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+{"text": "Calcium makes up just 2% of the human body, and 99% of it is sequestered in bones and teeth. The remainder exists within and around cells, influencing a variety of cellular processes, from transcription and cell survival to nerve signaling and muscle contraction. Calcium inhabits the intra- and extracellular space as a gradient, with extracellular concentrations some 1,000 times greater than those inside the cell. These gradients are maintained by calcium pumps. Calcium signaling operates mostly through voltage- and ligand-gated calcium channels , both in the plasma membrane of the cell and in the membranes enclosing intracellular organelles. Signaling is typically initiated by an influx of calcium across the plasma membrane or by the release of calcium from an organelle, such as the endoplasmic reticulum. In neuron-to-neuron signaling, calcium signaling helps convert electrical signals into chemical signals in the form of neurotransmitters. The arrival of an electrical signal at a nerve terminal opens the many calcium channels in the nerve terminal, admitting a stream of calcium ions. The increased intracellular calcium concentration in turn releases the resident neurotransmitters accumulated in the nerve terminal, converting the electrical signal into a chemical signal. As neurotransmitters bind to their receptors in the next target neuron, they change the cell's membrane potential, prompting the neuron to generate an electrical signal, thereby converting the chemical signal back into an electrical one and completing the signaling circuit. Since calcium dynamics mediates most neuronal information flow, it can be used as a general measure of neural activity.Through an elaborate network of electrical activity, the brain encodes, combines, and interprets signals to process information about the world. Simultaneous measurement of this activity in multiple brain locations has provided valuable insight into how neural networks function. But since electrical recordings can't pinpoint activity in the fine branches of individual neurons or pick up biochemical  signals, researchers are increasingly turning to approaches that measure calcium concentrations as a proxy for neuron activity. Now Mazahir Hasan et al. have created mice engineered to stably express two different kinds of fluorescent calcium indicator proteins (FCIPs) in the brain (the fluorescence produced by these proteins can be seen when the brain is viewed with a two-photon microscope). Because the indicators are incorporated into the mouse genomes, this approach offers the possibility of targeting specific cells (by using promoters that specify which cells the genes should be activated in), allowing researchers to map the activity of select neuron populations.Fluorescent proteins can be incorporated into a gene of interest to help researchers track that gene's protein in living tissue. FCIPs report calcium concentrations by changing fluorescence when they bind to calcium. Introducing calcium indicators into neural tissues was largely impractical, often failing to target specific cell types, until these genetically engineered indicators were developed in the late 1990s, allowing the desired specificity. While FCIPs have been used to good effect in worms, fruitflies, and zebrafish\u2014and just recently in mouse muscle\u2014they had not been stably and functionally expressed in the mammalian brain until now.To deliver the FCIPs to the mouse brain, Hasan et al. used a regulatory promoter called Ptet (in the tetracycline system), which offers the possibility of targeting the expression of the FCIPs in different neural populations. To test the functionality of the proteins, they used fluorescence microscopy to analyze neurons from mouse lines and found high levels of FCIP expression. The real test, however, was whether the FCIPs could fulfill their promise as a probe for calcium activity. When the authors electrically stimulated brain slices from a mouse line expressing moderate to high levels of FCIP , fluorescence increased rapidly following the stimulus. Significant changes in FCIP fluorescence were also observed when live mice responded to odor stimulation. That \u201cfast and robust\u201d FCIP signals were detected in live animals responding to sensory stimulation, the authors argue, proves the promise of FCIPs as a reporter on the activity of select neural populations in living systems. And since these indicator proteins retain stable functional expression over time (8- to 12-week-old mice continued to express the proteins), they could help researchers track neuronal activity over extended periods.While a variety of bugs remain to be worked out with FCIPs\u2014it's unclear, for example, why only the Ptet promoter generates high levels of FCIP expression in the brain and why not all neurons in a given population express the proteins\u2014Hasan et al. demonstrate that the tetracycline system supports stable expression of the calcium indicators. The FCIP approach avoids the complications of invasive techniques like surgically administering dyes and produces a more interpretable signal, since the cell populations are already known. Because FCIPS can be used in living animals, they can reveal where and when neurons are firing. And because FCIP mice can be crossed with mice containing mutations in genes important for neural function, this method could reveal how specific genes contribute to the construction of neural networks."}
+{"text": "PLoS Biology, Hajime Hirase and his colleagues at Rutgers University have done just that by focusing their microscope on the brains of living rats in order to examine how certain cells called astrocytes function in vivo.Did you ever wish you could peek inside someone's brain and see what was going on in there? In research reported in this issue of Glia is derived from the Latin for \u201cglue\u201d; these cells were thought to \u201chold it all together.\u201d) One type of glial cell, the astrocyte (named for its starlike shape), is the most populous cell in the brain and forms an intimate association with neurons and their synapses. It was thought that these cells played a supporting role in the brain, ensuring the proper chemical environment for synapses.In the longstanding quest to understand how the brain works, scientists have focused on neurons. Neurons conduct action potentials, electrical signals that transmit information in the nervous system. But the brain also contains several other types of cells called glia. (Recent research, however, has suggested that astrocytes and other glial cells may play a more significant role. When examining astrocytes cultured in the lab, scientists have observed behavior suggesting that astrocytes can communicate with neurons. Though astrocytes cannot propagate electrical signals like neurons do, they can sense the transmission of such signals at the synapse between two neurons. Furthermore, astrocytes are able to propagate a different kind of signal, a chemical signal based on the release of calcium ions. Calcium signaling is a mechanism of chemical signaling that has been observed in many other cell types. The exact properties of neuron\u2013astrocyte communication, however, are not clear because different preparations of these tissues have yielded different results. It has also not been established that this type of communication occurs in the living brain.To explore such questions, Hirase and colleagues have taken the next step by investigating the calcium signaling properties of astrocytes in the brains of living rats. To accomplish this feat, the researchers used a combination of two technologies. They monitored calcium signaling using a fluorescent dye called Fluo-4, which fluoresces in response to calcium ions. Then they used a special type of microscope called a two-photon laser scanning microscope to visualize the dye. Since this type of microscope uses a lower energy laser, it can image the dye in living tissue without causing harm.The researchers applied the dye to the brains of anesthetized rats, washed out the excess dye that had not penetrated into cells, and then imaged the tissue under the microscope. They first confirmed that they indeed were examining astrocytes and noticed that cells displayed a moderate level of baseline calcium signaling activity. They then used a drug called bicuculline to stimulate neurons and observed a significant increase in the calcium signaling activity of the astrocytes. Because bicuculline only affects neurons, this implies that the astrocytes are responding to the activity of the neurons. The researchers also found that neighboring astrocytes often also displayed coordinated calcium signaling activity, suggesting that the communication among astrocytes is facilitated by increased neuronal activity.This research confirms the complexity of astrocyte signaling functions in the living brain and demonstrates that astrocytes play far more than a supporting role in brain function. It also establishes an important experimental system for scientists seeking to understand how these distinct elements of the brain\u2014neurons and astrocytes\u2014work together. Though this research makes it clear that signaling exists both among astrocytes and between neurons and astrocytes, scientists have yet to understand the effect of this signaling. Some possibilities include regulation of synapse formation, modification of synaptic strength, or more complicated roles in information processing resulting from the coordination of neuronal activity. Future research using this and other systems will help reveal these functions."}
+{"text": "Pungitius pungitius), as well as cross-species transferability of SSR primers from three-spined (Gasterosteus aculeatus) to nine-spined sticklebacks. In addition, we examined the patterns and degree of SSR conservation between these species using their aligned sequences.Identification of genes involved in adaptation and speciation by targeting specific genes of interest has become a plausible strategy also for non-model organisms. We investigated the potential utility of available sequenced fish genomes to develop microsatellite  markers for functionally important genes in nine-spined sticklebacks  than for those randomly derived from genomic (35 out of 101) and cDNA (35 out of 87) libraries. Polymorphism was observed at a large proportion (65%) of the cross-amplified loci independently of SSR type. To develop SSR markers for functionally important genes in nine-spined sticklebacks, SSR locations were surveyed in or around 67 target genes based on the three-spined stickleback genome and these regions were sequenced with primers designed from conserved sequences in sequenced fish genomes. Out of the 81 SSRs identified in the sequenced regions , 57 exhibited the same motifs at the same locations as in the three-spined stickleback. Di- and trinucleotide SSRs appeared to be highly conserved whereas mononucleotide SSRs were less so. Species-specific primers were designed to amplify 58 SSRs using the sequences of nine-spined sticklebacks.Our results demonstrated that a large proportion of SSRs are conserved in the species that have diverged more than 10 million years ago. Therefore, the three-spined stickleback genome can be used to predict SSR locations in the nine-spined stickleback genome. While cross-species utility of SSR primers is limited due to low amplification success, SSR markers can be developed for target genes and genomic regions using our approach, which should be also applicable to other non-model organisms. The SSR markers developed in this study should be useful for identification of genes responsible for phenotypic variation and adaptive divergence of nine-spined stickleback populations, as well as for constructing comparative gene maps of nine-spined and three-spined sticklebacks. Recent advances in our understanding of the physiological and molecular functions of genes have paved the road for investigating functional genomic variation associated with adaptation and speciation in the wild ,2. ConseMicrosatellites or simple sequence repeats (SSRs) are highly abundant in eukaryotic genomes, accounting for 3-5% of the mammalian genomes ,9. OwingOne way to obtain SSR markers for specific genes and genomic regions in a given species is to use SSR primers developed for the closest relative with a sequenced genome. However, an obvious limitation of this approach is that mutations in SSR flanking sequences will inhibit cross-species amplification success - a problem that is likely to attenuate with an increasing divergence time . In geneFor the reasons elaborated above, development of SSR markers for target genes and genomic regions in non-model organisms is challenging. Yet, while the closest relative with a sequenced genome is too distantly related to the focal species, one can take an advantage of the increasing number of completed genomes for different species. For instance, as often used in species for which no direct species-specific sequence information is available, conserved sequences in specific genes and genomic regions of interest can be used to design primer sequences applicable to a wide variety of organisms [e.g. ].Danio rerio), three-spined stickleback (Gasterosteus aculeatus), medaka (Oryzias latipes), spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes)  coupled with 101 markers derived from genomic libraries (genomic SSRs) and 87 markers derived from ESTs  was used.Based on the literature on gene functions in teleosts, we selected 67 genes responsible for significant physiological - such as osmoregulation, thermal response, growth, disease and taste  - and developmental functions [e.g. ] Additi. Genomicdatabase  and mappGHRI and IGF-I, for which two and three regions were used, respectively , 0.15 U BIOTAQ DNA polymerase (Bioline), 5 pmol of each primer and approx. 40 ng of genomic DNA. The reactions were performed as follows: an initial degeneration step at 95\u00b0C for 3 min, followed by 30 s at 95\u00b0C, 30 s at 53-60\u00b0C and 60-120 s at 72\u00b0C for 35 cycles with a final extension at 72\u00b0C for 5 min .Two individuals of the Py\u00f6re\u00e4lampi were used for amplifying and sequencing the target genomic regions. One three-spined stickleback individual from the Baltic Sea  was used as a positive control. Using a primer pair for respective target regions  [[AB473819-AB473831] and Pungitius sp. (Omono type) [[AB300827-AB300851]. Out of the 38 sequences, six  were excluded from the analyses because of low BLAST hit scores and alignment problems. To develop SSR markers for functionally important genes, primer sets were designed based on the sequences of nine-spined sticklebacks using WebSat [GU553378-GU553434.The sequences in forward and reverse directions of two individuals were aligned using CLUSTAL W  as impleleback) [; GenBanko type) [; GenBank2, 0.2 mM dNTP (Finnzymes), 0.18 U BIOTAQ DNA polymerase (Bioline), 5 pmol of each primer and approx. 20 ng of template DNA. The reactions were performed as follows: an initial degeneration step at 95\u00b0C for 3 min, followed by 30 s at 95\u00b0C, 30 s at 53\u00b0C and 30 s at 72\u00b0C for 30 cycles with a final extension at 72\u00b0C for 5 min. Amplification success was determined by electrophoresis on 1.6% agarose gel. Secondly, for the loci that showed robust and specific amplification within the expected size range, polymorphism was investigated by genotyping 24 individuals from the Baltic Sea and the Lake 1 . For efficient screening, PCRs were carried out using the Qiagen Multiplex PCR Kit (Qiagen) in 10 \u03bcl reaction volumes containing 1\u00d7 Qiagen Multiplex PCR Master Mix, 0.5\u00d7 Q-Solution, 2 pmol of each primer and approx. 20 ng of template DNA. The reactions were performed by the following cycle: an initial activation step at 95\u00b0C for 15 min, followed by 30 s at 94\u00b0C, 90 s at 53\u00b0C and 60 s at 72\u00b0C for 30 cycles with a final extension at 60\u00b0C for 5 min. PCR products were visualized with a MegaBACE 1000 automated sequencer (Amersham Biosciences) and their sizes were determined with ET-ROX 550 size standard (Amersham Biosciences). Thirdly, genetic variability of the polymorphic loci identified with the 24 individuals was evaluated by genotyping a total of 24 individuals from each of the Baltic Sea, Lake 1 and Py\u00f6re\u00e4lampi populations using multiplex PCRs. For the SSR primers developed in nine-spined sticklebacks, polymorphism and genetic variability were evaluated following the procedures for the second and third steps. Since some of the SSR markers yielded clearer allele profiles at an annealing temperature of 55\u00b0C, this temperature was used for these loci instead of 53\u00b0C  with fluorescent labelled forward primers  and GTTT-tailed reverse primers . As a poHE) [FIS were estimated for each population to detect possible deviations from Hardy-Weinberg equilibrium with 10 000 permutations using FSTAT 2.9.3. Sequential Bonferroni corrections [Locus and population specific gene diversities (HE)  were estHE) ,86. Withrections  were apprections .Factors affecting the levels of genetic variation were evaluated with general linear models where allele number or heterozygosity was treated as a dependent variable, SSR marker origin (cf. three-spined and nine-spined sticklebacks), SSR type, SSR location and SSR repeat motif  as fixed factors and population as a random factor. These analyses were performed with JMP 5.TS conceived the study, contributed to the gene selection and localization, prepared the molecular data, conducted the analyses and wrote the manuscript. JR selected the genes and conducted the molecular work. YS made contributions to the gene selection and localization. JM advised on the statistical analyses and contributed to writing the manuscript. All authors read and approved the final manuscript.Genetic variability of three-spined stickleback SSR markers in three populations of nine-spined sticklebacks.Click here for fileList of three-spined stickleback SSR markers that were not polymorphic or successfully amplified in nine-spined sticklebacks.Click here for fileTarget genes and their function and location in the theree-spined stickleback genome, and PCR and sequencing primers for nine-spined sticklebacks.Click here for fileHomology of nine-spined stickleback sequences in the three-spined stickleback genome and comparative genomic location of SSRs in these species.Click here for file"}
