL at beach 3. Compared to previous and later routine samplings during the same bathing season, slightly elevated levels of intestinal enterococci at both beaches, 230 mpn/100 mL and 160 mpn/100 mL, respectively, were observed.Water samples taken on 20 August 2012 showed levels of Microcystis spp. at beach 4; intracellular microcystin levels were very low (<2 \u00b5g/L).Examination of the samples taken for cyanobacteria analyses by microscopy demonstrated the presence of Campylobacter spp. were present in the water samples taken at the official beaches (one sample per beach) on 23 August. Numbers appeared to be low, but could not be exactly determined due to overgrowth of the culture medium. Further identification to the species level was not done.NoV GI was detected in the subsurface sandy soil sample taken at sampling point A (beach 1). The other subsurface sandy soil samples and the water sample were NoV negative. Sequencing of the RT-PCR product from the subsurface sandy soil sample was impossible because the method for typing of RT-PCR products is less sensitive than the detection method, and the amount of RNA in the subsurface sandy soil sample appeared to be too low.Campylobacter infection in combination with the presence of a large seagull population resulted in choosing, what later appeared to be, the wrong direction. Following several leads and having various patient and environmental samples examined takes time, and in this case, led to the examination of water and subsurface sandy soil samples only over two weeks after the outbreak presumably started, thus diminishing the chances of detecting the etiological agent. Due to of the time gap between the onset of the outbreak and the environmental sampling, subsurface sandy soil sampling was performed in addition to the normal procedure of water sampling because sedimentation of virus particles during the delay period could have resulted in the presence of virus particles in the subsurface sandy soil [Following the reporting of many cases of gastroenteritis that seemed related to exposure to a recreational lake in The Netherlands during an exceptionally warm weekend in August 2012, several leads were investigated in order to discover the etiological agent and the source of the outbreak. Although the epidemiological investigation implicated the recreational lake as the most likely location of contracting illness, the different directions in which the investigation went, based on reported symptoms, demonstrate the difficulty in narrowing down the possible cause of recreational water related outbreaks. The results of the examination of patient material can ease the search for the etiological agent and the source; however, in this case, the reporting of a patient with a ndy soil . In thisndy soil . The detndy soil ,24, possndy soil . In a sundy soil , thus deThe epidemiological investigation showed that most of the patients were children. It has been demonstrated that, while swimming, compared to adults, children spend more time in the water and ingest a larger volume of water, thus increasing their exposure ,27 and rThe detection of the same NoV genogroup in subsurface sandy soil and in patients two weeks after the onset of the outbreak shows that NoV may be present in the environment for prolonged time through accumulation in sediment , and mayAn outbreak of gastrointestinal illness among over 100 visitors of a recreational lake in The Netherlands during an exceptionally warm weekend was caused by NoV. The results of the epidemiological investigation, combined with the detection of NoV GI in the stool samples of outbreak related patients and in the subsurface sandy soil of the beach where most patients swam, suggest that exposure to the recreational lake resulted in the outbreak. Although the primary contamination source has not been discovered, the most likely cause of contamination of the water is an infected human, because the lake is an isolated water body that is not subject to external sources of fecal contamination. An outbreak like this warrants active communication about human shedding of viruses during and after diarrheal episodes and the advice to refrain from swimming. The epidemiological curve and the timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the source in recreational water outbreaks."}
{"text": "The reliable prediction of Cu(II) hyperfine coupling constants remains a challenge for quantum chemistry. Until recently only density functional theory (DFT) could target this property for systems of realistic size. However, wave function based methods become increasingly applicable. In the present work, we define a large set of Cu(II) complexes with experimentally known hyperfine coupling constants and use it to investigate the performance of modern quantum chemical methods for the prediction of this challenging spectroscopic parameter. DFT methods are evaluated against orbital\u2010optimized second\u2010order M\u00f8ller\u2010Plesset (OO\u2010MP2) theory and coupled cluster calculations including singles and doubles excitations, driven by the domain\u2010based local pair natural orbital approach (DLPNO\u2010CCSD). Special attention is paid to the definition of a basis set that converges adequately toward the basis set limit for the given property for all methods considered in this study, and a specifically optimized basis set is proposed for this purpose. The results suggest that wave function based methods can supplant but do not outcompete DFT for the calculation of Cu(II) hyperfine coupling constants. Mainstream hybrid functionals such as B3PW91 remain on average the best choice. Modern correlated wave function methods (DLPNO\u2010CCSD and OO\u2010MP2) contend against density functional theory for the prediction of Cu(II) hyperfine coupling constants in a test set of realistic copper complexes. It is based on the primitives of the original 1994 Ahlrichs [8s5p3d] VTZ basis sets functions, addition of three tight and closely spaced s primitives obtained from the innermost exponent by successive multiplications with a factor of 2.5, and finally the addition of two p and one f polarization functions. The aug\u2010cc\u2010pVTZ\u2010J basis set of Hedeg\u00e5rd et\u2005al.s, one p, and one d functions. Its fully decontracted version, denoted aug\u2010cc\u2010pVTZ\u2010Junc, corresponds to a [25s18p10d3f2g] set with a total of 168 primitive functions ; this is the largest and most flexible basis set of the present study, and is therefore used as the reference for all others.Two types of basis sets proposed as appropriate for the calculation of core properties include CP(PPP), originally engineered for the calculation of M\u00f6ssbauer parameters,3)4]2+, obtained with the PBE0 functional. It is worth noting that all results discussed below have been confirmed for two other complexes ([Cu(dtc)2] and [Cu(acac)2]) using two additional functionals (TPSSh and B3PW91) and are reported in the Supporting Information . Several points become immediately apparent from Table\u2005s functions of the basis set. The aug\u2010cc\u2010pVTZ\u2010J (or Junc) and CP(PPP) basis sets are in close agreement with respect to AFC which is due to an important core\u2010shell spin polarization contribution of negative sign , a deficiency likely inherited from the parent Ahlrichs VTZ basis set.Table\u20052] and [Cu(acac)2] complexes when using the PBE0, B3PW91 and TPSSh functionals, as reported in the Supporting Information.The above results advocate the use of the purpose\u2010made aug\u2010cc\u2010pVTZ\u2010J basis set for the calculation of Cu HFCs, since it produces results indistinguishable from its fully decontracted version. Similar conclusions can be drawn from the results obtained with the and \u0394A representing the anisotropic difference (33\u2010A11A). The APDs are evaluated in the following manner:To assess the performance of the basis sets, the absolute percent deviations (APD) are evaluated with respect to experimental results for three relevant parameters: 3)4]2+[Cu4]2+ using the DKH2 Hamiltonian and appropriate corresponding basis sets. Here again aug\u2010cc\u2010pVTZ\u2010Junc serves as the reference. DKH\u2010adapted versions of the standard cc\u2010type basis sets similarly fail for the AFC term, but the DKH\u2010adapted versions and p shells. CP(PPP) can be considered adequate for the AFC term but it shows the same type of deviation as seen in the non\u2010relativistic calculations for both ASD and ASO components. Importantly, the contracted aug\u2010cc\u2010pVTZ\u2010J version deviates from the reference for the crucial Fermi contact term showing that it suffers from the contraction of s functions, whereas the ASD and ASO terms are practically the same as the reference values.An additional point concerns the robustness of the basis set with respect to scalar relativistic effects, which result in spatial contraction of 3)4]2+, employing the PBE0 and B3PW91 functionals (Tables\u2005S22\u2013S23). Applying the finite nucleus model has a very limited effect on the computed HFCs. Regardless of the functional used, the DK version of the cc basis sets still underperform and the aug\u2010cc\u2010pVTZ\u2010J keeps deviating from the \u2010Junc analogue while predicted HFCs with both CP(PPP) and DKH\u2010def2\u2010TZVP are close to those obtained with the reference. Overall, the finite nucleus model has a very limited effect on the calculated HFCs and we recover the main trends obtained without using it.The finite nucleus model available with the ORCA program package was applied with the DKH2 Hamiltonian for 2+ confirmed the expectation that aug\u2010cc\u2010pVTZ\u2010J and \u2010Junc recover massively more correlation energy than the CP(PPP), in line with the absence of appropriate correlation functions in the latter. All the above observations taken together would seem to suggest that the reference \u2010Junc basis set would be the safest choice, however this would be computationally cumbersome. Moreover, it is clear from Tables\u2005s is unnecessary. This led us to investigate modifications of the \u2010J basis sets that bring the results as close as possible to the reference while constraining the computational cost. The decontraction of s functions assures converged AFC values both in non\u2010relativistic and scalar relativistic calculations. However, close inspection of the Cu 1s orbital revealed that the three innermost s primitives remain practically unused and have near\u2010zero coefficients. The only condition under which we saw these functions making a noticeable, yet not significant, difference in HFC results was in DKH2 calculations that made use of the finite nucleus model, an approach that will not be further explored here. For the present purposes we therefore define a modified version of aug\u2010cc\u2010pVTZ\u2010J with decontracted s functions and removal of the three innermost s primitives  as a computationally optimal basis set for the calculation of Cu HFCs that is applicable to all methods considered in the present study.A further consideration in the context of the present study is how appropriate a basis set is for use with correlated wave function methods. Simple tests with MP2 and CCSD calculations on [CuNH4]2+ conf2.3In this section we compare the performances of density functional methods to predict reliable and accurate Cu hyperfine coupling constants. For that purpose, we exclusively employ our modified basis set, aug\u2010cc\u2010pVTZ\u2010Jmod, and conduct calculations on the complete set of copper complexes. Diverse density functionals are considered such as: GGA (PBE), hybrid  and range\u2010separated hybrid (CAM\u2010B3LYP); meta\u2010GGA  and hybrid meta\u2010GGA ; and double hybrid (B2PLYP). As shown in the previous section, no scalar relativity corrections need to be included in the calculations as they were proven to make no difference on average. On a more technical note, we observed that hybrid functionals are sensitive to the method used for calculating the spin\u2010orbit contribution. When the second flag of the SOC operator was set to compute the Coulomb term with the RI approximation, the calculated HFCs deviated remarkably from the experimental values. Setting this parameter to compute the Coulomb term exactly leads to good agreement between calculated and experimental results. When this flag was set to default (this study), the Coulomb term was computed semi\u2010numerically and the obtained hyperfine values are practically indistinguishable from those obtained by exact computation of the Coulomb term.3)4]2+. This is not the case for both GGA (PBE), meta\u2010GGA  and range\u2010separated (CAM\u2010B3LYP) functionals which deviate from experiment. While PBE and TPSS indeed provide low isoA term due to the underestimation of the core\u2010shell spin polarization contributions, the case of CAM\u2010B3LYP is quite different as it suffers from an incorrect description of the spin\u2010orbit contribution with underestimation from both pseudo\u2010contact and second\u2010order dipolar terms. The Minnesota functionals (M06 and M062X) do not reproduce the experimental values at all and behave quite erratically as no trend can be extracted from the predicted individual contributions. These observations are in line with previous studies[isoA 4]2+. For this parameter, the meta\u2010GGA functionals are also competitive with an average of 8\u2009% deviation. As previously mentioned, the other functionals are of poor quality for the prediction of 33A. Interestingly, similar trends are observed for the average value of the HFC, isoA, where deviations do increase to about 15\u2009% for the  group of functionals. Finally, the anisotropic parameter, \u0394A, is also in good agreement with experiment with an average of 11\u2009% deviation when looking at the hybrid functionals. The performances of the hybrid meta\u2010GGA, TPSSh, remain correct for both isotropic, isoA, and anisotropic, \u0394A, parameters. The worst performing functionals are M06 and M062X while the GGA and meta\u2010GGA functionals underperform compared to their hybrid counterpart.At this first glance, hybrid functionals reproduce very well the magnitude of the largest hyperfine components, For a complete evaluation of the performances of each functional, the calculations were subsequently performed for the complete set of 20 complexes (Tables\u2005S27\u2013S45) and the mean of each absolute percent deviation (MAPD) reported in Table\u2005From these mean absolute percent deviations, we can evaluate the ability of a given functional to predict reliable and accurate hyperfine coupling constants. Using our aug\u2010cc\u2010pVTZ\u2010Jmod basis sets, we obtain the following trend: B3PW91<TPSSh<B3LYP<PBE0<TPSS<PBE<CAM\u2010B3LYP<SCAN<M062X<M06, with B3PW91 having the lowest deviation from experiment and M06 the largest. Table\u20053)4]2+, Table\u2005To finalize the comparison of DFT methods, we included the double hybrid functional B2PLYP to our study and computed the HFCs for the complete set of complexes. This functional was first evaluated using the mean absolute percent deviations for the Fermi contact term and the three components of the spin dipolar contribution with respect to the ones obtained using the B3PW91 functional and the aug\u2010cc\u2010pVTZ\u2010Jmod basis set. In the specific case of 2+, the HFCs obtained with B2PLYP using the SOC contributions from B3PW91 are well above an acceptable error range over the complete set of complexes (Table\u2005S47). Table\u200533A, isoA and \u0394A making B2PLYP an unfit DFT method to predict Cu hyperfine coupling constants.While it seems promising for the case of [Cu(NH2.4nZ) series of basis sets. However, neither the standard basis sets identified in the benchmark DFT calculations as potential candidates for the calculation of copper hyperfine coupling constants, nor our final choice defined above (aug\u2010cc\u2010pVTZ\u2010Jmod) have dedicated auxiliary basis sets. It is noted that for the aug\u2010cc\u2010pVTZ\u2010J basis set it appears that the standard aug\u2010cc\u2010pVTZ/C is sufficient, both in non\u2010relativistic and DKH2 scalar relativistic calculations. In comparison, the aug\u2010cc\u2010pwCVTZ/C and the larger aug\u2010cc\u2010pVQZ/C auxiliary basis perform less well, demonstrating the low tolerances involved in adapting a correlation fitting basis set to a specific size and type of main basis. It is also noted that the def2/C basis sets are inadequate when either the aug\u2010cc\u2010pVTZ\u2010J or the smaller CP(PPP) were used as main basis sets for Cu. Modifications of standard basis sets to make them more appropriate for aug\u2010cc\u2010pVTZ\u2010Jmod did not show a systematic trend, therefore we opted instead for the automatic generation procedure encoded in ORCA,Most of the correlated wave function calculations required an auxiliary basis set for the resolution of the identity approximations that are used either optionally or by default, as for example in the OO methods and in DLPNO\u2010CCSD. Standard main basis sets often have custom\u2010made auxiliary bases for correlation fitting, denoted as /C. This is the case for the def2 family and the correlation consistent  hyperfine coupling constants.In this work we evaluated the performance of density functional theory and modern wave function based methods for the prediction of Cu hyperfine coupling constants using a large set of 20 complexes with experimentally known structures and resolved hyperfine coupling tensors. We defined a computationally optimal basis set for Cu, the aug\u2010cc\u2010pVTZ\u2010Jmod basis set, a modified version of aug\u2010cc\u2010pVTZ\u2010J with decontracted The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re\u2010organized for online delivery, but are not copy\u2010edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file."}
{"text": "Resilience contributes to the recovery of disaster victims. The resilience of Tibetan adolescents after the Yushu earthquake has not been properly studied. This study aimed to examine the current resilience and associated factors in Tibetan adolescent survivors in the hardest-hit area 5 years after the Yushu earthquake. This cross-sectional survey was conducted in the area hit the hardest by the Yushu earthquake. Data were collected from 4681 respondents in October and November 2015. Measurements included the participant characteristics, traumatic earthquake experience, the Connor-Davidson resilience scale (CD-RISC), and the social support appraisals (SS-A) scale. The individual datasets were randomized as 80% for the training set and 20% for the validation set. The mean resilience score of the Tibetan adolescent survivors was 55.0\u00b112.3. Thirteen variables were entered into the regression equation. The three dimensions of social support  were positively associated with resilience , among which support from others than family/friends was the strongest . Academic performance, activeness of participation in school activities, harmonious relationship with teachers/classmates, health over the last year, and regular physical exercise were positively associated with resilience . Being female and being extremely worried about their own lives were negatively associated with resilience (both P<0.05). In conclusion, among Tibetan adolescent survivors to the Yushu earthquake of 2010, support from others than family/friends was the strongest positive factor associated with resilience, while being female and extreme worry about their own lives were negative factors. These results expand our knowledge regarding resilience in Tibetan adolescent disaster survivors. China is an earthquake-prone country. From 2004 to 2018, 191 earthquakes occurred in China, of which 37 were above magnitude 6.0. Together they were responsible for 73,176 deaths and 486,231 injured . ResilieThe factors influencing resilience are usually divided into risk and protective factors . Those fIt is known that child, and adolescent survivors are a psychological stress-susceptible population to external adverse events. Xiao et al. ,34 showeThe Yushu earthquake area is situated in the relatively remote and undeveloped plateau mountainous area in western China, where the Tibetan culture is predominant. Five years after the earthquake, the current state of resilience among Tibetan adolescent survivors, given their unique geographical, cultural, social, and economic environment, is unknown, and the factors that have affected their resilience are still unidentified. Therefore, the aim of the present study was to examine the current resilience status and identify the associated factors of resilience in Tibetan adolescent survivors in the hardest-hit area 5 years after the Yushu earthquake. The results might provide useful insights for the post-disaster management and counseling of adolescents. Eventually, such data could allow better tailoring of the counseling to each individual.This was a cross-sectional study. The participants were recruited from four secondary schools (grades 7\u201312) in Gyegu town in October and November 2015. Gyegu town was almost completely destroyed by the earthquake. All participants met the following inclusion criteria: 1) Tibetan; 2) personally experienced the 2010 Yushu earthquake; 3) secondary school student of 12\u201318 years of age; and 4) willing, along with his/her guardians, to sign the written consent form for voluntary participation. Individuals with cognitive impairment or problems with hearing, verbal, and/or written communication were excluded. Ethical approval was provided by the Human Subjects Ethics Sub-committee of Sichuan University.In theory, adolescence is the transitional period between childhood and adulthood, usually between the ages of 12 and 22. In China, much of the research on Chinese adolescents has focused on the traditional middle school years, which are 12 to 18 years old, according to China's legal school age. Anyone over 18 is considered an adult. The main social place of Chinese middle school students is school, and their main social role is to study, and they have little contact with society. Those characteristics change dramatically after middle school since they begin working or go to university, which is considered in China as adult age. Therefore, the age of adolescents in this study is defined as middle school students aged 12\u201318 years; this definition is also used in other studies from China \u201340.The survey was conducted approximately five years after the disaster. Four parts were included: demographic and injury-related data, traumatic earthquake experience, Connor-Davidson resilience scale (CD-RISC), and Social support appraisals (SS-A) scale. The questionnaire was administered in Chinese, as Chinese is the language of instruction in schools in the Yushu area. Prior to the survey, we gained the permission and assistance of the local education management departments, as well as of the principals and class teachers of the schools. The participants were asked to complete a self-administered questionnaire in their classroom with the assistance of the researchers, who were two doctoral students and a psychology professional. All three researchers had received training for data collection . Researchers collected the questionnaires after checking each one for completeness. If a questionnaire was incomplete because of negligence, it was returned to the participant to be completed.Prior to the investigation, we explained the study purpose to the participants, assured them of the confidentiality of their personal data and the contents of the questionnaire, and obtained the consent of the participants and legal guardians. In the course of the investigation, the right of withdrawal was fully respected, and attention was given to the protection of the privacy of the participants during the entire research process. In each school, there were teachers and psychological professionals responsible for dealing with the psychological discomfort that the study may have caused. Non-research team members were not permitted to access the original data. In place of each participant\u2019s name, a code was used for data entry and analysis.Data included age, sex, single-child status, left-behind child , parental education level, relationship with family members, family financial conditions, living conditions, activeness of participation in school activities, academic performance (according to the results of the school\u2019s last academic year), academic stress, relationship with classmates, relationship with teachers, health over the last year, whether they undertook regular physical exercise , whether they had experienced stressful events , and whether they had received psychological counseling.Each participant\u2019s experience of the traumatic earthquake was assessed using twelve items: whether they were buried in the earthquake; whether they were injured/disabled in the earthquake; whether they had family members/classmates/teachers who died in the earthquake; whether they experienced severe property loss; whether they witnessed someone else being buried/injured or dying; whether they were extremely worried about their own lives; whether they were extremely worried about the health of family and friends; and whether they felt guilty because someone else was killed or injured in the earthquake.The CD-RISC was used to assess the level of individual resilience. The scale was developed by Connor and Davidson  and has Social support was evaluated using the SS-A scale developed by Vaux et al.  and reviStatistical analysis was performed using R software version 3.5.1 and SPSS 19 . The level of significance was set at 0.05 (two-tailed) for all statistical inferences.Participant characteristics and traumatic earthquake experiences were expressed as numbers (%). CD-RISC scores and SS-A scores were expressed as means and standard deviations. Pearson\u2019s correlations were calculated to evaluate the potential relationships between the CD-RISC and SS-A subscales.in = 0.05, \u03b1out = 0.10) was used to build a prediction model for resilience by incorporating the features selected in the LASSO regression model. Finally, the linear regression model was validated in the validation data set.The data were randomly divided into two parts, 80% as the training set and 20% as the verification set. First, using data from the participants included in the training set, the least absolute shrinkage and selection operator (LASSO) method was used to select the optimal associated features among the potential factors of resilience in Tibetan adolescent survivors. Features with non-zero coefficients in the LASSO regression model were selected . Second,A total of 5508 participants met the inclusion criteria, and 565 individuals from the four participating schools declined to participate. A total of 4943 questionnaires were administered, and 4681 (94.7%) valid questionnaires were collected. The mean age of the participants was 15.7\u00b11.8 years, and 2466 (52.7%) were female. Among the participants, 6.9% were burried, 14.6% were injured, 0.9% were disabled, 17.4% had family members who died, 33.9% had classmates or teachers who died, 42.9% experienced severe property loss, 36.9% witnessed someone else being buried, 62.9% witnessed someone being injured, 44.7% witnessed someone dying, 64.2% were extremely worried about their own life, 84.8% were extremely worried about the health of their family and friends, and 80.0% felt guilty because someone else was killed or injured. The participant characteristics and traumatic earthquake experience are shown in Tables The mean CD-RISC score was 55.0\u00b112.3 . Tables Of the participant characteristics, earthquake traumatic experience, and social support, 33 features were reduced to 21 potential factors on the basis of the training set  with non2 was 0.2731 (p<0.001).Resilience contributes to the recovery of disaster victims. The resilience of Tibetan adolescents after the Yushu earthquake of 2010 has not been properly studied. This cross-sectional survey aimed to examine the current resilience and associated factors in Tibetan adolescent survivors in the hardest-hit area 5 years after the Yushu earthquake. The results suggest that among Tibetan adolescent survivors to the Yushu earthquake of 2010, support from others than family/friends was the strongest positive factor associated with resilience while being female and extreme worry about their own lives were negative factors. These results expand our knowledge regarding resilience in Tibetan adolescent survivors.Tibetans enjoy good economic policies, so many adults work in their hometowns, and there are few left-behind children. Tibetans also did not implement the one-child policy, so there are fewer only children. Because the Yushu Tibetan area is located in a remote mountainous area, the education received by the older generations was mainly family education and temple education. In this paper, many of the respondents are the first generation of students in their families. Therefore, most of their parents' education background is primary school or below. In the univariable analysis, the scores of mental resilience of parents with college or above degrees were higher than those of parents with other educational levels, but the educational levels of parents did not enter the final regression equation.In the present study, the CD-RISC score (55.0) of Tibetan adolescent survivors was lower than that (69.6) found in a study by Yu et al. , who useBeing female was found to be a negative factor for resilience in the present study, as supported by Yu et al.  and StraGood health and performing regular physical exercise were found to be positive factors associated with resilience in our study, as supported by previous studies ,54. ReguThe positive relationship between academic performance and resilience found in this study is consistent with findings from de la Fuente et al. . To a ceThe item \u2018activeness of participation in school activities\u2019 was also an important factor for resilience. Of course, social life and school systems between China and the Western countries are very different. The main social place of Chinese middle school students is school, second only to home, their main social role is to study, and they have little contact with society beyond their fellow students, the teachers, and the school staff. Therefore, it might be considered that Chinese adolescents participating in school activities is like engaging in normal activities in a Western context. Returning to normal activities is seen as a way of being resilient . In addiIn the present study, all the measured dimensions of social support were positively associated with resilience, consistent with previous studies ,68. If pWe found that different earthquake traumatic experiences had different effects on resilience. Notably, \u2018feeling guilty about someone else being killed or injured\u2019 and \u2018extreme worry about the health of family and friends\u2019 were both positive factors of resilience for Tibetan adolescent survivors, while \u2018extreme worry about their own lives\u2019 was a negative factor. The three items were subjective responses to the earthquake and provided a reminder that the subjective responses of individuals to disasters are closely related to mental health, as supported by previous studies ,71. It iThis study has several limitations. First, the target population of this study included all the Tibetan secondary school students in Gyegu town who experienced the 2010 Yushu earthquake. Unfortunately, about 1200 potential participants were not included in this study because of the refusal of the principal of one junior high school. Although the general information of students in this school is not obviously different from that of other schools, according to the educational administration department, it might still lead to a bias. Second, the limitation of the cross-sectional study design makes it impossible to determine the causal relationship between variables, as well as the evolution of resilience in time. In addition, no data from before the earthquake are available. Third, the nature of quantitative research makes it difficult to understand how those factors influence the development of Tibetan adolescent survivors\u2019 resilience. Further qualitative research is necessary to explore this. Fourth, due to the specific characteristics of the Chinese culture, social system, and schools, the international generalizability of the conclusions is currently limited. Similar studies should be carried out in various countries. This would provide interesting results about the similarities and differences among different countries/cultures. Fifth, because of the logistics, legal, and ethical complexity of studies dealing with the psychological/psychiatric health of children, the present study did not examine the effect of counseling or psychiatric assessment after the trauma. This is planned for another study. Finally, the Tibetan Buddhist religious was not included in the study because religion is not talked about in school, but the questionnaire was complete and the spiritual item was included. Despite these limitations, this study revealed the associated independent factors of Tibetan adolescent survivors\u2019 resilience. As support from others than family/friends was found to be the most significant associated factor of the participants, and school-level items  were also found to be the associated independent factors, school-based, teacher-mediated intervention programs might be suitable and feasible to improve adolescents' resilience after a natural disaster. The results provide an empirical basis for the selection of the target population for intervention. Future studies should attempt to design, implement, and evaluate training programs for people with relatively low resilience. This study also showed that different disaster experiences had different effects on resilience, and subjective responses of individuals to disasters were independent influencing factors, especially \"extreme worry about their own lives\". In the process of post-disaster psychological aid, both the screening of negative psychological problems and the selection of psychological intervention populations should consider the subjective components of the earthquake trauma experience, especially those with extreme worry about their own lives should be paid more attention.Some of the included students were about 7 years of age when the earthquake struck, and they might have a different perception than those who were already adolescents at that time. During the study, we could observe that after the early fear and sadness, these children showed more gratitude, treasure in life, and hope for the future. They are generally more grateful for others' help, and they also want to help others in the future. They cherish their homes and families now. Although the earthquake brought great trauma to Yushu, it also brought in a new life. The construction assistance from all over the country not only made the whole city look brand-new but also increased communication with the outside world. Now Yushu is no longer a remote and backward town in western China, but a famous tourist city in China. The improvement of living conditions and economic conditions increased the hope and expectation for the future in those children.In conclusion, the findings provide insight into the context-specific factors associated with the levels of resilience among Tibetan adolescent survivors. The results suggest that their resilience needs to be improved despite the fact that many years have passed since the earthquake. This empirical knowledge base can be used to design post-disaster interventions and deploy much-needed resources and services to boost Tibetan adolescent survivors\u2019 resilience. More support should be directed particularly toward females and individuals who experienced the trauma of the earthquake and reported \u2018being extremely worried about their own lives\u2019.S1 Checklist(DOCX)Click here for additional data file.S2 Checklist(DOCX)Click here for additional data file."}
{"text": "Together for Healthy and Successful Schools Initiative (THSS).The benefits of youth engagement are well documented. In this paper, we examine youth engagement in America's Promise Alliance's Every School Healthy initiative, a part of the Robert Wood Johnson Foundation's Six community acceleration sites were selected through a competitive grant\u2010making process. Sites were required to describe youth engagement strategies. A case study design was employed to examine how sites conceptualize youth engagement as well as youth engagement strategies employed across 6 sites. Data sources included observations, team member debriefs, and document review.youth engagement and youth voice are conceptualized in educational settings, and readiness for youth engagement. Sites actively solicited and implemented youth engagement resources and strategies.There was variation in how By failing to engage young people, well\u2010intentioned adults miss important opportunities. Youth engagement presents an exciting opportunity for school leaders, policymakers, and program planners to gain a deeper understanding of the factors that influence individual and community health and wellbeing and, in turn, helps them to develop responsive policies and programs. Youth participation has been characterized in the literature as the process through which young people engage with and influence the organizations, institutions, and systems that impact their lives.Every School Healthy (ESH) initiative. ESH, by design, was intended to center young people employing the Whole School Whole Community Whole Child model (WSCC)In this paper, we explore the process of infusing youth participation in school\u2010community partnerships to accelerate school and community action to advance health through an illustrative case study of the America's Promise Alliance, A brief background on youth participation and voice is provided followed by an overview of the ESH initiative. The case study methodology employed is outlined and the results are presented and then discussed in the context of the extant literature. We conclude by discussing the limitations as well as the youth participation implications for school health.Youth participation in schools has been defined as a partnership or collaboration between students and diverse adult stakeholders in the school setting.youth voice in the ESH initiative in addition to community stakeholder engagement. Youth participation has been referred in the education literature as student or youth voice. Like youth participation and engagement, it is not consistently defined in practice. For example, it is not merely the collection of youth priorities or opinions by adults. More so, it refers to meaningful engagement in decision\u2010making and goal\u2010setting that leads to changes in school programs and policies that reflect youth priorities.Given the benefits of the youth participation and its alignment with the mission and values of America's Promise Alliance (APA), the organization sought to infuse In 2017, with funding from the Robert Wood Johnson Foundation, APA launched ESH. ESH is a multi\u2010year, multisector initiative that was designed to catalyze a national movement advancing school health. The initiative included 3 primary objectives and was grounded in the notion that all schools can be healthy schools. The first objective was focused on local action to advance health equity. The team recognized solutions to pronounced inequities in both health and education demand investment in local knowledge and decision\u2010making. As such, 6 unique school\u2010community partnerships from across the country were selected through a competitive application process. The second objective was to promote bidirectional capacity building and cross\u2010site learning, whereby practitioners from local sites as well as youth and partners, researchers, and school leaders were learning from one another\u2014both informing and adopting best practices. The learning laboratory, as it was developed, was then extended across sites to form a multisite learning community. Finally, broad dissemination was employed to further contextualize and amplify learnings. Real\u2010time sharing occurred with APA partners across the nation through webinar and local dialogues as well as, traditional earned media, listservs, and social media. At the center of this initiative were young people, students. As part of the application, process sites were asked to describe their plans for ensuring youth voice in their efforts. The level of youth engagement at each site was assessed. Using instrumental case study design, we describe the ranking process, and explore how youth engagement was conceptualized across sites. Youth engagement strategies and perceived technical assistance needs related to youth engagement are also examined. What emerged was a story about the promises and challenges associated with youth engagement as well as the need to explore organizational readiness for youth engagement.Instrumental case study design was employed to qualitatively examine how ESH sites conceptualize youth engagement as well as youth engagement strategies employed across 6 sites.The overall goal of observations was to explore youth engagement strategies as well as conceptualization of youth engagement and perceived technical assistance needs. Observations were planned and conducted by the youth engagement specialist at in\u2010person site visits, in addition, conference calls and remote meetings were observed. Comprehensive notes were taken during and at the culmination of each observation. Observation notes were typed and then analyzed thematically.Similar to the observations, document review was designed to explore how sites conceptualized youth engagement as well as strategies and technical assistant requests. Project documents included: ESH program descriptions developed by APA, applications and applicant narratives, evaluation reports, and learning agenda reports and grant reports from sites as well as materials released via websites, newsletters, and social media.Finally, themes were captured from debriefs with APA staff over the course of the project. In addition, debriefs with sites about their youth engagement technical assistance needs were conducted. The data collection and analysis, were continuous and emergent themes were shared with APA staff over the course of the initiative. Similarly, site\u2010specific themes were shared with sites after meetings and observations.Two types of data analysis were employed, holistic analysis of the whole case and embedded analysis of specific parts of the case.Each document was read multiple times to search for meaningful patterns,How youth engagement was assessed across the site application process is presented accompanied by youth engagement levels. We then highlight the approach to technical assistance strategies used by APA and its core partner organizations as well as youth engagement activities employed across the selected sites. The section concludes with an overview of the challenges of youth engagement as described by sites.Six acceleration sites were selected from a total of 138 eligible applications from 20 states. Figure\u00a0\u2026the campaign [initiative] will meet communities where they are when it comes to youth engagement. Some communities are ready for and have a need for infusing youth voice into their discussions, others want and need to have youth at their decision\u2010making table, and still others want to engage youth in pursuing inquiries that are needed for making decisions. [APA document]Youth engagement was scored separately from the overall application and did not impact the overall scoring. According to project documents, opportunities for youth involvement at both the national and local levels were intentionally built in. The goal was to work with sites to build their capacity for youth engagement throughout the process:Catalyze shifts in school, district, and broader community practices and policies that incorporate more youth voice in decision\u2010making processes and support youth as agents of change for healthy environments\u2026 [APA document]Increased knowledge, attitudes, and beliefs of direct stakeholders  of the factors that contribute to a healthy school environment and the key levers for systems\u2010level change. [APA document]The hope was that focusing on meaningful youth engagement would:According to project notes, the level of youth engagement was ranked for each application using content analysis and scoring. The content was coded based on the nature of the activities using the levels described in Table\u00a0The review process involved scanning application content to determine the presence of youth engagement activities described in the criteria outlined in Table\u00a0Of the 138 sites, 16 finalists were selected. Youth engagement scores varied among the 16 sites. The mean score was 1.6. Half of the 16 (50%), scored 1, 37.5% scored 2, and 12.5% scored 3. The APA team and core partners met to narrow the 16 applications to 6. All applicants were discussed and finalists were selected using a consensus decision\u2010making process with the group, that involved dot polling followed by group discussion. The process continued until the pool was narrowed to 6.How will youth be engaged in the decision\u2010making process?Will [youth] teams only provide feedback or will they be encouraged to lead the way?How will you ensure those [youth] most impacted will be at the [decision\u2010making] table? [review documents]The 6 finalists participated in telephone interviews with APA staff. Sites were asked a number of clarification questions associated with their youth engagement plans in an effort to further hone their level. For example, one site was asked:Interview responses were reviewed and coded using the process previously described. Scores were then adjusted. The 6 selected sites participated in telephone meetings with technical assistance (TA) providers including the youth engagement specialist.There are many terms that get used interchangeably when referring to how young people are involved with a program\u2014eg, Youth Engagement, Youth Participation, Youth Voice, Youth Leadership, Youth Empowerment. [project report]Technical assistance (TA) began with an introduction to youth engagement. Hart's ladderPlanning notes indicate that after the initial training individualized plans were developed based on level goals and TA activities outlined in Table\u00a0Some acceleration sites have strong programming in other areas . In this instance the youth engagement consultant [specialist] offers them support to modify the existing materials and curriculum so that it is appropriate for young people and the goals associated therewith. [internal initiative report]\u2026provided a 2\u2010day youth assessment training to young people from 2 high schools as well as a train\u2010the\u2010trainer workshop for site staff to provide these trainings in the future. [external initiative report]\u2026[TA materials] not only served as a resource but also provided a template for the \u201cState of Child Report\u201d that the \u2026 site was working on, wherein the young people talk about the biggest issues that impact young people in the city and the changes needed to solve those issues. [external initiative report]\u2026[TA] provided them with strategies, concepts, and framework that they had not thought through before. For example, one of the sites mentioned that they had not thought about how youth who are more vulnerable backgrounds  take more risk when they engage and make their voices heard as compared to youth who are less vulnerable. This led some sites to more careful planning in the first year [external initiative report]Another project director mentioned that the idea that the site could involve youth equipping them with basic research skills and data collection methodology so they can analyze and interpret their own data was new to the project director. [external initiative report]Level one sites, the goal was to provide a steady flow of information and to begin to explore strategies to plan.\u2026Interviewees said that engaging youth as leaders has the potential to bring new insights to the health and well\u2010being of young people, provide a broader understanding of young people's wellness, and increase their skills, confidence, and agency. Most importantly, engaging them made sure \u201cthat interventions and programs in school settings are actually addressing the needs of young people as they state them\u201d (site project director). [external initiative report]Overall, data indicate sites benefited from youth engagement frameworks and examples as well as brainstorming trouble shooting and direct training.Youth\u2010led Councils have been formed in 2 high schools and one middle school\u2026 All 3 student wellness councils have been trained in health equity and have chosen their top health issues to focus on next school year\u2026 [site report]All 6 acceleration sites have implemented or are in the process of implementing youth leadership collectives.Youth groups are referred to as action groups, ambassadors, councils, and advisors. The structure and function of youth leadership collectives vary across sites. Activities, for example, range from service and peer education to research and policy advocacy. Youth engagement activities are shown in Table\u00a0Youth engagement was not without its challenges. Sites described a number of impediments to establishing partnerships with young people and engaging them in meaningful ways. These challenges varied but in most cases were structural. Namely, community\u2010level factors such as transportation and organizational factors related to interagency collaboration, resource scale\u2010up, and staffing issues. Individual factors related to behavior, capacity, and life circumstances were also noted. Table\u00a0youth\u2010initiated activities and programs, there were instances when youth engagement was characterized as tokenism or assigned and informed.Consistent with the literature, we found there is a great deal of variation in how youth voice and youth engagement are conceptualized.structure, systems, strategy, skill, staffing, style, and shared values.There are multiple elements that comprise an organization. For example, McKinley lays out 7 organizational elements: Overall, sites found Hart's Ladder to be a framework by which to understand engagement and provided clear goals to target. Some sites used the graphic of the Ladder with youth to reflect on where they were with respect to programming. Exploring the rungs of the ladder together, youth and adults evaluated their engagement and were able to collaboratively generate partnership goals as well as strategies for arriving at their desired destination. Similarly, sites benefited from resources and TA activities aimed at examining how adultism operates within systems. Authentic youth engagement calls for adults ready to tackle ideology, systems and structures that proliferate adultism and impede engagement. At the end of the day, this work takes both time and intentionality. Organization readiness assessments may facilitate efforts to engage youth authentically.This work is not without limitation. We present an analysis of observational data and program documents coded by a single reviewer, which may have introduced bias. To compensate for this limitation study, findings have been reviewed by and discussed with APA staff to explore the extent to which finding reflect their lived experience. In addition, the triangulation of multiple data sources has helped to ensure validation.As we strive to implement WSCC strategies for improving outcomes,Consistent with the literature, we found efforts to create healthy environments for students that fail to meaningfully engage them to run the risk of employing adult\u2010centric approaches.In the context of COVID\u201019, we, adults, need to be particularly mindful of what it means to authentically engage young people. The goal of any social system is to maintain equilibrium. Youth engagement is complicated because it challenges norms within social systems, including schools. Today, systems are in crisis and having to adapt to a new landscape. As such, we run the risk of setbacks if we do not continue to both think and act intentionally about how we collaborate, partner, and engage with youth.This research was determined not to be human subjects research by the Boston University Charles River Campus Institutional Review Board.Linda Sprague Martinez is a research consultant for Americas Promise Alliance and the Community Clinic, Inc. She was previously a research consultant for RAND and Human Impact Partners."}