+{"text": "One of the major obstacles that must be overcome in the design of effective lentiviral vaccines is the ability of lentiviruses to evolve in order to escape from neutralizing antibodies. The primary target for neutralizing antibodies is the highly variable viral envelope glycoprotein (Env), a glycoprotein that is essential for viral entry and comprises both variable and conserved regions. As a result of the complex trimeric nature of Env, there is steric hindrance of conserved epitopes required for receptor binding so that these are not accessible to antibodies. Instead, the humoral response is targeted towards decoy immunodominant epitopes on variable domains such as the third hypervariable loop (V3) of Env. For feline immunodeficiency virus (FIV), as well as the related human immunodeficiency virus-1 (HIV-1), little is known about the factors that lead to the development of broadly neutralizing antibodies. In cats infected with FIV and patients infected with HIV-1, only rarely are plasma samples found that contain antibodies capable of neutralizing isolates from other clades. In this review we examine the neutralizing response to FIV, comparing and contrasting with the response to HIV. We ask whether broadly neutralizing antibodies are induced by FIV infection and discuss the comparative value of studies of neutralizing antibodies in FIV infection for the development of more effective vaccine strategies against lentiviral infections in general, including HIV-1. Infection with the primate and feline lentiviruses requires sequential interactions with a primary receptor and a co-receptor to trigger the conformational change in Env that allows fusion of the viral envelope and cellular membrane to proceed. Whereas CD4 is the primary receptor for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) \u20133, CD134The neutralizing response to HIV has been studied extensively and is reviewed elsewhere ,17. It i2.2.1.et al. in 1985, utilizes pseudotyped viruses that are generated by co-transfection of an env- and rev-deleted HIV backbone, together with the env clone of interest; the resulting pseudotypes are used to infect a transformed cell line expressing the appropriate viral receptors, such as TZMbl, a genetically engineered HeLa cell clone that expresses CD4, CXCR4 and CCR5 and contains a Tat-responsive reporter gene encoding the firefly luciferase enzyme [In order to quantify the level of neutralizing antibody in a sample of biological fluid, the sample is mixed with a known quantity of virus or viral antigen. The antibodies within the sample are allowed to interact with the virus before adding the mixture to an indicator cell or system in which the ability of the virus to grow or mediate an effect may be quantified. Thus, early HIV neutralization assays mixed infectious virus with serially-diluted sera before plating onto susceptible cells ,19. Howee enzyme ,23. Thise enzyme ,25. Othee enzyme . Howevere enzyme . A compae enzyme . Similare enzyme . It is p2.2.in vivo neutralization and vaccine protection was limited. Accordingly, PBMC-based neutralization assays were developed, measuring RT activity in the culture fluids to monitor inhibition of infection [env-deleted HIV provirus incorporating a luciferase reporter gene, pNL-Luc-E-R+ (11); and (ii) an FIV env gene expressed from the vector VR1012. The resulting HIV (FIV) luciferase pseudotypes were incubated with serial dilutions of each plasma sample in order to permit neutralization of the pseudotype to proceed, prior to the addition of substrate cells and further incubation. Infection with the residual, non-neutralized pseudotype was then quantified by measuring luciferase activity and the percent neutralization was calculated with reference to control wells containing no plasma. Plasmas were classified as strongly neutralizing (\u226580%), moderately neutralizing (60\u201379%), and weakly neutralizing (40\u201359%) [Novel isolates of FIV are conventionally isolated in primary cultures of mitogen-stimulated feline PBMC , cells wnfection . Given t(40\u201359%) .2.3.FIV infects cells by sequential interactions with the primary receptor CD134 ,13. Thus3.3.1.et al. [Anti-FIV antibodies can be detected as early as 6 weeks post infection in experimentally infected cats . Similaret al.  comparedet al. \u201344, whilet al. \u201348. Non-et al. ; howeveret al. , suggestet al. ,52). Whiet al. . Accordi3.2.Plasma samples were collected from 345 FIV sero-positive, naturally infected, cats across the United Kingdom and submitted to the Companion Animal Diagnostic Service at the University of Glasgow. The cats ranged from 5 months to 18 years of age, although the ages of many of the animals were either unknown or estimated by the cats\u2019 veterinarians. The study population also included FIV-infected cats held in cat protection shelters, strays awaiting adoption, or free-roaming cats. Cats within these groups are at greater risk of FIV infection, according to epidemiological studies . The FIV3.3.et al. found that while one patient achieved a neutralizing titer >1000 by 6 months post-infection, a second patient had no significant neutralizing antibody by 11 months post-infection [per se, a pseudovirion-based assay detecting significant neutralizing antibodies by 30 weeks post-infection while anti-V3 peptide reactivity was evident as early as 10 weeks post-infection [The neutralizing antibody response to HIV develops over time and there is considerable variation between patients in both the potency of the response and the time it takes to develop . For exanfection . The neunfection . Comparinfection ,47,55,56nfection , althougnfection ,58. The i.e., \u226580% at a 1:10 dilution) neutralized GL8 Env-bearing pseudotypes. As isolates within the UK are almost exclusively subtype A [Little is known about the breadth of neutralizing activity within sera from FIV-infected cats. Therefore, we conducted a pilot survey of neutralizing activity in cats naturally infected with FIV, screening 345 plasma samples for neutralizing antibodies against the GL8 isolate. The samples containing neutralizing antibody were collected from cats aged between 18 months and 14 years. Subsequently, samples which strongly neutralized GL8 were tested against other isolates to assess the breadth of cross-reactivity. Of the 345 plasma samples that were tested, 30 (8.7%) strongly  were found to recognize determinants on the viral transmembrane protein gp41. The antibodies bind to a highly conserved stretch of the transmembrane region of gp41 that lies immediately adjacent to the external surface of the viral lipid envelope, a region referred to as the membrane-proximal external region (MPER) (reviewed comprehensively in ). An ana3.6.Protection against FIV infection was first demonstrated by Yamamoto and colleagues using whole inactivated virus and cells prepared from the FL4 cell line, an IL-2 independent cell line persistently infected with the subtype A Petaluma strain of FIV (PET) ,95. Howein vivo are strain-specific and target V4 and V5 [Why do FL4-based vaccines fail to protect against heterologous strains of virus? One possibility is that the prototypic vaccines failed to induce neutralizing antibody against conformational epitopes that would be required to confer heterologous immunity; evidence to date would suggest that the majority of the neutralizing responses that develop 4 and V5 \u201369,101 w4 and V5 \u2013104, and4 and V5 . If this"}
+{"text": "Cluster randomised controlled trials (CRCTs) are frequently used in health service evaluation. Assuming an average cluster size, required sample sizes are readily computed for both binary and continuous outcomes, by estimating a design effect or inflation factor. However, where the number of clusters are fixed in advance, but where it is possible to increase the number of individuals within each cluster, as is frequently the case in health service evaluation, sample size formulae have been less well studied.We systematically outline sample size formulae  for CRCTs with a fixed number of clusters, to provide a concise summary for both binary and continuous outcomes. Extensions to the case of unequal cluster sizes are provided.k), the trial will be feasible provided the number of clusters is greater than the product of the number of individuals required under individual randomisation (In) and the estimated intra-cluster correlation (\u03c1). So, a simple rule is that the number of clusters (k) will be sufficient provided:For trials with a fixed number of equal sized clusters , irrespective of how many individuals are included within each cluster. Cluster randomised controlled trials (CRCTs), in which clusters of individuals are randomised to intervention groups, are frequently used in the evaluation of service delivery interventions, primarily to avoid contamination but also for logistic and economic reasons -3. WhilsHowever, when evaluating health care service delivery interventions the number of clusters might be limited to a fixed number even though the sample size within each cluster can be increased. In a real example, evaluating lay pregnancy support workers, clusters consisted of groups of pregnant women under the care of different midwifery teams ,7. The aThe existing literature on sample size formulae for CRCTs focuses largely on the case where there is no limit on the number of available clusters -5,11,12.For completeness we outline formulae for simpler designs for which the sample size formulae are relatively well known, or easily derived, as an important prelude. In so doing, the simple relationships between the formulae are clear and this allows progressive development to the less simple situation (that of binary detectable difference or power). It is hoped that by developing the formulae in this way the material will be accessible to applied statisticians and more mathematically minded health care researchers. We also provide a set of guidelines useful for investigators when designing trials of this nature.H0 : \u03bc0 = \u03bc1 where \u03bc0 and \u03bc1 represent the means of some variable in the control and intervention arms respectively; and where it is assumed that var(\u03bc0) = var(\u03bc1) = \u03c32. Suppose further that there are an equal number of individuals to be randomised to both arms, letting n denote the number of individuals per arm and letting d denote the difference to be detected such that d = \u03bc0 - \u03bc1, 1 - \u03b2 denotes the power and \u03b1 the significance level. We limit our consideration to trials with two equal sized parallel arms, with common standard deviation, two-sided test, and assume normality of outcomes and approximate the variance of the difference of two proportions. The sub-script, I , is used throughout to highlight any quantities which are specific to individual randomisation; and likewise the sub-script, C (for Cluster randomisation), is used throughout to highlight any quantities which are specific to cluster randomisation. No subscripts are used to distinguish cluster from individual randomisation for variables which are pre-specified by the user.Generally, suppose a trial is to be designed to test the null hypothesis \u03b2) and fixed sample size (n) per arm, the detectable difference, Id, with variance var(Id) = 2\u03c32I /nis:Following standard formulae, for a trial using individual randomisation, for fixz\u03b1/2 denotes the upper 100\u03b1/2 standard normal centile.where n individuals per arm, the power to detect a pre-specified difference of d, is 1 - I\u03b2, such that:For a trial with or equivalently:where \u03a6 is the cumulative standardised Normal distribution.\u03b2 to detect a pre-specified difference of d, is In, where:And, finally the required sample size per arm for a trial at pre-specified power 1 - \u03c32) of the proportions \u03c01 and \u03c02, by:Using Normal approximations, the above formulae can be used for binary outcomes, by approximating the variance :Suppose, instead of randomising over individuals, the trial will randomise the intervention over  results , the var\u03c1 is the Intra-Cluster Correlation (ICC) coefficient, which represents how strongly individuals within clusters are related to each other. Where the cluster sizes are unequal this variance inflation factor can be approximated by:where cv represents the coefficient of variation of the cluster sizes and C d(for fixed cluster sizes) becomes:where ter size . Thus, t\u03b2, to detect the pre-specified difference d, and where there are m individuals within each cluster, then the required sample size C n= km per arm, follows straightforwardly from equations 3 and 5 and is:and this is simply extended for varying cluster sizes using equation 6. To determine the required sample size for a CRCT with a pre-specified power 1 - I nis the required sample size per arm using a trial with individual randomization to detect a difference d, and VIF can be modified to allow for variation in cluster sizes (equation 6). This is the standard result, that the required sample size for a CRCT is that required under individual randomisation, inflated by the variance inflation factor [where n factor . The numassuming equal cluster sizes. This slight modification of the common formula for the number of required clusters (over that say presented in ), has roagain with rounding up to the average cluster size.Where a CRCT is to be designed with a completely fixed size, that is with a fixed number of clusters, each of a fixed size , then it is possible to evaluate both the detectable difference and the power, as would be the case in a design using individual randomisation. CRCTs of fixed size might not be the commonest of designs, but formulae presented below: are an important prelude to later formulae, might be useful for retrospectively computing power once a trial has commenced (and thus the size has been determined), and will also be useful in those limited number of studies for which the trial sample size is indeed completely fixed (for example within a cohort study) ,10.k per arm, with a fixed number of individuals per cluster m and with power 1 -\u03b2, then the detectable difference, Cd, follows straightforwardly from equation 1:For a CRCT with a fixed number of clusters I dis the detectable difference using individual randomisation and VIF might be either of those presented at equations 5 and 6. So the detectable difference in a CRCT can be thought of as the detectable difference in a trial using individual randomisation, inflated by the square-root of the variance inflation factor.where C \u03b2of a trial designed to detect a difference of d with fixed sample size C n= mk per arm, following equation 2, is:The power 1 - or equivalently, that:where again, VIF might be either of those presented at equations 5 and 6. So, power in a CRCT can be thought of as the power available under individual randomisation for a standardised effect size which is deflated by the square-root of the variance inflation factor.m) and determining the required number of clusters (k), implicitly assume that the number of clusters can be increased as required. However, in the design of health service interventions, it is often the case that the number of clusters will be limited by the number of cluster units willing or able to participate. So for example, in two general practice based CRCTs , the number of clusters was limited to the number of primary care practices that agreed to participate in the study. From an estimate of the number of clusters available, it is relatively straightforward to determine the required cluster size for each of the clusters. However, due to the limited increase in precision available by increasing cluster sizes, it might not always be feasible to detect the required difference at required power under a design with a fixed number of k clusters. These issues are explored below.Standard sample size formulae for CRCTs, by assuming knowledge of the cluster size (m) and consequently determines the number of clusters (k) required. For a pre-specified available number of clusters (k), investigators need instead to determine the required cluster size (m). Whilst this sample size formula is not commonly presented in the literature, it consists of a simple re-arrangement of the above formulae presented at equation 8 [k) the required sample size per arm for a trial with pre-specified power 1 - \u03b2, to detect a difference of d, is Cn, such that:The standard sample size formulae for CRCTs assumes knowledge of cluster size  available (using the ceiling function).this time rounding up the total sample size to a multiple of the number of clusters  available.again rounding up to a multiple of the number of clusters  becomes large, this precision reaches a theoretical limit:As the cluster size I nis the required sample size under individual randomisation, k is the available number of clusters, \u03c1 is the estimated intra-cluster correlation coefficient, and cv represents the coefficient of variation of cluster sizes. When this inequality does not hold, it will be necessary to re-evaluate the specifications of this sample size calculation. This might consist of a re-evaluation of the power and significance level of the trial, or it might consist of a re-evaluation of the detectable difference. Bounds, imposed as a result of the limited precision, on the detectable difference and power are derived below.for unequal cluster sizes. Here, k), and power 1 - \u03b2, the theoretical Minimum Detectable Difference (MDD) for an infinite cluster size is MDDd, where:For a trial with a fixed number of clusters  and the bound on precision (equation 18). This therefore gives a bound on the detectable difference achievable in a trial with a fixed number of clusters.\u03c01 is fixed (and \u03c02 > \u03c01), then the minimum detectable difference for a fixed number of clusters per arm k, is C d= \u03c02 - \u03c01 such that:For the case of two binary outcomes, where \u03c02 is:Re-arranging this as a function of a = - (1 + w), b = 2\u03c01 + w, w = \u03c1(z\u03b1/2 + z\u03b2)2/k. Solving this quadratic gives:where \u03c02 for two sided tests.Each of these two solutions to this quadratic will provide the limit on k), the theoretical Maximum Achievable Power (MAP) to detect a difference d is 1 - MAP \u03b2 where:For a trial again with a fixed number of clusters (MAP \u03b2where:which again follows from the formula for power (equation 2) and the bound on precision (equation 18). So the maximum achievable power is 1 - k.This therefore provides an upper limit on the power available under a design with a fixed number of clusters When designing a CRCT with a fixed number of clusters, researchers should be aware that such trials will have a limited available power, even when it is possible to increase the number of individuals per cluster. In such circumstances, it will be necessary to:In).(a) Determine the required number of individuals per arm in a trial using individual randomisation ((b) Determine whether a sufficient number of clusters are available. For equal sized clusters, this will occur when:I nis the sample size required under individual randomisation, \u03c1 is the intra-cluster correlation coefficient, and k is the number of clusters available in each arm. For unequal sized clusters:where cv is the coefficient of variation of cluster sizes.where I\u03c1k > n).(c) Where the design is not feasible and cluster sizes are unequal, determine whether the design becomes feasible with equal cluster sizes (i.e. if (d) Where the design is still not feasible:(i) Either: the power must be reset at a value lower than the maximum available power (equation 28),(ii) Or: the detectable difference must be set greater than the minimum detectable difference (equations 23 (continuous outcomes) and 26 (binary outcomes)),(iii) Or: both power and detectable difference are adjusted in combination.(e) Once a feasible design is found, determine the required number of individuals per cluster from equations 14  and 16 (for varying cluster sizes).Maximum achievable power for cluster designs with 10, 20, 30, 50 or 100 clusters per arm are presented in Figure Minimal detectable differences are also presented for both standardised effect sizes Figure  and propIn a real example, a CRCT is to be designed to evaluate the effectiveness of lay support workers to promote breastfeeding initiation and sustainability until 6 weeks postpartum. Due to fears of contamination, whereby new mothers indivertibly gain access and support from the lay workers, the intervention is to be randomised over cluster units. Cluster randomisation will also ensure that the trial is logistically simpler to run, as randomisation will be carried out at a single point in time, and midwives will have the benefit of remaining in either the intervention or control arm for the duration of the trial. The cluster units to be used are midwifery teams, which are teams of midwives who visit a set number of primary care general practices to deliver antenatal and postnatal care. The trial is to be carried out within a single primary care trust within the West Midlands. The nature of this design therefore means that the number of clusters available is fixed at the number of midwifery teams delivering care within the region.k = 20. Estimates of ICC range from 0.005 to 0.07 in similar trials [At the time of designing the trial, current breastfeeding rates, at 6 weeks postpartum, in the region were around 40%. National targets had been set to encourage all regions to increase rates to around 50%. It was known that 40 clusters are available (i.e. there are 40 midwifery teams within the region), so that the number of clusters per arm was fixed to I n= 385 . A similar design with 90% power would require 519 individuals per arm using individual randomisation. Again, because 515 \u00d7 0.005 = 2.57 < 20, this also means that 20 clusters per arm will be sufficient to detect an increase from 40% to 50% with 90% power . Equation 14 shows that under the assumption that \u03c1 = 0.005, either 22 or 30 individuals will be required per cluster (for 80% and 90% power respectively).Firstly, the feasibility check is implemented to determine whether the 20 available clusters per arm are sufficient to detect the 10 percentage point change assuming the lower estimated ICC (0.005). Where the power is set at 80%, the required sample size per arm to detect an increase in percentages from 40% to 50%, under individual randomisation, is >20, so the condition is not met at the 80% power level (and so neither at the 90% power level). Therefore, 20 clusters per arm is not a sufficient number of clusters, however many individuals are included within each cluster, to detect the required effect size at the pre-specified power and significance.Secondly, the feasibility check is evaluated to determine whether the 20 available clusters per arm is sufficient to detect the 10 percentage point change assuming the higher estimated ICC (0.07). However, in this case as 385 \u00d7 0.07 = 26.95 \u03c01 = 0.40, the minimum detectable difference is 0.12 (to 2 d.p.). That is, a change from 40% to 52%. To detect a change from 40% to 52% with 80% power, 189 individuals would be required per cluster. For a trial with 90% power, the minimum detectable difference is 0.14 (i.e. a change from 40% to 54%). To detect a change from 40% to 54% with 90% power, 146 individuals would be required per cluster.Since this latter design is not feasible, formulae at equation 25 allow determination of the minimum detectable difference (or maximum achievable power from equation 27). For a cluster trial with 80% power, and assuming a baseline event rate of In health care service evaluation cluster RCTs, pre-specifying the numbers of clusters available, are frequently used. That is, trials are designed based on a limited number of cluster units (e.g. GP practices) willing or able to participate ,7,9,10. For example, with just 15 clusters available per arm and an ICC of 0.05, power achievable for a trial aiming to detect an increase in percentage change from 40% to 50% is limited to about 62%, irrespective of how large the clusters are made. Cluster trials with just 15 clusters available per arm are not uncommon and a 10 percentage point change not an unrealistic goal in many settings. However, power levels as low as 60% are clearly sub-optimal, and might not be regarded as sufficiently high to warrant the costs of a clinical trial. Formulae provided here for minimum detectable differences show that to retain a power level in the region of 80%, triallists would have to be content with detecting a difference above a twelve percentage point change. Re-formulation of the problem in terms of minimum detectable difference can thus be used to compare the difference which is statistically detectable (at acceptable power levels) to that which is clinically, or managerially, important.Should the situation arise in which the postulated ICC suggests that it is not possible to detect the required difference (at pre-specified power), it might be tempting to lower the estimated ICC. Such an approach should be strongly discouraged, since loss of power will most likely result, potentially leading to a non-significant finding . Rather,Methodological limitations of the work presented here include the assumption of equal sized arms; equal standard deviations; Normality assumptions ; and lack of continuity correction for binary variables. Furthermore, CRCTs with a small number of clusters are controversial, primarily because the small number of units randomised open results to the possibility of bias and approximations to Normality become questionable. However, despite this, CRCTs with a small number of clusters are frequently reported. The Medical Research Council, for instance, has issued guidelines that cluster trials with fewer than 5 clusters per arm are inadvisable . Others k) will be sufficient provided:Evaluations of health service interventions using CRCTs, are frequently designed with a limited available number of clusters. Sample size formulae for CRCTs, are almost exclusively evaluated as a function of the average cluster size. Where no formal limits exist on the number of individuals enrolled within each cluster, increasing the numbers of individuals leads to a limited increase in the study power. This in turn means that for a trial with a fixed number of clusters, some designs will not be feasible, and we have provided simple guidelines to evaluate feasibility. A simple rule is that the number of clusters (For infeasible designs to retain acceptable levels of power, detectable difference might not be as small as desired, leading to the notion of a minimum detectable difference. Useful aidese memoires are that the detectable difference in a CRCT is that of an individual RCT inflated by the square root of the variance inflation factor; and the power is that under individual randomisation with the standardised effect size deflated by the square root of the variance inflation factor. A STATA function, clusterSampleSize.ado, allows practical implementation of all formulae discussed here and is available from the author.The authors declare that they have no competing interests.KH, JM and AS conceived the idea. KH wrote the first and subsequent drafts. AG and RJL helped developed the ideas. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/102/prepub"}
+{"text": "Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated  Epithelial-mesenchymal transition (EMT) is an essential phenotypic conversion that has been implicated in the initiation of metastasis for gastric cancer progression . When gaWnt signaling has historically been divided into two categories, namely, the canonical and the non-canonical signaling pathways. Wnt5a belongs to the non-transforming Wnt family that activates the non-canonical Wnt signaling. It is well known that Wnt5a has ample opportunities to influence diverse cell signaling, resulting in functional promiscuity on tumor initiation and progression. For example, in malignant melanoma and pancreatic cancer, Wnt5a promotes tumor progression . HoweverWnt5a is known as a highly regulated gene, and multiple transcription factors including NF-\u03baB, GLI, FOX, and SMAD are allowed to bind within the promoter region and play important roles in either promoting or repressing Wnt5a transcription under various cellular conditions [2+ pathway or the Wnt/planar cell polarity pathway to regulate oncogenesis and developmental processes [nditions . After pnditions . Normallrocesses . EGF hasrocesses . Althougrocesses , it remaWnt5a promoter to repress Wnt5a transcription. Results obtained in this study clearly establish a new relationship between EGF signaling and Wnt5a transcription in the context of EMT regulation, which could be essential in promoting EMT during invasion and metastasis.Recent studies including the results from our laboratory showed that Arf6 activation could be induced by EGF and act as a mediator of cell migration and invasion in various types of cancer cells \u201323. InteTo assess the effect of EGF on EMT of gastric cancer cells, SGC-7901 cells were treated with EGF (20 ng/mL) and harvested at indicated time points and the cellular morphologic changes were observed by phase-contrast microscopy. We found that EGF time-dependently induced mesenchymal-like morphologies in SGC-7901 cells Figure , and ledWe screened the SGC-7901 cells for mRNA expression for all the members of Wnt family and found that Wnt5a was the most abundantly expressed . In addiArf6 is well characterized in the EGF pathway, which has been associated with breast cancer invasion . We examA recent study has reported that ERK acts as a transcriptional repressor for several genes . We hypoTo probe the involvement of P-ERK in EGF-induced Wnt5a repression in SGC-7901 cells, we treated SGC-7901 cells with U0126 followed by stimulation with EGF. The results showed that U0126 treatment significantly rescued Wnt5a expression at both mRNA and protein levels after EGF stimulation Figure . To deteWnt5a promoter. Chromatin immunoprecipitation (ChIP) assays demonstrated that P-ERK was indeed specifically interacted with the Wnt5a promoter in SGC-7901 cells   Figure  could st) Figure  and stiln Figure  as well n Figure  in gastrn Figure . These rr = \u22120.288, P < 0.05)  was calculated as the intensity of the staining reaction multiplied by the percentage of positive cells , 27 Fig. Represe) Figure . Finally) Figure , suggestr Figure .Both canonical and non-canonical Wnt signalings are associated with the EMT process. Previous studies have shown that aberrant activation canonical Wnt/\u03b2-catenin signaling contributes to malignant progression of gastric cancer , 29. WntIn contrast to previous findings obtained from human breast cancer, which indicated that Wnt5a mRNA level and protein level are not coordinately regulated in same tissue sample , 31, we Wnt5a gene has been shown to encode two protein isoforms, which exert distinct activities in oncogenesis. The expression of the two Wnt5a isoforms is highly variable among individual cancer cell lines [Wnt5a transcription.ll lines . We notill lines \u201335, so iWnt5a transcription directly, thereby allowing EMT in gastric cancer cells. However, by analyzing the genomic sequence of Wnt5a, we didn\u2019t identify any potential Arf6-binding sites within the promoter region (data not shown). We therefore speculated that Arf6 might regulate Wnt5a transcription indirectly by other factors downstream of Arf6.Arf6, a member of the ADP-ribosylation factor (Arf) family, has emerged as a critical regulator of membrane trafficking and structural organization at the adhesion junction (AJ) membrane , 36. SinHere, we demonstrate that EGF induced a time-dependent increase in ERK activity, which was dependent on Arf6 activation. These results are similar with others\u2019 work using melanoma as well as breast cancer cells . In factWnt5a promoter region. More important, using ChIP and luciferase reporter assays, we were able to verify the importance of ERK within the nucleus, that is, through its direct binding to Wnt5a promoter, ERK negatively regulates Wnt5a transcriptional expression and serves