{"text": "Microcirculation refers to the smallest blood vessels within the body. During inflammation, changes can occur within these vessels which can further disease processes. Blood vessels in the gut are particularly vulnerable. Videomicroscopy devices are important for examining these changes. In animal experiments, intravital microscopy is the gold standard for evaluation. This technique allows for the visualization of these vessels within living animals. The changes that occur vary depending on the length of time the inflammation has been occurring for. Examples of these changes include changes in blood flow, vessel density and immune cell activation. This review discusses these changes in the context of various inflammatory conditions including infections of the intestine and pancreas, and non-infectious conditions of the bowel.Because of its unique microvascular anatomy, the intestine is particularly vulnerable to microcirculatory disturbances. During inflammation, pathological changes in blood flow, vessel integrity and capillary density result in impaired tissue oxygenation. In severe cases, these changes can progress to multiorgan failure and possibly death. Microcirculation may be evaluated in superficial tissues in patients using video microscopy devices, but these techniques do not allow the assessment of intestinal microcirculation. The gold standard for the experimental evaluation of intestinal microcirculation is intravital microscopy, a technique that allows for the in vivo examination of many pathophysiological processes including leukocyte-endothelial interactions and capillary blood flow. This review provides an overview of changes in the intestinal microcirculation in various acute and chronic inflammatory conditions. Acute conditions discussed include local infections, severe acute pancreatitis, necrotizing enterocolitis and sepsis. Inflammatory bowel disease and irritable bowel syndrome are included as examples of chronic conditions of the intestine. Microcirculation refers to the circulation of blood through microvessels with a diameter of less than 100 \u03bcm, which include arterioles, capillaries and venules. Larger vessels from the macrocirculation, including arteries and veins, supply blood to the microcirculation. The microcirculation has many roles within the body, as reviewed in detail by Guven et al. [The gastrointestinal (GI) tract is supplied by three major arteries, with the superior and inferior mesenteric arteries supplying the small and large intestine. A large number of anastomoses are present to facilitate continuous flow through the intestine. Intramural blood flow is not uniform, with the mucosal layer receiving 70\u201380% of the blood supply depending on food intake . Not allIn a state of homeostasis, macro and microcirculation are coupled to each other . Inflammation, either local or systemic, may result in microcirculatory disturbances and ultimately in the uncoupling of these circulatory systems. Because of the uncoupling of macro and microcirculation, therapies aimed at restoring microcirculatory parameters may not be effective at a microcirculatory level . Acute i\u00ae software to the semiautomated AVA3\u00ae technique noted that while the parameters are statistically related, further development is needed before the implementation of the fully automated software [Two key parameters are established for assessing the microcirculation. Capillary density and blood flow can be assessed directly using a variety of microscopy techniques. Assessment of the microcirculation in patients is limited to areas with superficial vasculature such as sublingual mucosal tissue. Accurate assessment of the microcirculation of internal organs, such as the intestine, remains challenging. Changes in the sublingual microcirculation are known to parallel those of the intestine, but this data may not always be representative of the intestinal microcirculation . Rectal software .The intestinal microcirculation can also be impacted by other factors which are not as easily addressed using imaging techniques. The gut is home to a rich, diverse microbiome which can vary during states of disease. Changes in bacterial populations have the potential to impact the immune response and affect the microcirculation. Amedei and Morbidelli reviewed the effects of various circulating metabolites produced by gut microbiota on endothelial cell function . One stuVarious imaging techniques are used to assess the microcirculation experimentally. Intravital microscopy (IVM) is an experimental technique that allows for the visualization of biological processes in vivo. The term encompasses various types of microscopy which can be used for in vivo visualization. IVM typically images tissue architecture and functions by fluorescently labeling components of interest. Various methodologies used in IVM are reviewed by Weigert et al. . IVM canHelicobacter pylori and rotavirus, as well as severe acute pancreatitis (SAP), necrotizing enterocolitis (NEC) and sepsis. Inflammatory bowel disease and irritable bowel syndrome are discussed as chronic inflammatory pathologies. The major changes found in each of these conditions are summarized in This review identifies some of the changes within the intestinal microcirculation during both acute and chronic inflammation. Acute pathologies addressed include various acute infections, such as infection with H. pylori is a common gastrointestinal pathogen, frequently infecting both the stomach and duodenum. As observed mainly by IVM, H. pylori infection can impact the microcirculation via changes in blood flow, leukocyte activity and the endothelial cells. Yoshida et al. demonstrated a significant increase in leukocyte adhesion in rat mesenteric venules after superfusion with H. pylori [H. pylori infection in mice resulted in a 30% reduction in mucosal blood flow as evaluated by laser Doppler [H. pylori infection was associated with a decrease in mucosal blood flow in patients who had undergone endoscopic mucosal resection [H. pylori infection discussed here are related to acute infection, it should be noted that this infection often becomes chronic, resulting in the formation of ulcers. Infection of the GI tract with an enteric pathogen can result in acute inflammation. Altered microcirculation is a common feature of infection by various pathogens, including bacteria, viruses and parasites ,70,71. E. pylori . An addi. pylori . H. pylo Doppler . Clinicaesection . While tInfection with rotavirus, a double-stranded RNA virus, serves as an additional example of microcirculatory changes during acute infection. Rotavirus is a common cause of gastroenteritis among children, resulting in vomiting and diarrhea. This infection can impact both the small and large intestine. Intestinal microvascular endothelial cells are able to recognize double-stranded RNA via Toll-like receptor 3, leading to production of proinflammatory interleukin-12 . InfectiSevere Acute Pancreatitis (SAP) is a serious illness featuring sudden inflammation of the pancreas, often secondary to gallstones or alcohol abuse. SAP has a mortality rate of approximately 40% and often progresses to sepsis and multiorgan failure . The GI Necrotizing Enterocolitis (NEC) is an inflammatory condition that primarily affects the intestine of preterm infants. The intestinal microcirculation of infants is particularly vulnerable to disease due to the variety of age-dependent changes that occur during the postnatal period . The defSepsis is a complex syndrome, defined as life-threatening organ dysfunction caused by the dysregulated host response to infection . With ovA systemic inflammatory response occurs early in sepsis. The innate immune system initiates a vigorous immune response, including the release of proinflammatory cytokines and the recruitment of leukocytes to the affected area. This immune response is dysregulated and can lead to tissue damage, edema and ultimately organ failure . PatientThe accurate identification of organ dysfunction is a current priority in sepsis research . Sepsis Hemodynamic resuscitation, a standard therapy for septic patients, is associated with reduced mortality . Obonyo Other microcirculation-targeted therapies for sepsis have reached the clinical trial stage, with limited success. Inhaled nitric oxide was unable to correct microcirculatory parameters in septic patients despite macrocirculatory stabilization as assessed by sidestream darkfield videomicroscopy of the sublingual microcirculation . AdminisInflammatory Bowel Disease (IBD) is an umbrella term that describes inflammatory disorders affecting the GI tract. There are two major types: Crohn\u2019s disease (CD) and ulcerative colitis (UC), which are distinguished primarily by the portion of the GI tract affected. While CD may impact any part of the GI tract, UC is typically restricted to the large intestine. In addition, CD is a transmural disease, affecting all layers of the gut, while UC is restricted to the mucosal layer. It is important to consider these specifications when considering the impact of these conditions on the intestinal microcirculation. Despite these differences, both diseases feature symptoms including abdominal pain, diarrhea, fatigue and weight loss. One recent global meta-analysis suggests that the prevalence of IBD exceeds 0.3% of the population in Westernized countries . IBD is Different patterns of vascular perfusion have been linked to different stages of IBD progression. Historically these changes were hypothesized to be secondary to histological changes. Animal models, which allow the investigation of very early changes during the disease course, have been essential to this research. In experimental UC induced by either trinitrobenzensulfonic acid or mitomycin-C, perfusion is increased in the early stages of disease and then reduced in chronic phases . These fChronic inflammation, characteristic of IBD, results in important changes within the intestinal microvasculature which may impact perfusion. Chronic inflammation and hypoxia within the GI tract upregulate various growth factors and other cytokines involved in angiogenesis. Vascular endothelial factor A (VEGF-A) has been linked to both inflammation and angiogenesis in samples from patients with IBD . IncreasBecause IBD patients experience chronic inflammation, there is involvement of the adaptive immune system. Lymphocyte homing is of particular relevance. Patients with IBD express high levels of chemokine ligand 20 (CCL20), which facilitates lymphocyte homing to chemokine receptor 6 in intestinal tissues ,36. IncrOnce patients enter remission, it is unclear what happens within the microcirculation. One macroscopic study indicated patients primarily experience a return to normal blood flow, with differences in flow possibly predicting treatment outcomes . A studyIrritable bowel syndrome (IBS) is a functional disorder of the GI tract which has a prevalence of approximately 12% in North America . IBS is Visceral hypersensitivity, as a result of autonomic dysfunction, has been suggested as a possible mechanism for IBS . FingertChronic, low level inflammation may play a role in IBS pathogenesis . ExperimMicrocirculatory dysfunction resulting from inflammatory illnesses is a serious clinical concern associated with negative outcomes including tissue damage and organ dysfunction. Despite the variety of clinical conditions discussed here, inflammatory conditions impacting the intestinal microcirculation have many common characteristics regardless of whether the inflammation is acute or chronic in nature . Microci"}
{"text": "In different models of asthma, PGE2, 15d-PGJ2, and LXs reduced lung inflammation and remodeling. Furthermore, these SPMs inhibited chemotaxis and function of several inflammatory cells involved in asthma pathogenesis, such as eosinophils, and presented an antiremodeling effect in airway epithelial, smooth muscle cells and fibroblasts in vitro. In addition, PGE2, 15d-PGJ2, and LXs are all able to induce macrophage reprogramming to an alternative M2 pro-resolving phenotype in vitro and in vivo. Although PGE2 and LXA4 showed some beneficial effects in asthmatic patients, there are limitations to their clinical use, since PGE2 caused side effects, while LXA4 presented low stability. Therefore, despite the strong evidence that these AA-derived SPMs induce resolution of both inflammatory response and tissue remodeling in asthma, safer and more stable analogs must be developed for further clinical investigation of their application in asthma treatment.Asthma represents one of the leading chronic diseases worldwide and causes a high global burden of death and disability. In asthmatic patients, the exacerbation and chronification of the inflammatory response are often related to a failure in the resolution phase of inflammation. We reviewed the role of the main arachidonic acid (AA) specialized pro-resolving mediators (SPMs) in the resolution of chronic lung inflammation of asthmatics. AA is metabolized by two classes of enzymes, cyclooxygenases (COX), which produce prostaglandins (PGs) and thromboxanes, and lypoxygenases (LOX), which form leukotrienes and lipoxins (LXs). In asthma, two primary pro-resolving derived mediators from COXs are PGE Asthma is a high prevalence chronic inflammatory pulmonary disease, the respiratory symptoms of which include cough, wheezing, shortness of breath, and chest tightness which leads to elevated morbidity, mortality, and disease social and economic costs \u20133. PulmoThe resolution of inflammation is an active and controlled process that reduces inflammation through the elimination of danger signals, leading to the restoration of tissue homeostasis and preventing the progression towards an uncontrolled chronic inflammatory state. Thus, catabolization and antagonization of pro-inflammatory mediators, a decrease in leukocyte numbers at inflammatory sites, and tissue repair are key events in the resolution process . It is n+ cells to M2 and T regulatory phenotypes respectively, sequestration and counter-regulation of pro-inflammatory mediators, apoptosis of polymorphonuclear cells with subsequent phagocytosis by M2 macrophages, and tissue repair , peptides , proteins , and nucleotides (i.e. adenosine and inosine) \u201319. Theye repair \u201322.Among the SPMs, the lipid mediators activate many aspects of the resolution process . These e(COX), especially COX-2 isoform, play a pivotal role in the conversion of AA into different pro-inflammatory lipid mediators, including prostaglandins (PG) and thromboxanes , a metabolite of PGD2 , mPGES-2, and cytosolic PGES (cPGES), which use PGH2 as substrate. The actions of PGE2 are mediated by four distinct 7TM receptors (EP1\u2013EP4)  time: the kinetics of PGE2 release can separate its pro-inflammatory and pro-resolving effects due to the presence of different targets  . Althougcontexts . So, wha targets , 32; ii)opposite , 33, 34;hance it .2 production by dendritic cells through reduction of COX-2 and mPGES-1 expression -induced lung eosinophilia  activation, but some of its effects are independent of this receptor , an enzyme that catalyzes PGHose mice . Furtherose mice . We prevhe lungs . The proulocytes . The inhinophils . Howeverof PPAR\u03b3 . 15d-PGJagonists , 89.2 reversed structural changes related to airway remodeling, including epithelial thickening, mucus exacerbation, and EM deposition, in both OVA and HDM murine models of asthma  and 15-LOX are the main LOXs involved in the metabolization of AA , resultiby 5-LOX , and in by 5-LOX . It is depi-LXB4 . LXA4 anted ALXR . ALXR isceptor 1 . Neverthentified .4 levels in BAL fluid, sputum, and whole blood compared to moderately asthmatic individuals. This reduction in LXA4 concentrations observed in severe asthma was associated with a higher degree of airway obstruction  cells and ILC2s are important in the control and exacerbation of asthma, respectively. NK cell depletion induced a persistent allergic airway inflammation in association with reduction of the LXA the BAL . LXA4 entivation . Meanwhitivation . LXA4 intivation .In vitro, LXA4 and 15-epi-LXA4 reduced IL-8 secretion induced by serum amyloid A in a human alveolar A549 cell line (4 increased basal proliferation and wound repair of human airway epithelial cells (4 reduced mucus production (4 reduced both LTE4- and IL-13\u2013primed ASM migration toward platelet-derived growth factor in vitro (4 inhibited connective tissue growth factor-induced human lung fibroblast proliferation in vitro (in vitro (4 reversed bleomycin-promoted fibrosis and lung damage in mice (ell line . Also, aal cells . In a mual cells  and LXB4oduction . ASM hypin vitro . Anotherin vitro . Moreovein vitro  and blocin vitro . Besides in mice . Altoget4, some clinical trials using this LX, its analogues, or LXA4 receptor agonist BML-111 were administered in asthmatic patients. The nebulization of LXA4 reduced LTC4-induced bronchoconstriction (4 (4 analog or BML-111, which is more potent and stable than LXA4 itself (4 analog\u00a0and BML-111 were well tolerated and presented no side effects (Due to the possible therapeutic application of LXAtriction ; howeverction (4  make its4 itself , improve4 itself  (Table  effects .2, 15d-PGJ2, and LXs with better physical-chemical properties, allowing greater stability and superior selectivity for specific receptors. Moreover, new analogs of AA-derived SPMs could also improve efficiency and reduce the required dose of glucocorticoid, the latter often leading to adverse effects and steroid-refractoriness, despite being the best asthma treatment so far.This mini-review presents several aspects of the pro-resolving effects of COX- and LOX-derivative mediators in asthma Figure\u00a01DI, MF, and DC contributed to the conception and design of the study, wrote the manuscript, discussed the content, and contributed to the manuscript revision. MM discussed the content and contributed to the manuscript revision. VC contributed to the conception and design of the study, wrote the manuscript, discussed the content, and contributed to the manuscript revision. All authors reviewed and/or edited the\u00a0manuscript prior submission. All authors contributed to the article and approved the submitted version.This work was supported by PrInt Fiocruz-CAPES Program N\u00b0 01/2020.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 paper presents a series of compression tests performed on a variety of high performance lightweight cellular materials conventionally used in energy absorption applications. Compressive tests were performed over a range of strain rates with a universal testing machine and a single stage gas gun. Experimental results revealed a dependency of the mechanical properties on the polymeric precursor, density, infill topology and strain rates. The dynamic strength of the investigated materials was determined through a material parameter identification study via the finite element (FE) method. Numerical results matched well with the experimental results and revealed a substantial enhancement in the compressive strength of the tested material from quasi-static to dynamic loading regimes by as much as 87%. The strength of 3D printed polymers was superior with respect to the tested polymeric foams. On the other hand, polymeric foams showed higher efficiency and energy absorption ability. Cellular materials such as honeycombs and foams are increasingly exploited in engineering applications due to their excellent ability to absorb energy, low weight and relatively low cost. The main applications for soft and semi-rigid foams include insulation, packaging and cushioning, whilst rigid foams and honeycombs are also employed in structural applications, as well as the core of lightweight sandwich structures. The selection of the material is application dependant and should be based on factors such as in-service force and pressure, duration and direction of the load, design constraints, environmental conditions and cost. Hence, a thorough understanding and evaluation of the mechanical behaviour under different loading conditions is necessary in order to determine the range of applications in which certain materials would have the best response. There is extensive experimental data available in the open literature on the mechanical properties of polymeric foams. For example, different peer reviewed articles document the compressive properties of polyurethane foams ,2,3,4, eIt is generally accepted and was experimentally confirmed in ,10,13,14 strains .In the last 30 years, additive manufacturing (AM) technology has been rapidly developed and is increasingly exploited for the creation of prototypes and complex shape parts. Thermoplastic polymeric materials such as ABS, PA, PLA and TPU can be processed using such techniques . When thOne of the fields in which AM could have enormous potential is in the construction of nano and micro Unmanned Aerial Systems (UASs). Yeong et al.  reviewedThis paper presents a series of compression tests performed on a range of lightweight materials conventionally used in energy absorption applications and 3D printed cellular solids. The experimental programme consisted of quasi-static compression tests performed with three different strain rates on thirty six different types of materials. The materials that showed the highest compressive strength, sensitivity to the strain rate, absorbed energy and efficiency were tested under dynamic conditions similar to a Taylor impact test. Hence, the finite element (FE) method was employed in order to quantify the strength at higher strain rates. The FE methodology involved the solution of an inverse problem where the material parameters were calibrated by matching the response of the virtual specimen with the experimentally observed response.A series of commercially available lightweight materials were procured and tested. The nominal thickness of the as-received panels was 25 mm. The physical properties of the investigated materials are listed in 3D printed specimens were prepared in-house using two different 3D printers (Makerbot Replicator 2X and Ultimaker 3 Extended) via the fused filament fabrication (FFF) method. A variety of raw materials  and different infill densities (10% and 30%) and infill topologies  were investigated. The experimental testing campaign was divided into two levels of testing. Level 1 consisted of conventional quasi-static compression tests on all materials listed in \u22121, 0.013 s\u22121 and 0.133 s\u22121, respectively. All tests were performed at room temperature. Quasi-static compression tests on 3D printed specimens were performed with cylinders of 20 mm diameter and 25 mm long. Although not recommended by the standard, this specimen geometry was chosen in order to compare the compressive properties of the specimens under quasi-static and dynamic conditions directly. It was necessary to print the skin to ensure the stability of the specimen and replicate periodic boundaries as in real structures.Uniaxial compression tests were carried out according to ASTM D1621-16 \u201cStandard Test Method for Compressive Properties of Rigid Cellular Plastics\u201d . Tests aThe compressive stress was determined by dividing the load by the initial cross-sectional area of the specimen;As cellular materials experience variations in the local strain field under compression, the average compressive strain was determined optically by tracking the relative displacement of two points drawn on the compressive platens.\u00ae Video GaugeTM software. The diameter of the specimen was chosen such that the cross-sectional area of the specimen was smaller with respect to the cross-sectional area of the tip of the Taylor bar. The length of the foam specimens was chosen to be the same as the thickness of the received billets in order to avoid cuts and bonding with adhesives. The length of 3D printed specimens was chosen to be the same as the other specimens in order to have a direct comparison throughout the testing campaign. The tip of the bar was lubricated with silicone grease to minimise frictional effects. The effect of the vacuum during the tests was neglected. For such testing conditions, the compressibility of the air would be insignificant compared to the structural stiffness of the tested materials.The dynamic characterisation of selected materials was carried out by firing the specimens onto a rigid bar anvil, in the manner of a Taylor test , using aIt could be observed that the global compressive behaviour of foams depended mainly on their precursor polymer and its relative density and the mechanical properties related to the cell structure. Generally, the higher the density, the higher the collapse stress  steeply .I, L, O, R) and ranged between 2.6 MPa and 5.3 MPa when tested at the slowest cross-head displacement rate. Soft and semi-rigid foams were the most compliant with a The highest collapse strength F, G, S, T) almost completely recovered their initial shape after removing the compressive load, regardless of the applied strain rate. As their structure was more compliant, low density PE and PVDF based foams are not used for structural applications, but mainly for their electro-magnetic and thermal properties, as well as acoustic insulation [A, B, C, D, E, J, K, L plastically deformed with a partial recovery of their initial shape after removing the compressive load. Foam Types H, I, M, N, O, P, Q, R plastically deformed without any shape recovery. Instead, small crumbles were observed on the surfaces of the latter types, indicating brittle crushing.Soft foams . The efEquation ): (1)W=\u222bA, B, C and AC, AD were similar to those observed for the other investigated materials.By plotting \u00ae RIST and Rohacell\u00ae IG. EPS foams had an envelope gradient 14% and 6% lower with respect to the former materials, respectively. The envelope gradient calculated for 3D printed PLA with a linear infill pattern was 0.505, i.e., the highest amongst the 3D printed materials. Nevertheless, its gradient was about 16% smaller with respect to Rohacell\u00ae RIST. The lowest ability to absorb energy was found for 3D printed CPE specimens, which were brittle and compliant. It also appears from It can be seen from A summary of the collapse stress vs. density is shown in Although PLA specimens had the highest collapse strength amongst the investigated materials, the plateau stress was unsteady and, when normalised with respect to the specimen density, similar to the normalised plateau stress of ABS specimens. Hence, the latter material was selected for dynamic compression tests.U and W progressively plastically buckled in a similar way to axially compressed tubes, whilst 30% filled specimens showed plastic deformation only near the impact face, which mushroomed. It also appeared that the 3D printed specimens with the highest core density produced the steepest reduction in velocity  showed the slowest deceleration and the highest compression strain amongst the investigated ones. The impact was almost completely elastic, and rebound velocity was about 42% compared with the initial velocity. Fluctuations in the rebound velocity could be attributed to elastic waves bouncing within the specimen itself. Amongst the tested materials, Type G was the only one able to recover its initial shape after impact. PET and PMI based foams pulverised upon impact at the impact face  mounted behind the tip of the bar, it was not possible to record any meaningful data. The momentum generated upon impact was too low to trigger the load cell, regardless of the set force threshold. Therefore, the dynamic strengths of the tested materials were calculated through a finite element (FE) study. FE can also simplify the problem as variations of specimen density, inertia effects and instantaneous acceleration are automatically computed by the solver.3; element aspect ratio between one and 1.8). The virtual specimens were set to impact a planar rigid wall with an initial velocity equal to 160 m/s. No friction was defined upon the contact between the specimen and the rigid wall.The simulations of the dynamic compression tests were carried out using the explicit LS-Dyna FE code . For eacAmong the different material models available in the FE solver, the Material Type 63 *MAT_CRUSHABLE_FOAM was selected for all the simulations of foam specimens. *MAT_CRUSHABLE_FOAM requires the user to input five parameters: density (RO), elastic modulus (E), Poisson\u2019s ratio (PR), a load curve LCID, which defines the yield stress vs. volumetric strain, tensile stress cut-off (TSC) and a rate sensitivity damping coefficient (DAMP). Nevertheless, when an LCID was defined, it was found that the variation of E and TSC did not affect the behaviour of the virtual specimen. For all simulations, the values of PR and DAMP were set to zero and 0.1, respectively, whilst the value of RO was equal to the density of the real specimen. A piecewise stress vs. volumetric strain curve a was useanalysed b.2; element aspect ratio between one and 2.2) with a thickness of 0.3 mm and 0.6 mm were used for the core and for the outer skin, respectively. The impact conditions were the same as those used for the crash simulations of the foams. Amongst the different material cards available in LS-Dyna, an elasto-plastic constitutive material model  was chosen. *MAT_24 requires the user to define an arbitrary stress vs. strain curve via the definition of effective plastic strain values and their corresponding yield stress values. In order to calibrate the models, the Young\u2019s modulus E and the yield strength SIGY were set as variables. The plateau stress was assumed constant at the value of The FE models of 3D printed specimens were created so as to reflect the actual geometry and topology of their real counterparts. O (Rohacell\u00ae 110RIST) is shown in As mentioned in the previous sections, an inverse approach was adopted to determine the dynamic strength of the specimens via the FE method. The material parameters were iteratively varied until a close correlation between the numerical and experimental results was found. In order to establish the predictive ability of the material model used, quasi-static compression tests were simulated using the stress vs. strain relationship sketched in The correlation between numerical and experimental results for quasi-static tests was good by using a simple elastic perfectly plastic stress vs. strain relationship with a Young\u2019s modulus equal to 160 MPa for all the simulations and YL (Rohacell\u00ae 110RIMA) under dynamic compression. The same approach was adopted for all the other impact cases  was experimentally investigated through a series of mechanical tests. The investigated materials were subjected to different loading conditions, from quasi-static regimes to impact loading. The experimental results revealed marked differences in the compressive response of the investigated material as far as the stress and strain were concerned. All the investigated materials were strain rate sensitive and showed enhanced strength with an increase in strain rate.Amongst the thirty six tested materials, ten were selected for the dynamic characterisation under dynamic conditions. A single stage gas gun was employed to fire cylindrical specimens onto a rigid bar anvil, in the manner of a Taylor test. High speed cameras were employed to record the deformation history of the specimen. The experimental results revealed an increase of the compressive strength of the materials with increasing the test strain rate. The mechanical properties were dependant on the material density, precursor material and infill topology. It was found that 3D printed materials can have higher collapse stress with respect to polymeric foams. On the other hand, the latter would be more efficient and able to absorb higher amounts of energy compared to the former class of materials for the same density. In order to determine the strength of the materials under dynamic conditions, the FE method was used. Numerical results matched well with the experimental results and revealed a further increase of the plateau stress by as much as 87% with respect to the strength calculated at quasi-static test rates."}
{"text": "Administration of liquid surfactant through an endotracheal tube for the treatment of respiratory distress syndrome has been the standard of care for decades. A skilled health care provider is needed to perform this procedure. In lower-income and middle-income countries (LMICs), healthcare resources are often limited, leading to increased mortality of premature infants, many of whom would benefit from surfactant administration. Therefore, having a simplified procedure for delivery of surfactant without the need for advanced skills could be life-saving, potentially diminish gaps in care, and help ensure more equitable global neonatal survival rates. Modifications to the standard approach of surfactant administration have been put into practice and these include: INtubation-SURfactant-Extubation (INSURE), thin catheter surfactant administration (TCA), aerosolized surfactant, and surfactant administration through laryngeal or supraglottic airways (SALSA). Although there is a need for larger studies to evaluate the comparative effectiveness of these newer methods, these methods are being embraced by the global community and being implemented in various settings throughout the world. Because the SALSA technique does not require laryngoscopy, a provider skilled in laryngoscopy is not required for the procedure. Therefore, because of the ease of use and safety profile, the SALSA technique should be strongly considered as a viable method of delivering surfactant in LMICs. Respiratory distress syndrome (RDS) is one of the most frequently diagnosed diseases in preterm infants in the neonatal intensive care unit (NICU) . ExogenoWhile INSURE requires endotracheal intubation, the TCA techniques are considered less traumatic to the airway than the traditional invasive methods of surfactant administration. These techniques still require operator expertise as laryngoscope insertion is needed . AerosolThe SALSA technique involves placement of a laryngeal mask or supraglottic airway into the posterior pharynx without the need for laryngoscopy. Liquid surfactant is then administered through the device . SurfactIn this manuscript we will review the pathophysiology of RDS and the impact that treatments such as surfactant and continuous positive airway pressure (CPAP) have had on the disease. The evidence, safety, efficacy, and outcomes for each of the newer methods of surfactant administration will also be reviewed and we will draw conclusions as to the applicability of these methods in the LMIC setting. We will conclude by providing instructions on how to perform the SALSA technique as it has unique and specific characteristics that make it ideal for the LMICs setting.RDS is the leading cause of respiratory disease in preterm infants , 26. ThePreterm infants produce limited amounts of surfactant and commonly require exogenous instillation of surfactant to decrease their oxygen requirement and ameliorate the symptoms of RDS. This is why surfactant therapy can be life-saving . Followip = 0.005)  . Similar= 0.005) , 39.While advances in therapies and available approaches in treatment of RDS have led to improved morbidity and mortality in premature infants, there is a lag to disseminate such technology from HICs to LMICs , 41. TheIn an effort to continue improving care and reducing infant mortality in LMICs, the WHO has recommended Sustainable Development Goals (SDG). This collection of 17 goals is designed to achieve a more sustainable future for all by 2030. One aim is for every country to reduce NMR to at least as low as 12 per 1,000 live births and end preventable deaths of newborns and children under 5 years of age . Thanks Continuous positive airway pressure (CPAP) is a simple, cost efficient, non-invasive respiratory support considered an optimal first line therapy for RDS. It aims to support functional residual capacity and prevent the need for mechanical ventilation (MV) , 46. CPADespite these studies coming from HICs, CPAP should be implemented in every LMIC seeking to decrease their NMR as it is an effective treatment for RDS. This can be supported by a systematic review which evaluated the feasibility and efficacy of CPAP in LMICs. Thukral et al. showed that implementing a CPAP program could reduce in-hospital mortality by up to 66% along with a decreased need for MV in preterm infants . BecauseWhen there is a need to administer surfactant, the traditional approach has been to deliver exogenous surfactant through an endotracheal tube (ETT). However, less invasive methods, which may be considered \u201coff label\u201d use based on the country, are now widely being used around the world .Neonatal endotracheal intubation is a life-saving and critical skill, but there are risks associated with it and this needs to be done by a neonatologist or health care provider with expertise in the procedure . This skIn areas without advanced neonatal care, a skilled health care provider capable of successful intubation may not be present in an urgent and timely manner when surfactant is needed in a critically ill infant. In these settings, less invasive methods could be an alternative.INSURE is the most studied mode of surfactant instillation. The prompt removal of an ETT after instillation of exogenous surfactant was both feasible, safe, and beneficial to the premature infant . INSURE via the ETT  of infants failed and required MV after using INSURE . This teSeveral terms are used to describe the variations of the TCA method. The most common are: Less Invasive Surfactant Administration (LISA), Minimally Invasive Surfactant Therapy (MIST), Take Care, and SurE . TCA invvia thin catheters was associated with lower likelihood of mortality, need for mechanical ventilation and bronchopulmonary dysplasia compared with InSurE , while death was not significantly different (10 vs. 7.8%), BPD at 36 weeks' postmenstrual age was lower in the MIST group (37.3 vs. 45.3%)  .Jena et al. of India, have shown that TCA administration can be applicable to an LMIC setting. They found that, compared with the INSURE group, TCA infants had less need for subsequent MV (19 vs. 40%), shorter duration of supplemental oxygen, shorter hospital stay, and a lower rate of supplemental oxygen at 36 weeks' corrected gestational age .The TCA method may be feasible in an LMIC setting, but requires a skilled provider to use a laryngoscope to pass a catheter through the vocal cords. Thus, its applicability is primarily within a NICU environment.p < 0.0001; RR: 0.51 90% CI: 0.41-0.63)  . In a sy41-0.63) .An advantage of aerosolized/nebulized surfactant is that it is the least invasive method to deliver surfactant and requires minimal skills to administer. However, as of the writing of this review, there is currently not a device that is commercially available, although a device is currently under review by the United States Food and Drug Administration. If a device were available, this method will require specific equipment that may be difficult or cost-prohibitive to obtain in LMICs, thereby limiting its application in this setting.SALSA is a less invasive method that has unique characteristics that makes it an ideal candidate for the LMIC setting. The SALSA technique consists of inserting a supraglottic airway device (SAD), commonly known by the trade name, Laryngeal Mask Airway (LMA), into the mouth and advancing it until the device is unable to be advanced further. Proper placement is indicated by listening for bilateral breath sounds and, if available, color change on a carbon dioxide detector. A piece of tubing connected to the surfactant syringe is then placed into the lumen of the device to deliver the surfactant in aliquots just above the vocal cords \u201323. FiguThe SALSA technique differs from traditional intubation, INSURE, and TCA in that there is no insertion of the device through the vocal cords, therefore, use of a laryngoscope is not required for proper placement of the device. In addition, insertion of the LMA is substantially easier and faster than the insertion of an ETT , and decIn the largest randomized controlled trial of SALSA, placement of the device took an average of 88 s range 12-500 s) and 67% of attempts were successful in <35 s . The dev-500 s anWith decreased need for MV, the SALSA technique may be beneficial for survival of infants in LMICs where access to MV is limited or not available. Because of this, SALSA was introduced in 2018 and has been widely used by physicians in the Republic of Azerbaijan. This has been one contributing factor to their recognition by WHO as one of the fastest progressors globally in meeting the SDG 2030 goals in reduction of the country's NMR , 73.n = 612) in Ethiopia found that only 39% of attendants at deliveries were medical doctors or general practitioners and only half (53%) of the births occurred at a referral hospital (According to the WHO, nearly 15 million babies are born prematurely each year. Preterm birth complications are considered the leading cause of death among children under 5 years of age. This accounted for 1 million deaths in 2015 . United hospital .Studies in adult and pediatric patients show that intubation at referring or community settings, where there is less access to a skilled airway management, increases the risk of poor outcomes, including death , 78, 79.INSURE and TCA methods for administration of surfactant do show a decrease in the need for MV. Because of this, these methods have been implemented as standard practice in many parts of the world. The need for a high degree of skill which is similar to intubation does make this method less desirable for areas where advanced neonatal care and the equipment required to provide this care is not available. Inhaled surfactant, although promising, still requires an approved device to determine whether it will be a widely accepted method, but the cost and additional resources required will likely not allow this method to be widely used outside of high resource settings.SALSA requires minimal skill and resources and is feasible in areas where advanced neonatal care is not widely available . This isAlthough we recognize the limitations of small, unblinded, clinical trials, and that larger trials are necessary to establish a complete safety and efficacy profile, SALSA should become a preferred alternative method to administer surfactant in infants that require this life-saving medication in the LMICs setting. Furthermore, it is ideal for use in situations where there is no provider skilled at intubation, CPAP is available, and/or a minimally invasive method of surfactant administration is desired. If health care providers are trained to administer surfactant using the SALSA technique, it may show benefits in reduction of oxygen needs, intubation and MV, but would also be expected to improve overall morbidity and mortality in newborns with RDS.Unlike intubation which requires significant training to acquire skills, placement of a SAD can be learned quickly with a higher successful rate at first attempt placement . To assihttps://www.youtube.com/watch?v=Iig9l4BgIy4 and https://www.youtube.com/watch?v=ioXGyfVLdyE.Detailed instructions on how to perform the SALSA technique are available in the following links: The benefits of surfactant are clear. The methods to deliver surfactant will, however, continue to evolve. Each of the techniques surveyed in this review have demonstrated safety and efficacy. It is the opinion of the authors that the SALSA technique is uniquely applicable to the LMICs setting. Establishing programs to implement the SALSA technique in LMICs may be advantageous to improve NMR in countries that lack skilled providers but have access to surfactant. One such initiative took place in Azerbaijan and was backed by the Ministry of Health . Other cSimilarly, the SALSA technique could be applied to community hospitals in HICs to help improve outcomes. This may include the prevention of transfers to tertiary care facilities, thus decreasing costs and health care utilization. Quality-improvement programs with stepped-wedge design could give more insight into the challenges of implementation across different hospital systems and how this could be improved. Leveraging social media's power to spread medical science and literature may also be of great benefit to shorten the time it takes to disseminate and implement evidence-based therapies from HICs to LMICs.Finally, it is recognized that at this time SADs are not widely available in most LMICs and that providers may not have experience with the device. As surfactant use becomes more widespread, an investment in education and a dissemination of the tools needed to deliver this medication is equally important. Another limitation is that currently available SADs are too large for infants weighing < ~1,200 g. Development of a cost-effective SAD designed to administer surfactant over a broad range of infant weights would benefit LMICs wishing to use surfactant in combination with CPAP to improve their infant mortality rates. When this occurs, it should be evaluated not only in a HIC setting, but in LMICs where we expect the greatest benefit will be found.SG, PF, and HZ: conception and design. HZ, PF, KR, DK, and SG: draft manuscript preparation. All authors reviewed and approved the final version of the manuscript.PF, KR, and SG have worked for and received payments from ONY Biotech in the past. The remaining 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. The reviewer AP declared past co-authorships with one of the authors KR and the absence of any ongoing collaboration with any of the authors to the handling editor.