as a master enforcer of gastric cancer cell EMT. It is believed that the status of ERK phosphorylation may regulate the trans-localization of ERK from the cytoplasm to the nucleus [Wnt5a transcription in both SGC-7901 and BGC-823 gastric cancer cells. ERK2, but not its isoform ERK1, is suggested to play a more important role in EMT process [Wnt5a promoter-binding ability of ERK, possibility that of ERK2, is dependent of its nuclear location, but not its kinase activity.ERK, an extensively studied protein known for cytoskeletal organization and focal adhesion renewal, has recently been found to be involved in transcriptional regulation . ERK is  nucleus . Interes nucleus , we noti process . Thus, oWnt5a transcriptional expression. These findings are of potential pathophysiological importance for understanding the integration of migration-related signaling and shed light on new therapeutic targets for gastric cancer.Although the current study has contributed to the mechanistic understanding the role of Wnt5a in EGF-induced gastric cancer cell EMT, the issue as to how Wnt5a precisely regulates EMT in gastric cancer cells is unlikely to be settled in this paper. Together, these data point to the ability of Arf6/ERK-dependent mechanism that leads to increased EGF-induced EMT of gastric cancer cells by inhibiting 2. Cells were grown on coverslips for fluorescence staining and on plastic dishes for protein extraction. Cells were made quiescent by serum starvation overnight followed by EGF  treatment.Human gastric cancer cell line SGC-7901, BGC-823 was obtained from the Cell Biology Institute of Chinese Academy of Sciences . SGC-7901 and BGC-823 cells were cultured in Dulbecco's modified Eagle's medium   supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone) and antibiotics (100 U/mL streptomycin and 100 \u03bcg/mL penicillin)  in a humidified incubator at 37\u00b0C with 5% COWnt5a cDNA was amplified from pBIG2r-Wnt5a plasmid using the following primer set, sense: 5\u2032-TTGCGGCCGCGCCACCATGAAGAAGTCCATTG GAA-3\u2032, antisence: 5\u2032-CCGGAATTCCTTGCACACAAA CTGGTCCACGATC-3\u2032. In these primers, Not I and EcoR I restriction site sequences have been underlined. The polymerase chain reaction (PCR) products were cloned into the pCMV-C-HA vector . Full-length Arf6 cDNA was amplified from pXS-Arf6 using the following primer set, sense: 5\u2032-CCGGAATTCGCCACCATGGGGAAGGTGCTATC- 3\u2032, antisence: 5\u2032-TCCCCGCGGAGATTTGTAGTTAG AGGTTAAC-3\u2032. EcoR I and Sac II restriction site sequences have been underlined. The PCR products were cloned into the pEGFP-N1 vector . The ERK2 WT (Genebank NM_002745.4) and TAYF mutation  sequences [The pBIG2r-Wnt5a plasmid and pXS-Arf6 plasmids (WT and T27N) were kindly provided by Dr. P. Michl  and Dr. Julie G. Donaldson , respectively. Full-length equences , the SV4The sequences of small interfering RNA (siRNA) for Arf6 were as follows: #1, 5\u2032-GUGGCAAAUAAUGAGU AAUTT-3\u2032, #2, 5\u2032-GCGACCACUAUGAUAAUAUTT-3\u2032, and #3, 5\u2032-GACGCCAUAAUCCUCAUCUTT-3\u2032; and the sequence of control siRNA was 5\u2032-UUCUCCGAACGUGUCACGUTT-3\u2032 . Cells were transfected with control siRNA or Arf6 siRNA pool  with Lipofectamine 2000, according to the manufacturer's instruction.Commercially HIV-based, GFP-labeled and puromycin resistant plasmids Wnt5a shRNA (HSH018531-4-HIVU6) and unrelated control shRNA (CSHCTR001-HIVU6) were obtained from GeneCopoeia . SGC-7901 cells were infected with those lentivirus plasmids by EndoFectin\u2122-Plus (GeneCopoeia) and selected by puromycin (0.4 \u03bcg/mL) 48 h after infection.SGC-7901 cells were seeded in a 96-well plate. Approximately 48 h later, when cells were 95~100% confluent, cells were incubated overnight in DMEM and wounding was performed by scraping through the cell monolayer with a 10 \u03bcL pipette tip. Medium and nonadherent cells were removed, and cells were washed twice with PBS, and new medium with or without EGF was added. Cells were permitted to migrate into the area of clearing for 48 h. For stably transfected cells, the monolayer cells were scratched and allowed to heal for 24 h in DMEM. Wound closure was monitored by visual examination under microscope .5/200 \u03bcL) were seeded into polycarbonate membrane inserts (8 \u03bcm pore size) in 24-transwell cell culture dishes. Cells were allowed to attach to the membrane for 30 min. The lower chamber was filled with 600 \u03bcL DMEM with 10% FBS. Cells were permitted to migrate for 12 h. After the incubation, stationary cells were removed from the upper surface of the membranes. The cells that had migrated to the lower surface were fixed and stained with 0.1% crystal violet. The area of stained cells was counted by pixel in photos taken by Nikon TS100 .For transwell migration assay, stably transfected SGC-7901 cells in exponential growth were harvested, washed, and suspended in DMEM without FBS. Cells . Equal amounts of RNA (1 \u03bcg) from each sample were used for cDNA synthesis using HiScriptQ RT SuperMix for qPCR . qPCR was performed on the ABI StepOne\u2122 Real-Time PCR System  using GoTaq qPCR Master Mix assay (Promega) and analyzed using StepOne Software v2.1 (Applied Biosystems). 2\u2212\u0394CT method was used to calculate gene expression levels. For sample loading control, GAPDH were tested. Primers used for qPCR amplification are available in Table S1.Sample protein extraction and concentration determination of whole cells were performed as previously described . Cytopla2HPO4, 2 mmol/L KH2PO4, 140 mmol/L NaCl, and 10 mmol/L KCl, pH7.2). At the end of this period, beads were captured by the magnet in a magnetic stand (Promega) and resuspended in 2 \u00d7 SDS sample buffer and subjected to immunoblotting analysis by using anti-Arf6 antibody.For detection of active Arf6, equal volumes of total cellular protein were incubated with GST-GGA3 for detection of active Arf6  beads captured on MagneGST Glutathione Particles (Promega) at 4\u00b0C with constant rotation for 30 min. The beads were washed three times with washing buffer , N-cadherin (1:50), Vimentin (1:200), P-ERK (1:200) antibodies at 4\u00b0C overnight followed by incubation with rhodamine-conjugated anti-rabbit or anti-mouse antibody for 1 h at room temperature within a moist chamber. After wash with PBS, the samples were mounted with DAPI Fluoromount G . Images were acquired using an Olympus BX51 microscope coupled with an Olympus DP70 digital camera.2O2 in methanol for 15\u2009min at 37\u00b0C. Antigen retrieval was performed by transferring the sections into Tris-acetate-ethylenediamine tetraacetic acid (EDTA) buffer (pH\u20098.0) inside a 700-watt microwave oven on full power for 5 min and half power for 10 min. After cooled down to room temperature, the sections were blocked in goat serum for 1 h and applied with Wnt5a  or P-ERK antibody  antibody at 4\u00b0C overnight. Then the sections were treated with the secondary antibody (1:1000) for 1 h at 37\u00b0C, and then washed in PBS. DAB substrate solution was applied to reveal the color of antibody staining. After counterstained with haematoxylin, the slides were mounted by neutral gum. Reagents for immunohistochemistry were all obtained from ZSGB-BIO . Wnt5a and P-ERK immunostaining was analyzed by evaluation of the percentage of tumor-stained cells and staining intensity, allowing assessment of an IRS [Tumor specimens used were obtained by the First Affiliated Hospital of Nanjing medical University, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Affiliated Zhongda Hospital of Dongnan University and the Second Affiliated Hospital of Nanjing Medical University . Fifty primary human gastric tumor samples were used for immunohistological staining in our Wnt5a/P-ERK expression correlation study. The paraffin section were deparaffinized and rehydrated. Peroxidase blocking was done with 3% Hf an IRS , 27. Bli7 cells were cross-linked with 1% formaldehyde. The nuclei were collected from the cells and the nuclear chromatin was.sonicated to an average length of about 1 kb. 20% of this chromatin solution was used as input and the remaining was pre-cleared by salmon sperm DNA/Protein A agarose slurry for 30 min at 4\u00b0C with rotation. The pre-cleared supernatant was then incubated with P-ERK antibody  or normal rabbit IgG  for 16 h at 4\u00b0C. The immune complexes were collected with salmon sperm DNA/Protein A agarose slurry. After wash with gradient stringent buffer, the eluted solution of the complexes was incubated for 16 h with 5 mol/L NaCl at 65\u00b0C and then with 0.5 mol/L EDTA, 1 mol/L Tris-HCl and 10mg/mL proteinase K at 45\u00b0C to reverse the cross-links. The IP-DNA was extracted using TIANamp Genomic DNA Kit  and confirmed by qPCR. Primers used for qPCR amplification were as follows: Wnt5a: 5\u2032-GGTTCGGTTTGTGTGGCTTC-3\u2032 (sense), 5\u2032-TGCGACATGTTTCCGAGTCA-3\u2032 (anti-sense); GAPDH: 5\u2032-TACTAGCGGTTTTACGGGCG-3\u2032 (sense), 5\u2032-TCGAACAGGAGGAGCAGAGAGCGA-3\u2032 (anti-sense).The chromatin immunoprecipitation (ChIP) assay was conducted based on the Millipore-Upstate ChIP protocol with a few modification . Briefly, 1.5 \u00d7 10GGTACCCGTGGCACGCGAGGAAGATT-3\u2032, 2: 5\u2032-GAAGATCTGCCCTGGTGCCAGGCGCTGC-3\u2032; 3: 5\u2032-GGGGTACCAAGCTTGGAAAGTGCACGCG-3\u2032, 4: 5\u2032-GAAGATCTAAGCTTCCGCTGCCGTTCCTCC-3\u2032; 5: 5\u2032-GGGGTACCCCAAGGCCAACTCCTACCCCT-3\u2032, 6: 5\u2032-GAAGATCTCGCGGCGAGCGGGGCGC-3\u2032. The six plasmids were named 12 (full length), 14, 16, 23, 25, 34 (short sections) according to their primers, respectively. In these primers, Kpn I and Bgl II restriction site sequences have been underlined. pRL-SV40 (Promega) plasmid containing a Renilla luciferase gene was co-transfected as control.The Wnt5a promoter  was syntWnt5a promoter reporter plasmid (full length or short sections) along with 12.5 ng of control plasmid expressing Renilla luciferase was co-transfected using lipofectamine 2000 (Invitrogen). The cells were then treated with EGF (20 ng/mL) or U0126 (10 \u03bcmol/L) for 48 h and collected for the luciferase assay using the Dual-Luciferase Assay Kit (Promega) on a GloMax 20/20 luminometer (Promega). Experiments were performed in triplicates and repeated at least three times.Cells were seeded in 24-well plates 24 h before transfection. The following day, 250 ng of the t test. P < 0.05 was considered to be significant (two tailed). Error bars represent standard error of mean (s.e.m.). In immunohistochemistry assay, the difference in expression level of Wnt5a and P-ERK was according to IRS score, Pearson correlation test was used to examine associations between Wnt5a and P-ERK protein expressions.Statistical analysis was performed using the SPSS statistical software program . Data were analyzed by Student's"}
+{"text": "Small nucleolar RNAs (snoRNAs) are one of the most ancient families amongst non-protein-coding RNAs. They are ubiquitous in Archaea and Eukarya but absent in bacteria. Their main function is to target chemical modifications of ribosomal RNAs. They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of chemical modification that they govern. Similarly to microRNAs, snoRNAs appear in distinct families of homologs that affect homologous targets. In animals, snoRNAs and their evolution have been studied in much detail. In plants, however, their evolution has attracted comparably little attention.In order to chart the phylogenetic distribution of individual snoRNA families in plants, we applied a sophisticated approach for identifying homologs of known plant snoRNAs across the plant kingdom. In response to the relatively fast evolution of snoRNAs, information on conserved sequence boxes, target sequences, and secondary structure is combined to identify additional snoRNAs. We identified 296 families of snoRNAs in 24 species and traced their evolution throughout the plant kingdom. Many of the plant snoRNA families comprise paralogs. We also found that targets are well-conserved for most snoRNA families.The sequence conservation of snoRNAs is sufficient to establish homologies between phyla. The degree of this conservation tapers off, however, between land plants and algae. Plant snoRNAs are frequently organized in highly conserved spatial clusters. As a resource for further investigations we provide carefully curated and annotated alignments for each snoRNA family under investigation.The online version of this article (doi:10.1186/s12864-016-3301-2) contains supplementary material, which is available to authorized users. Small nucleolar RNAs function as guides in site-specific RNA modification , 2. TheyIn vertebrates, mature snoRNAs are mainly produced from introns of precursors that can be both protein-coding mRNAs or non-coding \u201chost genes.\u201d In contrast, only a few snoRNAs are intronic in budding yeast and plants , 7. MoreThere is a tendency for polycistronic snoRNA precursors in general. In plants, however, polycistronic precursors are the standard \u201311. IndiBox C/D snoRNAs share the conserved sequence motifs C (RUGAUGA) close to the 5\u2019-end and D (CUGA) near the 3\u2019-end, which are tethered by a terminal stem-loop. In addition, internal C\u2019 and D\u2019 box can be found in many of the box C/D snoRNA. These motifs have the same consensus sequence as the C and D boxes, resp., but show a higher level of variation in both animals and plants. The assembly of box C/D snoRNPs involves the formation of a kink-turn (K-turn) motif , 17. ThiThe box H/ACA snoRNAs are distinguished by the presence of an ACA triplet at their 3\u2019-end and a characteristic hairpin-hinge-hairpin-tail secondary structure with the H box (ANANNA) located in the hinge region , 23.The conserved sequence motifs  serve as binding sites for protein components of the snoRNPs. Both classes of snoRNAs recognize their targets by complementary base pairing. The antisense elements of box C/D snoRNAs are located immediately upstream of the boxes D and D\u2019 and have a typical length of 10-15nt. The antisense elements of box H/ACA snoRNAs are located within interior loops that interrupt the hairpins, see e.g. .Beyond their function as guides for chemical modifications, a few snoRNAs are required for the cleavage of the ribosomal RNA precursors , among tBased on sequence similarity, snoRNAs fall into many well-defined families of homologous genes. As a consequence of the frequent segmental, chromosomal, and whole genome duplications in plant genome evolution, most plant snoRNA families have multiple paralogous members both in spatial clusters and spread throughout the genome .Despite their ancient ancestry as a class , the lonSeveral studies showed that many snoRNA families are conserved at phylum or even kingdom level in animals , plants Matching the situation in microRNAs , there iA survey from 2010 concludes that we are still far from a comprehensive picture of snoRNA evolution and many more snoRNAs of both known and novel families remain to be found . Recent Although there is good evidence for the conservation of many of the chemical modification sites on rRNAs and snRNAs between eukaryotic kingdoms , it is sIn this contribution we reconstruct the evolutionary history of snoRNAs in the plant kingdom. We focus on the identification of additional homologs in considered plant genomes and focuses on interesting patterns of conserved snoRNA families and regions of clustered snoRNAs. For each snoRNA family the evolution is systematically traced back to its last common ancestor.From the intial set of collected and curated snoRNA families, snoRNAs are mapped to all the plant genomes and family-wide alignments of all retained candidate sequences were calculated. Finally, a putative history of gains and losses of genes within each snoRNA family was constructed. The initial query set of 554 snoRNA genes was comprised of a collation of all available (plant) snoRNA databases. These sequences were assigned to 222 box C/D and 74 box H/ACA snoRNA families after manual curation and annotation of the box C/D and box H/ACA snoRNAs. We identified a total of 5116 additional homologs in the 24 plant species under consideration.Arabidopsis, rice, or Chlamydomonas are not shown in the heatmaps. For the heatmaps only the 110 snoRNA families that were found to be conserved in more than one species are selected.The phylogenetic distribution of the snoRNA families is shown in Figs. Several patterns are apparent. With the exception of the highly conserved U14 family and the snoR96 family that shows a much more scattered distribution, snoRNAs from land plants do not have identifyable homologs in green algae. Seven families of box C/D snoRNAs  are conserved in land plants. Among these U14 is present nearly ubiquitously. Missing sequences in single species (white cells) are most likely caused by unidentifiable homology due to rapid snoRNA evolution rather than representing true snoRNA losses.Four H/ACA snoRNA families  are present throughout the