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": "Conclusions: Pharmaco-resistance developed in approximately 20% of patients with PSE and was associated with younger age at stroke onset, stroke type and severity, status epilepticus occurrence, and seizure types.Objectives: The study aimed to explore the clinical predictors of pharmaco-resistance in patients with post-stroke epilepsy (PSE). Methods: Patients with epilepsy secondary to cerebral infarct or spontaneous intracerebral hemorrhage were included. The study outcome was the occurrence of pharmaco-resistance defined as the failure of adequate trials of two tolerated and appropriately chosen and used antiseizure medication schedules, whether as monotherapies or in combination, to achieve sustained seizure freedom. Results: One-hundred and fifty-nine patients with PSE and a median follow-up of 5 (3\u20139) years were included. The mean age of the patients at stroke onset was 56.7 (14.9) years, and 104 (65.4%) were males. In the study cohort, 29 participants were pharmaco-resistant. Age at stroke onset [odds ratio (OR) 0.97, 95% confidence interval (CI) 0.93\u20130.99;  Stroke is a leading disease worldwide, with an estimated incidence of 15 million cases annually ,3,4. PosThe aim of this study was to identify the clinical predictors of pharmaco-resistance in patients with PSE treated with antiseizure medications (ASMs).We retrospectively identified patients \u226516 years old who were referred to the Epilepsy Center of the Marche Polytechnic University, Ancona, Italy with a diagnosis of epilepsy secondary to cerebral infarct or spontaneous intracerebral hemorrhage and no history of seizures before stroke. According to the International League Against Epilepsy definition, seizures occurring within 7 days of the onset of stroke were classified as acute symptomatic seizures, seizures occurring beyond 7 days of the onset of stroke were classified as unprovoked seizures, and PSE was diagnosed as the occurrence of one or more unprovoked seizures . Patientt test, Mann\u2013Whitney test or \u03c72 test, as appropriate. Simple and multivariable logistic regression analyses were performed to identify the predictors of pharmaco-resistance. For multivariate analysis, sex, age at stroke onset, stroke type, occurrence of acute symptomatic post-stroke seizures, seizure type, and any variables with p values < 0.02 from comparisons of baseline characteristics were identified for statistical adjustment. Results were reported as odds ratio (OR) with associated 95% confidence interval (CI). Results were considered significant for p values < 0.05 (two sided). Data analysis was performed using STATA/IC 13.1 statistical package .Values were presented as mean (standard deviation) or median (interquartile range) for continuous variables and as the number (percent) of patients for categorical variables. Comparisons were made through the Student\u2019s Of 172 patients with PSE initially identified, 13 patients were excluded due to follow-up <12 months. Accordingly, 159 patients with PSE and a median follow-up of 5 (3\u20139) years were included in the analysis. The mean age of the patients at stroke onset was 56.7 (14.9) years, and 104 (65.4%) were males. Cerebral infarct occurred in 102 (64.2%) and cerebral hemorrhage in 57 (35.8%) patients. Blood pressure lowering agents were taken by 121 (76.1%), antidiabetic drugs by 30 (18.9%), and statins by 49 (30.8%) patients.p = 0.016) and severe stroke (p = 0.001) in comparison to responsive patients. Status epilepticus was the first epileptic symptom in 5 (3.9%) responsive and 7 (24.1%) refractory patients (p < 0.001), and focal to bilateral tonic seizures were more commonly observed among patients with drug-resistant epilepsy (p = 0.013).In the study cohort, 29 (18.2%) participants were pharmaco-resistant. Drug-resistance occurred in 13 out of 102 (12.8%) patients with ischemic and 16 out of 57 (28.1%) patients with hemorrhagic stroke. Baseline characteristics of the patients according to treatment responsiveness are shown in p = 0.044), history of intracerebral hemorrhage , severe stroke , status epilepticus as initial presentation of PSE , and focal to bilateral tonic-clonic seizures  had a higher likelihood of being refractory to ASM treatment  can favor refractoriness by decreasing drug sensitivity and cerebral drug uptake . Remarkal models ,17. The l models . Inductil models ,20,21, al models , has beel models . Sproutil models . The mall models . Overalll models .Common neurobiological factors can contribute to both epilepsy severity and pharmaco-resistance, and refractoriness can be inherent to disease severity . In thisThe predominant seizure type in patients with PSE was focal. Focal to bilateral tonic-clonic seizures were found in approximately one third of the cases, and associated with a higher risk of drug-refractoriness. In elderly patients with newly diagnosed epilepsy, seizure freedom was less commonly achieved by patients presenting with seizures evolving to bilateral convulsive than in patients with focal or generalized seizures ,34. A meAge at stroke onset was marginally associated with the risk of pharmaco-resistant PSE, with older age being a protective factor. Decreasing neuronal plasticity with age could smooth epileptogenicity and result in a reduced risk of pharmacoresistant epilepsy following stroke. This explanation has been previously advanced to explain the greater response to treatment observed among older adults . ConsistA not significant higher prevalence of acute symptomatic post-stroke seizures was found among drug-resistant patients. Seizures occurring early after stroke are thought to be a reaction of neuronal cells to acute cerebrovascular injury, and reflect transient cellular biochemical dysfunctions, including variations in calcium and sodium concentrations, glutamate release, and the breakdown of membrane phospholipids. Acute symptomatic seizures have also, however, been associated with an increased risk of late unprovoked seizures, indicating that they might be a hallmark of epileptogenesis . Our finWe did not find any relationship of sex with drug refractoriness. To date, conflicting evidence exists about the predictive role of sex. Sex did not emerge as a significant predictor of refractory epilepsy in two studies recruiting participants with epilepsy of any cause of all ages  and oldeThe current study has merit to provide evidence on the risk of drug-refractory PSE from the clinical perspective. Major strengths include the long-term follow-up of the patients and the real-world design, which allowed the generalizability of the findings to everyday clinical practice. Nonetheless, many shortcomings need to be considered. The retrospective analysis of data is prone to selection bias and misdiagnosis can occur even when medical reports are collected extensively. The inclusion of patients referred to an epilepsy clinic, which may represent a group preselected for age and severity, could limit the representativeness and generalizability of the findings to the general PSE population. Further, no information was available about the comorbidities and medications taken prior to stroke and during the acute phase, as well as whether vascular risk factors were adequately controlled after stroke. Therefore, we could not explore whether agents that have been suggested to reduce the risk of PSE may also influence the development of pharmaco-resistance . The unaThe available data on risks of acquiring PSE are not matched by knowledge on the condition itself and its prognosis, and there remains a large gap in the understanding of the causes and mechanisms of drug-refractoriness. The expected disease course is a reasonable question posed by patients and physicians. The identification of factors that may predict treatment response holds important practical implications to guide clinicians once a diagnosis has been made, and inform the health care system on the resources required when recovery from stroke is complicated by recurrent seizures."}
{"text": "The reporting of Coronavirus Disease 19 (COVID-19) mortality among healthcare workers highlights their vulnerability in managing the COVID-19 pandemic. Some low- and middle-income countries have highlighted the challenges with COVID-19 testing, such as inadequate capacity, untrained laboratory personnel, and inadequate funding. This article describes the components and implementation of a healthcare worker surveillance programme in a designated COVID-19 teaching hospital in Malaysia. In addition, the distribution and characteristics of healthcare workers placed under surveillance are described.th of March 2020 to the 20th of August 2020.A COVID-19 healthcare worker surveillance programme was implemented in University Malaya Medical Centre. The programme involved four teams: contact tracing, risk assessment, surveillance and outbreak investigation. Daily symptom surveillance was conducted over fourteen days for healthcare workers who were assessed to have low-, moderate- and high-risk of contracting COVID-19. A cross-sectional analysis was conducted for data collected over 24 weeks, from the 6A total of 1,174 healthcare workers were placed under surveillance. The majority were females (71.6%), aged between 25 and 34 years old (64.7%), were nursing staff (46.9%) and had no comorbidities (88.8%). A total of 70.9% were categorised as low-risk, 25.7% were moderate-risk, and 3.4% were at high risk of contracting COVID-19. One-third (35.2%) were symptomatic, with the sore throat (23.6%), cough (19.8%) and fever (5.0%) being the most commonly reported symptoms. A total of 17 healthcare workers tested positive for COVID-19, with a prevalence of 0.3% among all the healthcare workers. Risk category and presence of symptoms were associated with a positive COVID-19 test (p<0.001). Fever (p<0.001), cough (p = 0.003), shortness of breath (p = 0.015) and sore throat (p = 0.002) were associated with case positivity.COVID-19 symptom surveillance and risk-based assessment have merits to be included in a healthcare worker surveillance programme to safeguard the health of the workforce. Durinay 2020) . These aay 2020) . In low-ay 2020) , 13.th of January 2020 [Malaysia, an upper-middle-income country, reported its first COVID-19 patient on the 24ary 2020 . Initialary 2020 . As of mary 2020 .Following its designation as a COVID-19 hospital in Malaysia, the University Malaya Medical Centre (UMMC) COVID-19 Task Force conceived a comprehensive plan in ensuring the safety of its healthcare workforce while ensuring continuity of service. The strategies include a) cohorting COVID-19 patients to designated wards, b) development of clinical pathways and guidelines for the management of patients, c) ensuring adequate supply and adherence to PPE usage, d) extensive training of the healthcare workforce, and v) the development of a HCW surveillance programme.The HCW surveillance programme, in particular, leveraged a risk-based testing and isolating strategy that allowed for surveillance while balancing the use of resources. We aim to describe the components and implementation of the HCW surveillance programme in UMMC. In addition, we describe the distribution and characteristics of the HCWs placed under surveillance.th of March 2020 to the 20th of August 2020. Adherence to the surveillance programme by all HCWs is sanctioned as an administrative policy by UMMC to ensure the safety of its workforce. UMMC is a teaching hospital located in Kuala Lumpur and was one of the sixty hospitals designated to manage COVID-19 cases in Malaysia. UMMC has a total of 1,600 beds that caters for paediatric and adult patients, with a comprehensive range of speciality areas. Following its designation as a COVID-19 hospital, 153 beds across eight wards were allocated for COVID-19 patients, with 22 being Intensive Care Unit (ICU) beds. The hospital employed 5,826 HCWs, of which 1,987 staff were assigned to care exclusively for COVID-19 cases.This was a cross-sectional analysis of a HCW surveillance programme in UMMC over 24 weeks from the 6Ethical approval and waiver of written informed consent were obtained from the UMMC Medical Research Ethics Committee (MECID. No 202073\u20138862). The permission to collate data was obtained from the UMMC COVID-19 Taskforce. Dataset retrieved for this analysis did not contain any personal information, and there was no interaction with any HCWs. HCWs were not intentionally put at risk of exposure for the purpose of this study; they were existing professionals contracted to their routine work obligations at UMMC. There is standard practice for infection control for which HCWs have been trained, and appropriate PPE was provided.Fig 1 [The COVID-19 Operations Room, also known as a hospital emergency operation centre, was set up to prevent and manage COVID-19 outbreaks among UMMC HCWs through activities that focused on early detection and disease containment. The operations room workforce was categorised into four work teams, namely contact tracing, risk assessment, surveillance, and outbreak investigation. The flow of events following the identification of a COVID-19 positive case is illustrated in Fig 1 .The hospital\u2019s Infectious Disease team notified the Contact Tracing team whenever a new COVID-19 case was identified. A new COVID-19 case was either an existing inpatient who was newly diagnosed with COVID-19, a new admission diagnosed with COVID-19 referred to UMMC, or a HCW from UMMC infected with COVID-19. The activation of the contact tracing team would initiate a cascade of activities to gather and collate information pertaining to the movement of the infected person 48 hours before the diagnosis or the onset of symptoms. HCWs who had been in contact with the infected person would be listed as new contacts, while the non-HCW contacts were managed by personnel from respective District Health Offices (DHO) under the Ministry of Health (MOH).. The contacts were then classified into four different risk categories, namely \u2019no identifiable risk\u2019, \u2019low risk\u2019, \u2019medium risk\u2019 or \u2019high risk\u2019, based on the UMMC guidelines [S2 Appendix. For example, face shield, N95 mask, gloves, coverall suit, shoe cover and proper hand hygiene are recommended for aerosol-generating procedures. Detailed management algorithm of HCWs in high-, medium-, and low-risk groups are shown in . Nasopharyngeal swabs for RT-PCR were taken based on the level of risk and presence of symptoms. Those categorised as \u2019no identifiable risk\u2019 were not subjected to work restrictions nor placed under active surveillance.The list of new contacts was then relayed to the Risk Assessment team. The level of risk of the new contacts was assessed and stratified according to their risk level based on the \u201cassessment checklist for healthcare workers\u201d idelines . The UMMidelines . A \u2019low idelines . A \u2019mediidelines . The \u2019hiidelines . The recIn the next stage, HCWs classified as low, moderate, and high risk were placed under active surveillance and designated as \u2019Person Under Surveillance\u2019 (PUS). Their symptoms were monitored for 14 days from the date of their last exposure to a COVID-19 case by the surveillance team. A PUS who developed symptom(s) during the surveillance period was immediately referred to the Risk Assessment team for further management.All epidemiological data were collected from the COVID-19 operation room\u2019s centralised electronic database, which served as a digital repository for HCWs contact tracing, risk assessment and surveillance records. Additional retrievable data included sociodemographic information such as age, sex, comorbidities, and designation of the HCWs. All captured data were stored in a password-protected database which was only accessible to a few authorised personnel .th of April 2020. The outcome variable was HCWs with COVID-19, defined as a person with a laboratory confirmation for SARS-CoV-2 by reverse transcriptase-polymerase chain reaction (RT- PCR) [The exposure variables in this analysis were age, sex, job category, presence of comorbidities, risk category, and symptoms during surveillance. The symptoms, namely fever, cough, dyspnoea, sore throat, arthralgia, myalgia, gastrointestinal (GI) symptoms, and anosmia were self-reported. The symptom surveillance for anosmia was only started on the 4RT- PCR) .th of April 2020 onwards). The PUS would indicate if any symptoms were present and the type of symptoms (if any). Those who did not reply by afternoon each day were contacted by the surveillance team via text messaging or a phone call.During the initial phase, the surveillance team used an instant messaging system (WhatsApp application) to disseminate a standardised daily symptoms surveillance message to all PUS. This message enquired on any new symptom(s) for the day, any worsening of symptoms and the date of last exposure to COVID-19 patients. The selection of symptoms listed were fever, cough, breathing difficulty, myalgia, arthralgia, and any gastrointestinal symptom . The surveillance text message contained a link directing them to an online feedback form in the UMMC staff portal, and their responses were recorded in the database. Surveillance messages were sent at 6 am daily to elicit feedback on their general wellbeing. In the event that a PUS failed to reply, a second message was then sent at 10 am and a final reminder at 12 pm. Any PUS who failed to respond was contacted by the surveillance team via text messages or phone call.P <0.05. The prevalence of COVID-19 was calculated as the proportion of HCWs diagnosed as per case definition over the total number of staff in UMMC.Descriptive analysis was performed for the baseline characteristics. Pearson chi-square tests were used to compare the proportions of HCWs with and without COVID-19; Fischer\u2019s exact tests were conducted when more than 20% of the cells had expected frequencies below five [A post hoc analysis was conducted to calculate the sensitivity and specificity of the risk-based assessment in identifying cases and non-cases. All analyses were conducted using the IBM SPSS version 23 .Fig 2. From a total of 5,826 HCWs in UMMC during the study period, almost a quarter of them  were exposed to COVID-19 infection and had their risk assessed. After excluding HCWs who had \u2018no identifiable risk\u2019 and \u2018community exposure with casual contact\u2019, a total of 1,193 HCWs were placed under surveillance, of which 1,174 HCWs had work-related exposure. All 1,174 HCWs who had work-related exposure were included for analysis.The surveillance flowchart, along with the total number of HCWs included in the study, is detailed in Tables 1 and 2, respectively. Throughout the study period, a total of 17 HCWs (12 HCWs had work-related exposure and 5 HCWs had community exposure\u2013close contact) tested positive for COVID-19. This translates into a prevalence of 0.3%  COVID-19 case positivity among the HCW population in UMMC. As soon as each of these HCWs were tested positive, isolation measures were initiated, which constituted a 14-day admission to a COVID-19 designated ward regardless of the presence or absence of symptoms. Following these actions, no onward transmissions were recorded from infected HCWs. All HCWs who were placed under the surveillance program completed the stipulated surveillance duration. The mean daily response rate  was 96.3% (95% CI: 95.6\u201397.0) with a range from 88% to 100%.Baseline characteristics and surveillance outputs for all HCWs who had work-related exposure  are presented in Table 3 shows the univariable analysis for a potential association between variables of interest and case positivity of HCWs for COVID-19. There was no significant association between HCWs baseline characteristics and the positive COVID-19 test. The risk of exposure, however, was shown to be associated with positive COVID-19 tests (p<0.001). All of the 12 HCWs with work-related exposure who tested positive had at least moderate risk of exposure. Similarly, HCWs having symptoms were significantly associated with a positive COVID-19 test (p<0.001). When specific symptoms were investigated further, fever (p<0.001) and respiratory symptoms\u2013cough (p = 0.003), shortness of breath (p = 0.015) and sore throat (p = 0.002) were found to be significantly associated with case positivity while other symptoms such as arthralgia, myalgia, gastrointestinal symptoms, and anosmia had no significant association.. The ability to correctly identify HCWs who did not have COVID-19 or the specificity was around 72%. It should be noted, however, that not all HCWs in the low-risk category were tested. From the 832 HCWs in the low-risk category, only 282 HCWs (33.9%) were tested, with all of them yielding negative results. After excluding HCWs who were not tested, the specificity of this risk-based assessment was 46.1%. (= 282/612). Thus, the result needs to be interpreted cautiously.In the post hoc analysis, the risk-based assessment employed in this surveillance was found to have a sensitivity approaching 100% in identifying positive COVID-19 cases In this article, we describe how UMMC, a teaching hospital in an upper-middle-income country, adapted a surveillance system to safeguard the welfare of its HCWs against the COVID-19 pandemic. The HCW surveillance programme leveraged a risk-based testing and isolating strategy which do not require high resources for its operation.The establishment of the Operations Room represents the cornerstone of UMMCs COVID-19 prevention, containment and mitigation strategy. Throughout the 24 weeks, teams running the operations room tried to optimise the HCW surveillance system. The technological upgrade from the WhatsApp application (Phase 1) to the online portal-based surveillance system (Phase 2) was necessary to respond to the increasing number of PUS and in anticipation of the additional workload that ensued. This was because Phase 1 of the surveillance system required manual data entry that was time-consuming and laborious. Therefore, a partially automated mechanism, i.e. Phase 2 of the surveillance system, was developed by digitalising and integrating the surveillance processes into the existing UMMC portal for HCWs. This system allowed responses from the PUS to be gathered and analysed, producing a more streamlined workflow and organised database. Furthermore, the evolution from Phase 1 to Phase 2 not only improved the efficiency and sustainability of the surveillance system but was also resource-effective.The shift from a manual to an online reporting mechanism was met with some resistance from a small number of HCWs in the early stages. We received feedback that the portal-based surveillance monitoring was tedious as it required the PUS to log onto the UMMC portal and fill up the surveillance reporting form on their own. In order to promote acceptability and compliance, continuous education and training were done to familiarise users with the portal, keeping non-respondent rates at a minimal number. We also ensured that all PUS were given an adequate explanation on how and why the surveillance was done . These aEvery PUS who failed to respond to the daily surveillance messages was contacted. For PUS who did not respond for three consecutive days, their names were submitted to the hospital management for further action. The PUS compliance was further enhanced by email reminders from the Hospital Director. The effort and resources put into developing this surveillance system bridged a service gap in a time of need and serves as an investment for the future.In this study, the characteristics of the HCWs under surveillance was comparable to a study done in Wuhan whereby the younger aged population, females and also nurses predominated . The relIt is important to note the significant heterogeneity of symptoms experienced by the COVID-19 positive HCWs in different countries , 26, 27.The unique characteristic of our risk-based assessment is that the assignment of risk categories during the risk assessment is not dependent on the symptoms. This is important as there is growing evidence on asymptomatic and pre-symptomatic infection of COVID-19 . Our risGiven its feasibility and practicality, this risk-based assessment may be suitable to be implemented in healthcare settings with high numbers of COVID-19 cases and limited resources. Other low- and middle-income countries may benefit from our risk-based assessment as challenges with COVID-19 testing such as inadequate capacity, untrained laboratory personnel, and inadequate funding have been highlighted , 13. WhiThe decision to prescribe home surveillance or no work restriction is dependent on HCWs\u2019 risks. For example, HCWs with moderate and high-risk were put on home surveillance for 7 and 14 days, respectively, with specific protocol on testing for COVID-19. On the other hand, low-risk asymptomatic HCWs had no work restriction. The increased demand for workforce during this pandemic has posed challenges in the United Kingdom, with retired doctors being asked to consider returning to work and clinical staffs redeployed to areas of greatest need . During We have progressively developed a functional HCW surveillance system since the first COVID-19 case was admitted and made further modifications as the number of cases and new evidence surfaced. Besides that, we described the implementation of a HCW surveillance program and the surrounding issues and outputs that can be used to strengthen the program. Our study findings may potentially benefit other healthcare facilities where the resource for expanded screening is limited.As in any surveillance system, cases can be missed. Due to the risk-based assessment practice in our surveillance system, asymptomatic cases may have been missed, particularly for HCWs in the low-risk category. Airborne transmission of SARS-CoV-2 can occur under special circumstances, further raising the possibility of undiagnosed cases in our study . If testThe value of a well-designed surveillance programme and the importance to innovate methods in tandem with evolving situations is described in our article. The combination of contact tracing, risk assessment and symptom surveillance activities proved to be effective in containing the transmission of COVID-19 among HCWs while optimising the use of resources. Findings from our surveillance program suggest that risk-based assessment and symptom surveillance were associated with COVID-19 positivity, highlighting their roles and importance in a COVID-19 surveillance program to safeguard the health of the workforce in an upper-middle-income country.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file."}
{"text": "Gray literature from religious leaders about the Great Religions were also considered. Results from these sources contributing to the knowledge base of cultural, spiritual and religious dimensions of perinatal palliative care are considered in this paper.For perinatal palliative care (PPC) to be truly holistic, it is imperative that clinicians are conversant in the cultural, spiritual and religious needs of parents. That cultural, spiritual and religious needs for parents should be sensitively attended to are widely touted in the PPC literature and extant protocols, however there is little guidance available to the clinician as to how to meet these needs. The objective of this review article is to report what is known about the cultural, spiritual and religious practices of parents and how this might impact neonates who are born with a life-limiting fetal diagnosis (LLFD). The following religions will be considered\u2014Islam, Buddhism, Hinduism, Judaism, and Christianity\u2014in terms of what may be helpful for clinicians to consider regarding rituals and doctrine related to PPC. Data Sources include PubMed, Ovid, PsycInfo, CINAHL, and Medline from Jan 2000\u2013June 2020 using the terms \u201cperinatal palliative care,\u201d \u201cperinatal hospice,\u201d \u201ccultur Perinatal palliative care (PPC) is a specialized branch of pediatric palliative care that considers an interdisciplinary strategy for the care of newborns with life-limiting or life-threatening conditions. The World Health Organization defines palliative care as the \u201cprimary goal for the provision of a good quality of life for those with life-threatening diseases.\u201d This expands to palliative care for children and infants as a specialized field that extends to the family of children and infants. The WHO has set up principles of palliative care for children, and it includes the following points: \u201c(a) Complete care of the infant must be taken including mind, soul, and body; (b) Moral support to the family should be provided; (c) Palliative care should start when the decision for not providing any more intensive care has been made; and (d) Care should be implemented even when resources are limited\u201d .For PPC to be truly holistic, it is imperative that clinicians must be conversant in the cultural, spiritual, and religious needs of parents. The need for cultural, spiritual, and religious considerations to be sensitively attended to is widely touted in the literature on PPC and extant protocols; however, there is little guidance available to the clinician on meeting these needs.In recent times, most life-limiting fetal diagnoses (LLFD) are made during the prenatal period at a time that should represent hope and joy for parents and families. When faced with an LLFD, however, these hopes and dreams of a healthy child fade. It is at the margins of life and death that people consider the existential questions of life and their spirituality, and, at times, they may even question their religious beliefs. The death of their child will lead to maladaptive grief, long-term diminished quality of life, and symptoms linked to psychological morbidity if parents are unable to reconcile these very personal needs . ReligioWhile it is not possible for the clinician to be knowledgeable about all religious tenets and cultural norms encountered in practice, having the knowledge of the most common religions and how these religious tenets apply to infants with an LLFD or that are born at the margins of viability can assist parents greatly. Sadeghi et al.  identifiThe large variations in religious practice are known from the outset, and it is beyond the reach of this review to identify and accept all of these variations exhaustively. The objective of this review is to report on what is known about the cultural, spiritual, and religious practices of parents and how this might impact the neonates who are born with an LLFD. The following religions will be considered in terms of what may be helpful for clinicians to consider regarding rituals and doctrine related to PPC: Islam, Buddhism, Hinduism, Judaism, and Christianity.In addition, cultural and spiritual needs will be addressed.*,\u201d \u201cspiritual*,\u201d and \u201creligiou*.\u201d The inclusion criteria included all empirical and research studies published in English that focused on the cultural, spiritual, and religious needs of parents who opted to continue with their pregnancy, where the fetus had a life-limiting condition or had received PPC. Results from these studies contributing to the knowledge base of cultural and religious dimensions of PPC are considered in this review. Data sources for this review included PubMed, Ovid, PsycInfo, CINAHL, and Medline. The data from January 2000 to August 2020 were collected using the terms \u201cperinatal palliative care,\u201d \u201cperinatal hospice,\u201d \u201cculturIn addition to these published papers, sources discussing the principles of the Great Religions were also included to provide a background about religious doctrine, beliefs, and current global demographics.We live in a multicultural, multi-faith world, where religion is seemingly more important than it has ever been, with four out of five people identifying as belonging to an organized faith . ChristiReligion is somewhat simpler to define than spirituality. It considers beliefs, practices, and rituals and may also include beliefs about spirits, both good (angels) and bad (demons). Religion can be observed either publicly, as part of a community, or privately, and often as both . It is eIn a discussion paper, McGuirl and Campbell  claimed In the Southeast and East Asian community, religious and spiritual values may also directly affect the pregnancy decisions linked to genetics. Leung et al.  stated tIt is critical that clinicians are aware of the importance of inquiring about the social and religious affiliations of a patient, as these do not always correlate with the specified religious affiliation of the patient and these are important guiding principles in perinatal decision-making.Allah, translated literally as \u201cGod\u201d). Clinicians should be aware of the Five Pillars of Islam and work with families to understand how this belief may influence their decision-making when having a baby with an LLFD. For Muslims, the Five Pillars are five broad principles that allow them to live a good and responsible life. These include the following: The Declaration of Faith (Shahada); praying five times a day ; giving charity (Zakah); fasting during the month of Ramadan (Sawm); and a pilgrimage to Mecca at least once (Hajj). It is important that healthcare providers are cognizant and do not confuse Islamic traditions with cultural traditions.Islam impacts all aspects of daily living: from eating to clothing to health practices. Islam has been pursued for more than 1,400 years, originating from Mohammad in Mecca, Arabia, who is believed to be the last prophet sent by God , the pervasive belief is that curative medical interventions come from Allah, and healthcare professionals are the medium for delivering the will of Allah. The Quran provides guidance and is considered the source of healing for psychological and spiritual distress. If family members, including infants and children, require surgical or medical measures, the Quran can be used as an addendum to resolve theological, social, and cultural needs .To facilitate decision-making, rigorous ethical codes are required that adhere to the Islamic social and legal standards . When anin utero is confirmed. The infant can receive PPC if born alive , or the sum of one's actions (and inactions) in their current and previous states of existence. The sum of these karmic actions decides their fate in future existences, influencing how they will be reincarnated and what they will be reincarnated as. Karma, however, is not isolated to Hinduism and Buddhism: in America, 27% of the population were reported to have belief in reincarnation and \u201csome form of karma\u201d . Comprised of 6.9% of the global population, Buddhists recognize suffering as central to human existence. Suffering is unavoidable and life-limiting illness serves to prompt reflection on the ultimate meaning of life. Death is associated with rebirth (reincarnation), and having serene surroundings at the time of death and during the dying process is important to the dignity of the person and for their rebirth . Althougf karma\u201d . In 2014f karma\u201d .Essentially, when the death of their infant becomes imminent, Buddhist parents need to be able to focus on caring for the spiritual state of the infant rather than unnaturally prolonging the life of their child, as this will encourage a good rebirth. It is important for medication to not interfere with consciousness. When caring for Buddhist families, the clinician will benefit from demonstrating the \u201cright understanding\u201d  and an understanding of mindfulness (the whole-body-and-mind awareness of the present moment), which in turn creates good karma for the child and encourages the well-being of the child. In both its present state and as a living being, this will benefit the child in the current life and for the next life to come .Christianity is a monotheistic Abrahamic faith. Its ideology is based on the life and teachings of Jesus Christ of Nazareth, practiced by over 30% of the global population. Its followers, known as Christians, include many denominations: in the Western world, Roman Catholic; Protestant ; in the East, Eastern Catholic; Eastern Orthodox; Oriental Orthodox Church of the East (Nestorian); and in Nontrinitarian, Jehovah's Witness; Latter Day Saint; and Oneness Pentecostal.In Christianity, Jesus is considered the savior, and although beliefs vary between sub-denominations, most Christians view illness as a natural process of the body and even as a testing of their faith. While the death of an infant represents profound loss, most Christians view death as being part of the will of God . Table 4In a qualitative multicenter study, Boss et al.  describePrayer is an important component for Christians, regardless of the sub-denomination, and may be directed to one or all the holy trinity: God, the Holy Spirit, and/or Jesus Christ. Baptism, which is a Christian rite of entry into Christianity, almost always through the use of water, is another important ritual, usually performed near the time of birth. According to Birgit , the onuCaulfield et al.  have recAccess to emergency baptism services is important, because, in the early stages of grief of parents whose infant had died, 37% of the parents  said that their religious beliefs were challenged and that they questioned their faith . ParentsBrahman), but other gods and goddesses are still acknowledged by their followers. In the scriptures and wisdom heritages of the Vedic tradition, however, all of them have their individual origins  is the primary purpose. Similar to Buddhism, disease and illness are considered the result of karmic actions, and death is simply a passage because the atma has no beginning or end  affords personhood to the fetus at 180 days of gestation as the embryo begins to develop awareness and beings to feel pain (Dharmashastras (the treatises of Hinduism) view the termination of pregnancy (the \u201ckilling\u201d of a human fetus is referred to as \u201cbhrunahatya\u201d) as a sin; however, in less traditional Hindu families, termination may not be seen this way.In terms of infants born with an LLFD, the Clinicians should be aware that more importance is placed on the male child in Hinduism, given that they continue the lineage of the family, including the name. Therefore, it is more likely that the death of a male infant will be more acutely felt in traditional Hindu families than the death of a female infant . In the Rabbi. Psalms, as the last prayer of confession (vidui), are conducted at the bedside of the person , reconstructionist (an evolving civilization of the Jewish people), conservative , and orthodox . Jewish clergy are known as e person .Nefel\u201d: the same word is often applied to an abortus or an infant born with major life-incompatible defects. The word Nefel, in Jewish culture, refers to an infant who lacks sufficient hair and nail growth; is born in the eighth month of pregnancy; and survives for <30 days. Relevant social rules are different for a Nefel in Judaism compared to a well term infant ; however, nowadays this is mostly untrue. . It has been reported that Jewish parents still feel a keen sense of loss and grief when an infant dies before the thirty-day benchmark .Though used interchangeably, faith and spirituality are different from each other. Spirituality is more difficult to define because there are no common characteristics that were agreed upon. It is a broader concept that each individual defines for themselves . SpirituIn an explorative study about relevant sense-making activities, whether through prayer and/or meditation, first-time mothers established two key themes to help them deal with the life-threatening illness of their infants: (a) looking for meaning circles and (b) using intuitive/spiritual experiences to find meaning . SimilarTo help parents make meaning and meet their spiritual needs when faced with perinatal death, the parents should be encouraged to consider how their infant has touched other lives besides their own; this includes other family members, particularly grandparents, who would have had their own hopes and dreams for the child. Raingruber and Milstein  propose In 2017, Hawthorne et al. explored the disparities between gender, race/ethnicity, and religion in the use of spiritual and religious coping strategies by parents after the death of a child. The study found that non-Hispanic black mothers were more likely to use religious coping strategies than non-Hispanic white mothers. Christian parents used more religious coping practices than the other groups, and mothers reported considerably more use of religious and spiritual coping practices than fathers . This stCulture refers to the collective deposit of information, experience, opinions, principles, behaviors, meanings, hierarchies, faith, notions of time, responsibilities, spatial relationships, concepts of the world, and material objects and belongings acquired through person and community by a group of people over different periods of time .In 2008, Davies et al. conducted a survey demonstrating that almost 40% of healthcare professionals cited \u201ccultural differences\u201d as a barrier to providing palliative care. This was particularly so among ethnic minorities whose cultures were poorly understood, including Latino, Indian, and Native and African Americans. Cultural gaps extended to language barriers, religious differences, and mistrust of healthcare providers and care of health practitioners who did not have the same racial or cultural context and who did not recognize these differences . In a stNo matter how committed, highly trained, and dedicated the healthcare team is, when caring for parents and their infant with an LLFD, an expert understanding of how families function, spiritually, cross-culturally, and existentially, presents a considerable challenge in PPC.It is not surprising that families rely on their faith and spirituality in times of crisis to help them make sense of the path to the end of life. This analysis revealed why healthcare practitioners need to become familiar with the definitions of the moral and religious ideas that arise in their clinical field or specialization for parents and families. Regardless of the religious, spiritual, or cultural beliefs, all parents facing the death of their infant due to an LLFD require a compassionate, supportive, and knowledgeable healthcare service. Life and death are momentous occasions in the life cycle, and when the two occur in close proximity, beliefs and religious ceremonies become even more profound. All members of the healthcare team providing care to parents from the moment of the diagnosis through the palliation of to death of the infant require a unique skill set, including the ability to pay attention to, and the knowledge of, religious, spiritual, and cultural needs of the parents.VK conceptualized and designed the review, drafted the manuscript, and is solely responsible for data collection and interpretation of the literature data.The author declares 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": "Lower limb alignment is crucial in total knee arthroplasty (TKA). Previous