land plants, albeit only snoR2 was found in almost all species investigated here. The largest fraction of identified snoRNAs (76 box C/D and 20 box H/ACA families) are common to the flowering plants including both monocots and dicots. Target prediction employed by the snoStrip pipeline  suggestsArabidopsis, e.g. snoR107, 28 families appear to be specific to Oryza, e.g. snoR146a, and 131 families appear only in Chlamydomonas, e.g. CrACA02. Most of the Arabidopsis-specific snoRNAs have been reported to have their targets in ribosomal RNAs [Arabidopsis, Oryza, and Chlamydomonas. For other species much less extensive data have been reported in the literature, hence most of the snoRNA genes are annotated by homology.On the other hand, there are many families with a very narrow phylogenetic distribution: 27 families are found only in mal RNAs . Either A very interesting pattern is the large block of box C/D snoRNAs (20 families) that is only present in monocots. A similar pattern is not visible for box H/ACA snoRNAs. There is also no such pattern of dicot-specific box C/D snoRNAs or dicot-specific box H/ACA snoRNAs. Hence, it is very unlikely that the monocot specific families of box C/D snoRNAs are just an artefact caused by limitations in the homology search method. So they should be interpreted as true monocot innovations.Brassica rapa and Digitalis purpurea among dicots, as well as Triticum aestivum and Hordeum vulgare among monocots. By comparison with the Plant Genome Duplication Database [Finally, focussing on column-wise patterns we observe a systematically elevated number of snoRNA paralogs in some species. Examples include Database  this obsThere are several reasons why snoRNAs appear to be missing in some species or clades. First, we may see true gene losses. A second explanation is that they have diverged beyond our ability to detect and identify them by any of the available methods of homology search. This a likely explanation in particular for large phylogenetic distances. Third, incomplete genome assemblies can explain apparent gene losses. This explanation is plausible in particular for scattered, non-systematic \u201cwhite spots\u201d in the heatmaps.A. thaliana [A. thaliana [SnoRNAs that are encoded or positioned closely together in the same chromosomal region are considered as \u201csnoRNA clusters\u201d. In order to study the long-term integrity of those clusters we investigated representative examples: the 68 rice snoRNA clusters described in . Multiplthaliana . In thisthaliana .P. dactylifera), snoR7b is a more recent addition, incorporated in the dicot ancestor. Its homolog in A. thaliana was discussed in [The 10 genomic clusters involve 22 distinct snoRNA families. A subset of the clusters comprises highly conserved snoRNAs, whereas most of the rice clusters are not conserved in other species. Several snoRNA families have members in distinct clusters. Figure ussed in  as the \u201cA.thaliana [A. thaliana \u201cU32.2-U27.2-U80.2 cluster\u201d [A. thaliana [A.thaliana [A. thaliana [Details on the 9 other conserved clusters  in the terminology of ) are prothaliana , U24 wascluster\u201d , snoR22bthaliana  as the \u201cthaliana , both U6thaliana  as \u201csnoRSystematic prediction of snoRNA targets in rRNAs and snRNAs showed that known and many predicted targets are usually conserved when the snoRNA is conserved. The complete archive of rRNAs and snRNAs used for target prediction is provided as Additional file all plant snoRNA families is given in Figs. To draw a comprehensive picture of the snoRNA evolution in the 24 plant species we used the compational approach ePoPE . It implMany snoRNA families are deeply conserved in the plant kingdom. Surprisingly, only a few families can unambiguously be traced back to the ancestor of land plants. Some families are innovations that emerged later during plant evolution. We hypothesize that at least 8 snoRNA families are recent innovations, i.e. snoR59, U29, snoR72Y, snoR6, U31, snoR8, snoR23, and snoR7. This hypothesis is supported by a large group of monocot-specific snoRNAs. The strong conservation of some chemical modification sites in ribosomal RNAs, however, supports the idea that there is a core of snoRNA genes that are ubiquituously present in Eukarya and possibly even in Archaea. The small size, the relative fast rate of evolution, and limitations of available homology search techniques, however, make it hard to directly test this hypothesis. Surprisingly, homology search methods fail, with very few exceptions, to identify homologs of landplant snoRNAs in green algae. We suspect, however, that this rather a limitation of the state of the art in homology search.Despite these and many other limitations, several interesting patterns on snoRNA evolution in plants can be observed. Many snoRNA families have well-identifiable paralogs. Furthermore, distinction between evolutionarily old families and a collection of evolutionarily young innovations is observed see Figs. Arabidopsis or Oryza names for snoRNA families wherever possible based on the assumption that these are most widely used. A comprehensive table of synonyms is provided as Additional file The nomenclature of plant snoRNAs is often species specific and it respects only partially known orthology relationships at the level of individual snoRNAs families. In particular, this is the case where data go beyond the plant snoRNA database . In some1 format  and theWe considered only the rRNAs/snRNAs as potential targets. Ribosomal RNA sequences of the 24 plant and red algae species are downloaded from the SILVA database . The snRFrom the initial set of collected snoRNAs, the box motifs are annotated and categorized into box C/D and box H/ACA snoRNAs. The characteristic boxes  are annotated manually using the sequence patterns as constraints given in .Previous analyses from the Bachellerie laboratory showed conserved spacing between the box C/D core motif and the internal D\u2019/C\u2019 motif of the archaeal box C/D snoRNAs . AlthougHence, box motifs are annotated based on both known pattern of conserved nucleotides and likely spacer distances, usually 12nt, between the box C/D and D\u2019/C\u2019 motifs. Only snoRNAs with boxes that could be annotated with high certainty are selected for the initial query set. The sequences are then grouped into gene families based on known orthology and sequence similarity.In the next step all snoRNA families were mapped to all plant genomes. The list of all genomes with accession numbers is provided as Additional file The expected boxes and the anti-sense elements were annotated based on sequence alignments, and candidates were filtered for the presence of the boxes. The snoRNA fasta files along with coordinates of annotated snoRNAs are provided as Additional file Data were then aggregated to heatmaps showing the number of family members in each species. SnoRNA clusters were identified by proximities of genomic coordinates.The history of gains and losses in each snoRNA family was reconstructed using a Dollo parsimony approach implemented in the ePoPe programm .Arabidopsis, Oryza, and Chlamydomonas. A complete table of family names and their species-specific synonyms is provided as Additional file Since the nomenclature of plant snoRNAs only partially respects known or detectable sequence homology we used a unique internal family identifier throughout this study. These identifiers are re-translated to a consolidated family nomenclature that is based, in this order, on the nomenclature for 1https://en.wikipedia.org/wiki/Stockholm_format"}
+{"text": "The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. \u03b2-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the \u03b2-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level.HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation.Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level.We identified two different frameshift mutations positioned in the third exon of Our results show that a lack of natural stop codon due to the frameshift in exon 3 of \u03b2-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway.The online version of this article (doi:10.1186/s12881-017-0428-1) contains supplementary material, which is available to authorized users. They are common in tropical and subtropical regions where they provide protection against malaria, but are rather rare in Northern Europe. Some limited cases have been described in the indigenous British, Irish, Breton , 2, ScanHemoglobin disorders comprise two different groups, structural hemoglobin defects and the thalassemias, where the abundance of a globin chain is decreased. The classical recessive forms of \u03b2-thalassemia lead to a reduced production of normal \u03b2-globin chains. In contrast, dominantly inherited \u03b2-thalassemia is caused by some rare mutations resulting in the synthesis of extremely unstable \u03b2-globin .HBB gene encoding \u03b2-globin, spans 1.6\u00a0kb and comprises three exons, with the first and third one containing mainly noncoding sequences of the5\u2019 and 3\u2019 UTRs. 378 different \u03b2-thalassemia mutations have now been characterized worldwide [The orldwide . In veryorldwide . RNA surHBB mRNA to degradation, which depends on proper position of termination codon.The aim of the present study was to look for mutations in the \u03b2-globin gene in patients with hereditary hemolytic anemia suspected of thalassemia. Two cases (Patient 1 and Patient 2) were investigated and various frameshift mutations (c.375_376insCCAGT and c.349del) located in the third exon have been identified. In both patients the level of \u03b2-globin mRNA was substantially lower than in healthy subjects. By mimicking CCAGT insertion in cultured human cells we established the molecular mechanism by which the identified genetic changes diminish \u03b2-globin expression. Here, we show that newly identified mutations direct the Patient 1: A 6-year-old boy, born to non-consanguineous parents has been under medical supervision since birth because of hemolytic anemia of unknown etiology. He presented with splenomegaly and xanthoderma. Occasionally, the patient needed red blood cells (RBCs) transfusions. Laboratory tests indicated profound anemia and hyperbilirubinemia. Congenital dyserythropoietic anemias, hereditary spherocytosis and anemias due to deficiency of red cell enzymes were excluded. HbF and HbA2 levels were elevated and \u03b2-thalassemia was diagnosed. Compatible clinical presentation of hemolytic anemia was observed in mother and younger sister of the proband. His mother had splenectomy in childhood without significant improvement of anemia but she hasn\u2019t need RBC transfusion. The results of blood tests of all subjects are presented in Table\u00a0Patient 2: A 10-year-old boy with hemolytic anemia and hyperbilirubinemia of unknown etiology, born to healthy non-consanguineous parents, has been observed since birth. Initially, hereditary spherocytosis was diagnosed, based on the assessment of peripheral blood smear. Occasionally, he needed RBC transfusions. At the age of 8, splenectomy was performed but without beneficial effect. Hemoglobin electrophoresis revealed elevated level of fetal hemoglobin (4%) and hemoglobin A2 (4.3%), consistently with \u03b2-thalassemia diagnosis. There was no family history of blood diseases.Both patients were Caucasians of Polish origin. The patients' parents gave informed consent to the study. The study protocol has been approved by the Ethical Committee of the Medical University of Warsaw. The study was conducted according to principles laid out in the Declaration of Helsinki.Total RNA was isolated from peripheral blood samples using the PAXgene Blood RNA Kit . cDNA was amplified with the use of primers encompassing the coding sequence from exon 2 to 3\u2019UTR. qPCR amplification was performed using a LightCycler1.5 and LightCyclerFastStart DNA Master SYBR Green I .EMP55 (erythrocyte membrane protein p55).mRNA quantification of the \u03b2-globin genes was carried out using real-time PCR. The Pfaffl model  and the Genomic DNA was isolated from peripheral blood using the MagNA Pure Compact Nucleic Acid Isolation Kit I . Polymerase chain reaction (PCR) was used to amplify the promoter region, entire coding sequence and surrounding sequences of the \u03b2\u2013globin gene. DNA fragments generated by PCR amplification were sequenced directly as previously described . Primer XmnI  was used to verify the presence of the XmnI-G\u03b3 polymorphism .HBB was amplified using RSZ394 (gtcggtaccccatggtgcatctgactcctg) and RSZ396 (gacctcgagttccctttttagtaaaatattcag) primers and DNA isolated from human 293 cells as a template. Amplified DNA fragment was cloned into pcDNA5FRT/TO vector  digested with Acc65I and XhoI to give plasmid pRS478. Standard site-directed mutagenesis of pRS478 was performed to introduce the mutation identified in patient 1 (pPO9) or the mutation together with an additional nucleotide restoring the reading frame (pPO10). The following primers were used for mutagenesis: RSZ533 (gcaaagaattcaccccaccagtccagtgcaggctgcctatcag) and RSZ534 (ctgataggcagcctgcactggactggtggggtgaattctttgc) (to get pPO9) and RSZ535 (gcaaagaattcaccccaccagtccagttgcaggctgcctatcag) and RSZ536 (ctgataggcagcctgcaactggactggtggggtgaattctttgc) (to get pPO10).DNA cloning was performed using standard methods. 2). Stable transfected cell lines were obtained from human 293 TRex-FlpIn cells using a procedure described previously [Cells were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS (Invitrogen) under standard conditions  according to manufacturer\u2019s recommendation. Northern blotting was performed as previously . The priInitially we have quantified levels of globin mRNAs in both patients which revealed a decreased \u03b2-globin transcript level by more than 50% in the investigated patients and elevated levels of \u03b3- and \u03b4-globin compared to the control group Table\u00a0.Table 2RIn Patient 2 both \u03b3 - and \u03b4 -globin mRNAs were substantially more abundant than in controls, while in Patient 1 only \u03b3 -globin mRNA was up-regulated markedly and \u03b4-globin one showed a modest increase  or 157 (Patient 2) Fig.\u00a0.HBB mRNA. The mutations could somehow affect transcription of the gene, splicing or the mutated HBB mRNA could be degraded rapidly by RNA surveillance. Because, mutations are within a coding region we found the first possibility to be very unlikely. In order to analyse the effect of mutations on splicing and stability we focused on the Patient 1 case (c.375_376insCCAGT) and constructed three stable human cell lines expressing in an inducible manner wild-type HBB, its mutated version, or a variant containing the original Patient 1 mutation followed by an additional thymidine which restored the reading frame  tails. Regardless of the nature of the two HBB mRNA species, these results clearly show that frameshift caused by the c.375_376insCCAGT insertion, but not the insertion itself, activates an RNA degradation without affecting pre-mRNA processing.We considered three possible mechanisms which could be responsible for the observed low steady-state levels of the mutated ame Fig.\u00a0. An incoHBB Fig.\u00a0, howeverHBB Fig.\u00a0. The samRNA degradation plays an essential role in ensuring that only fully functional RNA molecules become translated by disposing of aberrant ones that are often mature but dysfunctional. Their expression at the protein level could disturb cellular functioning. Truncated or too long polypeptide chains could titer out other proteins, form inactive complexes, misfold and aggregate or otherwise hamper to the cell. Although numerous mechanisms do exist for the disposal of aberrant polypeptides, they could easily become overwhelmed by excessive production of their substrates. Thus, defective RNA molecules, which are often mature but dysfunctional, need to be degraded rapidly by variety of RNA surveillance pathways .HBB gene which introducing a premature stop codon inducing nonsense mediated RNA decay. It has been reported that NMD-promoting mutations located in exon 1 or 2 of \u03b2-globin gene subject the transcripts to rapid degradation [Some cases of \u03b2-thalassemias are caused by mutations in the radation . Such muradation . In geneHBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although mutations of Patient 1 and Patient 2 are not expected to induce NMD, the mutant version of mRNA is not detectable. For one of these mutations, experiments using reporters in 293 cell lines confirmed the degradation of the mutated transcript. Furthermore, restoring of the origin reading frame stabilized the transcript, which suggested that the destruction of the wild type HBB reading frame was crucial for the degradation of the defective mRNAs.In our study we identified two different frameshift mutations positioned in the third exon of http://globin.cse.psu.edu/) the majority of frameshift mutations in the exon 3 at codons 108\u2013141 do not produce a Hb variant detectable by electrophoresis, chromatography, or stability tests. In contrast, missense mutations downstream of codon 141 produce abnormal Hb. It seems likely that additional factors could bind to specific sequences created by the frameshift mutations to facilitate their identification and consequent mRNA degradation. It seems that effect of a given mutation, besides other factors, also depends on its position within the HBB gene. This phenomenon is currently unclear.Our understanding of the mechanisms controlling RNA quality is limited , and theHBB mRNA.The two mutations described have add new information regarding degradation of mutated \u03b2-globin mRNA and thus could facilitate the deciphering of the underlying mechanism. In particular, we have shown that moving the STOP codon by as few as twelve codons downstream suffices to destabilize the"}