studies have shown that robotics and personalized three-dimensional (3D) preoperative planning could improve postoperative alignment accuracy compared with conventional TKA, but comparison between the above two techniques has never been reported. The authors hypothesized that robotics may be superior to personalized 3D preoperative planning in terms of postoperative alignment in primary TKA, with similar patient-reported outcome measures (PROMs) but higher cost and longer operative time.A consecutive series of patients who received TKA in our center from September 2020 to January 2021 were enrolled retrospectively. After 1:2 matching, 52 and 104 patients were included and divided into study group for robotics and control group for personalized 3D preoperative planning, respectively. Multiple postoperative alignment angles were measured, and clinical features such as operation and tourniquet time, length of hospital stay and hemoglobin (Hb) were recorded. Knee Society Score (KSS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were used to evaluate clinical results.P\u2009<\u20090.05). It also had less outliers in hip\u2013knee\u2013ankle angle (HKA), FTC, lateral femoral component angle (LFC) and lateral tibial component angle (LTC) (P\u2009<\u20090.05). Hb loss of robotics group was significantly lower than control group (P\u2009<\u20090.001), while the operation and tourniquet time were longer (P\u2009<\u20090.001). There was no significant difference in KSS and WOMAC scores between two groups.Compared with control group, robotics group had significantly lower frontal femoral component angle (FFC) and frontal tibial component angle (FTC) absolute deviation (Compared with control group, patients in robotics group had significantly less malalignment, malposition, Hb loss, but similar PROMs. The operations in robotics group spent longer operation time and cost more compared with control group.Trial registration: The Chinese Clinical Trial Registry, ChiCTR2000036235. Registered 22 August 2020, http://www.chictr.org.cn/showproj.aspx?proj=59300.III.The online version contains supplementary material available at 10.1186/s13018-022-03115-3. Substantial studies have demonstrated that accurate alignment and prosthesis positioning in total knee arthroplasty (TKA) are closely related to satisfactory postoperative outcomes and prosthesis longevity \u20135. AlthoThe alignment and prosthesis positioning accuracy among navigation, PSI, robotics and conventional TKA were compared in the previous large sample meta-analysis , 14. A BTo achieve accurate alignment and prosthesis positioning with a cost-effective method, we reported previously a verified technique which used personalized 3D preoperative planning . It coulThe previous study confirmed that personalized 3D preoperative planning could improve resection accuracy compared with conventional TKA , while tMedical records and imaging data were retrospectively collected from a consecutive series of TKA performed in our center from September 2020 to January 2021. The robotics group included patients with knee osteoarthritis, who underwent TKA with the Skywalker\u2122 robotics system ; the 3D preoperative planning group included patients with knee osteoarthritis who underwent TKA with personalized 3D preoperative planning. Patients failed to achieve pre- and postoperative full-length weight-bearing radiographs (FLX) or did not meet Paley\u2019s criteria  were exc\u03b2\u2009=\u20090.95, \u03b1\u2009=\u20090.05). The power analysis by PASS 15.0 revealed that 48 in robotics group and 96 in 3D preoperative planning group would be an appropriate sample size. To reduce the influence of selection bias and potential confounding factors in this retrospective study, the gender, left or right, age, body mass index (BMI) and preoperative hip\u2013knee\u2013ankle angle (HKA) were selected to perform a 1:2 matching with the \u201cnearest\u201d method by R software . Finally, 52 robot-assisted TKAs and 104 personalized 3D preoperative planning TKAs were compared in this study.Previous literatures indicated that malalignment rates of robotics and 3D preoperative planning after TKA was about 2% and 18%, respectively , 19, 20. or the previous article [The personalized 3D preoperative planning  and Legi1 article  for moreThe Skywalker\u2122 robotics system and Advance\u00ae medial-pivot knee prosthesis  were used in robotics group. A patient-specific 3D model was formulated automatically after importing the patients' lower limb CT data into the Skywalker\u2122 robotics system. Multiple feature points were marked in the 3D model, such as the center point of femoral head, knee joint and ankle joint, the most prominent point of lateral femoral epicondyle, and the most concave point of medial femoral epicondyle. Then, the appropriate prosthesis positioning and alignment parameters were selected in preview to form a preoperative planning. During the surgery, the navigation markers made by radix lens  were installed and the patient's anatomical characteristics were registered to fit the preoperative plan. Under the help of optical measurement technology, the robotic arm with a cutting guide at the distal end automatically moved to the appropriate position and assisted the surgeons to complete accurate resection with saw.In both groups, nerve block anesthesia and medial parapatellar approach were conducted. A tourniquet was applied before skin incision and released after the closure of joint capsule. In order to reduce total blood loss, tranexamic acid was routinely used. No patella replacement was performed, and all cases were conducted following mechanical alignment. Discharge criteria included that there were no obvious swelling, no extension lag, active bending\u2009\u2265\u200990\u00b0, walking distance with assistance\u2009\u2265\u2009200\u00a0m and VAS pain score\u2009\u2264\u20094. Patients who met all the above criteria could be discharged. The hospital stay included postoperative rehabilitation programs education.2), operation time (min), tourniquet time (min), hospital stay (day) and hemoglobin (Hb) loss at 1 and 3\u00a0days after operation (g/L) were collected via the electronic medical record management system. Meanwhile, KSS and WOMAC scores were obtained preoperatively and 3, 6, 12\u00a0months after operation during outpatient follow-ups. Based on the guidance [Preoperative FLX (within 1\u00a0month before surgery) and FLX of the latest follow-up were collected. Preoperative HKA and postoperative frontal femoral component (FFC) angle, frontal tibial component (FTC) angle, lateral femoral component (LFC) angle, lateral tibial component (LTC) angle and HKA  was used to evaluate intra-rater and inter-rater consistency in FLX measurement. ICC values less than 0.5, between 0.5 and 0.75, between 0.75 and 0.9, and greater than 0.90 were indicative of poor, moderate, good and excellent reproducibility, respectively [P\u2009<\u20090.05 was considered statistically significant.The ectively . StatistAll baseline characteristics were similar between two groups after 1:2 matching (Table P\u2009<\u20090.05). The postoperative HKA and FFC were significantly different between two groups (P\u2009<\u20090.05), while the two groups had similar postoperative FTC, LFC and LTC. The two groups had similar absolute deviations from the target value in HKA, LFC and LTC, but the robotics group was significantly better in FFC and FTC absolute deviation (P\u2009<\u20090.05). Besides, the robotics group had significantly less outliers compared with the 3D preoperative planning group in HKA, FTC, LFC and LTC (P\u2009<\u20090.05)  Table .Table 2CP\u2009<\u20090.001), while the robotics group had lower Hb loss at 1 and 3\u00a0days after operation (P\u2009<\u20090.001)  and less outliers in HKA, FTC, LFC and LTC (P\u2009<\u20090.05), compared with the personalized 3D preoperative planning group  (Table The Hb loss at 1 and 3\u00a0days after operation was significantly lower in robotic group (P\u2009<\u20090.001) , which is the only prostheses that the robotics system could recognize. Although prostheses types differences had no effect on the comparison of alignment accuracy between robotics and the personalized 3D preoperative planning, this made the comparison of postoperative outcomes a little confused because various prosthesis designs might influence PROMs \u201334. SecoRobotics could significantly improve alignment accuracy, but the expensive start-up costs  discourCompared with 3D preoperative planning group, patients in robotics group had significantly less malalignment, malposition, Hb loss, but similar PROMs. The operations in robotics group spent longer operation time and cost more compared with 3D preoperative planning group.Additional file 1:\u00a0Detailed procedure of the personalized 3D preoperative planning."}
{"text": "Molecular markers play an important role in predicting clinical outcomes in pancreatic adenocarcinoma (PAAD) patients. Analysis of the ferroptosis-related genes may provide novel potential targets for the prognosis and treatment of PAAD.RNA-sequence and clinical data of PAAD was downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public databases. The PAAD samples were clustered by a non-negative matrix factorization (NMF) algorithm. The differentially expressed genes (DEGs) between different subtypes were used by \u201climma_3.42.2\u201d package. The R software package clusterProfiler was used for functional enrichment analysis. Then, a multivariate Cox proportional and LASSO regression were used to develop a ferroptosis-related gene signature for pancreatic adenocarcinoma. A nomogram and corrected curves were constructed. Finally, the expression and function of these signature genes were explored by qRT-PCR, immunohistochemistry (IHC) and proliferation, migration and invasion assays.The 173 samples were divided into 3 categories  and a 3-gene signature model  was constructed. The prognostic model showed good independent prognostic ability in PAAD. In the GSE62452 external validation set, the molecular model also showed good risk prediction. KM-curve analysis showed that there were significant differences between the high and low-risk groups, samples with a high-risk score had a worse prognosis. The predictive efficiency of the 3-gene signature-based nomogram was significantly better than that of traditional clinical features. For comparison with other models, that our model, with a reasonable number of genes, yields a more effective result. The results obtained with qPCR and IHC assays showed that ALOX5 was highly expressed, whether ALOX12 and CISD1 were expressed at low levels in tissue samples. Finally, function assays results suggested that ALOX5 may be an oncogene and ALOX12 and CISD1 may be tumor suppressor genes.We present a novel prognostic molecular model for PAAD based on ferroptosis-related genes, which serves as a potentially effective tool for prognostic differentiation in pancreatic cancer patients.The online version contains supplementary material available at 10.1186/s12935-021-02431-8. Pancreatic cancer has a low 5-year survival rate and is one of the cancers that have high mortality . There wFerroptosis, a type of cell death, plays a vital role in inhibiting tumorigenesis by removing cells that either has a deficiency or overabundance of key nutrients or cells damaged by environmental pressure . Unlike Recently, research has focused on the immune infiltration and its role in cancer. Previous studies have demonstrated that infiltrating immune cells can be isolated from tumors suggesting that tumor immune infiltration is a crucial biological process of cancers . FurtherWith the development of next-generation sequencing technology, gene transcription profiles in PAAD can be better understood. Based on The Cancer Genome Atlas (TCGA) and the comprehensive Gene Expression Omnibus (GEO) databases, many genes be found contribute to the development of PAAD. Recent studies have shown that CA9, CXCL9, and GIMAP7 genes specifically regulate the expression of FoxO1, thereby regulating immune infiltration in PAAD . FurtherIn this study, we identified ferroptosis-associated genes and constructed molecular subtypes of PAAD based on TCGA and GEO databases. Finally, we established a 3-gene signature prognostic model and verified its ability to predict the prognostic risk and immune infiltrates of PAAD. This serves as a potentially effective tool for prognostic risk prediction in patients with pancreatic cancer.Expression data and corresponding clinical follow-up information from the TCGA-PAAD data set were downloaded using the UCSC genome browser database. GSE62452 chip data sets with survival time were selected from Gene Expression Omnibus (GEO) database. The GSE62452 data set is Microarray gene-expression profiles of 69 pancreatic tumors and 61 adjacent non-tumor tissue from patients with pancreatic ductal adenocarcinoma. ([HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]). Henceforth, samples that lacked clinical follow-up information were removed and the expression of multiple gene symbols was considered the median value.Firstly, 60 ferroptosis-related genes were retrieved from the literature \u201318  method of the GSVA  package was used to identify the immune score-based relationships among the molecular subtypes in the TCGA-PAAD data set. The scores of 28 immune cells were assessed  and thenA total of 173 samples in the TCGA-PAAD data set were divided into a training set and a test set. To prevent the random allocation bias from affecting the stability of subsequent modeling, all samples were put back into random grouping, two hundred times in advance. Using the training data set, a univariate Cox proportional risk regression model was constructed for ferroptosis-related genes (n\u2009=\u200960) and survival data was constructed using the coxph function with survival R package. Since 58 ferroptosis-related genes had expression profile data in our data set, only these were selected for the univariate Cox regression analysis, and P\u2009<\u20090.05 was selected as the threshold for filtering. The R software package \u201cglmnet_4.10\u20131\u201d  was usedThe risk scores of each sample were calculated separately based on the expression level of the sample. KM-curve analysis showed significant differences between the high and low expression groups. Furthermore, we used the R software package timeROC_0.4  to conduTo identify the independence of the 3-gene signature model in clinical applications, we performed Cox regression analysis on the TCGA-PAAD training dataset. Based on the results of univariate and multivariate analyses, we used the TCGA-PAAD training dataset to construct a histogram. In addition, corrected curves were used to analyze the prediction accuracy of nomogram at 1, 3, and 5\u00a0years.PAAD tissues were derived from surgically resected specimens and snap-frozen in liquid nitrogen until RNA extraction. None of the patients received chemotherapy or radiation therapy before surgery. All patients signed informed consent forms provided by the Eastern Hepatobiliary Surgery Hospital. This study was approved by the Ethics Committee of the Eastern Hepatobiliary Surgery Hospital.RNA was extracted from tissues using the TRIzol reagent  and was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit . Quantitative PCR (qPCR) uses real-time fluorescence to measure the quantity of DNA present at each cycle during a PCR. Real-time qPCR analyses were quantified with SYBR-Green , and expression levels were normalized to GAPDH levels.Immunohistochemistry was performed by two-step method according to the instructions . Pancreatic cancer samples were fixed in 10% formalin, embedded in paraffin, and processed into 5-\u00b5m sequential sections. The samples were de-waxed with ethanol and blocked to inhibit the endogenous peroxidase activity. After this, samples were heated in a microwave for antigen retrieval, cooled to room temperature, and blocked using goat serum for 30\u00a0min at 37\u00a0\u00b0C. The samples were incubated overnight at 4\u00a0\u00b0C with rabbit anti-ALOX5 (ab169755), anti-ALOX12 (ab211506), and anti-CISD1 (ab203096)  (1:200), followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody  at 37\u00a0\u00b0C for 30\u00a0min. The samples were then stained with 3,3\u2032-Diaminobenzidine (DAB). Cell nuclei were stained blue with hematoxylin. The sections were then dehydrated, cleared with xylene, and mounted. ALOX5, ALOX12, and CISD1 expressions were determined by immunohistochemistry (IHC) using the streptavidin peroxidase method, with adjacent tissues serving as the controls. The experimental procedure was performed as per the manufacturer\u2019s instructions. Image-Pro Plus 6.0 Software  was used to analyze protein expression and perform statistical analysis of the results obtained by IHC.2 in a humidified incubator. The Panc 02.03 cell lines are derived from human primary pancreatic cancer and are epithelial-like cells. The Panc 02.03 cell lines were cultured in RPMI-1640  with 15% fetal bovine serum at 37\u00a0\u00b0C under 5% CO2 in a humidified incubator. Si-ALOX5 (No: CAT#: SR319325) was purchased from Origene . Transfection was performed using Lipofectamine 3000 reagent  according to the instructions and cell transfection efficiency was 82%.The human ALOX12 and CISD coding sequences were cloned into the pEZ-M03 Vector.The human PAAD cell line T3M4 and Panc 02.03 were provided by the National Collection Authenticated Cell Cultures . The T3M4 cell lines are derived from the metastatic lymph node tissue of human pancreatic cancer and are epithelial-like cells. The T3M4 cell lines were cultured in DMEM (Dulbecco\u2019 modified eagle medium) . Supplemented with 10% fetal bovine serum  at 37\u00a0\u00b0C under 5% COThe si-ALOX5 transfected in T3M4 cell lines, and ALOX12 or CISD transfected in Panc 02.03 cell lines. Forty-eight hours post infection, the cells were collected and seeded into 96-well plates at a concentration of 2000 cells per well. Cell viability was detected by Cell Counting Kit-8 assay  according to the manufacturer\u2019s protocol after 48\u00a0h. Te absorbance at 450\u00a0nm was measured using an automatic microplate reader . All Cell Counting Kit-8 assay were performed in five times.5 cells were added in the upper chamber. The cells were harvested, resuspended in serum-free media and placed into the upper chamber of a Transwell membrane filter  for the migration assays or in the upper chamber of a transwell membrane filter coated with Matrigel (Corning) for the invasion assays.The T3M4 and Panc 02.03 cells were added in the upper chamber. After incubation for 24\u00a0h at 37\u00a0\u00b0C, the Transwell chamber was removed. The cells were stained with methanol and 0.1% crystal violet, imaged, and the relative cell density was measured by ImageJ . ImageJ software was used to analyze and calculate the migration and\u00a0invasion area of the cells. The migration and\u00a0invasion index (%) depicts a proportion of an area where cells have invaded in percentage and is calculated as epithelium area divided by the total area of invaded cells area. The area describes the overall area (\u03bcm2) of invaded cells. Evaluation of invasive capacity was performed by counting invading cells under a microscope (40\u2009\u00d7\u200910). Five random fields of view were analyzed for each chamber. All cell migration and\u00a0invasion assays were performed in five times.The si-ALOX5 transfected in T3M4 cell lines, and ALOX12 or CISD transfected in Panc 02.03 cell lines. Forty-eight hours post infection, the cells were collected. For the migration assay, 800\u00a0\u03bcl DMEM with 20% serum was added to the lower chamber of a Transwell plate , and 1.5\u2009\u00d7\u200910All data were analyzed using the SPSS 21.0 statistical software program . Graphs were generated with GraphPad Prism 8.0 software . Student\u2019s t-tests were performed. For a two-tailed t-test, P\u2009<\u20090.05 was essential for considering the results to be statistically significant.After data preprocessing, there were 173 samples in TCGA-PAAD, with 88 samples in the training set and 85 samples in the test set. GSE62452 included 66 samples. The clinical statistical information of the samples is shown in Table The 173 samples were divided into 3 categories: C1, C2, and C3. The expression of prognostic ferroptosis-related genes in the three categories is shown in Fig.\u00a0With the help of the limma_3.42.2 package, 4,903 DEGs were identified between Cluster 1 and Cluster 2, among which 2,717 genes were up-regulated and 2,186 genes were down-regulated  (cell markers are from the reference ) and theThe R package survival coxph function is used to perform a univariate Cox proportional hazard regression model for ferroptosis-related genes (n\u2009=\u200960) and survival data in training set. Because only 58 ferroptosis-related genes are expressed in our expression profile data and we selected these genes for single-factor cox regression analysis, and selected P\u2009<\u20090.05 as the threshold for filtering. Finally, there were 10 genes related to prognosis. Next, LASSO regression was used to further compress the 10 genes to reduce the number of genes in the risk model. The changing trajectory of each independent variable is shown in Fig.\u00a0RiskScore\u2009=\u20090.289* ALOX5\u2009+\u2009(\u2212\u20091.359)*ALOX12\u2009+\u2009(\u2212\u20091.053)* CISD1.Further, we calculated the risk score of each sample based on the expression level of the sample. In TCGA-training set, KM-curve analysis showed that there were significant differences between the high and low-risk groups Fig.\u00a0C. The riAnalysis of the GSE62452 showed that the risk score distribution was consistent with that of the training set and that the high-risk score samples had a worse prognosis . It is the link that connects metabolism, redox biology, and human health. Accumulating evidence indicates that ferroptosis can be triggered to treat cancer to eradicate aggressive malignancies that are resistant to conventional therapies . It is cDue to the insidious and aggressive nature of PAAD, it is difficult to detect and prevent PAAD at an early stage. At the time of consultation, approximately 80% of patients have locally advanced or metastatic cancer . AlthougThe signature we constructed contained three genes, namely, ALOX5, ALOX12, and CISD1, all of which are closely related to tumors genesis and development. 5-Lipoxygenase (ALOX5) is a non-heme iron-containing dioxygenase that catalyzes the peroxidation of polyunsaturated fatty acids, such as arachidonic acid . ALOX5 iIn this study, we present evidence that ALOX5, ALOX12, and CISD1 may have prognostic value in PAAD. Specifically, our in vitro results suggest that ALOX5 may be an oncogene, while ALOX12 and CISD1 may be tumor suppressor genes in PAAD. The results of our study indicate that the established 3-gene signature model could be an effective prognostic tool for patients with PAAD. However, the limitations associated with this study emphasize the need for additional analysis before the clinical application of this signature. The samples used in our study were obtained retrospectively and the study solely focused on the prognostic value and clinical significance of ferroptosis. To validate our findings for clinical applications, we need to include prospective samples and evaluate the prognostic values of other biological processes characteristic to the development of cancer.In conclusion, we propose a 3-gene signature  predictive model based on ferroptosis-related genes in PAAD. Despite the many drawbacks of the current analysis, this model may serve as an interesting molecular diagnostic tool to assess the prognosis and possible risk factors of PAAD.Additional file 1.  Different expressed genes (DEGs) between three clusters.Additional file 2.  KEGG pathway analysis and GO functional enrichment analysis."}
{"text": "Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6)/JUMONJI DOMAIN-CONTAINING PROTEIN 12 demethylates H3K27me3 by recognizing a specific DNA motif. However, little is known about how REF6 activates target gene expression after recognition, especially in environmental responses. In response to warm ambient temperature, plants undergo thermomorphogenesis, which involves accelerated growth, early flowering and changes in morphology. Here we show that REF6 regulates thermomorphogenesis and cooperates with the transcription factor PHYTOCHROME INTERACTING FACTOR 4 to synergistically activate thermoresponsive genes under warm ambient temperature. The ref6 loss-of-function mutants exhibited attenuated hypocotyl elongation at warm temperature, partially due to downregulation of GIBBERELLIN 20-OXIDASE 2 and BASIC HELIX-LOOP-HELIX 87. REF6 enzymatic activity is necessary for warm ambient temperature responses. Together, our results provide direct evidence of an epigenetic modifier and a transcription factor working together to respond to the environment.Dynamic trimethylation of histone H3 at Lys27 (H3K27me3) affects gene expression and controls plant development and environmental responses. In  Arabidopsis, the main H3K27me3 demethylases are EARLY FLOWERING 6 (ELF6/JMJ 11) [H3K27me3, a conserved and facultative repressive histone mark found in euchromatin, is crucial for tissue-specific gene expression and developmental regulation in multicellular eukaryotes ,2. The d/JMJ 11) , JMJ13 [/JMJ 11)  and JMJ3/JMJ 11) ,11. ThesFLOWERING LOCUS C by the transcription factor BRASSINAZOLE-RESISTANT1 [Despite their functional redundancy, these H3K27me3 demethylases have distinct targeting mechanisms and biological functions . For insSISTANT1 , where iSISTANT1  and prevSISTANT1 ,14; DNA SISTANT1 ,16. REF6SISTANT1 , organ bSISTANT1 , leaf seSISTANT1  and seedSISTANT1 . InteresSISTANT1 , abscisiSISTANT1 , ethylenSISTANT1  and brasSISTANT1 . HoweverPlants have evolved diverse strategies to respond to natural environmental fluctuations ,24. AmbiYUCCA 8, TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 and CYTOCHROME P450 FAMILY 79B) [DWARF4 and BRASSINOSTEROID-6-OXIDASE 2) [FT promoter to mediate flowering responses [SPEECHLESS to restrict stomatal development under warm conditions [The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts as a central regulatory hub in thermomorphogenetic responses . PIF4 meILY 79B) ,30 and BIDASE 2)  to mediaesponses , and binnditions . In addinditions ,27.Arabidopsis [FLOWERING LOCUS C promoter [Multiple regulatory mechanisms are involved in the extensive network of plant thermoresponsive growth, including transcriptional regulation, RNA metabolism and protein stability . Among tbidopsis ,38. The bidopsis . JMJ13 abidopsis . JMJ30 ppromoter , and funpromoter .These reports revealed that histone methylation controls the balance between vegetative and reproductive growth at warm ambient temperature . Howeverref6 mutants is insensitive to warm ambient temperature. We then used hypocotyl elongation as a system to investigate the function of REF6 in targeting, H3K27me3 demethylation and activation of target genes. Three-day-old seedlings grown at 22\u00b0C were transferred to 22\u00b0C or 28\u00b0C for 3 days in long-day conditions  and REF6 complementation lines , were recorded after 3 days of treatment at 22\u00b0C or 28\u00b0C  at 22\u00b0C, the mutants exhibited markedly attenuated responses at 28\u00b0C compared with the wild-type seedlings . REF6-HA transgenic lines rescued the attenuated hypocotyl elongation of the ref6 mutants at 28\u00b0C -specific demethylases, namely JMJ11/ELF6 ,13, JMJ1REF6 expression and REF6 targeting activity at warm ambient temperature. The mRNA levels of REF6, as measured by reverse transcription-quantitative Polymerase Chain Reaction (PCR) (RT-qPCR), did not change at 22\u00b0C and 28\u00b0C , suggesting that REF6 expression is not induced by warm ambient temperature. To test whether elevated temperature affects the recruitment of REF6 to its target genes, we profiled the genome-wide localization of REF6 in wild-type Col using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) with anti-REF6 antibody [ref6-5 mutant was included as a negative control. Two biological replicates of REF6 ChIP-seq showed a high correlation  with each other . A total of 1954 and 2136 peaks covering 1834 and 1981 genes were bound by REF6 at 22\u00b0C and 28\u00b0C, respectively. These were highly correlated (r\u00a0=\u00a00.98) and overlapped \u00a0with each other . These results demonstrated that REF6 transcript levels, REF6 targeting and REF6 enzymatic activity are temperature insensitive.We then checked the H3K27me3 levels in wild-type Col and ref6 mutant influence target gene expression at warm temperatures. We therefore carried out an RNA sequencing (RNA-seq) analysis, using RNA from seedlings of wild-type Col and ref6-5 mutant treated at 22\u00b0C and 28\u00b0C (Supplementary Table 3). A total of 74 million (79%), 76 million (80%), 77 million (82%) and 76 million (82%) clean reads of highly correlated RNA-seq replicates were uniquely aligned to the Arabidopsis reference genome, and were mapped to 17\u2009092, 16\u2009981, 17\u2009189 and 17\u2009127 genes in the wild-type Col and ref6-5 mutants at 22\u00b0C and 28\u00b0C, respectively Reads Per Kilobase Million (RPKM\u00a0>\u00a01) (Supplementary Table 3). RNA-seq data from three replicate samples showed high correlation .Because REF6 is an H3K27me3 demethylase and H3K27me3 is associated with gene repression, we next investigated the relationship between REF6 targeting and gene activation, and whether the increased H3K27me3 levels in the ref6-5 mutants at 22\u00b0C and 28\u00b0C . For this analysis, we first calculated the fold change of transcript levels comparing 28\u00b0C to 22\u00b0C in wild-type Col and ref6 mutants  in wild-type Col at 28\u00b0C compared with 22\u00b0C, and inefficiently induced in ref6 mutants  encodes an enzyme that is involved in GA biosynthesis and temperature responses [BASIC HELIX-LOOP-HELIX 87 (bHLH87) was reported as a PIF4 downstream target in Arabidopsis [GA20ox2 and bHLH87 were upregulated by elevated temperature in wild-type Col, but were inefficiently induced in ref6-5 mutants with high levels of H3K27me3  with an independent batch of ChIP samples yielded consistent results . Evaluation of hypocotyl elongation at warm temperature in the wild type, double mutants and the corresponding parental lines showed that both ga20ox2 and bhlh87 are thermo-insensitive, similar to the ref6-5 mutant  that abolishes its enzymatic activity  [REF6 transcript levels comparable to those of wild-type Col were chosen for further analysis . Phenotypic analysis showed that REF6 H246A-HA ref6-1 transgenic lines did not rescue the attenuated hypocotyl elongation of the ref6 mutants at 28\u00b0C  . Transge8\u00b0C Fig.\u00a0A and B, bHLH87 and GA20ox2 . These results indicate that REF6 and PIF4 cooperate with each other to regulate some thermoresponsive genes.The bHLH transcription factor PIF4 accelerates hypocotyl growth at warm temperatures and acts as a central regulatory hub in ambient-temperature signaling, in which various signaling pathways modulate PIF4 activity, and PIF4 integrates hormone signaling in thermoresponsive regulation of growth ,26,28. BbHLH87, we performed electrophoretic mobility shift assays (EMSAs) using DNA fragments from bHLH87 that contain the CATATG and CTCTGTTT motifs . The DNA probes containing the CATATG motif and CTCTGTTT motif were incubated with maltose-binding-protein (MBP)-tagged PIF4 (PIF4-MBP) and Glutathione-S-transferase (GST)-tagged C-terminal REF6 (GST-REF6C). Consistent with the ChIP-seq data, PIF4 and REF6 can directly bind to bHLH87 DNA probes . Genetic analysis showed that pif4-101 ref6-5 plants displayed similar phenotypes to pif4-101 plants . Thermoresponsive hormone signaling in different plant organs should be coordinated to facilitate synchronized growth and acclimation to elevated temperatures [Plants actively sense temperature and, under warm ambient temperatures, they promote the biosynthesis and localization of signaling molecules, such as auxin, BR and GA, to optimize plant morphology in a coordinated manner ,47. Our bHLH87 locus under elevated temperature suggest that bHLH87 activation is regulated by both H3K27me3 demethylation and the thermoresponsive transcription factor PIF4, but neither REF6 nor PIF4 alone can activate bHLH87 expression  and DNA methylation ,16. Hereion Fig.\u00a0. The tar2/sec. Seedlings from each sample were scanned using ZEISS SteREO Discovery V20, and the hypocotyl length was measured using ImageJ software .Surface-sterilized seeds were sown on half-strength Murashige and Skoog (MS) medium supplemented with 0.8% (w/v) agar and 1% (w/v) sucrose. The plates were stratified at 4\u00b0C in darkness for 3 days, then transferred into a growth chamber  and germinated at 22\u00b0C for 3 days, then transferred to 28\u00b0C (treatment) or maintained at 22\u00b0C (control) at ZT0 (Zeitgeber Time) for another 3 days under a 16-h light/8-h dark cycle. The plants were grown under white fluorescent light with a fluence rate of \u223c75 \u03bcmol/mAbout 2.5\u00a0g of seedlings was used for ChIP assay, as previously described ,16, usinArabidopsis thaliana TAIR10 reference genome using Bowtie2 (version 2.2.1) [https://deeptools.readthedocs.io/en/develop/) with the \u2018\u2013normalizeUsing RPKM \u2013extendReads 200 \u2013binSize 50\u2019 options. Then the output BigWig files were visualized using the Integrative Genomics Viewer [All sequencing reads of ChIP-seq were aligned to the n 2.2.1)  with defn 2.2.1) . After fn 2.2.1) , only uns Viewer . MACS2 vs Viewer  was useds Viewer . Then ths Viewer  when a sq-value <\u00a00.05, log2 (fold change) >\u00a02.For RNA-seq data, all reads were mapped to the TAIR10 reference genome using HISAT2 (version 2.0.5)  with defChIP-seq and RNA-seq data from this article can be found in the Gene Expression Omnibus data library under accession number GSE181292.nwab213_Supplemental_FilesClick here for additional data file."}
{"text": "Based on detailed investigation of the reaction mechanism with several Tz, revealing up to 96\u2009% elimination after 2\u2005hours, we have developed a TCO\u2010caged prodrug with 750\u2010fold reduced cytotoxicity compared to the parent drug and achieved in\u2005situ activation upon Tz/TCO click\u2010to\u2010release.Bond\u2010cleavage reactions triggered by bioorthogonal tetrazine ligation have emerged as strategies to chemically control the function of (bio)molecules and achieve activation of prodrugs in living systems. While most of these approaches make use of caged amines, current methods for the release of phenols are limited by unfavorable reaction kinetics or insufficient stability of the Tz\u2010responsive reactants. To address this issue, we have implemented a self\u2010immolative linker that enables the connection of cleavable  Bioorthogonal activation of caged phenols enables strategies to control the function of (bio)molecules in biological environments. While previous methods have been limited by slow click kinetics or insufficient stability, we show that introduction of a self\u2010immolative linker affords stable trans\u2010cyclooctene conjugates, as demonstrated by the design of a prodrug that upon bioorthogonal reaction with tetrazines leads to >200\u2010fold cytotoxicity. Biocompatible bond\u2010cleavage reactions have expanded the repertoire of bioorthogonal tools substantially, providing methods beyond chemical ligation to control the function of (bio)molecules in biological environments and living systems.trans\u2010cyclooctenes (TCO) triggered by click reaction with 1,2,4,5\u2010tetrazines (Tz) has gained significant attention due to exceptional reaction kinetics and molecular versatility.et\u2005al., TCO modified with a carbamate moiety in an allylic position  undergoes a 1,4\u2010elimination upon inverse electron demand Diels\u2010Alder (IEDDA) reaction with Tz and subsequent post\u2010click tautomerization, ultimately leading to the click\u2010triggered release of an amine or amino\u2010functionalized molecule (click\u2010to\u2010release).2) of less than 0.001\u2005M\u22121\u2009s\u22121 have been observed for the IEDDA reaction of vinyl ethers and Tz.et\u2005al. have developed rTCO\u2010ethers and rTCO\u2010carbonates that react substantially faster with Tz (k2>10\u2005M\u22121\u2009s\u22121), leading to click\u2010triggered release of alcohols and phenols .The bioorthogonal elimination of N,N\u2019\u2010dimethylethylenediamine (DMEDA) can be used as a minimal SIL to achieve bioorthogonal elimination of phenols  that enables the use of carbamate linkages instead of ethers and carbonates. Here, we show that s Figure\u2005c. DependN\u2010methyl substituent with other functionalities moreover allows further modification, as very recently applied for the development of an rTCO\u2010caged fluorogenic resorufin derivative.DMEDA is commonly used as a self\u2010immolative linker or spacer in the design of cleavable and/or activatable compounds such as drug conjugates.p\u2010nitrophenyl (PNP) carbonate rTCO\u2010PNP (1) was treated with an excess of DMEDA and (following evaporation to afford crude intermediate 2) reacted with the PNP\u2010activated tyrosine\u2010BODIPY conjugate 3 to obtain the rTCO\u2010DMEDA\u2010caged compound 4 in an overall yield of 68\u2009%. Saponification and subsequent PEGylation of the intermediate acid 5 via HBTU\u2010mediated amide coupling yielded rTCO\u2010DMEDA\u2010Tyr\u2010BODIPY (6) with a yield of 76\u2009% over 2 steps  fluorophore that allows detection and relative quantification of all intermediates and products by HPLC using its characteristic absorption at a wavelength of 500\u2005nm.6 triggered by reaction with Tz was then studied by HPLC\u2010MS. According to the current mechanistic understanding,A that can tautomerize to the releasing 1,4\u2010DHP isomer B1 and the non\u2010releasing 2,5\u2010DHP isomer B2 ,[2 (8),9)6  of <100\u2005M\u22121s\u22121 have been reported for the click reaction of rTCO with DMT (7) and PA2 (8), pyrimidyl\u2010alkyl\u2010Tz such as 9 are known to be substantially more reactive, reaching k2 of >1000\u2005M\u22121\u2009s\u22121 (see Supporting Information for k2 determined in PBS at 37\u2009\u00b0C).[Click\u2010to\u2010release of 2 Figure\u2005a. The N\u2010x Figure\u2005a. Based igure\u20057),7, 14 6 Figure\u2005b. While pseudo\u2010release\u2019 that impedes accurate reaction monitoring.6 with Tz 7 and 8 in PBS at 37\u2009\u00b0C. Despite a tailing peak for the SIL\u2010intermediate S, all reactants, tautomers, and products could be separated and identified , the non\u2010releasing tautomer B2, and the SIL\u2010intermediate S (as the product of TCO\u2010release) that upon self\u2010immolation afforded phenol P with an overall yield of 89\u2009% after a reaction time of approx. 8\u2005hours. In agreement with our previous findings,B1 was found due to rapid 1,4\u2010elimination of this labile intermediate. The respective pyridazine dead\u2010end Ox was identified as the only non\u2010releasing byproduct  leading to \u20182 (8) is the only Tz reported so far that allows efficient release (>90\u2009%) of primary amines.A and shift the equilibrium to B1/B2, as shown for the reaction of 6 and 8  in Figure\u2005A was not detectable. Enhanced tautomerization leads to formation of instantaneously releasing B1 and slowly oxidizing B2, which can still tautomerize to B1 (via A), thereby enabling efficient release. In agreement with these mechanistic considerations, we observed 92\u2009% release of the uncaged phenol P and 8\u2009% of the respective oxidized dead\u2010end   and rTCO leads to formation of two initial click products  is either on the same (head\u2010to\u2010head) or the opposite side (head\u2010to\u2010tail) of the leaving group in allylic position of the TCO [3+ moiety) formed upon reaction of 6 with PymK (9) without provoking any pseudo\u2010release (for details see the Supporting Information). As expected, we observed immediate post\u2010click tautomerization (no A detected) and 1,4\u2010elimination (no B1 detected) leading to formation of the SIL\u2010intermediate S and stable B2 in a ratio of 7/3, which confirms favored click orientation with NH3+ in head\u2010to\u2010head position , revealing a first\u2010order rate constant (k1) of 1.0\u2005h\u22121 for the cyclization of the DMEDA\u2010carbamate and a respective half\u2010life (t1/2) of 42\u2005min .In contrast to the symmetric tetrazines s Figure\u2005a. DependO Figure\u2005b. Simila2 Figure\u2005a. Ammonie Figure\u2005c. As a rn Figure\u2005d. The unn Figure\u2005d, which CA4, Figure\u2005CA4 have motivated the development of several therapeutic strategies, in particular by applying prodrugs such as CA4\u2010phosphate  to overcome the poor water\u2010solubility of the parent drug.To demonstrate application of the Tz/TCO\u2010DMEDA strategy for the bioorthogonal release of phenols, we designed a prodrug of the anti\u2010mitotic and anti\u2010angiogenic agent combretastatin A\u20104 (CA4 was enabled by PNP\u2010activation of the free phenolic OH\u2010group yielding PNP\u2010CA4 (10)  was first treated with an excess of DMEDA and then reacted with PNP\u2010CA4 (10). Saponification of the methyl ester followed by HBTU\u2010mediated coupling of the intermediate acid with 2\u2010aminoethanesulfonic acid (taurine) afforded sulfo\u2010cTCO\u2010DMEDA\u2010CA4 (12) in an overall yield of 34\u2009%  Figure\u2005a. To fac2 Figure\u2005b. To inc% Figure\u2005b.12 by reaction with tetrazines 7 and 9 was monitored by HPLC\u2010MS as described above. The relative abundance of all intermediates and products was calculated based on the UV absorbance at 254\u2005nm and confirmed via quantification of released CA4 by external calibration (see Supporting Information). In agreement with data previously reported,12 with DMT (7) resulted in 96\u2009% release of CA4 already after 2\u2005hours, reaching an endpoint of 98\u2009% CA4 and 2\u2009% of the respective oxidized dead\u2010end after a total reaction time of approx. 4\u2005hours  did not lead to favored formation of the initial click product with the NH3+\u2010substituent (K) in head\u2010to\u2010head position, as indicated by an overall CA4 release of 50\u2009%. Accordingly, 50\u2009% of non\u2010releasing stable B2 with K in head\u2010to\u2010tail position was detected , which we hypothesize is due to CA4 being a better leaving group than the tyrosine\u2010BODIPY conjugate P  was needed to reduce the cell count by 50\u2009% (IC50) over the course of a 72\u2005h treatment . In situ reaction of 12 with DMT (7) and PymK (9) at a non\u2010toxic Tz concentration of 5\u2005\u03bcM restored drug cytotoxicity via Tz\u2010triggered bioorthogonal release of CA4, shifting the IC50 from 900\u2005nM to 4.1\u2005nM (220\u2010fold) and 6.4\u2005nM (140\u2010fold), respectively  alone, only very low signal was detected upon bioorthogonal release of CA4 via in\u2005situ reaction of both compounds , matching the CA4 control and its expected impact on tubulin polymerization.Based on these results we investigated the cytotoxicity of prodrug t Figure\u2005d, demons2H, NH3+) and their effect on post\u2010click tautomerization and 1,4\u2010elimination. We achieved >95\u2009% release of the phenolic drug CA4 upon Tz\u2010triggered cleavage of prodrug 12 in PBS within 2\u2005hours. Notably, we have observed a 750\u2010fold lower cytotoxicity of the prodrug compared to CA4, also demonstrating high stability of TCO\u2010DMEDA\u2010phenol conjugates. In situ reaction of 12 with Tz confirmed efficient bioorthogonal release of CA4 resulting in a substantially (up to 220\u2010fold) increased cytotoxicity. Based on these results, we aim to combine TCO\u2010DMEDA\u2010caging with next\u2010level chemical tools for bioorthogonal bond\u2010cleavage to control post\u2010click tautomerization and elimination, thereby accelerating the Tz/TCO click reaction while achieving fast and complete release of phenolic drugs.In summary, we show that DMEDA can be applied as a minimal self\u2010immolative linker for the design of bis(carbamate)\u2010linked TCO\u2010caged phenols that can be cleaved and thereby activated by bioorthogonal reaction with tetrazines. The mechanism of the overall bond\u2010cleavage process upon IEDDA\u2010ligation with selected symmetrical and unsymmetrical Tz was investigated in detail, with emphasis on Tz\u2010substituents modified with directing groups  for preparative RP\u2010HPLC or a Luna 10\u2005\u03bcm Silica (2) 100\u2005\u00c5, LC Column 250\u00d721.2\u2005mm (Phenomenex) for preparative NP\u2010HPLC purifications. Silica gel 60 (40\u201063\u2005\u03bcm) was purchased from Merck. HPLC\u2010MS (LCMS) analysis was performed on a Nexera X2 system (Shimadzu) comprised of LC\u201030AD pumps, a SIL\u201030AC autosampler, a CTO\u201020AC column oven, and a DGU\u201020\u2005A5/3 degasser module. Detection was done using an SPD\u2212M20\u2005A photo diode array, an RF\u201020Axs fluorescence detector, an ELS\u20102041 detector (JASCO) and an LCMS\u20102020 mass spectrometer (ESI). All separations were performed using a Waters XSelect CSH C18 2.5\u2005\u03bcm (3.0\u00d750\u2005mm) column XP at 40\u2009\u00b0C and a flowrate of 1.7\u2005mL/min. Used gradients : Acidic HPLC conditions (acetonitrile/0.1\u2009% formic acid): 0\u2005min, 5\u2009% \u2013 0.15\u2005min, 5\u2009% \u2013 2.20\u2005min, 98\u2009% \u2013 2.50\u2005min, 98\u2009%; buffered HPLC conditions : 0\u2005min, 5\u2009% \u2013 0.15\u2005min, 5\u2009% \u2013 2.2\u2005min, 98\u2009% \u2013 2.5\u2005min; 98\u2009%. 1H and 13C NMR spectra were recorded on a Bruker Ascend 600\u2005MHz spectrometer at 20\u2009\u00b0C. Chemical shifts (\u03b4) are reported in ppm relative to tetramethylsilane and calibrated using solvent residual peaks. HRMS analysis of aqueous or acetonitrile solutions of the compounds (sample concentration: 10\u2005ppm) was carried out on an Agilent 6545 Q\u2010TOF mass spectrometer equipped with an Agilent Dual AJS ESI source. The mass spectrometer was connected to a liquid chromatography system comprised of an Agilent G7167B multi sampler, an Agilent G7120\u2005A binary pump with degasser and an Agilent G7116B oven (Agilent). A SecurityGuard Cartridge (Phenomenex) was used as a stationary phase. Data evaluation was performed using Agilent MassHunter Workstation Qualitative Analysis 10.0. Stopped\u2010flow measurements were performed using an SX20\u2010LED stopped\u2010flow spectrophotometer (Applied Photophysics) equipped with a 535\u2005nm LED to monitor the characteristic tetrazine visible light absorbance (520\u2010540\u2005nm).Synthesis: Detailed experimental procedures for the synthesis of compounds 2\u20136, 10\u201312 and further intermediates are provided in the Supporting Information, including compound characterization data .Click\u2010to\u2010release monitoring (HPLC): Click\u2010to\u2010release reactions were performed in HPLC vials at 37\u2009\u00b0C using the temperature\u2010controlled HPLC autosampler . For acidic HPLC conditions, the aqueous solvent was prepared by addition of 2.5\u2005mL of formic acid to 2.5\u2005L of HPLC\u2010water to yield a final concentration of 0.1\u2009% formic acid. For buffered HPLC conditions, the aqueous solvent was prepared by addition of 625\u2009\u03bcL of 10\u2005M ammonium formate  to 2.5\u2005L of HPLC\u2010grade water followed by adjusting the pH to 8.4 by addition of 25\u2009% aqueous ammonia . Since its pH declines over time, this volatile buffer was freshly prepared each day. HPLC\u2010grade acetonitrile was used without any additives. Stock solutions of rTCO\u2010DMEDA\u2010Tyr\u2010BODIPY (6) and sulfo\u2010cTCO\u2010DMEDA\u2010CA4 (12) were prepared at a concentration of 20\u2005mM in DMSO. Tetrazine stock solutions of DMT (7), PA2 (8) and PymK (9) were prepared at a concentration of 10\u2005mM in DMSO.6): The stock solution of 6 was diluted with PBS to a concentration of 100\u2005\u03bcM in an LCMS vial . Tetrazine stock solutions were diluted with PBS to a concentration of 200\u2005\u03bcM in an LCMS vial , and the click\u2010to\u2010release reaction was initiated by addition of the diluted TCO solution (125\u2005\u03bcL) to obtain starting concentrations of 50\u2005\u03bcM TCO and 100\u2005\u03bcM Tz. The samples were immediately incubated at 37\u2009\u00b0C in the autosampler and subjected to serial HPLC analysis. All measurements were conducted in triplicates.rTCO\u2010DMEDA\u2010Tyr\u2010BODIPY (12): The stock solution of 12 was diluted with DMSO  to give a 10\u2005mM stock solution. Tz stock solution (10\u2005\u03bcL) was added to PBS , and the click\u2010to\u2010release reaction was initiated by addition of the stock solution of 12 (5.24\u2005\u03bcL) to obtain starting concentrations of 50\u2005\u03bcM TCO and 100\u2005\u03bcM Tz (in 10\u2009% DMSO/PBS). The samples were immediately incubated at 37\u2009\u00b0C in the autosampler and subjected to serial HPLC analysis. All measurements were conducted in triplicates.sulfo\u2010cTCO\u2010DMEDA\u2010CA4 : An approx. 20\u2005mM stock solution of 4rTCO\u2010PEG7) (extinction coefficient 510\u2005M\u22121\u2009cm\u22121 at 520\u2005nm), quantifying the decrease in tetrazine absorbance upon reaction with TCO. The initial DMSO stock was diluted with PBS to prepare solutions for stopped\u2010flow analysis at a final TCO concentration of 1\u2005mM. Stock solutions (20\u2005mM) of DMT (7), PA2 (8), and PymK (9) in DMSO were prepared. Serial dilution into PBS gave solutions for stopped\u2010flow analysis at a Tz concentration of 100\u2005\u03bcM. The reagent syringes of the stopped\u2010flow spectrophotometer  were loaded with tetrazine and TCO solutions and the instrument was primed. Stopped\u2010flow measurements were done in sextuplicate for each Tz. Reactions were conducted at 37\u2009\u00b0C and recorded automatically at the time of acquisition. Data sets were analyzed by fitting an exponential decay using Prism 6 (GraphPad) to calculate the observed pseudo\u2010first\u2010order rate constants that were converted into second\u2010order rate constants  by dividing through the TCO concentration.Cell viability assays: HT1080 human fibrosarcoma cells (ATCC) were cultivated in EMEM  supplemented with 10\u2009% fetal bovine serum and 1\u2009% antibiotic/antimycotic solution  at 37\u2009\u00b0C and 5\u2009% CO2. HT1080 cells were seeded into 96\u2010well plates (triplicates for each group) at 10.000 cells per well and allowed to grow overnight. The medium was removed and a dilution series of sulfo\u2010cTCO\u2010DMEDA\u2010CA4 (12) or the parent drug CA4 in growth medium  was added to the cells . For release experiments the same concentrations of 12 were used, while a stock solution of DMT (7) or PymK (9) was added to obtain final concentration of 5\u2005\u03bcM of the respective tetrazine. Incubation was carried out for 72\u2005h. Cell viability was assessed by replacing the medium with 100 \u03bcL of PrestoBlue solution  followed by incubation for 30\u2005minutes at 37\u2009\u00b0C. Read\u2010out of the fluorescence signal was carried out using a PerkinElmer EnSpire Multimode Plate Reader and data processing was done in GraphPad Prism. Following the same procedure, cells were treated with DMT (7), PymK (9), or 1,3\u2010dimethylimidazolidin\u20102\u2010one (=byproduct of the self\u2010immolation process) with concentrations of up to 10\u2005\u03bcM, revealing no significant effect on cell viability.Prodrug stability and cell imaging: Additional data and experimental details regarding (i) the stability of prodrug 12 in PBS and cell growth medium and (ii) confirmation of the mechanistic basis of cytotoxicity upon click activation of 12 via fluorescence microscopy after microtubules staining are included in the Supporting Information.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re\u2010organized for online delivery, but are not copy\u2010edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file."}
{"text": "Mandevilla Lindl. is an important genus of the Apocynaceae family, not only as ornamental plants but also for its medicinal uses. In Brazil, Mandevilla species are indicated to treat asthma and skin infections, their anti-inflammatory potential and wound healing properties are also reported in the literature. Concerning their chemical composition, this group of plants is a conspicuous producer of pregnane glycosides. Mandevilla dardanoi is an endemic species from the Brazilian semiarid region not studied by any phytochemical methods. In view of the medicinal potential of Mandevilla species, this study aimed to isolate new pregnane glycosides from M. dardanoi. To achieve this main goal, modern chromatography techniques were employed. Five new pregnane glycosides, dardanols A-E, were isolated from the roots of M. dardanoi by HPLC. Their structures were determined using extensive 1D and 2D-NMR and mass spectrometry (MSn and HRESIMS) data. The cytotoxicity and the anti-inflammatory potential of these compounds were evaluated. The first was evaluated by measuring proinflammatory cytokines and nitric oxide production by stimulated macrophages. Dardanols were able to inhibit the production of nitric oxide and reduce IL-1\u03b2 and TNF-\u03b1. The current work demonstrates the chemodiversity of Brazilian semiarid species and contributes to amplifying knowledge about the biological potential of the Mandevilla genus. Mandevilla Lindl. is one of the largest neotropical genera of the Apocynaceae family, this group comprises approximately 170 species + , indicating 13 indices of hydrogen deficiency (IHD) (Compound cy (IHD) .1H and 13C NMR data  corresponding to an acetal group hydrogen, \u03b4H 5.41 , an olefinic hydrogen, \u03b4H 4.58  and 3.81 , oxymethinic hydrogens, 1.06  and 0. 94 , methyl hydrogens, associated with their respective carbons at \u03b4C 104.2 (C-16), 122.1 (C-6), 78.9 (C-20), 77.5 (C-3), 16.9 (C-18) and 19.7 (C-19), are in agreement with the values described for the aglycone illustrol, a seco-norpregnane derivative, isolated from M. illustris and reported only once 25D +18 . Its molecular formula was determined to be C50H78O17 by HRESIMS, with m/z 973.5086 [M + Na]+ , indicating 12 hydrogen deficiency indices. The 1H and 13C NMR data were similar to compound 1 being assigned to illustrol-type aglycone , 4.78 , 5.15 , and 5.40 , as well as the presence of methyl hydrogen signals at \u03b4H 1.51 , 1.34 , 1.39  and 1.28 , and of the hydrogens of the methoxy groups at \u03b4H 3.33 , \u03b4H 3.39 , \u03b4H 3.46  and \u03b4H 3.38 , indicated the existence of four osidic units in compound 2.Compound aglycone . When co1H and 13C NMR data of the osidic units also resembled compound 1, allowing us to establish 2 as illus-trol-3-O-\u03b2-L-diginopyranosyl-(1-4)-\u03b1-L-sarmentopyranosyl-(1-4)-\u03b2-D-cymaropyranosy-(1-4)-\u03b2-L-4 acetoxylsarmentopyranosyl, a new natural product named dardanol B.The 3 was isolated as a white powder with a positive optical rotation of [\u03b1]25D +13 . Its molecular formula was determined to be C58H94O21 by HRESIMS, with m/z 1109.6252 [M \u2212 H2O + H]+ , indicating 12 hydrogen deficiency indices. The NMR data resembled that of the aglycone illustrol; however, some differences were observed for the carbons at \u03b4C 38.9, 46 and 62.5, assigned to C-8, C-13 and C-17, respectively, with C-8 and C-17 being deprotected and C-13 protected when compared to the chemical shifts of these carbons in compound 1. These data were associated with one lower hydrogen deficiency index compared with compound 1, suggesting the opening of the C-14-O-C-16 epoxide. The signal at \u03b4H 6.88 (s), uncorrelated in the HSQC spectrum, was assigned to the OH located at C-14. This signal showed correlations in the HMBC spectrum with the signals at \u03b4C 46.0 and 110.1 that were assigned to C-13 and C-14, respectively. Additionally, in the HMBC spectrum, we observed correlations of the signal at \u03b4H 1.40 (3H-18) with the carbons at \u03b4C 110.1 (C-14) and in the HMBC spectrum at \u03b4C 46.0 (C-13) and with \u03b4C 62.5, which was assigned to C-17. The signal at \u03b4H 5.17, whose corresponding carbon in the HSQC spectrum was \u03b4C 105.8, was assigned to C-16. The signal at \u03b4H 5.17 correlated with \u03b4C 46.0 (C-13) and with \u03b4C 62.5 (C-17). A correlation of this signal with \u03b4C 71.4, assigned to C-21, was also observed. A correlation was also observed with the signal at \u03b4C 54.5 that was assigned to methoxy bound at position 16. The coupling constant of H-17  and a singlet for H-16 demonstrated near 90-degree angulation between these hydrogens, and thus, the aglycone was defined as shown in 1 data and high-resolution mass spectrometry confirmed the presence of five 1\u20134 bonded osidic units identical to compound 1 and inserted in C-3 -\u03b1-L-sarmentopyranosyl-(1-4)-\u03b2-D-cymaropyranosy-(1-4)-\u03b2-D-oleandropyranosyl-(1-4)-\u03b2-L-4-acetoxylsarmentopyranosyl, a new natural pro-duct named dardanol C.To reject the possibility that the aglycone had been formed in the process of separating the compounds, a direct infusion on the ESIMS/MS was performed with the crude ethanolic extract. The presence of the ion at mpound 3 . Thus, c4 was isolated as a white powder with a positive optical rotation of [\u03b1]25D +17 . Its molecular formula was determined to be C28H44O8 by HRESIMS, m/z 531,2923 [M + Na]+ , indicating 7 hydrogen deficiency indices. The NMR data for the aglycone of this compound were similar to compound 3, and it could be concluded that it was of the same type as OH at C-14 and OCH3 at C-16. In the 13C NMR spectrum, it was possible to observe six carbons at \u03b4C 99.0, 33.2, 79.4, 67.4, 71.4 and 20.4 in addition to a signal for methoxyl at \u03b4C 55.3 compared with the literature, and the other osidic units of compounds 1\u20133 were marked as diginopyranose. The H-1\u2032 coupling constant at 11.6 and 4.0 Hz was compatible with \u03b2-glycosidic bonding, and the C-2\u2032 value at \u03b4C 33.5 allowed the configuration of the osidic unit to be assigned as L-diginopyranosyl. The molecular mass of this compound was also found in the analysis of the crude extract by ESIMS/MS, which again rejected the possibility of artifacts. Thus, compound 4 was identified as seco-illustrol-3-O-\u03b2-L-diginopyranosyl, a new natural product named dardanol D.Compound 5 was isolated as a white powder with a positive optical rotation of [\u03b1]25D \u221219 . Its molecular formula was determined to be C58H92O21 by HRESIMS, with m/z 1147.6037 [M + Na]+ . The 1H and 13C NMR data showed signals characteristic of pregnane glycosides. The hydrogen signals at \u03b4H 5.00 , 5.89  and 4.55  together with carbons at \u03b4C 93.5, 109.7 and 74.7 are in agreement with the values described for the velutinol aglycon, reported only in M. velutinus 25D + 14 ; 1H and 13C NMR data, see m/z 1117.5918 [M + Na]+ .Dardanol B (2): white powder; [\u03b1]25D + 18 ; 1H and 13C NMR data, see m/z 973.5086 [M + Na]+ .Dardanol C (3): white powder; [\u03b1]25D + 13 ; 1H and 13C NMR data, see m/z 1109.6252 [M \u2212 H2O + H]+ .Dardanol D (4): white powder; [\u03b1]25D + 17 ; 1H and 13C NMR data, see m/z 531,2923 [M + Na]+ .Dardanol E (5): white powder; [\u03b1]25D \u2212 19 ; 1H and 13C NMR data, see m/z 1147.6037 [M + Na]+ .Mandevilla dardanoi by modern chromatographic techniques and characterized by comprehensive spectroscopic data. Among them, compounds 3 and 4 contained a novel seco-pregnane-type aglycone. Dardanols A, B, C and E showed anti-inflammatory potential by inhibiting the production of nitric oxide and reducing the pro-inflammatory cytokines IL-1\u03b2 anFive new pregnane steroidal glycosides (dardanols A\u2013E) were isolated from the ethanolic extract of d TNF-\u03b1 in stimulated macrophages. These findings enrich the knowledge of the chemodiversity and biological potential of Caatinga species."}
{"text": "Because of the well-defined steps in its progression (adenoma - Tis - T1 invasive cancer- T2 advanced cancer with metastases), colorectal cancer (CRC) represents a model for investigating the effects of angiogenesis throughout tumor development.\u00a0\u00a0The aim of this study is to determine the role of VEGF expression by immunohistochemistry (IHC) in normal epithelium, dysplasia, carcinoma of colorectal specimens and to correlate the same with tumor grade, stage, nodal status, and metastasis.Introduction:\u00a0Colorectal cancer is one of the malignancies in which angiogenesis has been implicated and its importance at different stages of malignant disease. Neovascularization begins when the angiogenic switch is turned on and when angiogenesis activators outweigh angiogenic inhibitors. The process of blood vessel formation is regulated through several growth factor systems. The vascular endothelial growth factor (VEGF) system is one of the important ones and may consequently be a key system in relation to different aspects of colorectal carcinoma treatment.Methods: This is a retrospective study on paraffin blocks of 50 colon cancer specimens, 40 adenoma specimens, and 10 normal colonic mucosa specimens. Immunohistochemical stain for VEGF was done on the sections along with controls. Monoclonal antibody detected against VEGF antigen was observed in the cytoplasm of the tumor cells and the intensity of\u00a0VEGF expression in individual tumor cells was scored on a scale of 0 (no staining) to 3 (strong intensity), and the percentage of cells with VEGF staining at each intensity was estimated from 0 to 100. Pearson\u2019s Chi-squared test and Mann-Whitney test were used to determine significant clinicopathological differences between VEGF expression in positive and negative tumors.Results:\u00a0In normal epithelium, VEGF immunoreactivity was seen in all 10 cases with high intensity. Among adenomas, VEGF expression was seen in 26 (65%) of the 40 cases. Out of which in tubular adenomas VEGF expression was seen in 13 cases (60%) and negative in eight cases (40%). In tubulo villous adenoma, VEGF expression was seen in nine cases (60%) and negative in six cases (40%). Villous adenomas showed VEGF expression in all four cases (100%). In adenocarcinoma, VEGF expression was seen to be expressed in 42 cases (84%) and negative in eight cases (16%) and expression was\u00a0higher in low-grade carcinomas (70%) compared to high-grade carcinomas. A significant difference in the expression of\u00a0VEGF among adenomas and carcinomas was observed\u00a0with higher intensity present in adenoma when compared to carcinoma.Conclusion:\u00a0Expression of\u00a0VEGF could be considered as an early carcinogenic factor in\u00a0colorectal carcinomas as it is expressed in higher intensity in the precancerous lesion and low-grade and stage 1 adenocarcinoma. Hence, we infer that early colorectal carcinomas are an important model for targeted therapy with antiangiogenic factors for VEGF.\u00a0 Colorectal cancer (CRC) is the third most common cancer occurring worldwide with an estimation of one million new cases diagnosed each year\u00a0. In receThis is a retrospective study on 100 paraffin blocks of 50 colon cancer specimens, 40 adenoma specimens, and 10 normal colonic mucosa specimens received in the Department of Pathology at Sri Ramachandra University over a period of three years. Immunohistochemical stain for VEGF was done on the sections along with controls.Permission from the Institutional Ethics Committee was obtained prior to commencing the study (IEC NO: CSP-MED/13/JUN/07/34). Gross findings were recorded and clinical data were obtained from the medical record section and available local area computer network in service. Paraffin blocks\u00a0made after routine processing of resected colectomy specimens were used for the same. Blocks with sections containing\u00a0tumors, adenomas, and normal colonic mucosa\u00a0were chosen for the immunohistochemistry (IHC) study. This IHC study was conducted on an estimated sample size of 50 cases of histopathology-proven colonic carcinoma, 40 adenomas, and 10 normal colonic mucosa.Hematoxylin and eosin (H&E) stained sections were used to grade colon cancer by architectural and cytological features. Other histopathological features observed were adjacent dysplasia and adenomas. Immunohistochemical staining for VEGF was performed on all 100 cases. Monoclonal antibody detected against VEGF antigen was observed as brown color\u00a0in the cytoplasm of the tumor cells and the intensity of\u00a0VEGF expression in individual tumor cells was scored on a scale of 0 (no staining) to 3 (strong intensity) . Pearson\u2019s Chi-squared test and Mann-Whitney test were used to determine significant clinicopathological differences between VEGF expression in positive and negative tumors. Differences were considered statistically significant when p value was\u00a0<0.05.We studied holoprosencephaly (HPE) proven cases of 50 colorectal carcinomas, 40 colonic adenomas\u00a0which included 21 tubular adenomas, 15 tubulovillous adenomas,\u00a0and 4 villous adenomas diagnosed in the Department of Pathology, Sri Ramachandra Medical College and Research Institute over a period of three years. We included 10 normal colonic tissues away from the lesion\u00a0from the CRC specimens.In our study of colon cancers, the age of the patients ranged from 27 to 85 years. The highest incidence was noted in the fifth decade. In adenomas, the highest incidence was noted in the sixth decade of life.\u00a0We found that colon cancer occurred predominantly in men as compared to women with a male-to-female ratio of\u00a03.1 : 1 and in adenomas also males were affected predominantly than females with a ratio of\u00a02.3:1. Rectum was the most common site involved in both colonic cancer as well as in adenomas . The size of the CRCs ranged from 2.5 to 12 cm out of which up to 5 cm was 48% and more than 5 cm was 52%.\u00a0In adenomas size ranged from 0.2 to 8 cm out of which in tubular adenomas size ranged from 0.2 to 2 cm and villous adenoma was slightly bigger in size and ranged from 0.3 to 8 cm. There is no definite predilection for large tumors.\u00a0In CRCs, grade 1 was categorized as well differentiated, grade 2 as moderately differentiated, and grade 3 as poorly differentiated. In this study grade 2 is more common\u00a0(69%) followed by grade 1 (18%).In our study most of the tumors (50%) belong to pT3  of the TNM  classification, followed by pT2 - (44%) (tumor invades muscularis propria). Regional lymph node metastasis was seen in 48% with N1 - 36%  and N2 - 12% (metastasis in more than four lymph nodes). Nodal metastasis was absent in 52%. Clinically no distant metastasis (cM0) was noted in all the cases except for one case.\u00a0When the comparison was made for stage, stages 1 and 3 predominated over stages 2 and 4. The high stage was noted predominantly in males and more common in the age group of >60 years.We analyzed the expression of VEGF in normal colonic epithelium, tubular adenoma, tubulovillous adenoma, villous adenoma, and carcinoma. We noted that the staining of VEGF immunohistochemical stain had taken up in the cytoplasm of allcases .In normal epithelium, VEGF immunoreactivity was seen in all 10 cases with a high intensity of 3+\u00a0Figure\u00a0.Among adenomas, VEGF expression was seen in 26 (65%) of the 40 cases with an intensity of 2+ in 15 cases and\u00a03+ in 11 cases. In tubular adenoma, 13 cases showed positivity with an intensity of 2+ in\u00a0nine cases and 3+ in four cases . The H score ranged from 40 to 300 in villous adenomas and 70 to 300 in tubular adenomas Table\u00a0.According to the age, VEGF was expressed high in the age group of 61-70 in all the adenomas and there is no significant difference in the expression of VEGF according to the site.In colorectal carcinomas, VEGF expression when compared based on the age of the patients, showed positivity in 18 cases (58.1%) among the age group <45 years\u00a0and positivity in eight cases (42.1%) among patients >45 years of age. Thus there is no significant difference with VEGF expression and age of the patients (p =\u00a00.273). When VEGF expression was compared to the size of the tumor, 11 cases (42.3%) were positive in tumors of size up to 5 cm and 15 cases (62.3%) were positive in tumors >5 cm with a p-value of 0.153 which was statistically insignificant.The clinicopathological data of VEGF expression is given in Table\u00a0The VEGF expression was analyzed with grade and TNM staging in colorectal carcinomas; the data are summarized in Table\u00a0In our study, expression of VEGF was seen positive in 7/10 (70%) cases in grade 1 with an intensity of 3+ and showed almost equal positivity in grades 2 and 3 with an intensity of 2+ (47% and 52%)\u00a0(p=0.443) Figures\u00a0-7.Expression of VEGF was also analyzed for pT staging, 13 cases of pT2\u00a0stage tumors were positive with an intensity of 3+ in eight cases\u00a0and 2+ in five cases.\u00a0Some 12 cases of pT3 were positive with an intensity of 2+\u00a0in eight cases and 3+ in four cases and out of three cases in pT4 only one case was positive which showed an intensity of 2+ (p=0.600) Figure\u00a0.In nodal metastasis, VEGF was positive in eight (44.8%) cases/18 cases in N1\u00a0and\u00a0three (50%)/six cases in N2 were positive with variable intensity and a\u00a0p-value of 0.684, which was not significant.\u00a0When the expression of VEGF and stage were compared, stage 1 showed maximum expression in 13 cases (65%), H scores ranging from\u00a010 to 300, stage 2 showed expression in four cases 44.4%) H score ranging from 100 to 300, stage 3 (40%) in eight cases score ranging from 30 to 300, and stage 4 in one case (100%) with a score of 280\u00a0(p=0.303). In our study, the p-value was higher so there was no correlation with the\u00a0grade and stage of the tumor \u00a0, moderately differentiated (grade II), and poorly differentiated (grade III). In this study grade 2 was more common (69%) followed by grade 1 (18%) which correlated with the study done by Yalcin et al.\u00a0. In our In this study we studied the expression of VEGF in normal colonic epithelium, tubular adenoma, tubulovillous adenoma, villous adenoma, and carcinoma.\u00a0In normal epithelium VEGF immunoreactivity was seen in all the 10 cases with a maximum intensity and a score of 300 which correlated with the study of Hanrahan et al.\u00a0. In his In adenomas, VEGF expression was seen in 25 (64.1%) cases out of which VEGF expression was seen in 12 cases (60%) of tubular adenomas. The VEGF expression in tubulo villous adenoma\u00a0was seen in nine cases (60%) and villous adenoma showed VEGF expression in all four cases (100%). There was a significant difference between\u00a0the expression of VEGF in villous adenoma and tubular adenoma (p=0.05). The H score ranged from 40 to 300 in villous adenoma and 70 to 300 in tubular adenoma and in carcinomas expression was seen in 51% of cases.From our study there was a slight increase in the expression of VEGF in colonic adenomas than the carcinomas with a mean of 216 in adenoma and 195 in carcinoma whereas in the study conducted by\u00a0Abdou et al.\u00a0,\u00a0the VEGMolecular studies on transgenic mouse models have shown that angiogenesis begins in the premalignant phase of tumorogenesis, when dysplastic lesions acquire an increased microvasculature\u00a0. Thus thIn colorectal carcinomas, VEGF expression was seen in 18 cases (58.1%) <45 years of age and positive in eight cases (42.1%) >45 years. There was no significant difference in age with VEGF expression (p = 0.273). When VEGF expression was compared to the size, 11 cases (42.3%) were positive among tumors up to 5 cm and 15 cases (62.3%) were positive among tumors > 5 cm (p=0.153) which was statistically insignificant. Similar findings were observed by Abdou et al.\u00a0\u00a0and BendTumor grade and stage in relation to VEGF positivity were analyzed for correlation. The VEGF was expressed high in grade 1 with a median score of 280 and\u00a0a score of\u00a0210 in grade 2 and a median score of 180 in grade 3. Statistical data showed a p-value of 0.684. Conflicting studies were published considering VEGF expression and tumor differentiation. The VEGF expression was significantly more intense in poorly differentiated tumors in comparison with the well-differentiated ones in CRCs. In the present study, there was an\u00a0inverse relationship between tumor grade and VEGF expression though it was insignificant. Similar findings were observed by Uner et al.\u00a0\u00a0and AbdoIn our study when the expression of VEGF and stage was compared a low expression was seen in advanced stage tumor. Stage 1 showed a maximum expression of 13 cases (65%), stage 2 showed an expression in four cases (44.4%), stage 3 (40%) in eight cases, and stage 4 in one case with a p-value of\u00a00.303. According to Zheng et al.\u00a0, HanrahaGeorge et al.\u00a0\u00a0and SaadIn view of the association of high VEGF expression in normal epithelium away from the tumor, adenomas, tumors of low grade, early stage, and negative nodal status by immune histochemical assessment, it could be suggested that VEGF may be an early carcinogenic factor in CRC. VEGF may be required for CRC development in early stages but as tumors become less differentiated, more aggressive, and deeply invading, it is no longer in need of VEGF that appears to be downregulated. So other angiogenic factors could have the upper hand in this stage of aggression and advancement of CRCs.From our study reduced expression of VEGF\u00a0in tumors with high grade and advanced stage necessitates further correlation with the molecular studies in CRCs in this part of India.From the above findings of our study, we conclude that VEGF could be considered as an early carcinogenic factor in\u00a0colorectal carcinomas as it is expressed in higher intensity in precancerous lesions like adenoma and low-grade and stage 1 adenocarcinomas. Hence we infer that early colorectal carcinoma\u00a0is an important model for targeted therapy with antiangiogenic factors for VEGF.\u00a0The decreased expression of VEGF\u00a0in higher grades and stages of adenocarcinomas suggests that it has a reduced role in tumor progression. Hence we suggest that the reduced role of VEGF in tumors with high grade and advanced stage necessitates further study of the molecular pathology of CRC in this part of India."}
{"text": "The proliferation of Internet-of-Things has promoted a wide variety of emerging applications that require compact, lightweight, and low-cost optical spectrometers. While substantial progresses have been made in the miniaturization of spectrometers, most of them are with a major focus on the technical side but tend to feature a lower technology readiness level for manufacturability. More importantly, in spite of the advancement in miniaturized spectrometers, their performance and the metrics of real-life applications have seldomly been connected but are highly important. This review paper shows the market trend for chip-scale spectrometers and analyzes the key metrics that are required to adopt miniaturized spectrometers in real-life applications. Recent progress addressing the challenges of miniaturization of spectrometers is summarized, paying a special attention to the CMOS-compatible fabrication platform that shows a clear pathway to massive production. Insights for ways forward are also presented. Conventional high-performance spectrometers are typically based on bulky and costly systems with large dispersive components, long optical path length, and movable mechanisms. To meet the requirements of various application scenarios where the portability, cost, robustness, and power consumption are paramount metrics, such as portable or wearable sensing devices for healthcare, food safety monitoring5, smartphone-based spectrometers, drone-based remote sensing6 and space exploration8, substantial progresses have been made in miniaturizing spectrometers while maintaining adequate performance during the past decades. The projected market for mini- and micro- spectrometers has gone up to about 900 million dollars9, which stimulates considerable research efforts from both academia and industry. To name a few, Fraunhofer ENAS announced its goal of batch producing \u201cone gram spectrometer\u201d for smartphone integration at the cost of around one dollar10, whilst Rockley Photonics released a whitepaper aiming to realize high-yield manufacturable on-chip spectrometers for \u201cclinic on the wrist\u201d health monitoring11. Clearly, efforts are driven toward exploring miniaturized spectrometers that are mass-manufacturable for the consumer market.Optical spectrometer is one of the most essential instruments in numerous fields, including chemical engineering, materials analysis, astronomical science, medical diagnosis and biological sensing20. While some work has indeed advanced the state-of-the-art in miniaturized spectrometers, they may suffer from insufficient performance and low technology readiness levels in connection with commercialization. For instance, ultra-compact spectrometers at the scale of dozens of microns based on single compositionally engineered nanowires have been demonstrated, representing the smallest footprint on record14, which for sure, is recognized as a milestone for miniaturized spectrometers. However, a resolution of 5~10\u2009nm is far away from being adequate for applications like glucose monitoring, not to mention gas analysis. Moreover, such technology requires customized epitaxial growth and electron-beam lithography based post-processing, which inevitably results in low yields and high cost at current technology readiness level. Similar concerns also apply to those designs based on colloidal quantum dot18 and perovskite quantum dot19 given the complexities of their fabrication processes.In principle, the miniaturization of spectrometers inevitably leads to performance trade-offs in size, operating bandwidth, measuring speed, spectral resolution, dynamic range (or insertion loss), etc. Thus, to achieve commercial products, it is vital to tailor the spectrometer design to suit specific application scenarios. By now, a wide variety of miniaturized spectrometers have been demonstrated, involving diverse architectures, working principles and material platforms, as well as varying technology readiness levels for large-scale manufacturing21. Besides, the silicon photonics platform features an ultra-high refractive index contrast, facilitating the realization of small device footprints. These advantages can be further extended by introducing other CMOS compatible materials, such as, silicon nitride for its ultra-low propagation loss, broadband transparent spectral window, and insensitivity to temperature variations22. Furthermore, by means of heterogeneous integration approaches, III-V gain materials can be heterogeneously or monolithically integrated within the same chip package to implement high-performance light sources and photodetectors24, enabling densely integrated spectrometer on-chip systems.To ensure low cost, miniaturized spectrometers must demonstrate a path toward high-volume manufacturing. This generally means lithography-based fabrication and high-level integration. Being compatible with the mature CMOS technologies, silicon photonics provides a promising solutionDifferent to other review articles summarizing the general trends on miniaturized spectrometers, this paper pays special attention to the integrated spectrometers that hold great promises for massive fabrication at low cost and specifies their application perspectives. In the following chapter, we will first give a brief market analysis of miniaturized spectrometers and in the chapter after that, application-oriented analysis of miniaturized spectrometers will be provided, including bio-medical monitoring and industrial sensing. In chapter 4, an in-depth technological analysis of miniaturized spectrometers that can be potentially massively manufactured will be performed. The outlook toward developing next-generation integrated spectrometer will be given in chapter 5. Finally, we draw the conclusions in chapter 6.26. The predicted penetration rates and market values of compact spectrometers in different applications and segments are shown in Fig. 19. The research field has the largest value in 2019 whereas the rapid growth occurs at consumer and biomedical markets almost from 300 to 400% within five years.Due to the unique light-matter interaction, optical spectroscopy has proven to be an efficient technique to non-invasively analyze compositions of chemical materials, gases, biological tissues, food, etc. The applications of spectrometers were predominantly limited to labs, tests, and metrology, whilst they are now sharply penetrating to consumer market in healthcare monitoring, and food safety. This trend is largely attributed to the development of miniaturized spectrometers, as evident by the rapid growth of the market volume of micro- and nano-spectrometers which arises from 655 million US dollars to 922 million in just three years between 2016 and 201927. The rapidly expanding market imposes strong stimulations for both academia and industry players to join this field. Hamamatsu launched a mini spectrometer with a size comparable to fingertip and a weight of only 0.3\u2009g. The device works between 650\u2009nm and 1050\u2009nm with a spectral resolution of about 15\u2009nm, which is suitable for food inspection, light-level measurement and component analysis28. NeoSpectra-Micro developed an integrated near infrared spectral sensor (1250\u20132500\u2009nm) with 16\u2009nm resolution that can be used in a wide variety of material sensing applications for both qualitative and quantitative analysis29. Continuous efforts are also being poured into developing low-cost chip-scale spectrometers that can be integrated into consumer electronics such as smartphones. For instance, Fraunhofer-ENAS announced its goal of batch producing \u201cone gram spectrometer\u201d for smartphone integration at a cost of around one dollar10. Samsung patented their smartphone-embedded IR spectrometers and prepared to integrate them in Galaxy S11 for skin moisture detection and CO2 measurement27. In 2020, trinamiX and Viavi Solutions had announced a joint force in building a near infrared spectrometer module targeting for consumer devices. Very recently, Rockley Photonics, a silicon photonics enterprise focusing on telecommunications, released a whitepaper about highly manufacturable on-chip spectrometers for \u201cclinic on the wrist\u201d health monitoring11.Among those micro- and nano-spectrometers, chip-scale spectrometers are shown to play a dominant role in the rapid market growth in coming years, due to their superior integrability into portable devices such as smartphones, wearable devices and drones, which, on the other hand, is the main driving force behind the development of compact spectrometers. A recent report foresees that chip-scale spectrometers will have a disruptive increase in market volume from less than 2 million US dollars in 2019 to over 1.6 billion US dollars in 2024, as shown in Fig. 2All these examples herald the rapid development of chip-scale spectrometers in the future, especially for the consumer and biomedical applications. It is foreseeable that the next-generation integrated spectrometers will be developed on the basis of silicon photonics to enjoy massive productibility at low cost and targeted for the adaptability in ecosystems of intelligent devices such as smartphone and wearable devices4, while for the detection of biomarkers, such as blood glucose or lactate, a sub-nanometer resolution is desired to identify their wide signature absorption peaks (~30\u2009nm) to ensure the measurement accuracy31. Therefore, it is critical to establish a set of key figures and specify their application-oriented metrics. In this chapter, we discuss the application prospects for integrated spectrometers in different fields, elaborating their key figures of merits in terms of resolution, bandwidth, etc.Chip-size integrated spectrometers offer great potential for large-scale, in-field, and real-time spectroscopic sensing. Nevertheless, in general, the footprint reduction of a spectrometer consequentially give rise to certain performance degradation regarding its operation bandwidth, resolution, measuring speed, dynamic range, or signal-to-noise ratio, making it crucial to customize the spectrometer design for specified application scenarios. For example, in some cases such as the portable spectroscopies for chemical sensing, a resolution on the scale of 10\u2009nm can be considered acceptable31, i.e., to recognize certain chemical substances or functional groups based on the interaction between electromagnetic radiation with vibrational molecules. Among them, since the MIR technique is not suitable for on-chip integration due to the lack of appropriate integration platforms , it is not within the scope of this paper. Here we focus on the NIR and Raman spectroscopy techniques that are applicable for chip-size integrated spectrometers and summarize their key spectroscopic performance metrics required for different application scenarios.By now, the most popular but demanding application scenarios of the integrated optical spectrometers include biomedical sensing for health monitoring and disease diagnosing, chemical sensing for pollution monitoring, gas leak detection and industrial emission screening etc. These detections rely on different vibrational spectroscopies such as near-infrared , mid-infrared , and Raman spectroscopy  techniques to measure the relevant markers35. For instance, the detection of the glucose concentration in blood or saliva can provide diagnostic or monitoring information for diabetes, while measuring the electrolyte level in sweat helps screen for dehydration. These biomedical measurements are commonly achieved by NIR spectroscopy techniques. Since the absorption bands in NIR region are prominently generated by a large number of overtones and combinations of fundamental vibrations of -CH, -NH, and -OH functional groups, the information of various biomarkers can therefore be elucidated accordingly. Benefitting from the maturity of spectrometer integration platform, NIR spectroscopy with portable devices has gradually become a research hotspot, showing promising prospects for in-vivo biomedical sensing.The sensing of biomarkers in either exhaled breath or biofluids, e.g., blood, breath, sweat, saliva, urine, is of great importance for bioanalysis and biomedical diagnosis36. In theory, by identifying the spectral signatures at different bands, quantitative information of corresponded chemical compounds can be retrieved, e.g., the overtones of -CH stretch are associated with carbohydrates while the overtones of -NH stretch relate to proteins. However, given that these peaks in NIR region are rather broad and do overlap, specified spectrometer metrics, as well as additional calibration processes, such as partial least squares regression (PLSR), are needed to extract the mixed bio-information with reasonable accuracy38. Specifically, such NIR spectrometers should be designed to feature high resolution in particular wavelength regions where the minimum peak overlaps with water or other molecules exist, as well as high sensitivity since the measurable light (either reflected by or transmitted through living tissues) is always rather weak.Figure 45. The informative peaks of the glucose molecule come from the vibrations between -OH and -CH in either long wavelength-NIR  or short wavelength-NIR , representing the two main observe windows on which current analytical methods are based. Relatively, the spectral signatures in LW-NIR are more pronounced than those in SW-NIR which enable higher precision for in-vitro measurement, while the absorption index in SW-NIR bands is notably lower, allowing a deeper penetration depth for in-vivo testing. In Fig. We first take the measurement of blood glucose as a representative example. As one of the earliest applications of NIR spectroscopy in medical diagnosis, researchers have shown great interests in non-invasive systems for in-vivo blood glucose measurements to alleviate the pain and high cost of conventional invasive methods52. Since these compounds have lower concentrations in blood or cells compared to glucose, current measurement approaches are mostly based on in-vitro sample testing, which rely on several hundreds of nanometers wide bandwidths with about 1\u2009nm resolutions in the LW-NIR range. Note that in ref. 53, the detection of deoxygenated hemoglobin (Hb) oxygenated hemoglobin (HbO2) applies only two wavelengths at 730\u2009nm and 850\u2009nm because the absorption spectra at these wavelengths differ significantly between Hb and HbO2, and the ratio is then calculated to determine the concentrations. However, this measurement is not accurate on the patients with abnormal hemoglobin structure, abnormal hemoglobin levels.Besides glucose, NIR spectroscopy has also been demonstrated to extract information about other metabolites, including lactate, urea, glutamine, Ammonia, and (de) oxygenated hemoglobin etc.The above performance requirements can already be met by some commercialized mini spectrometers but are still quite challenging for chip-scale integrated spectrometers. To realize wearable non-invasive devices for in-vivo biomedical sensing, further evolvements in both instrumentation and spectral analysis algorithms are desired.51. For instance, the strong infrared absorption of water (H2O) always severely interferes the spectroscopic detection and limits the scope of applications whilst Raman spectroscopy is insensitive to water. Therefore, for chip-scale spectrometer, NIR and Raman (800~2500\u2009nm) and their combination spectroscopies are well competent for realizing the high-efficiency chemical detection in a great variety of complicated industrial scenarios59.Besides biomedical applications, optical spectroscopic sensing has also been proven as a versatile and powerful tool for high-efficiency industrial chemical detection of various substances. Among them, NIR and Raman spectroscopy are widely adopted and are regarded as a couple of complementary techniques60. So far, the gas detection has been widely demanded by industrial applications, including monitoring the hazardous gases for production safety management, detecting the emission of greenhouse gases and volatile organic compounds (VOCs) for environmental protection61. It can be concluded that for industrial applications except agriculture and food analysis, it is always desirable for the spectrometers to have very high resolution (<1\u2009nm) with a bandwidth over 100\u2009nm, which, as mentioned above, remains a great challenge for integrated spectrometers at this moment.Figure To summarize, the key spectroscopic requirements for both biomedical and industrial chemical sensing applications are listed in Table From the perspective of fundamental technology route, the optical spectrometers can be roughly classified into two groups: wavelength de-multiplexing based (WdM) and wavelength multiplexing (WM) based. As suggested by their names, the WdM spectrometers need to de-multiplex, or in other words, split the incident signals\u2019 spectra, either spatially or temporally, and measure the intensity of individual channel. On-chip WdM spectrometers are typically implemented by dispersive structures, in which different wavelengths exhibit different propagation properties and consequently arrive at detectors distributed at different spatial locations or reach a single detector at different time. Alternatively, WdM spectrometers can be implemented by using an array of narrowband filters or a single tunable narrowband filter, whose spectral responses determine the spectral contents arriving at the detector, as shown in Fig. Being the most straightforward spectroscopic approach, integrated WdM spectrometers don\u2019t require complex computational sources, thus have attracted numerous research attention to be demonstrated on silicon photonics platform. Till now, gratings are the most widely adopted elements for dispersive structures based WdM spectrometers. The fundamental principle is to create multi-path interference effects between the incident signal and make different wavelengths arrive at different locations. Depending on how the multi-path interference is created, gratings based spectrometers can be further divided into planar concave grating (PCG) and arrayed waveguide grating (AWG).62. The main research focus since then have been given to improve the spectrometer\u2019s performance by optimizing the gratings, including using Bragg reflectors64 or photonics crystal reflectors65, coating grating with silver films66, Retro-reflecting V-shaped facets67. etc., as plotted in Fig. PCG is the planar version of the classic diffraction grating spectrometer . The light enters from an input aperture into the free propagation region (a slab waveguide section) after which it diffracts and hits the curved grating on the other end, which reflects as well as focuses different wavelengths at different output apertures located along a so-called Rowland circle as shown in Fig. 40. Compared with PCG, AWG is relatively simpler in design and resolution improvement. However, it has larger footprint as it incorporates two free propagation regions and an array of long waveguides. AWG is also more vulnerable to fabrication variations that induce phase errors along the arrayed waveguides, this is why AWG typically exhibit stronger channel crosstalk than PCG. Various innovations have been created to combat those problems. For instance, the footprint reduction can be achieved by replacing the free propagation regions with multi-mode interferometers  with varying armlength differences, spectrometers with spectral resolutions from 0.13\u2009nm to 20\u2009nm can be easily synthesized81.Another unique advantage of narrowband filter based spectrometer compared with dispersive gratings lies in the tunability of its operation wavelength, particularly for resonant filters, which makes scanning spectrometer that uses a single filter with ultra-compact footprint and large dynamic range possible. However, for filters with very narrow linewidth , tuning the wavelength for a broad optical range will require a significant amount of power consumption and the range will typically be limited by its free spectral range. Therefore, the trend of combining dispersive gratings and narrowband filters for spectrometers with both broad bandwidth, high spectral resolution and small channel count is arising. The