+{"text": "Self-evaluated physical and mental health was significantly improved for the total group; the relationship with partner, the subjective sexual ability, and the quality of life that were measured with QOL1 and QOL5 questionnaires were all significantly improved. VA or Hippocratic pelvic massage is technically a simple procedure corresponding to the explorative phase of the standard pelvic examination, supplemented with the patient\u2019s report on the feelings provoked followed by processing and integration of these feelings, but ethical aspects are complicated. Acupressure through the vagina/pelvic massage must be done according to the highest ethical standard with great care, after obtaining consent and the necessary trust of the patient within the framework of the local laws. It must be followed by conversational therapy and further holistic existential processing.This is a pilot study of 20 female patients with a long history of sexual problems (mean is 8.92 years) who received vaginal acupressure (VA) with a quantitative and qualitative evaluation: 56% experienced help and none reported setbacks, 89% rated the treatment to be of high quality, and 89% rated it as valuable. After the treatment, most reported their problems to be less serious and their general quality of life improved. Only 17% reported minor or temporary side effects. VA was found statistically and clinically significant ("}
+{"text": "NASA cleanrooms are certified by particle counts and are humidity-controlled, temperature-regulated, and oligotrophic in nature for assembling spacecraft subsystems. Microorganisms, which are not part of the cleanroom certification metrics, should not be overlooked when assessing the cleanliness of the facility since they can enter through soil or air, shed from humans, adapt to the oligotrophic conditions, and subsequently could contaminate spacecraft. These biogenic particles need to be identified to extend our knowledge of biological contamination for future NASA mission use. This study collected particles from the cleanroom and estimated the distribution of fallout microbial cell and inert dust particles using microscopy and molecular techniques. Aluminum coupon-based polycarbonate filter assemblies were deployed in the spacecraft assembly cleanroom facility to collect fallout particles. Epifluorescence and electron microscopy showed that particles varied in size and structure, and displayed live/dead biological and inert particle signatures from sources that include spores and fungal hyphae. Additionally, correlative epifluorescence and field emission scanning electron microscopy, combined with energy-dispersive X-ray analysis  methods, differentiated whether microbes adhering to particles were live/dead cells or inert particles. This visualization approach allowed for the classification of microorganisms as being standalone (free-living) or associated with a particle, as well as its characteristic size. Furthermore, time-course microscopy was used to determine the microbial cell growth and confirm the biological/molecular identification. Routine investigation of cleanroom biological and inert fallout particles will help to determine the biological load of spacecraft components and will also have direct relevance to the pharmaceutical and medical industries. One of the main objectives for NASA\u2019s current and future missions is to prevent forward and back contamination of exploring planets. The goal of this study is to determine the association of microorganisms with the inert, natural cleanroom fallout particles and to ascertain whether microorganisms prefer to adhere to a particle size. A novel microscopy technique was developed, and by utilizing various molecular techniques, particles and associated microbial phylogeny were characterized. An accurate assessment of the microbes associated with cleanroom particles is necessary to protect the health of the people who occupy the room for long duration for aeronautical, medical, and pharmaceutical industries. Fallout particles vary greatly in size (ranging from 0.1 \u03bcm to 1000 \u03bcm), morphology, and elemental composition, and can originate from sources such as human, cosmetics, cargo, and particles transported in the cleanroom environment from the external environment, which varies by geographic location8. Therefore, it is necessary to understand the size distribution and chemical composition of natural fallout particles in critical indoor environments such as intensive care units (ICUs), spacecraft assembly facilities, and other industrial cleanrooms9. However, reports on the biological burden associated with cleanroom natural fallout particles are not available. Even though National Aeronautics and Space Administration (NASA) cleanrooms have been maintained with controlled airflow circulation, temperature, humidity, and rigorous cleaning, they are not sterile12.Microorganism(s) associated with fallout particles are a critical concern for fabrication of sensitive products with highly liable surface particulate contamination, including medical devices, pharmaceutical products, electronic supplies, and spacecraft assembly15. Since 2000, in addition to the NASA spore assay, advanced next-generation sequencing methods such as targeted gene(s)17, as well as DNA microarray and shotgun metagenome sequencing18, were carried out to identify the microbial burden and their distribution on the cleanroom microbial ecosystem19, but no studies on the characterization of biological materials associated with fallout particles have been completed. In the past, particulate and microbial contamination were not assessed in tandem but analyzed separately using two distinct sets of witness coupons20. Therefore, research needs to be conducted to estimate the distribution of microbial communities and their association with cleanroom particles from the same set of materials.Since Viking, NASA has been assembling unmanned robotic spacecraft in cleanroom environments and continuously monitoring microorganisms to mitigate the forward contamination of other planets and bodies in the solar system21. In addition, visual characterization of particles were commonly captured using epifluorescence and field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray analysis (EDXA) since these approaches provide information on both morphology and organic elemental composition22. However, electron microscopy is limited to visualizing only the morphology of the target and cannot be used to discern biological particles from individual inert particles. In this communication, \u201cinert particle\u201d is defined as the particle that did not show any carbon signature when EDXA analysis was performed. Few studies utilized both microscopy and molecular biological techniques to concurrently link the microorganism to the environmental dust particles and determine whether microorganisms were associated with particles or were free-living24. Recent advances in microscopy techniques such as correlative light and electron microscopy procedures have been utilized to image the same particles in the same sample with two different microscopy25. This integrated novel approach will help to understand whether the particles in question are biological or inert particle by characterizing morphology (microscopy), chemistry (EDXA), and biology (growth) of the contaminants. Particles were collected in multiple NASA cleanrooms, including the Jet Propulsion Laboratory (JPL) assembly facility cleanrooms, using various microbiology and molecular biology techniques28. This is the first time a study was systematically conducted to characterize microorganisms of NASA spacecraft cleanroom particles and its direct association with inert fallout particles.Large sets of science data were available on the indoor/outdoor airborne microbes from bulk materials that employed traditional culture-dependent and molecular approaches to identify the microbial diversity in environments specific to cleanrooms29. This study has applied the lessons learned from the prior study on an entirely new set of deployed coupons with the goal of characterizing microbial association with particles and its distribution profile in International Organization for Standardization (ISO) 5 and ISO 7 spacecraft assembly cleanroom environments. The main objective of this study is to develop a correlative epifluorescence microscopy (EFM) based technique combined with FESEM-EDXA (cEFM-FESEM-EDXA) to acquire information on the same field of image on the viability (live/dead) of microbial cell, morphology of the structure, and elemental composition of the fallout particle. Furthermore, microorganisms associated with particles were isolated and identified.Previously, we devised a witness coupon to collect fallout particles using polycarbonate (PC) filters and confirmed the presence of microbial cells using epifluorescence imaging and ImageJ analysis3 of air during the period of this study even though this facility certification is ISO-7.The JPL spacecraft assembly facility examined in this study is maintained with cleaning regimens at frequencies appropriate to the current level of activity of the cleanroom facility. At the time of the sampling events, a significant amount of assembly activity of critical spacecraft hardware occupied the clean room. Thus, cleaning frequency for facility at the time of sampling was daily. Daily cleaning regimens of cleanroom facility maintenance include the replacing of tacky mats, wiping surfaces, and vacuuming/mopping floors using clean room-certified sanitizing agents . All personnel who enter the cleanrooms must take appropriate actions to minimize the influx of particulate matter. Specific entry procedures varied depending on the certification level of the clean room and the presence or absence of mission hardware. General precautions include donning of cleanroom-certified garments to minimize exposure of skin, hair, and the regular clothing of the personnel. General precautions also include prohibition of the usage of cosmetics, fragrances, body spray, and hair gels before entry into the cleanroom. The air of both facilities was filtered through high-efficiency particle air (HEPA) filters. The total number of particles of the size >0.5\u2009\u00b5m were <10,000/ftet al.29 to secure sterile 0.2-\u00b5m pore size polycarbonate (PC) membrane filters. These witness coupons were deployed to trap fallout particles and replaced every two weeks for four consecutive sampling periods in the JPL spacecraft assembly facility cleanrooms . The entire PC filter was imaged and reconstructed by the MosaiX function in the AxioVision software . These images were further processed to determine the particle distribution with the ImageJ programThe PC filter was aseptically detached from the aluminum frame and mounted on carbon stubs for carbon coating. A 5-nm thin film of carbon was deposited on the surface of each PC filter using a vacuum coating unit . Samples were observed under high vacuum at 5\u201320\u2009kV, with a 6\u20138\u2009mm working distance using the secondary electron (SE) detector mode on the FEI Sirion scanning electron microscope . Electron microscopy images were recorded at magnifications ranging from 100x to 20,000x. To examine the particle chemical composition, EDX analysis was carried out at each field using a Phoenix EDXA tower, and the spectra were captured using Genesis software.Bacillus pumillus SAFR032 spores were purified as per established protocol31. Appropriate aliquots of the spore suspensions were trapped onto PC filters (n\u2009=\u20096), carefully placed on to the trypticase soy agar (TSA) plate, incubated for 180\u2009mins at 30\u2009\u00b0C, and every 30\u2009mins, one PC filter each  was stained with BacLight and observed under epifluorescence microscopy32.Time-lapse microscopy was validated using a known microorganism before being used to characterize fallout particles. n\u2009=\u20092) were isolated by placing the PC filters onto Reasoner\u2019s 2\u2009A (R2A) agar and incubated at 30\u2009\u00b0C for 16 hrs. One of these pre-enrichment treated PC filters was transferred into a fresh R2A agar via replica-plating33 and incubated at 30\u2009\u00b0C for 5 days, which provided enough time to form well-developed microbial colonies. The other PC filter was used for the EFM method to count viable cells after BacLight staining as described above. The microbial colonies developed on R2A plates were subsequently picked and archived in the semisolid R2A slants (agar media diluted 1:10) for further study. Once a culture was confirmed to be pure, the UltraClean DNA kit  was used to extract the DNA. Next, polymerase chain reaction (PCR) was performed to amplify the 1.5\u2009kb 16S rRNA gene to identify bacterial strains using the forward primer, 27\u2009F (5\u2032-AGA GTT TGA TCC TGG CTC AG-3\u2032) and the reverse primer, 1492\u2009R (5\u2032-GGT TAC CTT GTT ACG ACT T-3\u2032)35. The PCR conditions were as follows: denaturation at 95\u2009\u00b0C for 5\u2009mins, followed by 35 cycles consisting of denaturation at 95\u2009\u00b0C for 50\u2009sec, annealing at 55\u2009\u00b0C for 50\u2009sec, extension at 72\u2009\u00b0C for 90\u2009sec, and finalized by extension at 72\u2009\u00b0C for 10\u2009mins. The sequences were assembled using SeqMan Pro from the DNAStar Lasergene Package . The bacterial sequences were searched against EzTaxon-e database. The identification was based on the closest percentage similarity (>97%) to previously identified microbial type strains. The 16S rRNA sequences of the isolates were deposited in the NCBI GenBank under accession no.: MN006145 \u2013 MN006154.Microorganisms associated with the fallout particles trapped on PC filters  or viable/intact particles (green fluorescence). Second, images of the fallout particles were captured using FESEM to determine the associated particle size, and its chemical composition was confirmed using EDXA. The shortfall of the EFM method alone is that it does not give ample resolution to determine whether the green fluorescence comes from one or more closely packed particles. The use of FESEM eliminated this deficiency and allowed the study to determine the fraction of particles that are multiples for each particle size. The FESEM observations further informed the bin size selection as displayed in Supplementary Table\u00a036 was spiked onto the PC filters, and control PC filters were used to measure clean backgrounds  inoculumnds Fig.\u00a0. Not surnds Fig.\u00a0. Moreovends Fig.\u00a0. Since tnds Fig.\u00a0, the bioA total of 24 PC filters were exposed to an ISO 7 environment, and 8 PC filters were exposed to an ISO 5 environment. Fallout particles were collected after a two-week duration for four consecutive months in JPL cleanrooms. The FESEM microscopy analysis of the PC filters revealed various sizes and shapes of the particles, but EDX chemical analysis was required to differentiate biological materials  from inert particles Fig.\u00a0. By empl22. Geogenic particles, such as quartz (Si-O)  Fig.\u00a0, alumino-O) Fig.\u00a0, or fly -O) Fig.\u00a0 were comParticles collected from location \u201cc\u201d in ISO 7 and \u201cd\u201d in ISO 5 were analyzed in detail due to the high human traffic as well as more activities in assembling flight hardware. The elemental compositions of 25 out of 70 individual particles captured in the ISO 7 location \u201cc\u201d were displayed in Fig.\u00a0To determine whether the EFM images were biological or inert in nature, the EFM filters were directly analyzed using the FESEM/EDXA approach (cEFM-SEM-EDXA); the experimental protocol is shown in Fig.\u00a0In some red EFM images, yellow-stained particles were noticed Fig.