dispersive gratings are intended to have low-resolution and split the spectrum into multiple channels with large bandwidth, while a narrowband filter finely scan each entire channel by tuning its center wavelength. Recently, the combination of a AWG and ring resonators was demonstrated for 0.1\u2009nm resolution and 25.4\u2009nm bandwidth with only 9 channels as shown in Fig. The fundamental principle of WdM spectrometers requires the spectrum to be split into multiple channels, which limits the power at each detector and the dynamic range of the spectrometer. In contrast, WM spectrometers pre-process the entire spectrum of the incident signal by certain type of modulation and reconstruct the spectrum using proper algorithms to post-process the modulated spectrum. Based on the specific modulation, WM based spectrometers can be categorized as Fourier transform spectrometers (FTSs) and Computational spectrometers (CSs).82. Compared with WdM spectrometers that commonly measure different spectral components separately in the temporal or spatial domain, the multiplexing measurement of FTSs can provide much better SNR with higher optical throughput captured by the detectors, especially when the detector noise is dominant, which is identified as the Fellgett\u2019s advantage (multiplex) and the Jacquinot\u2019s advantage . According to the Rayleigh criterion, the spectral resolution \u03b4\u03bb and bandwidth \u0394\u03bb of FTSs are determined by the maximum OPD introduced to the signal and the number of OPDs, respectively. For conventional free-spaced FTSs, the OPD modulation can be easily performed using a movable mirror as shown in Fig. FTSs modulate the incident light via tuning the optical path difference (OPD) to obtain the interferograms and then convert them back to the wavelength domain by performing Fourier transform in the post-processing stage, as shown in Fig. 10a83 implements 32 MZIs for a resolution of 0.04\u2009nm, but the bandwidth is limited to only 0.75\u2009nm. The degree of polarization is exploited to further enhance its performance at a given channel count. A Dual-polarization SH-FTS that can process both fundamental transverse electric and transverse magnetic modes was successfully demonstrated in 201915. In detail, the dual-polarized SH-FTS is composed of MZIs with different waveguide widths and arm imbalances, where each MZI can contribute two sampling OPDs, one from the TE mode and the other from the TM mode. As a consequence, the same number of MZIs can provide twice OPD compared with a single polarization operation, as shown in Fig. 84 and machine learning85.SH-FTS are based on a simple and stable multi-interferometer configuration composed of a spatial array of imbalanced MZIs, each of which produce a linearly increasing amount of OPD to the signal, together they could produce a digitized interferogram of the input signal. This kind of configuration can easily produce a very large OPD that turns into high spectral resolution, but it suffers from a similar problem with WdM based spectrometers: inherent trade-off between spectral performance  and channel count. For example, the demonstration in ref. 86, as shown in Fig. 16. After that, the research attention has been given to further increase its resolution. The fundamental reasons for this low resolution include short waveguide length and low power injection to the heater. Increasing waveguide length will result in large propagation loss and high electrical resistance for the heater. By using advanced waveguide design to reduce the optical loss as well as heater structures to receive higher power injection, Li et al. successfully increase the waveguide length from 3\u2009cm in ref. 16 to 10\u2009cm in ref. 87, and the spectral resolution is increased to sub-nanometer. Later, Li et al. combines multiple techniques to further increase the resolution to 0.16\u2009nm, including Michelson interferometers (MI) structure to double the OPD, an optimized heater and air trenches to achieve higher thermal efficiency, a novel multiple interferometers approach is employed which combines balanced MI with N statically imbalanced MIs, thereby increasing the OPD of a single MI by factor of N\u2009+\u20091, as shown in Fig. 10f17.The multi-interferometer configuration with a single-aperture input allows the single-shot interference capture for rapid spectral measurement, while sacrificing the Jacquinot (throughput) advantage. Since the incident light is required to be split equally into every MZI through tree-structured cascaded Y-junctions, the actual throughput for interference modulation at every OPD dramatically decreases with the interferometer number. Temporal heterodyne FTS (TH-FTS), on the other hand, is more analogous to conventional free-space based FTS, as the OPDs are modulated in time domain, and a single detector is used to capture the interferogram. The measurement time is sacrificed in order to maintain Jacquinot (throughput) advantage. Recently, two different mechanisms of achieving temporal modulation of the OPD are demonstrated. First one is similar with SH-FTS in the sense that the OPDs are also in the form of varying physical length difference, but optical switches are employed to dynamically change the physical pathway and realize the temporal modulation of OPD1 and \u03bbM refer to the starting and ending wavelength of the spectrum, respectively. For signal processing purpose, the equation can be discretized asRecently, a new scheme of WM based spectrometer named computational spectrometer has drawn extensive study, which utilizes an array of propagation channels with distinct spectral responses, each of which can be interpreted as sampling coefficients of the incident spectrum at each wavelength point, as shown in Fig. Similarly, if the signal passes through N channels with distinct F, the equation has the form ofNxM, this equation can still be reasonably solved at the case N\u2009\u226a\u2009M, obeying the following rule:For narrowband filters based spectrometer, F is a matrix with only one nonzero component at each row, thus the equation can only be solved when N\u2009=\u2009M. While for computational spectrometers, with advanced algorithms and proper F88. In 1997 a holographic-sensor was designed using random gratings89. Applying this concept in signal acquisition and imaging processing has also been a well-established technique starting from early of 2000, identified as compressed sensing90. In the field of optical spectrometer, this concept was originally adopted only to combat with the non-ideality of narrowband filters94. In other words, these spectrometers still consist of narrowband filters, but optical span outside filters\u2019 passbands is not zero due to fabrication imperfections, causing unwanted sampling at other wavelength span. Only till 2013, researchers theoretically verified the possibility to employ intentionally designed filters with random transmittances for improving spectrometer\u2019s performance, as shown in Fig. 11b95. The main efforts since then have been put to find integrated filters or dispersive media with desirable spectral responses in order to provide good performance (large M) with only a small number of channels . Generally speaking, the channel spectral responses should meet following requirements:Each channel\u2019s spectral response should be linearly independent with the others to provide uncorrelated spectrum sampling.Each channel\u2019s spectral response should contain adequate sharp features to provide high sampling resolution as the minimum optical distance between two distinguishable points in the spectral response determines the spectral resolution of this filter.The channels should be compact, low loss and be able to enjoy massive production.Using this concept for reconstructing signals in a specific domain is not novel. Back in 1987, researchers already proposed to design a velocimeter (a device reconstructing velocities) using random patterns13. Thanks to its high level of randomness, the first two requirements were met and the spectrometer with about 30 channels can produce a spectral resolution of 0.75\u2009nm within 25\u2009nm bandwidth. The use of silicon photonics indeed enables massive production for such spectrometer, but the large amount of air holes introduces significant loss to the signal  for sub-nm resolution, which is unacceptable for consumer electronics.99, the total detection time for such path-reconfigurable spectrometer can be within one second even for hundreds of detection channels.Computational spectrometers are believed promising for achieving a balanced performance with compact footprint and low power consumption, as it can significantly reduce the number of channels compared with dispersive gratings or narrowband filters based spectrometer, and do not demand power consumption compared with tunable filters spectrometers or TH-FTS. But current demonstrations still require the incident signal to be split over 30~50 sampling channels and the channel count has to scale up in order to achieve better performance, which reduces the power intensity in each channel, resulting in a worse SNR and smaller dynamic range. One potential solution is to replace the passive optical power splitting with active path scanning, that is, to actively configure different optical paths (sampling channels) for the incident signal over time, rather than splitting it over space, as shown in Fig. 101, which is an emerging technology allowing the on-chip optical signal to be manipulated at run-time to enable various applications. As shown in Fig. Currently demonstrated spectrometers, irrespective of platforms and working principles, are all fixed in terms of performance, such as the center wavelength, operation bandwidth and spectral resolution. While it is highly desirable to develop a universal spectrometer that can dynamically tune its performance in order to suit requirements of different applications or working scenarios. Ideally, all of the spectrometers\u2019 performance indicators are desired to be programmable, while in practice, it would be valuable enough to develop a spectrometer with adjustable bandwidth and resolution. This idea can be possibly realized in incorporation with programmable photonic integrated circuits (PICs)103, can also be explored to enhance spectrometer\u2019s performance. Currently, WdM is widely adopted for on-chip optical communication to increase the data volume and has been proved to be able to cover a broad bandwidth, such as C+L-band WdM104 and O+C-band WdM105. Analogically, parallelism can also be introduced into the design of spectrometer systems as shown in Fig. 107.Parallelism, which has always existed in the design of optical transmission system since the arise of wavelength division multiplexing (WdM) technologies109. III-V materials, in contrast, feature direct and tunable bandgap, thus can achieve high absorption coefficients at different spectral bands. Moreover, they can also serve as gain materials for optical emitters, such as lasers110 or LEDs111. Currently, high-density cost-efficient III-V-on-Silicon integration relies on hybrid integration approaches, such as, flip-chip bonding112, die/wafer-to-wafer bonding113, and micro-transfer printing114. Therefore, it is predictable that hybrid integrated platforms will play an important role in the future commercialization of integrated spectrometers.Besides the design of spectrometer system itself, to realize full functionalities, it is desired to also implement high-performance light sources and photon detectors within the same chip package, which requires the introduction of other materials on the basis of silicon photonics. Currently, the mainstream integrated photodetectors are based on germanium, as it\u2019s already a standard fabrication process in many CMOS foundries. However, Ge-on-Si photon detectors always suffer from high dark currents and low absorptivity attributed to its nature of indirect bandgap, and normally cannot cover the spectral range beyond NIR116. An additional design dimension could bring unique advantages for 3D nano-printed spectrometers, such as ultra-compact footprint. A wide operation bandwidth can also be expected thanks to the various ultra-broadband 3D optical coupling strategies117. However, at current stage, 3D nano printing only supports passive polymer structures, making external light sources and photo detectors indispensable. Therefore, by implanting the 3D nano-printed spectrometer onto the hybrid integrated III-V-on-Silicon PICs, it is possible to enjoy the advantages from both sides, representing a promising development direction.Beyond silicon photonic platforms, polymer-based 3D lithography offers new possibilities for massive producible miniaturized spectrometers. Based on the laser-induced polymerization effect, current commercial 3D nano printer can implement arbitrary three-dimensional pattern with <200\u2009nm feature size. A variety of demonstrations of 3D-nanoprinted spectrometers have been reported based on different principles ranging from free-space optics to computational reconsctructionWith the rapid development of Internet-of-Things and popularity of intelligent portable device, the demand for low-cost, lightweight, miniaturized spectrometers has never been so imperative. Various efforts have been made to miniaturize spectrometers, yet they somewhat lack technological readiness for commercial products. Being compatible with CMOS technology, silicon photonics is widely accepted to be one of the most promising platforms for mass-manufacturable photonic integrated circuits. Therefore, in this paper we review recent progresses in integrated spectrometers that leverage silicon photonics technology, including their market trends, application-oriented requirements with a special focus on biomedical sensing and industrial detections, and technological evolution that covers mainstream structures for silicon integrated spectrometers. Four possible research directions are foreseen for developing next-generation integrated spectrometers. We expect to see expanding integration of chip-scale spectrometers into consumer productions within the next few years, providing cost-effective and reliable services to users worldwide."}
{"text": "In this short review, we summarized the results obtained with an assay to detect influenza virus-specific antibodies that mediate ADCC, which was developed and evaluated within the framework of the IMI-funded project \u201cFLUCOP\u201d. HA-specific ADCC mediating antibodies were detected in serum samples from children and adults pre- and post-vaccination with monovalent, trivalent, or quadrivalent seasonal influenza vaccines, or following infection with H1N1pdm09 virus. Additionally, using chimeric influenza HA proteins, the presence of HA-stalk-specific ADCC mediating antibodies after vaccination and natural infection with H1N1pdm09 virus was demonstrated. With serum samples obtained from children that experienced a primary infection with an influenza B virus, we showed that primary infection induces HA-specific ADCC-mediating antibodies that cross-reacted with HA from influenza B viruses from the heterologous lineage. These cross-reactive antibodies were found to be directed to the HA stalk region. Antibodies directed to the influenza B virus HA head mediated low levels of ADCC. Finally, vaccination with a recombinant modified vaccinia virus Ankara expressing the HA gene of a clade 1 A(H5N1) highly pathogenic avian influenza virus led to the induction of ADCC-mediating antibodies, which cross-reacted with H5 viruses of antigenically distinct clades. Taken together, it is clear that virus-specific antibodies induced by infection or vaccination have immunological functionalities in addition to neutralization. These functionalities could contribute to protective immunity. The functional profiling of vaccine-induced antibodies may provide further insight into the effector functions of virus-specific antibodies and their contribution to virus-specific immunity. These outbreaks of influenza are caused by influenza A viruses (IAV) of the H1N1 and H3N2 subtypes, and influenza B virus (IBV). Two lineages of IBV have been distinguished that have cocirculated since the 1970s, the B/Victoria/2/87-like (B/Vic) lineage and the B/Yamagata/16/88-like (B/Yam) lineage, which differ based on genetic and antigenic properties of their hemagglutinins  by seasonal influenza viruses, which is known as antigenic drift. Because HA is the main target for the development of virus-neutralizing (VN) antibodies and the major component of influenza vaccines, the composition of the vaccines needs to be updated almost annually to guarantee optimal effectiveness . The curUpon influenza virus infection, virus-specific antibody and T cell responses are induced. Virus-neutralizing antibodies directed to the viral HA are considered an important correlate of protection from infection, provided that they match the influenza virus causing the infection antigenically. For the detection and quantification of virus-specific serum antibodies the hemagglutination inhibition (HI) and VN assays are commonly used. HI antibody titers are an accepted proxy for VN antibodies and are used to determine the efficacy of seasonal influenza vaccines. HI antibody titers are considered a good correlate of protection , althougvia a different mechanism than preventing receptor interaction, namely by preventing conformational changes of HA that take place in the endosome in a pH dependent fashion. These antibodies additionally exert other biological activities that contribute to protective immunity, which are described below. Because the HA-stalk is relatively conserved, even across various subtypes of IAV, stalk-specific antibodies are highly cross-reactive.VN antibodies are typically directed to epitopes located in and around the RBD of the variable globular head domain of HA and thus can block binding of the virus to its receptor on host cells. Most HI antibodies are strain-specific and do not recognize variants that have accumulated one or more amino acid substitution in vicinity of the RBD . Becausevia Fc-receptors and contribute to protective antiviral immunity, as was shown for influenza virus , antibody-dependent complement deposition (ADC) and antibody dependent cellular cytotoxicity (ADCC). All of these activities are mediated za virus \u20139, humanza virus \u201312, and za virus \u201315. The In the framework of the Innovative Medicines Initiative (IMI) funded project \u201cFLUCOP\u201d, which aimed at the standardization and development of assays for assessment of influenza vaccines correlates of protection, we developed and used an assay for the detection of antibodies with ADCC activity that would be suitable for standardization. Here, we compiled antibody-dependent cellular cytotoxicity (ADCC) data obtained in four separate studies to get a comprehensive overview of the induction of ADCC-mediating antibodies after influenza virus infection, or immunization with monovalent, trivalent, or quadrivalent influenza vaccines \u201319.22.1All data shown here were obtained with serum samples from subjects that were previously enrolled in other studies; ethical permission was therefore previously obtained.Serum samples were obtained from 9- and 10-year-old children before and after infection with the H1N1pdm09 virus  . In addiThe serum panels included in this study are summarized in 2.2Mycoplasma species, and were only cultured for a limited number of passages. In brief, microtiter plates were coated with recombinant protein  and after blocking the plates with bovine serum albumin (BSA), specific serum antibodies were allowed to bind at a serum dilution of 1:160. This dilution was found to be optimal and in the linear range of the dose response curve. A serum pool from uninfected children, intravenous immunoglobulins  and a high-positive serum were included in the experiments as negative and positive controls. After the serum incubation, 1x10(5) NK92.05-CD16 cells were added per well in the presence of a monoclonal antibody to CD107a, (degranulation marker), golgistop and golgiplug. After incubation for 5 hours at 37\u00b0C, the cells were stained for flow cytometric analysis. Live NK92.05-CD16 cells were identified by LIVE/DEAD and CD56 staining as described previously; CD56-PE (BD Biosciences) was used in a concentration of 1\u00b5g/ml, aqua live/dead (Thermo Fisher) was used per manufacturer\u2019s instruction  as previously described . NK92.05truction , 18. Sub2.3For the detection of ADCC-mediating antibodies to influenza A and B viruses, several antigens were used as previously described \u201319. Puri2.4Differences between two groups were assessed by either Mann-Whitney U test or Wilcoxon rank test, dependent on whether unpaired or paired samples were used. Differences between multiple groups were assessed by Kruskal-Wallis test with multiple comparisons. Linear regression on log-transformed ADNK activation percentages was performed for the serum samples from MVA-H5 vaccinated participants.33.1To get a complete overview of the induction of ADCC-mediating antibodies by influenza virus infection or influenza vaccination, we compiled datasets from four different studies Table\u00a01.3.2To determine whether a primary infection with influenza virus induced ADCC-mediating antibodies, ADCC was measured in a unique sample set of sera from 5 children obtained before and after primary infection with H1N1pdm09 influenza virus , and selected study subjects were boosted with an additional vaccine 1 year later. Serum samples were obtained prior to vaccination and throughout the trial. ADCC-mediating antibodies targeting the full-length homologous H5 protein (A/Vietnam/1194/04) were detected prior to vaccination in some participants, although exposure to an H5 IAV was not deemed likely . By measuring ADCC-mediating antibodies after adjuvanted inactivated trivalent vaccination in 24 adults  in combination with recombinant proteins bound to a solid phase. Activation of NK cells cination . The usecination , 30.We developed this assay in the framework of the IMI project \u201cFLUCOP\u201d, aimed at the standardization and development of assays for assessment of influenza vaccines correlates of protection. Although we found that results obtained with the developed ADCC degranulation assay were highly reproducible, the assay still depended on biological components (NK cells) and is relatively low-throughput. For that reason, reporter assays based on cells that express luciferase upon engagement of Fc\u03b3RIIIa have been developed. Recently, results obtained with the degranulation assay were compared with those obtained with a reporter assay, which showed that the results strongly and significantly correlated . The repIt is clear that virus-specific antibodies, induced by either infection or vaccination, have immunologically relevant functionalities, in addition to neutralization of virus infectivity by blocking glycoprotein \u2013 receptor interactions; ADP-, ADC- and ADCC-mediating antibodies are likely to contribute to protective immunity , 10\u201315. The importance of functionality profiling was recently demonstrated in a phase I clinical trial, in which chimeric hemagglutinin-based universal influenza virus vaccines were used to induce broad immunity. This was achieved by targeted boosting of HA-stalk-specific antibodies; because the HA stalk is conserved, these antibodies contribute to heterosubtypic immunity. In this trial, not only did the authors demonstrate the presence of broadly-reactive stalk-specific antibodies, they also showed functionality in neuraminidase inhibition and ADCC reporter assays. Passive transfer of these antibodies in mice showed that they were indeed an independent correlate of protection . The indTogether, we showed that influenza virus infection and influenza vaccination led to the induction of ADCC-mediating antibodies, which can be rapidly recalled by re-infection or re-vaccination. ADCC-mediating antibodies often target epitopes in the relatively conserved HA-stalk, potentially important to achieve broadly-reactive immunity. We conclude that complete functional profiling of the antibody responses is crucial to provide insight into effector functions of virus-specific antibodies and their contribution to virus-specific immunity. In addition to profiling antibody responses in blood, it is important to additionally assess the contribution of IgA at the mucosal sites. The availability of reliable assays for the measurement of functional antibodies will aid the evaluation of existing and novel influenza vaccines.RDdV and GFR conceptualized the manuscript. KH organised clinical samples for the study. All authors contributed to the writing of the manuscript. KH contributed to the study design."}
{"text": "Life-space mobility (LSM), as the extent of mobility within one\u2019s environment, is a\u00a0key for successful aging and has become a\u00a0relevant concept in gerontology and geriatric research. Adequate assessment instruments are needed to identify older persons with LSM restrictions, and to initiate, adapt or evaluate intervention strategies.To systematically identify, describe and analyze the psychometric properties of LSM questionnaires, with a\u00a0special focus on their availability in the German language.A\u00a0systematic literature search was conducted in PubMed, PsycINFO, Cochrane Library, CINAHL, and Web of Science. Studies that examined at least one psychometric property of LSM questionnaires published up to August 2021 were included and evaluated based on the consensus-based standards for the selection of health measurement instruments (COSMIN) guidelines.This study included 37 validation studies describing 13 different LSM questionnaires. Methodological quality and comprehensiveness of validations were heterogeneous. Based on comprehensive and high-quality results, four LSM questionnaires stood out: the University of Alabama at Birmingham life-space assessment (UAB-LSA), life-space assessment in persons with cognitive impairment (LSA-CI), interview-based and proxy-based versions of the life-space assessment in institutionalized settings (LSA-IS), all of them available in the German language.This systematic review provides a\u00a0concise overview of available LSM questionnaires and their psychometric properties to facilitate the selection for use in clinical practice and research. The UAB-LSA and LSA-CI for community settings and the interview-based or proxy-based LSA-IS for institutional settings were found to be the most appropriate LSM questionnaires.The online version of this article (10.1007/s00391-022-02035-5) contains supplementary material, which is available to authorized users. Mobility is a\u00a0key factor for successful aging. In clinical practice and research, assessing mobility is important for identifying individuals at risk or with mobility limitations, developing and adapting intervention strategies, and evaluating intervention effectiveness. Various mobility instruments have been developed, with a\u00a0focus on assessing motor capacity ; howeverDespite technical developments such as the global positioning system (GPS)\u00a0to objectively measure life-space parameters , LSM hasSelecting an appropriate questionnaire for clinical practice and research is challenging. Setting and target population as well as practical aspects , and the methodological quality of the instrument determined by psychometric properties  must be considered. The use of instruments validated in the appropriate language and cultural setting represents a\u00a0mandatory methodological criterion, which has so far not been documented for LSM questionnaires in the German language. In addition, previous reviews of LSM measurement tools focused only on specific LSM questionnaires  or did nThus, the primary aim of the present systematic review was to provide an overview on currently available LSM questionnaires and their psychometric properties to facilitate the selection for use in clinical practice and research. A\u00a0secondary aim was to identify LSM questionnaires validated for use in the German language.A\u00a0systematic literature search was conducted in accordance with recommendations from the Preferred Reporting Items for Systematic review and Meta-Analysis (PRISMA) protocols  and the We performed a\u00a0complete database search with no language restrictions in CINAHL, Cochrane Library, PsycInfo, Pubmed, and Web of Science up to August 2021. Search terms included combinations of variants for the keyword life-space along with different terms for assessment, validation, and psychometric properties (see Table\u00a0S1), as recommended elsewhere .Titles, abstracts and full texts of studies identified by the search were screened for eligibility by two independent reviewers . Any disagreements were solved by a\u00a0third reviewer (KH). Reference lists of relevant articles and reviews, and grey literature were also screened for additional studies for inclusion.Studies eligible for inclusion provided empirical evidence for validity, reliability, sensitivity to change, and/or feasibility of a\u00a0questionnaire designed to assess more than two spatial aspects of mobility (e.g. not only indoor vs. outdoor), usually described as life-space mobility (LSM). Each version of a\u00a0LSM questionnaire with relevant changes to the assessment procedure or the information collected were considered as a\u00a0separate instrument.Data extraction was performed by two independent reviewers . Information on main features of the identified questionnaires  was extracted from the original article of each questionnaire. Information on different aspects of feasibility, validity, reliability, and sensitivity to change was gathered from all included studies. The methodological quality of the included studies was rated by three reviewers  using the COSMIN risk of bias checklist, considering additional references for sample size rating , 11. DisAn additional focus of the narrative analysis was lain on the availability of LSM questionnaires validated in the German language.The literature search yielded 37\u00a0studies eligible for inclusion see Fig.\u00a0, in whicA\u00a0detailed description of these questionnaires is provided in Table\u00a0S2. The majority of them consider not only spatial  but also contextual aspects  of mobility. Nine questionnaires have initially been developed for community-dwelling older persons  \u201321, 24, n\u202f=\u200919) were identified for evaluating the psychometric properties of UAB-LSA [Figure\u00a0 UAB-LSA  in diffe UAB-LSA \u201345, addi UAB-LSA , 46, 47  UAB-LSA  are alsoSample sizes of the validation studies ranged from\u00a05  to 2147 Floor or ceiling effects were analyzed in 16\u00a0studies, with all reporting absence of such effects \u201348, 50. r\u202f>\u20090.5 for similar  and r\u202f>\u20090.30\u20130.50 for related constructs  for evaluation. Concurrent validity was assessed in seven studies [Almost all studies included a\u00a0validity analysis. Construct validity was assessed in 30\u00a0studies \u201348, 50,  studies , 49, 50. studies , 24, con studies , 28, 36, studies , 28.Reliability results were evaluated in terms of test-retest reliability in 22\u00a0studies, showing predominantly acceptable to good results with intraclass correlation coefficients or Kappas of >\u202f0.7 . Poor teOnly eight studies analyzed sensitivity to change of the LSM questionnaires , 47, 48.The methodological quality of the included validation studies assessing the different psychometric properties was predominantly moderate to high across studies (Table\u00a0S3). Overall, methodological quality of validity studies was predominantly negatively affected by low sample sizes that were not statistically justified , 42, 50 The LSA-CI and both versions of the LSA-IS were comprehensively validated in the German language, with overall good results on feasibility, validity, reliability, and sensitivity to change \u201323, 48. This systematic review provides a\u00a0concise overview of currently available questionnaires to assess LSM and their psychometric properties with comprehensive information in the supplements. To the best of our knowledge, this is the first review to systematically analyze such a\u00a0wide range of LSM questionnaires, with the aim of providing clinicians and researchers with practical knowledge on LSM questionnaires to help them select an instrument appropriate for their purpose.A\u00a0large number of different LSM questionnaires were identified. Given the importance of LSM-associated factors with aspects of successful aging  , 15 and Apart from appropriateness of the instrument for the target population, which also covers the availability in the specific language, its feasibility plays an important role in both clinical routine and research. Although feasibility aspects of the LSM questionnaires were rarely reported or only as side notes, presented results for the completion rates/times and floor/ceiling effects suggest that they are accepted and not time consuming and cover an adequate range of respondents\u2019 LSM.Most information was provided for the validity of the LSM questionnaires, more specifically for their construct validity showing predominantly hypothesis-confirming associations with physical, cognitive, psychosocial, financial, environmental, and/or sociodemographic variables. Given the overall complexity of real-life mobility , it was Among the different LSM questionnaires, the UAB-LSA stands out due to its widespread use and the availability of validated versions in various languages and populations. Potential limitations of the UAB-LSA are its relatively long, retrospective assessment period of 4 weeks and its focus on the community setting, which may complicate its use in studies focusing on more short-term LSM (changes), in populations with cognitive impairment due to possible recall bias or institutional settings. To promote recall of LSM, the recommended LSA-CI covers a\u00a0shorter assessment period of 1\u00a0week and has shown to be feasible, valid, reliable and sensitive to intervention-induced changes in community-dwelling older persons with and without cognitive impairment , 48. TheOverall, recommendations for use based on comprehensive results on adequate psychometric properties with high methodological quality could only be identified for the UAB-LSA  and the Future studies on psychometric properties of LSM questionnaires should focus on feasibility aspects, select variables for construct validity analyses based on a\u00a0conceptual LSM framework and evaluate the ability to detect changes in LSM. Future developments might combine the accuracy of sensor-based assessments with aspects evaluated by questionnaires concerning independence of mobility.Several life-space mobility (LSM) questionnaires are available for clinicians or researchers to assess spatial aspects of mobility in everyday life; however, only four questionnaires provided a\u00a0comprehensive validation including good feasibility, validity, reliability and sensitivity to change. Given the comprehensive validation and overall good psychometric properties, the UAB-LSA and LSA-CI can be recommended for use in community-dwelling older persons and the two versions of the LSA-IS (interview-based and proxy-based) for use in institutionalized older persons. Each of these four LSM questionnaires is available in the German language.Most identified LSM questionnaires showed acceptable to good psychometric properties, although validation strategies were partly limited to single psychometric properties in a\u00a0number of studies.Based on the available evidence, we recommend the most comprehensively validated instruments with overall good psychometric properties: UAB-LSA and LSA-CI for community-dwelling older persons and the LSA-IS as self-based or proxy-based version for institutionalized older persons.All recommended questionnaires are validated for use in the German language.The supplemental documents include information on the search terms (Table S1), characteristics (Table S2) and psychometric properties (Table S3) of included assessment instruments, and the available assessment forms and manuals in German language"}
{"text": "During the COVID-19 pandemic, different SARS-CoV-2 variants of concern (VOC) with specific characteristics have emerged and spread worldwide. At the same time, clinicians routinely evaluate the results of certain blood tests upon patient admission as well as during hospitalization to assess disease severity and the overall patient status. In the present study, we searched for significant cell blood count and biomarker differences among patients affected with the Alpha, Delta and Omicron VOCs at admission. Data from 330 patients were retrieved regarding age, gender, VOC, cell blood count results , common biomarkers , ICU admission and death. Statistical analyses were performed using ANOVA, the Kruskal\u2013Wallis test, two-way ANOVA, Chi-square, T-test, the Mann\u2013Whitney test and logistic regression was performed where appropriate using SPSS v.28 and STATA 14. Age and VOC were significantly associated with hospitalization, whereas significant differences among VOC groups were found for WBC, PLT, Neut%, IL-6, creatinine, CRP, D-dimers and suPAR. Our analyses showed that throughout the current pandemic, not only the SARS-CoV-2 VOCs but also the laboratory parameters that are used to evaluate the patient\u2019s status at admission are subject to changes. Among these categories, variants of interest (VOI) are those that present characteristics that warrant continuous monitoring and further evaluation, while variants of concern (VOC) are the variants that are proven to be more virulent, more transmissible, more pathogenic and at some extent may evade treatment options and/or the immune responses due to vaccination or previous infection , coagulative parameters , biochemical parameters  and inflammatory parameters . Other parameters, such as procalcitonin, KAccording to the dynamic nomenclature for SARS-CoV-2 lineages  and the The laboratory diagnosis of COVID-19 was performed on nasopharyngeal samples by real-time PCR, using either the NeuMoDxTM SARS-CoV-2 assay  or the Abbott Molecular Real-Time assay . The assay selection for each case depended upon the priority given during the everyday clinical practice for the respective sample. Testing of samples with higher priority was performed using the NeuMoDxTM SARS-CoV-2 assay.Viral RNA was extracted from nasopharyngeal samples using the MagMAX Viral/Pathogen Nucleic Acid Isolation kit in the automated KingFisher Flex platform  according to the manufacturer\u2019s protocol. A total of 10 \u03bcL of the extracted RNA was used for the library construction with the QIAseq FX DNA Library Core Kit and the QIAseq SARS-CoV-2 primer panel . Library quantification was performed with the KAPA SYBR FAST Universal qPCR kit , and the samples were paired-end sequenced on the NextSeq500 Illumina platform with an additional step of heat denaturation of libraries before loading. The resulting sequenced reads were 150 bp\u2014long, and they were quality-controlled using the FastQC software v.0.11.9 .https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2 last accessed on 5 March 2023).Raw sequencing data were first processed using Trim galore for adapter and quality trimming. The cleaned reads were then mapped to the SARS-CoV-2 reference genome  using Minimap2 tool with a minimal chaining score equal to 40. From this process, only the paired-end sequences were retained, while any other  were removed. The produced alignment was processed  using the samtools/bedtools software tools suite. GATK Haplotype Caller  was used to detect and filter nucleotide mutations present in over 90%  of the sequences at each position of the reference genome. The detected mutations were used to create consensus sequences for each sample (bcf tools consensus), which were used as input to the Pangolin tool for phylogenetic identification and assignment of different SARS-CoV-2 strains. Ultimately, all identified mutations were annotated using the SnpEff tool and the NC_045512.2 (v.5.0) database  of EMBL-EBI (accession number PRJEB44141). Sample accession numbers are provided as \u00ae, Brea, CA, USA). The suPAR was determined by the suPARnostic\u00ae TurbiLatex assay  on the Cobas\u00ae 8000 c702 analyzer  using K3-EDTA plasma samples. The measurement of IL-6 was performed on the Cobas\u00ae e 411  in serum samples. D-dimers were measured on the ACL TOP 750 CTS  coagulation analyzer in plasma samples retrieved from whole blood, drawn into buffered sodium citrate  tubes after centrifugation at 3000 rpm for 10 min.Blood cell analysis was performed on XN 1000 hematology analyzer , using whole blood samples in tubes with K3EDTA (Ethylene Diamine Tetra Acetic acid) as anticoagulant. Measurements of urea, creatinine, SGOT, SGPT and CRP were done in serum samples on the DxC 700 AU instrument  and the Kruskal\u2013Wallis test  in order to examine the association of lab indexes and COVID-19 subtypes for the entire group of patients, and moreover, we performed the same analysis separate for male and female patients. Patients were categorized based on their age into three groups . Two-way ANOVA was performed to explore the interaction between age groups and COVID-19 subtypes on the values of lab indexes. Two-way ANOVA was also performed to examine possible associations between the age of the patients and the interactions between clinical outcomes and COVID-19 subtypes. Chi-square tests were performed to examine the association between COVID-19 subtypes and the clinical outcomes of interest . T-tests  were performed to evaluate the association between the age of the patients and the clinical outcomes. Finally, logistic regression models were performed to explore the effect of age on clinical outcomes, with and without adjusting for the different COVID-19 subtypes. All analyses were performed using IBM SPSS v.28 and STATA 14 .The study group included 330 patients  with a mean age of 55.2 years . Overallp-value = 0.020). More specifically, higher WBC values were found in Omicron cases (7.59) compared to Delta (6.12) . More specifically, Alpha showed a higher % of neutrophils (69.6) than Omicron . More specifically, statistically significant differences between Delta (0.61) and Alpha (1.07) were observed . Omicron presented higher PLT counts (247) than Alpha  and Delta  as well . Participants with the Delta subtype (5.92) had higher creatinine levels compared to participants with the Alpha (1.01) and the Omicron (1.67) subtypes . Participants with the Alpha subtype (48.3) had higher IL-6 levels compared to participants with the Omicron (31.9) subtype . The two-way ANOVA analysis proved that this difference occurred mainly in the patients of the Alpha subgroup. In the subgroup of Omicron, the Neut% was almost the same for patients >70 and patients 50\u201370 y. Similarly, in the Delta subgroup, the Neut% was almost the same for patients of 50\u201370 y and patients <50 y. Alpha patients also had higher Ig values (1.07) compared to Delta patients , and Delta patients presented higher creatinine values (5.92) than both Alpha  and Omicron  patients.The two-way ANOVA analysis proved that the Neut% difference was mainly due to the group of participants aged >70 y. In the group of participants aged 50\u201370 y, the difference of Neut% between Alpha and Omicron was almost zero. Additionally, in the younger age group (<50 y), the patients with Alpha had lower Neut% values compared to patients with Delta. We also observed that the main effect of age was statistically significantly associated with the Neut%  association between SARS-CoV-2 VOC and hospitalization was found. Indeed, 44.4% of the Omicron patients admitted to our hospital needed hospitalization, whereas 75% of Alpha and 70.6% of Delta patients were hospitalized. A significant association with ICU admission (p = 0.002) was also found. 30% of the patients with Alpha needed ICU admission, whereas only 16% of Omicron and 10% of Delta patients were admitted to the ICU. This similarity between Omicron and Delta highlights that even though Omicron cases were less likely to be hospitalized, severe Omicron cases could end up in the ICU with similar probabilities to Delta. No association was observed between VOC and death as a final outcome.A statistically significant . Delta-infected male patients had higher creatinine values (1.51) than Alpha patients . Among females, Omicron patients had higher WBC counts (7.71) than Delta  and lower IL-6 values  than Alpha  and Delta . CRP values were higher for female patients with the Delta (5.10) than for those with the Omicron  variant.Among male patients, those infected by Omicron had higher WBC (7.5) and PLT (233) counts than those infected by Delta  (p < 0.001)  A. As exp< 0.001) B. ParticNo statistically significant interactions between hospitalization and VOC on age were found. In other words, age was statistically significantly associated with hospitalization , but this association did not change for different SARS-CoV-2 VOCs. That means that despite the VOC, older patients are more likely to be hospitalized. No statistically significant interactions between ICU admission and VOC on age were detected. The same was observed for death and VOC on age.In the logistic regression analysis, however, age was statistically significantly associated with the hospitalization status of patients when adjusting for VOC. If the age was increased by one year, the risk of hospitalization increased by 6% for patients with the same VOC. Moreover, for patients of the same age, those with the Alpha VOC had a 4-fold higher risk of hospitalization compared to patients with Omicron, and those with Delta had a 3.8-fold higher risk of hospitalization compared to patients with Omicron. Age was significantly associated with death when adjusting for VOC. If the age was increased by one year, the risk of death increased by 8.3% for patients with the same VOC.p < 0.001). We observed that D-dimers differed significantly among the three VOCs . Patients with Omicron had higher mean values of D-dimers compared to patients with the other VOCs. Two-way ANOVA analysis proved that the main reason for this difference was due to the group of participants aged >70 y, where these differences were greater compared to the other age groups (p < 0.001). Two-way ANOVA analysis proved that the main reason for this difference was caused by patients with Omicron, where the differences between the three age groups were higher.Statistically significant interactions were found among VOC and age groups on D-dimers  were observed. The suPAR values differed significantly among the three VOCs (p < 0.001), and patients with Omicron had higher mean values of suPAR compared to the patients with the other VOCs. Two-way ANOVA analysis proved that the main reason for this difference was (due to) the group of participants aged >70 y, where these differences were greater compared to the other age groups (p < 0.001). Two-way ANOVA analysis proved that the main reason for this difference was the patients with Omicron, where the differences between the three age groups were greater, and also the Delta subgroup patients aged <50 y who had higher suPAR values compared to patients aged 50\u201370 y.Statistically significant interactions among VOC and age group on the suPAR  emerged and prevailed within the range of two weeks. Delta sub-variants, however, continued to be detected at lower rates. Omicron 2 (BA.2 and BA.2.9) emerged in February 2022 and became rapidly predominant within the next months .During the pandemic, it has been evident that infections with SARS-CoV-2 are associated with a wide range of symptoms. Moreover, a large study from England  showed cAt first glance, our results showed that together with the evolution of SARS-CoV-2, its interaction with the human organism is subject to changes, and this is reflected in variations regarding the values of different useful biomarkers for COVID-19. Some of our results seem to be confirmatory, like the fact that Omicron patients were less likely to be hospitalized than Alpha and Delta patients. Indeed, Alpha and Delta patients were four- and 3.8-fold more probable to be hospitalized than Omicron, respectively, and this underlines the value of molecular epidemiology surveillance to clinical practice and patient management. On the other hand, the elevated Omicron ICU admissions or the lack of significant association between VOCs and death should be considered, also taking into account that in our sample, the vaccination status and information on comorbidities were not available. The same limitations could also partially explain the fact that in our study, age was related to hospitalization and death but not to ICU admission.Interestingly, in our study group, Omicron was related to elevated PLT counts and D-dimer values in the elderly, suggesting that thromboembolic events may be more likely to occur in such patients. According to estimates, thrombosis accounts for one in every four fatalities around the world, making it one of the main causes of mortality in the globe . After 2SuPAR is the soluble form of uPAR, and elevated levels can be found in infections , neoplasmatic and autoimmune diseases. It has already been shown that high levels of suPAR are observed in COVID-19 patients and especially those with severe or critical illness, who had longer hospitalization and higher demands of oxygen therapy . SuPAR pFrom the early stages of the pandemic, the need to interpret the results of fast and easy-to-perform blood tests in the context of COVID-19 severity and/or outcome was evident. For this reason, several systematic reviews aimed to collectively assess the results of various individual research works. In a systematic review by Tjendra et al. , severalIn a later systematic review and meta-analysis , lymphopIn a systematic review and meta-analysis regarding the role of IL-6 in COVID-19, it was shown that higher levels of IL-6 were associated with poor clinical outcomes . More spAnother systematic review and meta-analysis investigated the role of biomarkers and attempted an estimation of threshold values that could be related to adverse clinical outcomes . HypoalbIn a systematic review and meta-analysis by Zhang et al. , the rolIn a study by Wang et al. , the corThe need for creating a risk tool based on the results of routine blood biomarkers and basic patient information that would ensure the best possible triage for COVID-19 patients led to the development of LUCAS, a COVID-19 mortality prediction calculator . LUCAS uOur study presents some limitations mainly because of its retrospective nature. Obviously, the outcome and the laboratory test results of COVID-19 patients may be influenced by viral coinfections and/or superimposed bacterial and fungal infections, especially during hospitalization . Even thDespite these limitations, to our knowledge, this is the first study to compare blood parameters among patient groups affected by different VOCs documented by whole genome sequencing data. These findings contribute to the growing literature on COVID-19 as different variants emerge. Overall, our results showed that each VOC combines with a unique \u201claboratory\u201d identity. This fact may affect the utility of laboratory markers for monitoring COVID-19 activity and progression. In the same context, the therapeutic guidelines based on laboratory values could be adjusted for distinct VOCs. Altogether, perpetual updating of knowledge about the distinctive laboratory values of each VOC is vital for clinicians setting the personalized and targeted therapy of all COVID-19 patients as an ultimate goal.Our data, within the context and limitations of a real-world study, show that during the course of the pandemic, not only SARS-CoV-2 variants but also the laboratory parameters that are used to evaluate the patient\u2019s status at admission may be subject to changes. The regular monitoring of VOIs and VOCs may provide essential information to clinicians about the circulating variants and their clinical importance. Medical doctors, however, should also be aware that the usefulness of some biomarker values may vary as variants evolve over time. Therefore, uninterrupted research and continuous medical education are needed to provide the best healthcare services to patients affected by COVID-19."}