\u00a0. HoweverBacillus pumillus SAFR032, were spotted onto the PC filter, and the spore germination was tracked using BacLight staining. The spores on top of the PC filters were revived when placed onto a TSA agar plate and germinated normally  associated with these particles were isolated from the R2A agar plate, purified, and sequenced for their 16S ribosomal gene for the phylogenetic affiliation. The sequencing results of this study confirmed the identity of the spore-forming bacteria as Bacillus zhangzhouensis and Bacillus horneckii  was examined by growing them using a time-course microscopy technique. Spores of a cleanroom isolate, lly Fig.\u00a0. The spolly Fig.\u00a0. After 9lly Fig.\u00a0. Two PC lly Fig.\u00a0 and S6A lly Fig.\u00a0 showed nlly Fig.\u00a0. This mikii Fig.\u00a037. The rBased on ImageJ analyses, the diameter size of the cleanroom particles ranged from 0.5\u2009\u03bcm to 500\u2009\u03bcm  showed a positive correlation between human presence and elevated bioaerosol counts. Since the bioaerosol presence could not be confirmed using microbiological and DNA-based methods for the presence of microorganisms, visualization using correlative microscopy methods performed during this study should be considered39. The goal of this study is to determine the association of microorganisms with the inert, natural cleanroom fallout particles and to ascertain whether microorganisms prefer to adhere to a particular particle size. A novel microscopy technique was developed, and by utilizing various molecular techniques, particles and associated microbial phylogeny were characterized. By providing an accurate and confident assessment of the microbes associated with cleanroom particles, it is possible to protect the health of the people who occupy the room for long duration in assembling critical components. The outcome from this study would help to address the distribution of microbes to particles on the spacecraft and the associated environment, as well as help other pharmaceutical and medical industries. Multiple studies have used SYTO9 and PI for the estimation of live and dead biological particles and electron microscopy to characterize morphology and elemental composition; however, no report is available where all of these techniques were used together40. Here, for the first time, we designed and implemented live/dead staining combined with correlative epifluorescence, electron microscopy (c-EFM-SEM-EDXA), and DNA sequencing technology to characterize live microbial particles isolated from the JPL-related spacecraft assembly facilities.One of the main objectives for NASA\u2019s current and future missions is to prevent forward and backward contamination23. Previous studies have shown microbial ecology and diversity in spacecraft assembly facility cleanrooms utilizing traditional and molecular microbiology methods; however, those techniques were for characterizing molecular microbial community analyses in bulk samples collected from surfaces, but none of them characterized individual particles44. The particles characterized in this study utilizing microscopy techniques ranged from 0.5\u2013500 \u03bcm in diameter and displayed morphologies that varied from carbonaceous tubular structures to regular- and irregular-shaped biological and inert particles. Additionally, EDX analyses of the large inert particles (10\u2013500 \u03bcm in diameter) showed that Al and Si groups were dominated and that the shapes of particles varied from triangular to irregular. Notably, fiber-like individual carbonaceous inert particles were dominated by tubular structures ranging from 50\u2013200 \u03bcm in diameter. Interestingly, biological particles were associated with sizes ranging from 1\u201350 \u03bcm. A spherical or tubular-shape biological particle, such as bacteria, fungal spore, and hyphae, were also observed with C and O signatures as well as minor amounts of other essential elements . Furthermore, soil-derived natural fallout SiO2 particles were also found in the cleanroom with other elements such as Al, Mg, and Na.In general, airborne microbes can be associated with inert particles of various morphology and sizes45. Recent developments on advanced correlative microscopy have created a powerful biological research tool to identify specific particles (either live or dead) that have been pre-selected using light microscopy in conjunction with SYTO9 and PI staining prior to imaging the same particles by electron microscopy. Results from correlative microscopy in this study suggested that samples have not cross-contaminated or particle removed during sample preparation, processing, transporting, and imaging. Furthermore, the characterization of such samples contributed significantly to understanding the morphological characterization of cleanroom fallout particles.The standard particle characterization method, using both light and electron microscopy methods, was useful in characterizing the particles, but incapable of distinguishing between biological and inert particlesFirmicutes were also reported to be the most prominent groups of bacteria detected in NASA cleanroom and assembly facility associated surfaces44. Furthermore, the dominant fallout Bacillus spore-forming particles found in this study are considered likely to impact spacecraft, thus posing the greatest risk of forward contamination46. It is also reported that endospore-forming bacteria as noticed in this study would be the most possible survivors of spacecraft disinfection47. The isolation of B. zhangzhouensis, which falls into the same phylogenetic clade as that of B. pumilus, an extraordinary UV-resistant spore-forming bacteria48, begs the question about the UV-resistant characteristics of the spores associated with the particles in this study. In addition, the isolation of B. hornekiae, a space vacuum surviving endospore-former49, from the particles in this study indicates that it should be characterized for various decontamination and sterilization regimes. The live biological fallout particles as observed via microscopy were cultured on the specific media needed to perform downstream DNA sequencing for the species level identity. These particle-associated biological materials were archived for further analyses, which would enable us to understand their resistance characteristics toward various cleaning and sterilization techniques.This study\u2019s isolation of bacterial endospores belonging to the members of In conclusion, correlative epifluorescence and electron microscopy coupled with energy dispersive x-ray analysis (cEFM-SEM-EDX) and ImageJ processing are useful for the evaluation of fallout particles and their corresponding size distribution. The proposed method is precise and rapid. Furthermore, the recommended method could be very useful for investigating the behavior of the particles themselves, according to their morphology and size, on the spacecraft assembly cleanroom facilities.Supplementary Dataset 1"}
+{"text": "N-Nitrosodimethylamine (NDMA) is a strong carcinogen. The compositions of the by-products are important for evaluating the treatment efficiency and understanding the UDMH degradation mechanism to achieve UDMH mineralization. The intermediate and end products of UDMH treatment with different oxidants were investigated by using a simple and fast method, solid-phase micro-extraction (SPME) in combination with gas chromatography\u2013mass spectrometry (GC-MS). The effects of several parameters  were studied to optimize analyte recovery. The best response can be attained by the 65 \u00b5m PDMS/DVB fibre at pH 7 during 10 min after desorption of 1 min in the GC inlet. The intermediate and final oxidative products of UDMH wastewater treatment with different oxidants  were investigated. The results showed that the UDMH treatment with O3 could lead to high yields of NDMA. Metal catalytic ozonation could largely minimize the formation of NDMA. No NDMA was observed in the final decontaminated samples after treatment with Fe2+/H2O2. The NDMA formation and degradation mechanism were discussed based on the intermediates. This study is expected to provide useful information for controlling NDMA formation during UDMH wastewater treatment.The majority of unsymmetrical dimethylhydrazine (UDMH) treatments produce lots of toxic by-products, among which  It is notable that NDMA is an ineluctable by-product of most methods, which is a strong carcinogen with a cancer risk level for drinking water 0.7 ng l\u22121. An acceptable destruction scheme must eliminate UDMH without forming other toxic products. The efficiency of oxidants proposed for detoxification has also been estimated mainly based on the composition of UDMH degradation products. Moreover, the UDMH degradation mechanism and NDMA formation mechanism are important for NDMA controlling. The knowledge about the compositions of the intermediates and the end products of UDMH degradation is useful for NDMA formation and reduction mechanism. So, it is necessary to investigate the compounds of UDMH with different reagents.Unsymmetrical dimethylhydrazine (UDMH) has been widely used as a high-energy propellant for military applications. UDMH is known as a primary eco-toxicant with the maximum permissible concentration in water as low as 0.02 mg l2+/H2O2) \u20135, potas2+/H2O2) , chlorin2+/H2O2) , ozone [2+/H2O2) \u201311 and F2+/H2O2) \u201314) for Numerous investigations have been carried out regarding the environmental transformation effects of UDMH in water and in soil. Various analytical methods have been carried out, such as the high-performance liquid chromatography (HPLC)-MS method \u201317, gas 3, Mn2+/O3 and Fenton reagents. The NDMA formation and destruction mechanisms have been discussed. The results of this study are expected to provide useful data for controlling NDMA formation during UDMH wastewater treatment , TMT , Dimethylamino-Acetonitrile (MSDS) , 1-methyl-1,2,4-triazole (MT) , DMF , CH3OH , CH3COOH , HCHO , DMA , NDMA , NaCl , Fe2SO4 \u00b7 7H2O  and H2O2  were commercially available. Ozone was generated by a laboratory ozonizer with an oxygen source .UDMH , FDMH , CHIn all of the experiments, deionized water was used to prepare the solutions. Measurement of the pH was done by a pH meter . The degradation samples were treated with an SPME device  and were analysed by a GC/MS .2.2.\u22121) was added to the reaction chamber. Then, the reaction chamber was flushed with ozone .UDMH ozonation procedure: 200 ml of UDMH (1 g l2+ procedure: 200 ml of UDMH (1 g l\u22121) and 0.755 g of MnSO4 were added to the reaction chamber in order. Then, the reaction chamber was flushed with ozone.UDMH ozonation with catalyst Mn2+/H2O2) procedure: 100 ml of UDMH (1 gl\u22121), 4.08 ml of 30% H2O2, 7.8 ml of FeSO4 \u00b7 7H2O (0.5 mol l\u22121) were added to the reaction chamber in that order. An appropriate amount of distilled water was added to make a volume of 200 ml, so that the concentration of dimethyl hydrazine in the flask was 1000 mg l\u22121. At 20 min, 50 ml of the reaction solution was filtered and analysed. And the reaction mixture was added with 30% H2O2 (3.06 ml) and FeSO4 \u00b7 7H2O (5.85 ml).UDMH oxidation by Fenton reagents  was added. The small beaker was placed on a magnetic stirrer. After the sodium chloride was completely dissolved, the SPME fibre was immersed in the solution with different times for adsorption equilibrium. Finally, the SPME fibre was inserted into the GC inlet by the SPME syringe.2.4.\u22121. The GC oven programme was as follows: 35\u00b0C for the first 5 min, followed by 10\u00b0C min\u22121 ramping to 15\u00b0C. The split ratio was 1 : 1.The optimal injector temperature was 200\u00b0C. The separation was performed on a DB-225S (30 m \u00d7 250 \u00b5m \u00d7 0.25 \u00b5m) capillary column. The column was maintained at a constant flow of UHP-grade helium at 1.5 ml minm/z. The ion source was held at 230\u00b0C and the electron multiplier was set at 70 eV.The MS was operated in the full scan mode from 19 to 550 2.5.The peak area was calculated by an RET integrator. The minimum integral is 0.1 of the maximum area, which is used as the integral parameter to ensure that the peak with a smaller area can be effectively integrated. Repeatability of the applied method was confirmed by the analysis of selected samples in triplicate. RSDs for replicates did not exceed 15%.3.3.1.The objective of this chapter is to screen UDMH in as many degradation oxidative products as possible and to obtain the best responses based on SPME-GC/MS. The effects of several sampling and sample parameters were studied. These involved SPME fibre coating type, effects of added salt, SPME sampling pH and time and optimization of GC injection/desorption.3.1.1.Polymeric coatings used in SPME vary in their layer materials and thicknesses, which affect SPME sensitivity towards specific chemical groups of compounds. The main coating materials, which are polydimethylsilane (PDMS), divinylbenzene (DVB), polyethylene glycol (CW), polyacrylate (PA) and activated carbon , are currently widely used. The most suitable SPME fibre coating for sampling of UDMH and its degradation products from water were selected from 65 \u00b5m PDMS/DVB, 85 \u00b5m CAR/PDMS, 85 \u00b5m PA and 100 \u00b5m PDMS tested for identifying the main UDMH degradation oxidative products. Extractions with SPME were carried out from the samples at 30 min. As shown in 3OH, UDMH, NM, TMT, CH3COOH, MSDS, NDMA, DMF, MT, HCHO) in the fresh sample. Except for HCHO, they could all be detected. The identified transformation products are listed in 3OH and CH3COOH were unstable and poor, others were selected for further studies.There were 12 kinds of UDMH and their oxidative products  the recovery decreased. It was obvious that the peak recovery occurred with the salt concentration of NaCl (30%). The salt concentration of NaCl (30%) was therefore chosen to prepare the samples.3.1.3.pH plays a significant role in compound distribution between cation and neutral molecule, which affects the extraction effect. So the effect of pH on the peak area of degradation products was studied at 5.0, 7.0 and 10.0. The results are presented in As we can see from 3.1.4.SPME is often used for equilibrium sampling, resulting in an equilibrium between the analytes in the aqueous phase and the fibre, and also including any other matrix capable of binding the analyte. The extraction time was determined based on sufficient response and reproducibility within a practical time frame. Several sampling times  were studied to determine the practical SPME sampling time. Mass detector responses versus SPME sampling time are presented in 3.1.5.Studies have shown that excessive desorption time will cause the fibre coating to degrade slowly in a high-temperature environment, affecting the service life of the extracted fibre. But, too short a desorption time cannot fully release the target component, resulting in reduced desorption efficiency and material residue.In this experiment, different desorption times  were selected for the experiment. The results are shown in 3.2.ik is the confidence factor, generally 3, is is the standard deviation of the sample measurement reading. c is a sample content value and Generally, the revised definition of the method detection limit (MDL)  is an es\u22121, 3.3 mg l\u22121, 5.0 mg l\u22121 and 7.1 mg l\u22121, respectively. The MDL of FDMH and NDMA is as low as 20 \u00b5g l\u22121. The MDL of TMT as low as 6.9 \u00b5g l\u22121.As shown in 3.3.3, Mn2+/O3, Fe2+/H2O2) to treat UDMH wastewater. The compositions of the fresh UDMH solutions and solutions after treatment with oxidative reagents were analysed using GC-MS based on SPME.Here, we selected three oxidative reagents  H atom abstraction from a C\u2212H bond of the methyl group, (ii) HO\u00b7 addition to amine nitrogen, and (iii) HO\u00b7 addition to nitrosyl nitrogen.However, there was no NDMA formation observed in the end products. This indicates that the degradation of NDMA can be a result of the HO\u00b7. We suppose that it may be initialled by H-abstraction from the \u2212CHMinakata  et al. pAccording to their quantum mechanical calculation results, H-abstraction from C\u2013H bond of the methyl functional group of NDMA is the dominant initial reaction pathway as induced by HO. So it is necessary for the treatment method to add the Fenton reagents to the intermediate products. NDMA can be totally destructed.The results are compared on identification of UDMH degradation products in wastewater with the previously published data ,12,14,334.The main intermediate and end products of UDMH after treatment were qualitatively analysed by a new sample and fast method, SPME-GC/MS. The effects of several sampling and sample preparation parameters were studied. These involved SPME fibre type, salt addition, pH, sampling time and desorption time. It was determined that the 65 \u00b5m PDMS/DVB SPME fibre coating provides the highest selectivity for detection of UDMH oxidative products and provides the best combination of convenience and sensitivity achieved at pH 7, sampling time 10 min and desorption time 1 min.3, Mn2+/O3, Fe2+/H2O2 have been investigated. The results showed that the treatment with O3 produces large amounts of NDMA. Metal catalytic ozonation (Mn2+/O3) could largely minimize the formation of NDMA. This is mainly due to the fact that the system generates oxidative free radicals , which plays a key role in H-atom abstraction from the \u2212NH2 group of UDMH but not in O-addition, finally leading to high yields of TMT. The NDMA in the end products may be formed from TMT oxidation. The intermediate products after treatment with Fe2+/H2O2 contain NDMA. NDMA can be destructed by further oxidation via HO. The ozone plays a key role in NDMA formation, while the HO\u00b7 makes a significant contribution to NDMA destruction.The intermediate and end products of UDMH after treatment with O"}