{"text": "PURPOSE: To explore the relationship between ATM, ATR and CAT polymorphisms and prognosis of lung cancer patients received platinum-based chemotherapy.METHODS: 404 patients with lung cancer who received platinum-chemotherapy were enrolled and DNA typing was performed. Cox regression analysis and stratification analyses was performed to assess relationships between OS and PFS with SNPs genotypes. The prognosis of lung adenocarcinomaand squamous cell carcinomapatients was analyzed with The Cancer Genome Atlas (TCGA) database according to the grouping of CAT expression.RESULTS:CAT rs769217 was significantly related to PFS of patients with lung cancer who received platinum-chemotherapy. In the Additive model, rs769217 was associated with PFS . In the Dominant model, CT and TT genotypes led to lung cancer progression 0.738 times more than CC genotype. In stratification analyses of association between CAT rs769217 polymorphisms and PFS, the HR of patients at stage IV in additive model was 0.73, and HR was 0.745 (p = 0.034) in dominant model. For OS analyses, HR was 0.672 in the older lung cancer patients (>55 years old) in additive model. Meanwhile, in the Dominant model, it was found that the older patients with CT and TT genotypes had better prognosis, and the risk of death after receiving platinum-based chemotherapy was 0.692 times that of patients with CC genotype (p = 0.037). TCGA data shows that LUAD patients with high CAT expression have longer OS (p = 0.020).CONCLUSION:CAT rs769217 is significantly related to PSF of platinum-based chemotherapy in lung cancer patients and may be a biomarker for predicting the prognosis of lung cancer patients with platinum-based chemotherapy. ATM (ataxia telangiectasia mutant gene), ATR (ataxia telangiectasia and Rad3 related) and CAT  polymorphisms may be related to the prognosis of lung cancer patients receiving platinum-chemotherapy[Lung cancer is still the main cause of cancer death in the worldwide. There were an estimated 2,206,771 new cases and 1,796,144 cancer deaths of lung ancer worldwide in 2020 according to GLOBOCAN 2020. Platinumotherapy\u201310.via their downstream targets[ATM and ATR may be potential targets to affect platinum chemosensitivity[ATM and ATR SNPs may regulate kinase to enhance the DNA-damaging effect of Pt-based chemotherapy in cancer cells[ATM and ATR kinases were the key mediators of DNA damage response (DDR), which induce cell cycle arrest and facilitate DNA repair m targets\u201313. Obvinsitivity,15. Resensitivity. ATM andcer cells,16,17.2O2, reducing the production of ROS in cells[CAT rs769218 is related to the prognosis of gastric cancer patients receiving platin and fluorouracil-based adjuvant therapy, but the correlation between the prognosis of lung cancer patients and CAT polymorphism has not been reported[ATM, ATR and CAT with platinum-based chemotherapy outcome of lung cancer patients.Platinum-based chemotherapy causes cancer cells death through inducing oxidative stress to highly toxic level. Emergin in cells. Studies2) or carboplatin (AUC 5), which were both administered on Day 1 every 3\u00a0weeks, in combination with pemetrexed (500\u00a0mg/m2) on Day 1 every 3\u00a0weeks, gemcitabine  on Days 1 and 8 every\u00a03 weeks, paclitaxel (175\u00a0mg/m2) on Day 1 every 3\u00a0weeks, docetaxel (75 mg/m2) on Day 1 every 3\u00a0weeks, or navelbine (25\u00a0mg/m2) on Days 1 and 8 every 3\u00a0weeks. Patients did not undergo surgery, targeted therapy, radiotherapy, or other anti-tumor therapy before chemotherapy. A physical examination as well as a detailed inquiry into each patient\u2019s medical history was carried out. Patients with serious concomitant diseases that might greatly affect their physical condition were excluded. The study protocol was approved by the Ethics Committee of Institute of Clinical Pharmacology, Central South University , and all subjects were provided with written informed consents. We applied this study for clinical admission in the Chinese Clinical Trial Register (registration number: ChiCTR-RO-12002873)[All patients in this study were recruited from Xiangya Hospital of Central South University and Hunan Cancer Hospital , from 2012 to 2019. All patients were diagnosed with NSCLC by histopathological examination and confirmed the absence of driver genetic alterations that could be targeted. All patients received first-line platinum-based chemotherapy regimens for two to six cycles: cisplatin (75\u00a0mg/m12002873).http://pngu.mgh.harvard.edu/purcell/plink/)to detect and control the quality of SNPs data, requiring that the MAF  \u22650.05, the call rate\u226590%, and conform to Hardy-Weinberg equilibrium. Finally, we selected 7 SNPs of ATR, ATM, and CAT for follow-up  and stored at \u221220\u00b0C according to the instructions. Each DNA sample was genotyped by Sequenom Mass Array Genotype Platform . After polymerase chain reaction (PCR), the product was purified by resin and analyzed by the Mass Array system (Sequenom). Then weollow-up .p-values were two-sided, p < 0.05 were supposed to be significant. All the above analyses were performed by The SPSS version 25.0 . UCSC Xena  was used to analyze the prognosis of LUAD and LUSC patients in TCGA database.OS  was defined as the time from treatment initiation until either the date of death, the date of last follow-up or the date of end of analysis. PFS  was defined as the time from treatment initiation until disease progression or death, whichever occurred first. Kaplan-A total of 404 lung cancer patients received platinum-based chemotherapy were included in this research. There were 175 patients with age \u226455\u00a0years old, accounting for 43.32%. Most of the patients were male, accounting for 77.48% (313). 40.10% (162) patients have the habit of smoking. 97.52% of the patients (394) were in stage III or IV. 50% of the patients (202) were adenocarcinoma and 42.33% (171) were small cell carcinoma .CAT rs769217 was significantly related to PFS. In the additive model, rs769217 was associated with PFS, HR = 0.747, 95% CI = 0.581\u20130.960, p = 0.023. In the dominant model, CT + TT genotypes led to lung cancer progression 0.738 times more than CC genotype. In the recessive model, the p-value > 0.05, which is not significant . In the Dominant model, the HR of patients in stage IV was 0.745, p = 0.034 (p = 0.031 of the older lung cancer patients (>55\u00a0years old) was found in the Additive model. Meanwhile, in the Dominant model, it was found that the older patients with CT and TT genotype had better prognosis, and the risk of death after receiving platinum-based chemotherapy was 0.692 times that of patients with CC genotype (p = 0.037)  .In 2019, about 2 million people worldwide died from lung cancer, more than any other cancer. Annuallin vitro studies lead to acute apoptosis that involves induction of oxidative stress but is largely DNA damage-independent[DNA is usually considered as the main target of platinum chemotherapeutic drugs, but the cisplatin used dependent. Celluladependent. Howeverdependent. ROS candependent.CAT rs769217 may affect the PFS of lung cancer patients receiving platinum-based chemotherapy , is a key antioxidant enzyme in the bodies defense against oxidative stress[CAT rs1001179 polymorphism and prostate cancer[CAT rs769217 can affect the prognosis of patients with biliary tr an act cancer (BTC), that knockdown of CAT induced chemoresistance through elevation of ROS level and activation of Nrf2-ABCG2 pathway in BTC cell lines[CAT polymorphism on the prognosis of patients with lung cancer receiving platinum chemotherapy has not been reported. CAT polymorphisms can affect the expression of CAT mRNA in tumor tissues[CAT expression is significantly longer (p = 0.020, p = 0.048 respectively; CAT expression in tumor cells, thus regulating ROS and making tumor cells sensitive to platinum-chemotherapy, but the specific mechanism needs to be further explored. Therefore, CAT can be used as a potential target to enhance the sensitivity of platinum-chemotherapy, nanocarriers of platinum and CAT enhance the cytotoxicity of drug resistant cancer cells[CAT expression (CAT genes. In conclusion, our study showed that CAT rs769217 is significantly related to PSF of platinum-based chemotherapy in lung cancer patients. CAT rs769217 may be a biomarker for predicting the prognosis of lung cancer patients with platinum-based chemotherapy.Our results suggest that otherapy . CAT genve stress,42. Prevve stress\u201345. Two te cancer,47. At tte cancer\u201350. Therell lines. But ther tissues, and acccer cells. In addiression . The exp"}
{"text": "Regulated cell death (RCD) is a regulable cell death that involves well-organized signaling cascades and molecular mechanisms. RCD is implicated in fundamental processes such as organ production and tissue remodeling, removing superfluous structures or cells, and regulating cell numbers. Previous studies have not been able to reveal the complete mechanisms, and novel methods of RCD are constantly being proposed. Two metal ions, iron (Fe) and copper (Cu) are essential factors leading to RCDs that not only induce ferroptosis and cuproptosis, respectively but also lead to cell impairment and eventually diverse cell death. This review summarizes the direct and indirect mechanisms by which Fe and Cu impede cell growth and the various forms of RCD mediated by these two metals. Moreover, we aimed to delineate the interrelationships between these RCDs with the distinct pathways of ferroptosis and cuproptosis, shedding light on the complex and intricate mechanisms that govern cellular survival and death. Finally, the prospects outlined in this review suggest a novel approach for investigating cell death, which may involve integrating current therapeutic strategies and offer a promising solution to overcome drug resistance in certain diseases.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01267-1. In multicellular organisms, the process of generating new cells and removing damaged or unwanted cells maintains a dynamic balance during the development and maintenance of homeostasis. Thus, cell death is essential for life. In 1972, Kerr et al. first proposed a new term \u2018apoptosis\u2019 and described its morphological features, which has been a classic pattern of programmed cell death .To date, several studies have linked iron (Fe) and copper (Cu) with multiple forms of RCD. Newly discovered RCDs, ferroptosis and cuproptosis are dependent on these two transition metal ions respectively, which distinguish them from other RCDs , 5. FerrTo determine the effects of Fe and Cu on RCDs, we summarized almost all possible modes of iron and copper to impart significant cell damage and elaborated the mechanisms of iron- and copper-mediated diverse RCDs, including ferroptosis, cuproptosis, apoptosis, autophagy, necroptosis and pyroptosis Fig. . In addi2+, Cu 1+) and oxidized  states to perform numerous biological functions, including redox reactions, electron transport, oxygen transport and energy metabolism [Iron and copper belong to the first series of transition metals, which also includes chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni) and zinc (Zn). Iron and copper have similar characteristics, are both essential nutrients, are involved in fundamental biological processes and play a crucial role in health and disease , 9. In ltabolism \u201312.Iron and copper can also influence the functions of proteins upon binding to these proteins. On the one hand, Fe and Cu must be bound and protected within the active sites of proteins . For exaThe structure of the tyrosinase active site consists of a dual-core copper center and histidine residues . TyrosinIn addition, their relationship is close. Copper may positively influence the transport of iron. Systemic copper deficiency blocks iron transport and accumulates in tissues, ultimately generating cellular iron deficiency . ConversIt should be mentioned that everything has both sides. Iron and copper are beneficial to life in moderate amounts, but excesses or deficiencies of these metal ions are harmful. On the one hand, when there is too much iron in the body, excess iron forms a labile iron pool, confers cell toxicity, and affects cell damage, which can lead to cancer, hematological diseases, brain injury and other chronic and commonly encountered diseases . In addiIron and copper have many of the same or different modes that stimulate a range of indirect negative effects, provoking cell impairment and eventually cell death . Then, wOxidative stress is a state of oxidation-antioxidant imbalance and generates large amounts of reactive oxygen species (ROS). Moderate ROS levels assist in the control of cell proliferation and differentiation, but high concentrations of ROS are harmful to normal and cancer cells , 27. Whe2+ and Cu+ transform hydrogen peroxide into hydroxyl radicals (OH\u22c5). OH\u22c5 is one of the most reactive species found in nature and is extremely toxic.+ + H2O2 \u2192 Cu2+ + OH\u2212 + OH\u22c5Cu\u2212based Fenton reaction\u2003Cu2+ + H2O2 \u2192 Fe3+ + OH\u2212 + OH\u22c5Fe\u2212based Fenton reaction\u2003FeThe first mechanism by which Fe and Cu induce oxidative stress is through the Fenton reaction , 33, 37.The second way of inducing oxidative stress is the consumption of antioxidants. There are two kinds of antioxidants. One class of them is low-molecular-weight antioxidants, including glutathione (GSH), ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), carotenoids, flavonoids, and other antioxidants that are capable of chelating metal ions to inhibit their catalytic activity and reduce ROS . The othEvidence of copper and iron leading to antioxidant deficiency can be clearly seen in the case of GSH. GSH plays a critical role in removing ROS and is a substrate for multiple enzymes that remove ROS. Copper and iron can catalyze GSH oxidation, which oxidizes reduced GSH to oxidized glutathione disulfide (GSSG) to reduce the concentration of GSH . The depSome studies have revealed that transition metal ions, including Cu, Fe, Aurum (Au) and Manganese (Mn), can inhibit the proteasome , 41, 42.Iron has a separate function driving lipid peroxidation, while copper has separate functions, including breaking DNA directly, protein ubiquitination and driving protein lipoylation and aggregation Table\u00a0.Table 2ICopper could influence DNA indirectly through ROS. Meanwhile, copper could also act on DNA directly in another way.Copper could bind to DNA to form a bis- copper(II) complex, which may be inserted into the minor groove in the DNA. This DNA\u2013copper complex is oxidized in the presence of an activator, especially hydrogen peroxide. Then, hydrogen bonds of the DNA\u2013copper complex are hydrolyzed, and DNA is cleaved , 56. AnoIron complexes that could bind and damage DNA were undetected. Instead, iron even assists DNA synthesis and repair and works as a cofactor of multiple enzymes, including multiple DNA repair enzymes  and ribonucleotide reductase .In addition to regulating the proteasome, copper also has another way to impact protein degradation. Recent work detected that Cu+ promoted multiple proteins ubiquitination and degradation by positive allosteric activation of the E2 conjugating enzyme clade UBE2D1\u2013UBE2D4 . SeveralStudies have indicated that Cu(II) is effective at aggregating proteins . MeanwhiIt has been presented previously that aggregating proteins are not critical for Cu cytotoxicity. In vitro, Cu(II) caused bovine albumin (BSA) to aggregate in a time- and concentration-dependent manner. It is possible that this aggregation is the result of covalently cross-linked multimers because Cu(II) is able to oxidize \u2013SH groups in different molecules. In addition, Cu(II) plays an important role in the nonamyloid aggregation of H\u03b3D crystallin in cataract disease. Copper ions alter the conformation of hexagonal D-crystallin, causing light-scattering aggregates with high molecular weights. Surprisingly, the interaction of Cu ions with H\u03b3D crystallin also promotes protein folding . In otheNonetheless, Tsvetkov et al. found that Cu drives protein lipoylation and aggregation, resulting in proteotoxic stress and further inducing cell death . This ilIn ferroptosis, overloaded iron(II) produces a large number of ROS via Fenton reaction, promotes the production of lipid ROS, and then promotes ferroptosis . SpecifiIron and copper mediate many forms of cell death. One or both metals could induce ferroptosis, cuproptosis, apoptosis, autophagy, necroptosis and pyroptosis in different ways Table\u00a0.Table 3IIron and copper are able to induce ferroptosis , 6. Ferr3+, free Fe2+ ions show higher toxicity to induce ferroptosis. Fe3+-Compared to Fe3+ is reduced to Fe2+ by STEAP3 , and then Fe2+ is released into the cytosol through divalent metal transporter 1 (DMT1) [2+ forms the labile iron pool (LIP), which plays a significant role in ferroptosis [The transferrin (TF) complex binds with the membrane protein TFRC (transferrin receptor) and is imported into cells through endocytosis . In endo1 (DMT1) . Excess roptosis . In addiroptosis .Generally, cells manage iron through absorption, output, utilization, and storage . When inPolyunsaturated fatty acids (PUFAs), such as adrenoyl (ADA) and arachidonoyl (AA), are the easiest lipids to oxidize in ferroptosis . With th\u2212 is a membrane transport protein and consists of SLC7A11 and SLC3A2. System Xc\u2212 transports glutamate outside and cystine inside cells at a ratio of 1:1 [System Xco of 1:1 . Cystineo of 1:1 . Hence, 2+ directly binds to the C107 and C148 cysteine residues of the GPX4 protein, which induces GPX4 protein aggregation. Then, the aggregates are recognized by the autophagic receptor TAX1BP1 (Tax1 binding protein 1) and degraded by the autophagy pathway. Subsequently, ferroptosis occurs in response to autophagy [In general, Fe is the ferroptosis-inducing factor. Interestingly, a study found that Cu also induced ferroptosis . Gao et utophagy .Tsvetkov et al. discovered a new form of RCD termed cuproptosis . They reThe copper transporters SLC31A1 (CTR1), ATP7A and ATP7B are responsible for regulating the level of copper in cells . SLC31A1In addition, there are useful tools that can bind to copper ions and help them enter cells, named copper ionophores . These iGSH binding to copper is an important way to reduce copper accumulation. Tsvetkov et al. also found that the GSH synthase inhibitor BSO induced cuproptosis.The TCA cycle can control cell fate and function, serving as an important route for oxidative phosphorylation and regulating redox, biosynthetic and bioenergetic balance . TsvetkoLipoylation is a posttranslational modification in which lipoic acid is attached to proteins. This modification is unique to the pyruvate dehydrogenase complex (PDC) in the TCA cycle . In mitoFDX1 is a mitochondrial reductase that reduces Cu2+ to its cuprous and more toxic form, Cu1+. In addition, FDX1 binds and is inhibited by elesclomol . HoweverAlthough ferroptosis inhibitors do not inhibit copper-induced cell death in cells, excessive copper leads to a loss of Fe-S cluster proteins under the regulation of FDX1 \u00a0Fig. 3)3). TheseIn addition, GSH is a known copper and iron chelate that can reduce metal ion toxicity. GSH serves as the principal substrate for GPX4 and acts as a suppressor of ferroptosis. The induction of cuproptosis by the GSH synthase inhibitor BSO shows that GSH serves the same function in cuproptosis. These observations raise the possibility of crosstalk between Cu and Fe.Apoptosis is a classic RCD that can be induced by both external and internal factors. External apoptosis is initiated by cell membrane proteins known as death receptors . IntrinsThere are a number of reports about Fe- and Cu-mediated apoptosis\u00a0Fig. A. In thoIron and copper can trigger intrinsic apoptosis. In osteoblasts, iron overload induces intrinsic apoptosis via ROS. Tian et al. noted that excess iron effectively causes apoptosis in osteoblasts in vitro. Iron overload leads to an increase in ROS that decreases and depolarizes MMP, increases Bax and cleaved caspase-3, and reduces Bcl-2. Changes in Bcl-2 and Bax expression also promote mitochondrial membrane permeability . In addi2+ can also modify the permeability of mitochondrial membranes, cause mitochondrial membrane depolarization, decrease mitochondrial membrane potential, reduce cytochrome\u2005c oxidase activity, and eventually induce intrinsic apoptosis [Cupoptosis , 66, 101poptosis . Rochforpoptosis .Except for intrinsic apoptosis, Fe and Cu also induce external apoptosis. Dehydroabietic acid (DHC)-Fe(III) and DHC-Cu(II) trigger mitochondrial intrinsic and extrinsic apoptosis. These complexes activate caspase-9, increase Bax, reduce Bcl-2 (intrinsic pathway), and facilitate caspase-8/caspase-4 and Fas (extrinsic pathway) . DHC- coSeveral studies have documented that iron mediates autophagy\u00a0Fig. B, a procCu complexes also lead to autophagy , 73, 74.Fe also triggers necroptosis\u00a0Fig. C, with fSeveral pathways contribute to pyroptosis, including the Caspase-1 activation-mediated classical pyroptotic pathway, Caspase-4/5/11-dependent nonclassical pyroptotic pathway, Caspase-8-dependent pyroptotic pathway and Caspase-3-dependent pyroptotic pathway .It is now well established from several studies that iron is a crucial inducer of pyroptosis\u00a0Fig. D. Zhou eCopper can also induce pyroptosis by relying on ROS accumulation . In hepaSeveral types of cell death, including apoptosis, autophagy, necroptosis, pyroptosis and cuproptosis, are related to ferroptosis. However, the cross between cuproptosis and other forms of RCD has not been reported.Ferroptosis is associated with apoptosis. p53 participates in the regulation of both ferroptosis and apoptosis. P53 is an important regulator of apoptosis, and a large number of apoptotic factors are dependent on P53 activation to regulate apoptosis. In addition, p53 can trigger apoptosis by provoking mitochondrial translocation and accelerating cytochrome c release directly . p53 alsOther studies have shown that autophagy also plays a role in the occurrence of ferroptosis , 115. AcNecroptosis and ferroptosis often co-occur in diverse diseases. In hemorrhagic stroke, ferroptotic (activating phospho-ERK1/2) and necroptotic cell death (increasing RIP1 and RIP3 mRNA expression and activating phospho-RIP1) simultaneously occur . Basit ePyroptosis and ferroptosis often occur simultaneously in diverse diseases as well. In colorectal cancer, NFS1 knockout combined with oxaliplatin causes PANoptosis  by increasing ROS . PyroptoFerroptosis is also related to cuproptosis. It was recently revealed that the ferroptosis inducers sorafenib and erastin promote cuproptosis by enhancing copper-dependent lipoylated protein aggregation in primary liver cancer cells . In a luAlthough none of the studies reviewed the cross between cuproptosis and apoptosis, autophagy, necroptosis and pyroptosis, some clues offer the possibility. A review summarized the promotion effects on RCDs of tumor suppressor p53, including apoptosis, ferroptosis, parthanatos, programmed necrosis, and autophagic cell death. They stated that p53 might also play a role in cuproptosis because the gene regulates the biogenesis of iron-sulfur clusters and the copper chelator glutathione, which are two critical components of the cuproptotic pathway . AnotherBased on the importance of copper and iron in diseases, a multitude of strategies have been developed to regulate intracellular copper and iron levels. One of the major roles of those agents is developing novel anticancer therapies. Iron and copper are vital for tumorigenesis and cancer progression. In particular, cancer cells have been shown to have higher iron requirements than normal cells, often referred to as \u2018iron addiction\u2019 . Two polIron and copper targeting strategies have been used extensively Table\u00a0. For exaIron (Fe) and copper (Cu) are the first series of transition metals that are essential nutrients, are involved in fundamental biological processes and play a crucial role in health and disease. Moderate Fe and Cu are beneficial to life, but excesses or deficiencies are harmful. Therefore, Fe and Cu are sometimes toxic to cells. Iron and copper own or co-own modes that lead to cell impairment and eventually cell death.In this review, we described some modes of iron and copper that may be deleterious to cell growth directly or indirectly. We found that iron and copper are able to impair cells through excessive ROS and proteasome inhibition. In addition, iron can drive lipid peroxidation, which leads to ferroptosis, while copper can bind and break DNA and bind and activate E2D2-inducing protein ubiquitination and degradation.and drive protein lipoylation and aggregation, inducing cuproptosis as well.Notably, it is important to tell the real reason for DNA damage: iron and copper influence ROS, evocating DNA damage indirectly, or copper binds and breaks DNA directly. In addition, it is also noteworthy to judge whether protein ubiquitination induced by copper harms cells. Copper promotes target polyubiquitination and can thus regulate the degradation rate of many proteins that are also favorable for cell growth. For example, protein ubiquitination induced by copper is helpful in development and head formation in Drosophila . Cu+ canIron and copper mediate diverse forms of cell death. Both Fe and Cu can induce extrinsic and intrinsic apoptosis, autophagy, ferroptosis and pyroptosis. Only iron is capable of inducing necroptosis, while copper is able to trigger cuproptosis. From the studies discussed thus far, copper has more functions to induce cell death, which acts on proteins compared to iron. We hypothesize that copper may also trigger necroptosis or ferroptosis by impacting protein structure and function, which may provide a new research direction.Types of cell death may be related to one another, and iron- and copper-mediated cell death is likely to be interrelated as well. Ferroptosis, as an independent mode of cell death, is related to apoptosis, autophagy, necroptosis, pyroptosis and cuproptosis, which have been extensively reported. However, only very few findings have offered clues regarding the cross between cuproptosis and other forms of RCD. There are several ways of cross talk between RCDs. First, causal relations between autophagy and ferroptosis, ferroptosis and necroptosis and ferroptosis and cuproptosis are present. Two different types of autophagy, ferritinophagy and lipophagy, producing excessive free iron ions and fatty acids, respectively, result in ferroptosis. Ferroptosis contributes to the occurrence of necroptosis. Ferroptosis inducers and regulatory genes also contribute to the occurrence of cuproptosis. Second, there are some common points between apoptosis and ferroptosis, pyroptosis and ferroptosis and cuproptosis and multiple RCDs. IFN-\u03b3 works as both apoptosis- and ferroptosis-inducing factors. Pyroptosis and ferroptosis have the same point, lipid peroxidation. p53 has promoting effects on RCDs, including apoptosis, ferroptosis, cuproptosis, and autophagic cell death. HMGB1 also plays an important role in cuproptosis and autophagy. Third, necroptosis, pyroptosis and ferroptosis, as well as ferroptosis and cuproptosis, often cooccur in diverse diseases. Fourth, pyroptosis also acts cooperatively with ferroptosis to lead to\u00a0many\u00a0diseases. The cross-talk between ferroptosis and cuproptosis with other cell death provides the possibility of joint application of existing treatment schemes and helps to solve drug resistance issues in some diseases, which may provide a new research direction.Cell death has advantages and disadvantages for individuals. On the one hand, the low viability of normal cells compromises individual survival. On the other hand, tumor cell death is beneficial to life prolongation. In this study, we described iron- and copper-induced cell death. Saving normal cells alive or killing cancer cells by regulating iron- and copper-mediated cell death may be a smart approach. In addition, the pattern and relationships between ferroptosis and other forms of death involvement in diseases determine the drugs that can be adopted to prevent uncontrolled cell death.Does copper also contribute to necroptosis? What is the relationship between cuproptosis and different types of cell death? Is it synergy or antagonism? Whether similarities and differences between copper and iron can help us explore the detailed mechanisms of cell death mediated by them. Whether these various modes of cell death can be integrated into a complete regulatory network still requires further exploration."}
{"text": "Do patients with breast cancer tend to discontinue their cardiovascular therapy at the same time they discontinue adjuvant hormone therapy?In this population-based cohort study of 5493 patients, the highest risk of discontinuing cardiovascular therapy was observed at the time when patients discontinued adjuvant hormone therapy. Furthermore, patients who discontinued adjuvant hormone therapy were at a higher risk of both breast cancer mortality and cardiovascular mortality.These findings suggest that multiple disease\u2212focused interventions are warranted to prevent discontinuation of treatment for other diseases and to improve overall survival in patients with breast cancer. This cohort study investigates whether patients with breast cancer who discontinue adjuvant hormone therapy tend to discontinue their cardiovascular therapy concomitantly, examines disease-specific morality by discontinuation of adjuvant hormone therapy, and identifies factors associated with discontinuation of therapy among patients with breast cancer. A large proportion of patients with breast cancer concomitantly use adjuvant hormone therapy and cardiovascular therapy.To examine the relative risk of discontinuing cardiovascular therapy during the periods before and after discontinuation of adjuvant hormone therapy.This population-based cohort study included all women aged 40 to 74 years in Stockholm, Sweden, who were diagnosed with breast cancer and concomitantly using adjuvant hormone therapy and cardiovascular therapy. Patients were enrolled from July 1, 2005, to August 31, 2020, with a median follow-up of 7.2 years. Data were analyzed from November 3, 2021, to May 12, 2022.Discontinuation of adjuvant hormone therapy.The main outcome was discontinuation of cardiovascular therapy  within 1 year before and after discontinuation of adjuvant hormone therapy. Incidence rate ratios with 95% CIs were estimated using Poisson regression. Furthermore, hazard ratios (HRs) with 95% CIs for cause-specific mortality were estimated using Cox proportional hazards regression models, comparing those who discontinued and continued adjuvant hormone therapy.A total of 5493 patients with breast cancer who concomitantly used cardiovascular therapy were identified; 1811 who discontinued adjuvant hormone therapy were individually matched to 1 patient each who continued therapy by year of breast cancer diagnosis, age at diagnosis, and use of the same cardiovascular therapy. Most patients (4070 [74.1%]) were aged 60 years or older at diagnosis. At the time when patients discontinued adjuvant hormone therapy, 248 (12.2%) concomitantly discontinued their cardiovascular therapy. During follow-up, a higher discontinuation rate of cardiovascular therapy was also observed among those who discontinued adjuvant hormone therapy. Consistently, adjuvant hormone therapy discontinuation was associated with an increased risk of death not only due to breast cancer  but also cardiovascular disease . Stratifying the analyses on baseline type of adjuvant hormone therapy yielded consistent results.In this cohort study of data from population-based registers in Sweden, patients who discontinued adjuvant hormone therapy were also more likely to discontinue cardiovascular therapy, especially at the time when they discontinued adjuvant hormone therapy. These findings suggest that clinicians should shift from single- to multiple-disease focus to prevent discontinuation of therapies for other diseases among patients with breast cancer. However, nonadherence to long-term cardiovascular therapy remains a concern for the expanding population of patients who survive breast cancer.18 Despite previous studies having identified a few shared risk factors for discontinuation of adjuvant hormone therapy and cardiovascular therapy,15 little is known about whether discontinuation of these 2 therapies happens simultaneously.Improved survival in patients with breast cancer has brought increased incidence of comorbidities. Cardiovascular disease is one of the most frequent comorbidities among patients with breast cancer.We hypothesized that patients who discontinue adjuvant hormone therapy were more likely to discontinue cardiovascular therapy and thus had higher mortality due to cardiovascular disease. Specifically, our study aims to (1) investigate whether patients with breast cancer who discontinue adjuvant hormone therapy tend to discontinue their cardiovascular therapy concomitantly; (2) examine breast cancer\u2013specific mortality and cardiovascular disease\u2013specific morality by discontinuation of adjuvant hormone therapy; and (3) identify shared and unshared factors associated with discontinuation of adjuvant hormone therapy and cardiovascular therapy.STROBE) reporting guideline.The regional Ethical Review Authority in Stockholm, Sweden, approved this cohort study. Informed consent is not required for large-scale, registry-based studies in Sweden, provided that the study is deemed ethical by the Ethical Review Authority. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology ,24 Multi-Generation Register,25 National Cancer Register,26 and Cause of Death Register.27 The Stockholm-Gotland Quality Register for Breast Cancer  includes all patients with breast cancer diagnosed in the Stockholm-Gotland region and provides detailed information on tumor characteristics and treatments, with 99.9% of incident breast cancer cases being recorded.20 The Prescribed Drug Register contains prescription records nationwide since July 2005, with 99.7% completeness.21 The Cause-of-Death Register (1952 onward) assesses the underlying cause of death according to strict rules by the patient\u2019s usual physician or the physician last seeing the patient before death and records a specific underlying cause of death for 96% of individuals.27Using unique personal identification numbers,Through linkage between the Stockholm-Gotland Quality Register for Breast Cancer and the Prescribed Drug Register, we identified 13\u2009053 women diagnosed with nonmetastatic and estrogen receptor\u2013positive breast cancer at 40 to 74 years of age from July 1, 2005, to August 31, 2020, in Stockholm, Sweden, who initiated adjuvant hormone therapy  code L02BA01] and/or aromatase inhibitors [ATC code L02BG]). Among them, we further identified 5493 patients with breast cancer who concomitantly used cardiovascular therapy, defined as filling at least 2 prescriptions of cardiovascular drugs , statins (ATC code C10AA), or aspirin (ATC code B01AC06) after adjuvant hormone therapy initiation and before end of follow-up , death, emigration, completion of 5-year adjuvant hormone therapy, or end of the study period , whichever came first. Analyses for cardiovascular therapy discontinuation were additionally censored at occurrence or recurrence of cardiovascular comorbidities after start of follow-up, including heart failure , arrhythmias (ICD-10 codes I47-I49), ischemic heart disease (ICD-10 codes I20-I25), and cerebrovascular diseases (ICD-10 codes I60-I69).Discontinuation of adjuvant hormone therapy and cardiovascular therapy was defined as failure to refill a prescription of the corresponding therapy within 6 months after a previous prescription.ICD-10 code C50 and death due to cardiovascular disease through ICD-10 codes I00 to I99 in the Cause-of-Death Register. Patients were followed up until death, emigration, or end of the study period , whichever came first. The median follow-up time was 6.5 years.The main underlying cause of death was used to identify death due to breast cancer through 24 Use of symptom-relieving drugs, including analgesics (ATC code N02), gastrointestinal tract drugs , antidepressants (ATC code N06A), or hypnotics and/or sedatives (ATC code N05C), was defined as having 1 prescription of corresponding drugs during the first 6 months of adjuvant hormone therapy. Prediagnosis hormone replacement therapy was defined as a systematic use of estrogen and/or progesterone  with at least 1 prescription within 6 months before breast cancer diagnosis, excluding patch and vaginal cream preparations. Participation in mammography screening was defined as attendance of mammography within 2 years before breast cancer diagnosis. Family history of breast cancer and major cardiovascular events was obtained by linking the Multi-Generation Register to the National Cancer Register and National Patient Register, respectively. Charlson Comorbidity Index score before breast cancer diagnosis was calculated using main diagnoses retrieved from the Swedish Patient Register.28Information on divorce and income at diagnosis was retrieved from the LISA register.29Data were analyzed from November 3, 2021, to May 12, 2022. To investigate whether adjuvant hormone therapy and cardiovascular therapy tend to be discontinued concomitantly, we first identified 1811 patients who discontinued adjuvant hormone therapy and used cardiovascular therapy within 1 year before the therapy discontinuation. We compared the discontinuation rate of cardiovascular therapy at adjuvant hormone therapy discontinuation with other periods . Poisson regression with a clustered sandwich estimator was used to account for lack of independence due to patients using more than 1 cardiovascular therapy.2 tests to compare baseline characteristics between those who continued and discontinued adjuvant hormone therapy. Poisson regression with clustered sandwich estimator was used to estimate the association, adjusting for matching variables, tumor size, lymph node status, tumor grade, progesterone receptor status, surgery type, chemotherapy, radiotherapy, baseline hormone therapy type, prediagnosis use of cardiovascular therapy, prediagnosis major cardiovascular event, and Charlson Comorbidity Index score at diagnosis. This analysis was stratified by 4 time bands in relation to adjuvant hormone therapy discontinuation: \u221212 to \u22123 months, \u22123 months, 3 months, and 3 to 12 months.To further investigate the association between discontinuation of adjuvant hormone therapy and cardiovascular therapy, we used a matched cohort. For each patient who discontinued adjuvant hormone therapy, we randomly sampled 1 patient who continued the therapy at the first patient\u2019s discontinuation date and used the same cardiovascular medication. Those who discontinued and continued medications were individually matched on year of breast cancer diagnosis and age at diagnosis (\u00b13 years). We used \u03c7Kaplan-Meier curves were further constructed for breast cancer\u2013specific and cardiovascular disease\u2013specific mortality for those who discontinued adjuvant hormone therapy and matched patients who continued therapy. Marginal Cox proportional hazards regression models with clustered sandwich estimator were used to compare cause-specific mortality between patients who discontinued and matched patients who continued therapy, adjusting for matching variables, tumor size, lymph node status, tumor grade, progesterone receptor status, surgery type, chemotherapy, radiotherapy, baseline hormone therapy type, prediagnosis use of cardiovascular therapy, prediagnosis major cardiovascular event, and Charlson Comorbidity Index score at diagnosis. All the aforementioned analyses were stratified by baseline type of adjuvant hormone therapy.Finally, we identified shared and unshared factors associated with discontinuation of adjuvant hormone therapy and cardiovascular therapy among patients who concomitantly used these 2 therapies. Marginal Cox proportional hazards regression models with clustered sandwich estimator were used, adjusting for age at breast cancer diagnosis, diagnosis calendar period, tumor size, lymph node status, tumor grade, progesterone receptor status, surgery type, chemotherapy, radiotherapy, baseline hormone therapy type, prediagnosis use of cardiovascular therapy, prediagnosis major cardiovascular event, and Charlson Comorbidity Index score at diagnosis. All analyses were performed using SAS, version 9.4 (SAS Institute Inc); Stata, version 17.0 (StataCorp LLC); or R, version 1.4.1106  at a 2-tailed \u03b1 = .05.Of the 13\u2009053 patients who initiated adjuvant hormone therapy, 5493 (42.1%) concomitantly used at least 1 of the following cardiovascular therapies: 4966 (38.0%) used cardiovascular drugs, 1861 (14.3%) used statins, and 968 (7.4%) used aspirin. In the full cohort of patients who concomitantly used cardiovascular therapies, 4070 patients (74.1%) were diagnosed after 60 years of age and 4046 of 5176 (78.2%) used the corresponding cardiovascular therapy before breast cancer diagnosis . Table 1At discontinuation of adjuvant hormone therapy, 248 of 2030 patients (12.2%) also discontinued their cardiovascular therapy . The discontinuation rate of cardiovascular therapy was the highest at the time of discontinuing adjuvant hormone therapy compared with other periods of follow-up, with an indcidence rate ratio (IRR) of 197.63 . Stratified analyses by baseline type of adjuvant hormone therapy yielded consistent results, with IRRs of 199.35  and 194.81  for aromatase inhibitors users and tamoxifen users, respectively.During the period 1 year before and after discontinuation of adjuvant hormone therapy, the incidence of discontinuing cardiovascular therapy was 0.82 per 1000 person-days among discontinuers and 0.41 per 1000 person-days among continuers. Discontinuers were more likely to discontinue cardiovascular therapy, with IRRs of 1.83  and 2.31  within 3 months before and after discontinuation, respectively . SimilarOur findings suggest that patients with breast cancer who discontinued adjuvant hormone therapy were also more likely to discontinue their cardiovascular therapy. In particular, patients with breast cancer tended to discontinue these 2 therapies at the same time. Moreover, those who discontinued adjuvant hormone therapy were at a higher mortality risk due not only to breast cancer but also cardiovascular disease.30 Elderly patients now account for most patients who survive breast cancer, and the number is projected to double by 2040.32 Such a large number highlights the clinical importance of monitoring use of cardiovascular medications among patients with breast cancer.Our findings further suggest that more than 2 in 5 patients with breast cancer concurrently used cardiovascular therapy and adjuvant hormone therapy. Many patients with breast cancer, especially elderly patients, present with multiple chronic diseases and concomitantly use several long-term medications.We also found that among concomitant users of adjuvant hormone therapy and cardiovascular therapy, the discontinuation of these 2 therapies tends to happen concomitantly. More than 1 in 9 patients with breast cancer discontinued their cardiovascular therapy when they discontinued their adjuvant hormone therapy. This finding suggests that, once oncologists detected discontinuation of adjuvant hormone therapy, they should ask patients about their adherence to cardiovascular therapy. That is, oncologists should shift from single- to multiple-disease focus to prevent discontinuation of treatment by patients with breast cancer, which is likely to be achieved through an extended cardio-oncology approach and cooperation.34 showing that nonadherence to cardiovascular therapy before breast cancer diagnosis is associated with nonadherence to adjuvant hormone therapy.In addition to concomitant discontinuation, we found that patients who discontinued adjuvant hormone therapy were also more likely to discontinue cardiovascular therapy in other time periods, both before and after adjuvant hormone therapy discontinuation. This result is consistent with results of a previous study36 indicating that nonadherence to cardiovascular medications following breast cancer diagnosis is associated with a higher level of low-density lipoprotein and an increased risk of a cardiac event. To potentially prevent these unnecessary deaths, interventions focusing on multiple medications adherence are urgently needed. Furthermore, while the association with cause-specific mortality did not significantly differ by hormone therapy type, it is important for future studies with a larger sample size to replicate this analysis considering the difference in efficacy and adverse effects profile between tamoxifen and aromatase inhibitors.38As expected, we found that adjuvant hormone therapy discontinuation was associated with an increased mortality not only due to breast cancer but also cardiovascular disease, after adjusting for tumor characteristics, treatments, prediagnosis major cardiovascular event, and prediagnosis use of cardiovascular therapy. Our result suggests that the concomitant discontinuation is highly likely to translate to higher mortality. This aligns with previous studiesDiscontinuation of adjuvant hormone therapy and cardiovascular therapy may share common risk factors and underlying mechanisms. In our study, we found that nonparticipation in mammography screening, divorce, low income, and use of symptom-relieving drugs are associated with both discontinuation of adjuvant hormone therapy and discontinuation of cardiovascular therapy. These shared factors may serve as potential targets for interventions that may simultaneously improve adherence to more than 1 life-saving medication, thus having greater cost-effectiveness. For example, we found that patients who used symptom-relieving drugs were at higher risk of discontinuing both therapies, suggesting that only prescribing symptom-relieving drugs seems to be not enough to prevent therapy discontinuation once patients have therapy-related symptoms. Other additional interventions, such as better clinician-patient communication, are needed to improve adherence for both adjuvant hormone therapy and cardiovascular therapy.Despite that, we also identified several unique factors specifically associated with discontinuation of adjuvant hormone therapy or discontinuation of cardiovascular therapy. For example, women with a family history of breast cancer were less likely to discontinue their adjuvant hormone therapy, but no difference was found for discontinuation of cardiovascular therapy. These unique factors suggest that discontinuation of different therapies also has its own unique mechanism and thus should be investigated separately to build a full picture of discontinuation of specific therapy.This study has several limitations. First, using prescription data from the register may underestimate the discontinuation rate, because prescription refill does not necessarily guarantee that a patient used the drug. Second, misclassifying patients who used aspirin as nonusers is possible because the Swedish Prescribed Drug Register does not capture over-the-counter medications. However, given the fact that low-dose and/or long-term use of aspirin is prescribed, we anticipate this misclassification is likely minimal. Third, although we censored patients at disease progress of both breast cancer and cardiovascular disease and adjusted for Charlson Comorbidity Index score at diagnosis and cancer treatments, we cannot rule out the possibility that the concomitant discontinuation of adjuvant hormone therapy and cardiovascular therapy could have been a result of clinical decision-making. While this may partially contribute to the observed association in our study, it is unlikely to substantially account for all associations we have reported. Fourth, the study did not have sufficient power to conduct a stratification analysis by timing of initiating cardiovascular therapy due to limited sample size for patients who initiated cardiovascular therapy after breast cancer diagnosis. Fifth, caution is needed when generalizing our findings to other countries, since our study population included only patients with breast cancer diagnosed in Sweden.In this cohort study of patients with breast cancer concomitantly using adjuvant hormone therapy and cardiovascular therapy, our results call for extra vigilance for concomitant discontinuation of adjuvant hormone therapy and cardiovascular therapy in clinical practice. Shared risk factors identified in our study may help to develop targeted interventions to simultaneously improve adherence to both therapies and subsequently improve overall survival among patients with breast cancer."}
{"text": "Introduction: A soft pneumatic muscle was developed to replicate intricate ankle motions essential for rehabilitation, with a specific focus on rotational movement along the x-axis, crucial for walking. The design incorporated precise geometrical parameters and air pressure regulation to enable controlled expansion and motion.Methods: The muscle\u2019s response was evaluated under pressure conditions ranging from 100-145\u00a0kPa. To optimize the muscle design, finite element simulation was employed to analyze its performance in terms of motion range, force generation, and energy efficiency. An experimental platform was created to assess the muscle\u2019s deformation, utilizing advanced techniques such as high-resolution imaging and deep-learning position estimation models for accurate measurements. The fabrication process involved silicone-based materials and 3D-printed molds, enabling precise control and customization of muscle expansion and contraction.Results: The experimental results demonstrated that, under a pressure of 145\u00a0kPa, the y-axis deformation (y-def) reached 165\u00a0mm, while the x-axis and z-axis deformations were significantly smaller at 0.056\u00a0mm and 0.0376\u00a0mm, respectively, highlighting the predominant elongation in the y-axis resulting from pressure actuation. The soft muscle model featured a single chamber constructed from silicone rubber, and the visually illustrated and detailed geometrical parameters played a critical role in its functionality, allowing systematic manipulation to meet specific application requirements.Discussion: The simulation and experimental results provided compelling evidence of the soft muscle design\u2019s adaptability, controllability, and effectiveness, thus establishing a solid foundation for further advancements in ankle rehabilitation and soft robotics. Incorporating this soft muscle into rehabilitation protocols holds significant promise for enhancing ankle mobility and overall ambulatory function, offering new opportunities to tailor rehabilitation interventions and improve motor function restoration. Assistive robots have long been introduced into the field of rehabilitation. Despite the contentious progression of inflexible robots, these machines still suffer from numerous limitations that prove challenging to overcome. These limitations primarily stem from the materials utilized in their construction, which possess a substantial Young\u2019s modulus, and the inherent constraints imposed by their structures. Simultaneously, rehabilitation exercises often require high flexibility and freedom of movement. Traditional rehabilitation exoskeletons, predominantly composed of rigid components such as linkages and hinges, tend to increase the weight borne by patients, thus inducing discomfort. Moreover, the attainment of multi-degree-of-freedom motion requires additional drive components and intricate structures . ConsequIn contrast to rigid rehabilitation robots, which have inherent limitations, soft robots offer distinctive advantages in terms of safety and adaptability. The abundance of degrees of freedom in soft robots effectively compensates for many of the limitations encountered with their rigid counterparts, allowing greater range of motion and versatility . Soft reComposed of flexible materials that mimic biological properties, soft rehabilitation robots offer high adaptability, flexibility, and safety, specifically regarding human-computer interaction and pneumatic-driven actuators . This deOver the years, artificial muscles have garnered increasing interest, with their initial prototypes dating back several years . One notA two-dimensional design of elastic muscles was proposed . In its A bubble artificial muscle (BAM) was proposed, which differs from the radial pleated muscle. This artificial muscle restricts its expansion by placing metal rings on plastic tubes, forming circumferential pleats, and dividing them into several units. This design achieves a similar contraction rate of 45%\u201350% . The RobIn the realm of rehabilitation technology, significant advancements have been made in the development of exoskeleton-assisted systems, particularly in the field of upper limb rehabilitation . One notFurthermore, recent developments have focused on incorporating synchronized fingertip haptic stimulation into exoskeleton-assisted hand rehabilitation systems. These innovative approaches have demonstrated promising results, particularly in enhancing attention levels and user engagement, especially when heavier grasping weights are involved. Including haptic stimulation has proven to be instrumental in improving rehabilitation outcomes and promoting active user participation . The colA comprehensive approach to wrist rehabilitation utilizes a compact and low-profile soft robotic wrist brace constructed from ethylene-vinyl acetate material. Integrating eight soft origami-patterned actuators onto a commercially available brace significantly enhances its functionality. Furthermore, the adoption of blow molding techniques enables cost-effective mass production of these actuators, ensuring the scalability and reproducibility of the device . A serieThe experimental analysis unveils noteworthy enhancements achieved by the robotic limb system compared to traditional hand drilling methods. Particularly significant is the reduction in vibration transmission by an impressive range of 40%\u201360%, all while maintaining satisfactory time performance. These findings underscore the potential of the developed system to enhance worker safety, minimize the incidence of injuries, and optimize workplace ergonomics and worker wellbeing . The comAn investigation introduces a wearable system that integrates various assistive technologies for individuals with upper-limb impairments . The sysA novel 4-degree-of-freedom (DOF) lower limb rehabilitation robot has been introduced, offering flexion/extension (F/E) training for three limb joints and adduction/abduction (A/A) training for the hip joint . This inA soft-pneumatic actuator-driven exoskeleton designed specifically for hip flexion rehabilitation is presented in this study. Comprehensive testing and evaluation have confirmed the effectiveness of the exoskeleton in assisting hip flexion movements, generating substantial torque, and reducing muscle effort. These findings underscore the exoskeleton\u2019s potential as a valuable tool for facilitating efficient and effective hip flexion rehabilitation and alleviating muscle burden . In anotMoreover, a bio-inspired controller that leverages motor primitives has been proposed for a lower limb exoskeleton. This controller effectively compensates for torque deficiencies and accommodates variations in gait characteristics, thus enhancing motor performance and synchronization between the human and exoskeleton system. The effectiveness of the motor primitive-based controller has been demonstrated in addressing motor deficiencies during lower limb movements . A novelFocusing on the limitations of rigid rehabilitation robots and the advantages of soft robots in rehabilitation. Despite integrating sensor systems, rigid rehabilitation exoskeletons still pose safety risks due to their hardness, high density, and inability to deform in response to external forces. In contrast, soft rehabilitation robots, with their flexibility and adaptability, have found utility in specific rehabilitation applications. However, existing soft actuators, such as artificial muscles actuated pneumatically, are susceptible to damage and limitations in performance. Therefore, there is a need for improved soft actuators that overcome these drawbacks. The paper aims to explore advancements in wearable systems and lower limb rehabilitation robots, specifically focusing on ankle rehabilitation. The paper aims to propose a compact and low-profile soft robotic ankle brace and investigate the development of a supernumerary robotic limb to mitigate injuries and reduce the lower limb\u2019s joint load. The goal is to enhance individuals\u2019 functional abilities and improve rehabilitation outcomes through innovative technologies and approaches tailored to ankle rehabilitation.x-axis, as this movement is pivotal in supporting the patient\u2019s walking ability. Incorporating the soft muscle into the rehabilitation process is expected to assist patients in regaining ankle mobility and enhancing their overall ambulatory function.In the human leg, four muscles are crucial in facilitating ankle rotation movement: Peroneus brevis, Peroneus longus, Extensor digitorum longus, and Tibialis anterior . HoweverX-axis, as exemplified in In this study, a novel soft muscle design is proposed to replicate the complex movements of the ankle joint. As illustrated in The present study employs a systematic approach for selecting geometrical parameters to achieve optimal performance of the soft pneumatic muscles. A rigorous benchmarking process was undertaken, considering various factors such as motion range, force generation, and energy efficiency. The resulting model showcases promising motions that hold potential applications in post-stroke patient rehabilitation and physical therapy settings. To gain a comprehensive understanding of the soft muscle\u2019s behavior, an in-depth analysis was carried out. This analysis evaluated the muscle\u2019s motion under different conditions, specifically on predetermined air pressure values. By examining the muscle\u2019s response across various pressures, valuable insights were obtained regarding its performance characteristics. The selected geometrical parameters, critical to the soft muscle\u2019s functionality, are visually illustrated in Do), chamber height (H), pitch (P), and chamber thickness (T), as outlined in The design represents a single chamber but also contains a multi-air pillow is better to mention it as a multi-air pillow constructed from a flexible elastomeric material, specifically silicone rubber. Instead of a traditional reinforcement layer, chamber rings provide structural integrity. The controlled expansion of the soft muscle is achieved through the pressurization of these chambers with air. Key parameters governing the behavior of the soft muscle include the chamber length (L), outer diameter . Where the only material constant required by the simulation software is C30.Here, For the purposes of simulating the model in this study, finite element analysis was performed using ANSYS. The prototype was subjected to simulations ranging from a pressure of 105\u00a0KPa to 145\u00a0KPa. Then, the material parameters derived from the curve fitting process were subsequently integrated into the FEA software (ANSYS) to establish the finite element analysis. Detailed information regarding the specific material parameters of the hyperelastic model can be found in z-axes and quantitatively assess the distribution of stress within the structure of the soft muscle. To achieve this objective, the muscle underwent a meticulous examination with a wide range of pressures applied, ranging from 105 to 145\u00a0kPa in increments of 5\u00a0kPa. The pressure application was thoughtfully implemented, selectively targeting the inner surfaces of the muscle while ensuring that the outer surfaces were maintained at a constant atmospheric pressure.This rigorous simulation study systematically evaluated the mechanical response and performance of a soft pneumatic muscle for ankle rehabilitation under various loading conditions. The primary objective of this comprehensive analysis was to determine the maximum deformation along the x, y, and The findings obtained from this extensive simulation analysis, as visually presented in Y-axis (Y-def) exhibited a substantial measurement of 165\u00a0mm, clearly indicating a significant elongation primarily occurring in the Y-direction.From this meticulous analysis, several notable findings have emerged, providing valuable insights into the performance of the soft muscle with respect to deformation and stress. Remarkably, at a pressure of 145\u00a0kPa, the deformation along the x and z-axes were negligibly small, measuring 0.056\u00a0mm and 0.0376 mm, respectively, indicating minimal elongation along these axes. This observation emphasizes the specificity of the soft muscle response to pressure actuation in the intended direction. In addition, the controllability of the muscle at different pressure values was evident, as the elongation range spanned from 0\u00a0mm to 165\u00a0mm. This remarkable adaptability and controllability enable precise regulation of the ankle\u2019s rotational angle, thus accommodating individual patient needs and facilitating customized rehabilitation protocols.This outcome underscores the robustness and efficacy of the soft muscle design, particularly in facilitating ankle flexion and extension. On the contrary, the deformations along the The visual representation in The soft muscle fabrication process for ankle rehabilitation is based on advances in soft robotics technology and molding techniques. These methods have attracted significant attention and recognition across various areas, including medical devices, prostheses, and wearable exoskeletons, with various areas attracting this method. Soft muscles have inherent qualities of flexibility and adaptability, making them very promising for rehabilitation applications where patient comfort and safety are paramount. The main purpose of designing soft muscles for ankle rehabilitation is to create devices that faithfully replicate the behavior of natural muscles and tendons, providing the required strength and range of motion for ankle movements such as flexion, extension, inversion, and eversion.In order to achieve this goal, the correct selection and optimization of the shape and silicone material properties of the mold and silicone material are essential. Because of its elasticity, durability, and biocompatible properties, silicone is an ideal material for prolonged contact with the human body. In addition, silicone enables silicone to be easily molded into custom shapes, allowing soft muscle development to be tailored to an individual\u2019s specific needs and anatomical structure. It also allows for customization. Incorporating air pillows into soft muscle designs precisely controls muscle expansion and contraction. By manipulating the air pressure in the soft muscle, the appropriate level of support or resistance can be imparted during ankle exercises, thus facilitating patient-specific rehabilitation protocols. Additionally, the presence of air pillows increases the comfort of soft muscles, allowing them to adapt to the wearer\u2019s movement accurately. Furthermore, air pillows enhance its overall compliance, allowing it to seamlessly adjust to the wearer\u2019s movements, minimizing any discomfort or inconvenience.The manufacturing process includes the use of silicone-based materials and the creation of a hollow soft muscle model using two distinct 3D printed molds, as shown in Based on previous experiments and empirical evidence, the most effective adhesive for bonding two silicone components was the silicone material itself, as shown in An experimental platform was developed and implemented with meticulous attention to detail to investigate the deformation of soft muscle during actuation with different pressures ranging from 100 to 145 (Kpa) for ankle rehabilitation. The experimental setup consisted of several critical components, each serving a specific purpose to ensure accurate and reliable results. The soft muscle was first securely fixed at one end in a 3D environment, allowing for unrestricted movement and deformation. This freedom of movement was essential for observing muscle behavior under different pressure conditions. To facilitate precise measurements of the muscle\u2019s displacement, a 1 m \u00d7 1\u00a0m cubic cell was employed as the workspace, with a grid sheet positioned behind the muscle as a reference to ensure accurate displacement measurements throughout the experiment, as shown in To deliver pressurized air and secure the muscle in place, cantilever support was attached to the muscle and suspended at the top of the cubic cell. The cantilever support was designed to be dual-purpose, essential in securing the muscle and providing the necessary pressure head changes. The pneumatic system incorporated into the platform allowed for regulating the pressure and flow rates delivered to the muscle, enabling precise control over the muscle\u2019s response to pressure head variations. Accurate capture of the muscle\u2019s movement was essential for analyzing its behavior during the experiment. To accomplish this, a high-resolution RealSense D435i camera was employed to record video footage of the muscle\u2019s displacement in the X, Y, and Z directions. The camera\u2019s high resolution provided an accurate and comprehensive record of the muscle\u2019s response under varying pressure conditions, which was synchronized with other experimental parameters using LabVIEW software. The camera was positioned at a fixed distance from the muscle. It was oriented perpendicular to the grid sheet to ensure accurate measurement of the muscle\u2019s displacement in all three dimensions.Deep learning position estimation models were used to locate and measure the soft muscle end tip position x y z, marked with a red mark, to investigate the relationship between pressure head changes and soft muscle elongation for ankle rehabilitation. The algorithm defined displacement relative to the calibrated end tip\u2019s first position. Before starting the measurement process, the end tip position was located using an initial calibration procedure. The observed position changes were then plotted with respect to the change of pressure, as shown in In the realm of artificial muscle systems research, a comprehensive comparison was conducted between McKibben artificial muscles, Festo pneumatics, and an actuator specially designed within this study. The evaluation focused on several critical parameters, including actuation speed, force generation, durability, ease of control, and fabrication. These factors are instrumental in determining the design, operation, and overall efficacy of artificial muscles, particularly in applications such as ankle rehabilitation.Actuation speed was a key consideration. The actuator developed in this study was engineered to respond swiftly to control inputs, mirroring the rapid actuation characteristic of McKibben muscles. In contrast, slower actuation speeds were observed in Festo actuators, a trait shaped by their distinct design and operational principles. In terms of force generation, McKibben artificial muscles have been widely recognized for their high force-to-weight ratio. The actuator developed in this study, while not precisely matching the force generation of McKibben muscles, was found to generate a substantial force aptly suited for the intended applications. Festo actuators, conversely, were found to offer a range of force generation capabilities, primarily determined by the specific model, but a high force output is generally provided.The durability of these artificial muscles was also examined. Both McKibben muscles and Festo actuators were found to exhibit robustness in a variety of applications. The actuator developed within this study, constructed from durable silicone and featuring an innovative design, also demonstrated promising durability, as evidenced by repeated testing cycles. Ease of control emerged as another vital parameter. The control of McKibben muscles can prove complex owing to their non-linear behavior. Festo actuators, despite being easier to control, often necessitate complex control systems. This complexity was mitigated in the developed actuator through design for easy control, a feature augmented by a LabVIEW-based control scheme.Fabrication was another domain where the developed actuator excelled. It was designed with a strong focus on simple fabrication and assembly, as detailed in this paper. In contrast, both McKibben and Festo actuators were found to require more complex fabrication and assembly processes, which can pose significant challenges to their production and implementation.Finally, McKibben artificial muscles and Festo pneumatic actuators each demonstrate their own strengths and have proven their effectiveness in various applications, the soft actuator developed in this study exhibits promising characteristics. These attributes position it as a potential candidate for targeted applications, particularly in the field of ankle rehabilitation.x-axis rotational movement during walking. The study achieved controlled expansion, accurate measurement, and comprehensive analysis of the muscle\u2019s performance by integrating precise geometrical parameters, air pressure regulation, and advanced experimental techniques. Rigorous finite element simulation and experimental investigations provided compelling evidence of the soft muscle design\u2019s adaptability, controllability, and effectiveness, establishing a solid foundation for future advancements in ankle rehabilitation and soft robotics. The experimental results revealed noteworthy findings. Under a pressure of 145\u00a0kPa, the soft muscle exhibited a substantial deformation along the y-axis (y-def) measuring 165\u00a0mm, emphasizing its significant elongation primarily in the y-direction. Conversely, the deformations along the x-axis and z-axis were minimal, measuring only 0.056\u00a0mm and 0.0376 mm, respectively, indicating negligible elongation along these axes. This observation underscores the specific and targeted response of the soft muscle to pressure actuation in the intended direction. Furthermore, the findings demonstrated the controllability of the muscle across different pressure values, showcasing an elongation range spanning from 0\u00a0mm to 165\u00a0mm. This remarkable adaptability and controllability enable precise regulation of the ankle\u2019s rotational angle, accommodating the unique needs of individual patients and facilitating tailored rehabilitation protocols. The comprehensive understanding gained from this study significantly contributes to soft robotics and offers valuable guidance for future research and development in medical applications. The experimental platform developed for investigating the soft muscle\u2019s deformation under various pressure conditions proved a reliable and accurate tool, enabling precise control and measurement of the muscle\u2019s response. Overall, the results obtained from this study enhance our understanding of the intricate relationship between pressure head changes and soft muscle elongation for ankle rehabilitation. The measured values reinforce the performance and robustness of the soft muscle design, providing a solid foundation for further advancements in ankle rehabilitation and soft robotics. This research opens up new possibilities for improving ankle mobility and overall ambulatory function, leading to enhanced rehabilitation outcomes in clinical practice.In this research study, a soft pneumatic muscle was successfully developed and evaluated to replicate the intricate ankle motions necessary for effective rehabilitation, specifically emphasizing the crucial"}
{"text": "Soft robotics, a recent advancement in robotics systems, distinguishes itself by utilizing soft and flexible materials like silicon rubber, prioritizing safety during human interaction, and excelling in handling complex or delicate objects. Soft pneumatic actuators, a prevalent type of soft robot, are the focus of this paper. A new geometrical parameter for soft artificial pneumatic muscles is introduced, enabling the prediction of actuation behavior using analytical models based on specific design parameters. The study investigated the impact of the chamber pitch parameter and actuation conditions on the deformation direction and internal stress of three tested soft pneumatic muscle (SPM) models. Simulation involved the modeling of hyperelastic materials using finite element analysis. Additionally, an artificial neural network (ANN) was employed to predict pressure values in three chambers at desired Cartesian positions. The trained ANN model demonstrated exceptional performance. It achieved high accuracy with training, validation, and testing residuals of 99.58%, 99.89%, and 99.79%, respectively. During the validation simulations and neural network results, the maximum errors in the x, y, and z coordinates were found to be 9.3%, 7.83%, and 8.8%, respectively. These results highlight the successful performance and efficacy of the trained ANN model in accurately predicting pressure values for the desired positions in the soft pneumatic muscles. Significant advancements in soft pneumatic actuators, commonly known as pneumatic artificial muscles (PAMs), have led to notable progress in soft robotics. The origins of soft robotic muscles can be traced back to the 1950s when McKibben actuators were introduced as linear actuators, laying the foundation for the subsequent development of pneumatic muscles . The intPneumatic artificial muscles (PAMs) have emerged as a powerful technology in soft robotics, playing a crucial role in assistive muscle and upper limb rehabilitation applications. PAMs possess unique properties that enable safe and seamless interactions between humans and robots, making them highly suitable for performing intricate or delicate tasks. These versatile actuators find applications in diverse domains, including mobile robots, wearable devices, and exoskeletons, contributing to realizing assistive and rehabilitative functionalities ,4. LeverA seminal study ,8 presenIn contrast to the extensive field of conventional robotics, soft robotics has emerged as a distinct and promising research domain, reshaping the landscape of robot design and motion. Soft robotics represents a paradigm shift from traditional approaches, as it leverages the inherent properties of its actuators to achieve motion without relying on external torque or conventional motors ,12,13,14The deformation behavior of soft robots is influenced by various factors, including the selection of materials ,23,24, fSoft pneumatic muscles, also known as flexible artificial pneumatic actuators, are designed to mimic the actuation properties of human muscles ,32. ThesSignificant advancements have been made in the field of fluidic actuators, contributing to the development of innovative solutions for various applications in robotics and automation. One noteworthy example is the bidirectional electrohydrodynamic pump (BEDP), which has gained attention due to its high symmetrical performance and its potential application in tube actuators. The BEDP utilizes the principles of electrohydrodynamics, employing an electric field to induce fluid flow in a conductive medium. This technology enables precise control and efficient actuation, making it a promising candidate for integration into artificial pneumatic muscle systems for human assistive devices . AnotherA proposed strategy, referred to as echo-state Gaussian process-based nonlinear model predictive control (ESGP-NMPC), addresses challenges related to trajectory tracking using pneumatic muscle actuators (PMAs) in rehabilitation robots ,43,44. TThis study aims to design and determine the geometrical parameters for the SPM and soft parallel muscle, model the soft actuators using finite element analysis (FEA), and investigate the influence of varying the parameter P with sequential values of pressures. Additionally, the study focuses on applying these soft muscles for rehabilitation purposes. Furthermore, an (ANN) is employed to establish both forward and inverse kinematics for the SPM and soft parallel muscle models, enabling the estimation of pressure values given the endpoint Cartesian coordinates . The manuscript\u2019s structure is organized as follows: Human assistive devices play a vital role in enhancing the quality of life for individuals with physical disabilities or impairments. These devices encompass a broad range of technologies designed to aid individuals in overcoming mobility challenges, improving functional capabilities, and promoting independence. Soft pneumatic muscles, as described in our study, have promising applications in the field of human assistive devices for upper limb support due to their inherent flexibility, adaptability, and safety characteristics.One prominent application of soft pneumatic muscles is in the development of wearable exoskeletons for upper limb assistance. These devices provide external support and assistance to the wearer\u2019s arms and hands, helping them perform daily tasks that may have been difficult or impossible due to physical limitations. Soft pneumatic muscles can be integrated into upper limb exoskeletons in order to provide a more natural and compliant interaction between the device and the human body, reducing the risk of injury and discomfort while improving overall functionality.The proposed soft muscle will be suitable to fit a wearable exoskeleton design for the shoulder joint and incorporates three parallel soft pneumatic muscles, strategically arranged to enable efficient actuation and a wide range of motion. Each muscle is designed to be identical and consists of three chambers, allowing for tailored pressure application and control in each chamber, as shown in In addition to the shoulder actuation, another SPM is integrated into the design to operate and assist the human forearm and enable elbow joint rotation. This single-chamber muscle, also constructed from flexible elastomeric material, can be pressurized to induce controlled bending and extension of the elbow joint, effectively assisting the user in daily tasks that require forearm movement and manipulation. The muscle\u2019s geometry and pressure control can be optimized to provide the desired range of motion, force output, and responsiveness, considering the user\u2019s specific needs and physical abilities.The combination of the three parallel soft pneumatic muscles for shoulder actuation and the additional muscle for elbow joint rotation presents a comprehensive solution for upper limb assistance. This soft wearable exoskeleton design offers a more natural and intuitive interaction between the device and the human body, closely replicating the complex biomechanics of the upper limb. By incorporating soft pneumatic muscles into the exoskeleton, users can benefit from enhanced mobility, improved force output, and a greater sense of embodiment, ultimately leading to a better user experience and satisfaction with the assistive device.Soft pneumatic muscles are regarded as attractive for soft robotics applications due to their flexible and inflatable nature, enabling a wide range of motions and forces. Typically, a SPMdesign consists of three parallel muscles, thereby facilitating stimulating motion in multiple movement spaces. By incorporating a model with more than one chamber, achieving larger workspace movements for actuated muscles becomes possible. This study devised a three-chamber model, with each chamber designed to be identical. The selection of geometrical parameters was undertaken following a rigorous benchmarking process to ensure the optimal performance of the soft pneumatic muscles. The resulting model exhibits motions with potential applications in post-stroke patient rehabilitation and physical therapy. An in-depth analysis was conducted to examine the motion under different conditions at predetermined air pressure values. The geometrical parameters utilized in the models are visually represented in The soft artificial pneumatic muscle (SPM) model comprises three chambers constructed from a flexible elastomeric material, specifically silicone rubber, and utilizes chamber rings instead of a reinforcement layer. These chambers are pressurized with air or fluid to induce controlled muscle inflation and prevent unintended expansion. The relevant muscle parameters, including the length of the chamber (L), outer diameter (Do), the height of the chamber (H), pitch (P), and chamber thickness (T), with a constant number of rings, are presented in Soft robotics encompasses a wide range of materials employed in the manufacturing process, which contribute to the remarkable characteristics of these robots, including their flexibility, controllability, and human-safe nature. Recent advancements in soft robotics have focused on various aspects, including material selection, construction geometries, control systems, modeling techniques, and production methods . The fieIn order to comprehensively characterize the mechanical behavior of the silicon rubber material, a series of mechanical tests were conducted, including a uniaxial test performed under stable conditions. The test followed the ASTM No. D412 specifications, utilizing dumbbell-shaped specimens, as depicted in Upon completing the experimental phase, the most suitable hyperelastic model was determined through curve fitting analysis. The Yeoh 3rd-order model was identified as the best fit for the silicon rubber material. The Yeoh model can be represented by the following equation:The selection of silicon synthetic rubber as the material for fabricating the soft pneumatic muscle (SPM) was based on its exceptional hyperelastic performance, high-pressure resistance, and desirable mechanical properties, particularly its stress\u2013strain behavior. The Yeoh 3rd-order hyperelastic model was identified as the best fit for the silicon rubber material through a rigorous curve-fitting process. This model was subsequently integrated into the FEA software, ANSYS, to facilitate further analysis. The non-linear finite element analysis was carried out using ANSYS Workbench, primarily investigating various geometric aspects. To initiate the simulations, the CAD models of the soft pneumatic muscles were directly imported into the ANSYS Design Modeler, enabling the subsequent analysis of their behavior.The simulation was designed to characterize the muscle\u2019s response under different conditions comprehensively. This included evaluating the maximum deformation length in the x, y, and z directions and mapping the distribution of stress and strain within the structure. A pressure range from 100 to 160 kPa was applied to capture a wide range of scenarios, incrementing in steps of 10 kPa. Specifically, the pressure was selectively applied to two of the three chambers while the remaining chamber was kept at a constant pressure.By conducting simulations for three distinct models, each featuring different pitch chamber values , valuable insights were gained into the performance and behavior of the SPM under varying pressure conditions. The results obtained from this extensive analysis, as visualized in This comparison aims to analyze the influence of pitch variation on the deformation and stress behavior of the soft pneumatic muscle (SPM) when applying pressure. Specifically, the study focuses on altering the pressure in two chambers while maintaining a constant pressure in the third chamber. Throughout this comparison, the height (H = 20 mm) and thickness (T = 7 mm) of the SPM are kept consistent. The analysis reveals noteworthy findings in terms of deformation and stress. At a pitch value of P = 30 mm, the y-axis deformation (y-def) is measured to be 113 mm. When the pitch value is increased to P = 34 mm, the y-def increases to 130 mm. Further raising the pitch to P = 38 mm results in a higher y-def of 163 mm as shown in Similarly, the stress experienced by the SPM exhibits a corresponding variation. At P = 30 mm, the stress is measured to be 0.83 MPa, whereas, at P = 38 mm, the stress escalates to 2.2 MPa. The increase in pitch leads to increased stress experienced by the SPM. It is worth noting that the deformation effect in the x and z axes is minimal in this comparison and hence does not significantly contribute to the overall analysis. The visual representation in  further ANNs have found extensive applications in soft robotics for controlling the behavior of soft robots, making them a powerful tool for modeling complex nonlinear systems ,37. ANNsThe soft pneumatic muscles\u2019 (SPMs) workspace was investigated using forward kinematics, established through FEA software (ANSYS) workbench simulations. This analysis provided a comprehensive understanding of the total workspace position and orientation of the SPMs in Cartesian coordinates. The workspace assessment was crucial in evaluating the range of motion and capabilities of the SPMs across a full 360-degree span. The obtained workspaces demonstrated the SPMs\u2019 ability to perform various motions, including extension, bending, and twisting, within the Cartesian coordinate system. As depicted in The kinematic model of soft pneumatic muscles (SPMs) encompasses both forward and inverse kinematics. Forward kinematics calculates the X, Y, and Z coordinates of the tip center point based on a given pressure value, while inverse kinematics determines the input pressure required to achieve a desired tip position . In thisFor training the ANN, we utilized a dataset consisting of 66 samples, which included finite element analysis (FEA) results for pressures P1, P2, and P3, as well as deformation in the X, Y, and Z axes. To ensure a robust and accurate model, we adopted a Bayesian regularization (BR) approach. This type of ANN is particularly suitable for quantitative studies with smaller datasets as it can handle complex relationships without compromising power or precision. To assess the model\u2019s performance, the dataset was divided into training, validation, and test sets, with a recommended split ratio of 70%, 15%, and 15%, respectively. The optimal ANN structure, comprising one hidden layer with ten neurons, was determined through iterative adjustments during training to minimize the mean squared error (MSE). After 300 iterations, the best-performing ANN achieved a validation MSE of 2.8655, demonstrating its accuracy in predicting pressure values for desired tip positions.The inverse kinematics modeling employed an ANN with three inputs representing Cartesian X, Y, and Z coordinates, and three outputs representing the corresponding pressure values. Using the MATLAB neural network toolbox, the ANN was trained by iteratively adjusting the network output to optimize validation performance, as quantified by the lowest MSE. After 300 iterations, the trained ANN demonstrated an MSE of 2.8655, indicating its accuracy in predicting pressure values for desired tip positions. The training, validation, and testing process of the ANN is depicted in To validate the ANN\u2019s performance, a comparison was made between its results and finite element analysis (FEA) simulations. The observed maximum errors in the X, Y, and Z coordinates were 9.3%, 7.83%, and 8.8%, respectively, demonstrating the network\u2019s capability to accurately predict future values. These results provide validation for the reliability and performance of the trained ANN, underscoring its effectiveness in predicting the required pressure values to achieve desired tip positions in the SPMs. Leveraging the predictive capabilities of the ANN enables the precise control and manipulation of SPMs, contributing to the advancement of soft robotics and its broad range of applications.A comprehensive comparison was conducted between the mean absolute error (MAE) associated with two predictive models: finite element analysis (FEA) and artificial neural network (ANN). The primary objective was to predict the behavior of soft pneumatic muscles by considering key performance parameters such as force output, range of motion, and pressure response. Visual representation in the form of a bar chart was employed to illustrate the disparities in accuracy between the models, ultimately revealing the ANN model\u2019s consistent superiority in prediction accuracy. This observation highlights the enhanced capability of the ANN model in capturing the intricate and nonlinear relationships between design parameters and muscle performance. The MAE values obtained for the ANN model were 0.2251, 0.3739, and 0.3761 for the x, y, and z directions, respectively, as indicated in the corresponding In this study, the development and analysis of three soft pneumatic muscles (SPMs) were conducted in order to explore their potential use in human assistive devices for individuals with disabilities. The SPMs\u2019 geometric characteristics were meticulously designed and presented in"}