+{"text": "Viruses manipulate numerous host factors and cellular pathways to facilitate the replication of viral genomes and the production of infectious progeny. One way in which viruses interact with cells is through the utilization and exploitation of the host lipid metabolism. While it is likely that most\u2014if not all\u2014viruses require lipids or intermediates of lipid synthesis to replicate, many viruses also actively induce lipid metabolic pathways to sustain a favorable replication environment. From the formation of membranous replication compartments, to the generation of ATP or protein modifications, viruses exhibit differing requirements for host lipids. Thus, while the exploitation of lipid metabolism is a common replication strategy, diverse viruses employ a plethora of mechanisms to co-opt these critical cellular pathways. Here, we review recent literature regarding the exploitation of host lipids and lipid metabolism specifically by DNA viruses. Importantly, furthering the understanding of the viral requirements for host lipids may offer new targets for antiviral therapeutics and provide opportunities to repurpose the numerous FDA-approved compounds targeting lipid metabolic pathways as antiviral agents. Cellular metabolism encompasses numerous pathways that mediate the synthesis and degradation of energy-providing compounds and molecular building blocks. Importantly, thanks to a number of landmark studies and the seemingly universal importance of the metabolism for diverse viruses, the topic of cellular metabolism is enjoying renewed appreciation amongst virologists. In this review, we will discuss the interplay between cellular lipid metabolism and DNA virus infection, with a particular focus on the synthetic pathways.Lipid metabolism comprises multiple cellular pathways that mediate the synthesis and breakdown of compounds such as fatty acids, sterols, and other molecules. Similar to other metabolic pathways, many of the main players have been elucidated; however, the regulatory complexity of lipid metabolism is still an active area of investigation.The transcriptional regulation of lipogenesis occurs through the activation of several key transcription factors. Perhaps most notable of these are the sterol regulatory element-binding proteins (SREBPs) of which there are three proteins that are transcribed from two genes. The isoforms SREBP-1a and SREBP-1c are splice variants of a single gene, whereas SREBP2 is encoded at a distinct locus. Canonically, SREBP-1a acts as an activator of all sterol response element (SRE)-containing promoters, whereas SREBP-1c and SREBP-2 induce the expression of genes involved in fatty acid synthesis and cholesterol synthesis, respectively (reviewed in choline [3H] choline-containing lipids in newly synthesized viral particles, they did not observe any change in cellular phospholipid synthesis. More recent reports, however, have observed increased de novo phospholipid synthesis upon HSV-1 infection myristic acid than with [3H]palmitic acid, suggesting an inability of these particular viral proteins to be directly palmitoylated by pre-existing palmitic acid [With the exception of herpesviruses, poxviruses represent perhaps the best studied DNA virus family in the context of lipid metabolism. An early study of vaccinia virus (VACV) discovered that multiple viral proteins are modified by the cellular process of fatty acid acylation . Specifitic acid . This grtic acid ,92,93,94tic acid . The weltic acid ,96,97. Gtic acid .VACV, like many of the herpesviruses, also utilizes cholesterol-laden lipid rafts during entry. Cholesterol depletion of BSC40 via methyl-\u03b2-cyclodextrin treatment profoundly reduced VACV infection . OrynbayFollowing entry, VACV has been demonstrated to further manipulate cellular lipids by increasing de novo fatty acid synthesis . GresethLipids provide a backbone for the synthesis of numerous lipid-derived biosynthetic compounds and second messengers, including but not limited to prostaglandins, phosphoinositides, steroids, and diacylglycerol. While all of these compounds affect virus\u2013host interactions, these effects are pleiotropic, tissue-specific, and further complicated by the ability of lipid derivatives to modulate immune responses. Thus, the complex regulation of virus\u2013host interactions by fatty acid and cholesterol derivatives warrants an independent and comprehensive review. In contrast, in this review, we focus on 25-hydroxycholesterol (25HC), a cholesterol derivative with a broad and uniform antiviral activity. 25HC is an oxidation product of cholesterol synthesized by cholesterol 25-hydroxylase (CH25H) . The expOne of the first described functions of 25HC was the attenuation of cholesterol synthesis ,123. CurAdditional antiviral mechanisms of 25HC are likely to exist. Liu et al. reported that the pretreatment of cells with 25HC significantly reduced the entry of multiple enveloped viruses by directly modifying cell membranes to attenuate viral protein-dependent fusion processes . HoweverSimilar to select other oxysterols, 25HC is an LXR agonist ,132, alt-/- macrophages, suggesting that the ability of 25HC to antagonize SREBP2 processing was involved in the suppression of IL-1\u03b2 [-/- mice displayed significantly better survival and reduced pathology relative to wild-type control mice following a challenge with multiple influenza strains [-/- mice, suggesting that the ability of 25HC to potentiate TLR-mediated inflammatory signaling may indeed have pathological consequences in the context of infection.Moreover, several recent studies have reported an ability of 25HC to modulate inflammatory signaling pathways. Reboldi et al.  found thof IL-1\u03b2 . Accordiof IL-1\u03b2 . Interesof IL-1\u03b2 . Specifiof IL-1\u03b2 . Finally strains . These fDiverse viruses co-opt cellular metabolic pathways to facilitate virus infection. DNA viruses, most notably herpesviruses, are capable of inducing profound changes to cellular lipid synthesis. The resulting lipids have thus far been demonstrated to support viral entry, envelopment, egress, and signaling . Perhaps the most conserved use of lipids between viruses is the requirement for cholesterol-laden lipid rafts during viral entry. In some instances, specific lipids appear to be critical to viral replication. For instance, HCMV infection induces a marked increase in 26-carbon fatty acids which are incorporated into the viral membrane, and VACV encodes numerous proteins whose palmitoylation is required for efficient replication. While this review emphasized the critical roles of lipids during herpesvirus infection, it is likely that the importance of lipids in the context of other DNA virus infections will be unveiled in the coming years. Further, viruses routinely exploit host transcription factors to regulate viral gene expression. Interestingly, the ability of DNA viruses to usurp lipid-regulating transcription factors (such as SREBPs and LXRs) to directly regulate viral gene expression has not been reported and remains a curious possibility to be explored in future studies. Importantly, our understanding of the ways in which viruses hijack host lipids is currently superficial at best, and many of the mechanistic processes that underlie the viral manipulation of cellular lipid metabolism remain unknown. A better understanding of these mechanisms may offer new targets for antiviral therapeutics and provide opportunities to repurpose the numerous FDA-approved compounds targeting lipid metabolic pathways as antiviral agents."}
+{"text": "Background: Accumulating evidence suggests inhibiting neuroinflammation as a potential target in therapeutic or preventive strategies for Alzheimer's disease (AD). MAPK-activated protein kinase II (MK2), downstream kinase of p38 mitogen activated protein kinase (MAPK) p38 MAPK, was unveiled as a promising option for the treatment of AD. Increasing evidence points at MK2 as involved in neuroinflammatory responses. MMI-0100, a cell-penetrating peptide inhibitor of MK2, exhibits anti-inflammatory effects and is in current clinical trials for the treatment of pulmonary fibrosis. Therefore, it is important to understand the actions of MMI-0100 in neuroinflammation.Methods: The mouse memory function was evaluated using novel object recognition (NOR) and object location recognition (OLR) tasks. Brain hippocampus tissue samples were analyzed by quantitative PCR, Western blotting, and immunostaining. Near-infrared fluorescent and confocal microscopy experiments were used to detect the brain uptake and distribution after intranasal MMI-0100 application.Results: Central MMI-0100 was able to ameliorate the memory deficit induced by A\u03b21\u221242 or LPS in novel object and location memory tasks. MMI-0100 suppressed LPS-induced activation of astrocytes and microglia, and dramatically decreased a series of pro-inflammatory cytokines such as TNF-\u03b1, IL-6, IL-1\u03b2, COX-2, and iNOS via inhibiting phosphorylation of MK2, but not ERK, JNK, and p38 in vivo and in vitro. Importantly, one of the reasons for the failure of macromolecular protein or peptide drugs in the treatment of AD is that they cannot cross the blood\u2013brain barrier. Our data showed that intranasal administration of MMI-0100 significantly ameliorates the memory deficit induced by A\u03b21\u221242 or LPS. Near-infrared fluorescent and confocal microscopy experiment results showed that a strong fluorescent signal, coming from mouse brains, was observed at 2 h after nasal applications of Cy7.5-MMI-0100. However, brains from control mice treated with saline or Cy7.5 alone displayed no significant signal.Conclusions: MMI-0100 attenuates A\u03b21\u221242- and LPS-induced neuroinflammation and memory impairments via the MK2 signaling pathway. Meanwhile, these data suggest that the MMI-0100/MK2 system may provide a new potential target for treatment of AD. The pathological hallmarks of AD are amyloid-\u03b2 (A\u03b2) plaques and neurofibrillary tangles, accompanied by neuroinflammation characterized by the activation of microglia, and astrocytes \u20136. IncreThe p38 mitogen-activated protein kinase (MAPK) signaling pathway, a cascade contributing to neuroinflammation, could play a key role in AD pathophysiology, and preclinical and clinical trials have evaluated the pharmacological effects of p38 MAPK\u03b1 inhibitors in the brain . BachsteMMI-0100, a recently discovered cell-permeable peptide inhibitor of MK2, consists of 22 amino acids in the sequence YARAAARQARAKALARQLGVAA. YARAAARQARA is a cell-permeable peptide (CPP) named PDT-4, and it provided more specificity to inhibit MK2 . Phase 1A major challenge of protein and peptide drugs for the treatment of neurodegenerative diseases is the delivery of these drugs over the blood\u2013brain barrier (BBB). Solanezumab's failure in the Phase III trial inspired us to determine whether brain-targeted drugs can cross the BBB  . Intrana2 cages  with free access to tap water and food in a room. The temperature of the room was maintained at 22 \u00b1 2\u00b0C and was accompanied by a 12-h light\u2013dark cycle (8:00 a.m.\u22128:00 p.m.). All the protocols in our experiments were approved by the Ethics Committee of Lanzhou University (permit number: SYXK Gan 2009\u20130005).Male Kunming mice, a Swiss strain, 8\u201310 weeks old, weighing 20\u201324 g, were obtained from the Experimental Animal Center of Lanzhou University . All mice were housed in 20 \u00d7 30 cmAn 8-mm 26-gauge stainless steel guide cannula was implanted in the lateral ventricle or bilateral hippocampus according to our previous reports , 24. EacAt the end of the experiments, each mouse was sacrificed and brains were then dissected, and the position of each cannula was determined by injecting methylene blue into the cannula. Data exhibiting the diffusion of methylene blue in the ventricles were analyzed in the statistical evaluation. In addition, the bilateral hippocampus position was checked by hematoxylin\u2013eosin (H&E) staining . All mic1\u221242 peptide was purchased from Hangzhou Chinese Peptide Biochemical Company. A\u03b21\u221242 was first dissolved in hexafluoroisopropanol and frozen at \u221280\u00b0C, and it was directly lyophilized to form a powder, which is A\u03b21\u221242 oligomer. A\u03b21\u221242 was dissolved in 5% DMSO in saline, kept at \u221220\u00b0C, and was diluted in saline immediately before injected (800 pmol/mouse). Chronic injection of the A\u03b21\u221242 oligomer at 14 days prior to the start of the experiment is a widely used AD model (MMI-0100 (YARAAARQARAKALARQLGVAA) was synthesized by a standard Fmoc-based solid-phase synthetic method, and refer to our previous reports for detailed methods . PurifieAD model \u201329. LPS MMI-0100 or vehicle was infused into the brain by using a 32-gauge stainless steel syringe needle placed in and projecting 0.5 mm below the tip of the cannula for 2 min. This needle was connected via a PE tube to a 5-\u03bcl Hamilton syringe mounted to a Microdrive pump (KD Scientific). To allow the drug to diffuse, the infusion syringe remained in place for 1 min. For the lateral ventricle, injection volume is 2 \u03bcl, and for the bilateral hippocampus CA1, injection volume is 0.5 \u03bcl/side. After drug administration, each mouse was subjected to behavioral testing immediately. In addition, the selection of the doses was based on the previous reports and our preliminary observations .For nasal application, mice were anesthetized with isoflurane and infused with MMI-0100  using a 10-\u03bcl pipette. MMI-0100 was dissolved in 10% \u03b2-cyclodextrin for nasal application. Each of the applications to the left and right rhinarium, respectively, lasted about 1 min. Mice returned to their home cage and behavioral experiment was performed 2 h after intranasal application. To minimize non-specific stress responses during the application procedure, holding of the mice for 2\u20133 min was practiced for at least 3 days prior to the experiment.The behavior experiments were carried out 15 min after i.c.v. injection of MMI-0100, or 2 h after intranasal infusion of MMI-0100. The cytokines were measured 15 min after i.c.v. injection of MMI-0100.The procedures for the novel object recognition (NOR) and object location recognition (OLR) tasks were described in our previous report , 30. SchIn the training phase, two identical objects were placed on opposite sides of the home cage. The training trial was ended when the mouse had explored the two identical objects for 10 s within 5 min. Animals were eliminated if TET in the training phase did not reach 10 s within 5 min. In the test phase, a novel object, and a familiar object from the training trial were placed in the same positions as in the training phase, and the test phase lasted for 5 min. The test phase was ended when the mouse had explored the two objects for a total of 25 s, or after 5 min had passed, the test phase was terminated.In the training phase, two identical objects were placed in distinct corners of the home cage. The training trial was ended when the mouse had explored the two identical objects for 10 s within 5 min. Animals were eliminated if the TET in the training phase could not reach 10 s within 5 min. In the test phase, one of the objects was moved to a new corner, which was randomly chosen, and the test phase lasted for 5 min. The test phase was ended when the mouse had explored two locations of the objects for a total of 25 s, or after 5 min had passed, the test phase was terminated.1\u221242 injection for 14 days or LPS infusion for 3 days; 15 min after injection of MMI-0100 or vehicle, the hippocampus was removed from mice. Real-time RT-PCR was performed according to a previous report . Total proteins were determined using a bicinchoninic acid protein assay kit . IL-1\u03b2, IL-6, and TNF-\u03b1 in the hippocampus tissue were measured using ELISA kits , according to the manufacturer's instructions.Western blotting was carried out as per the manufacturer's instructions . Protein was extracted with RIPA buffer containing protease inhibitor . The protein concentration was determined using a BCA protein assay kit . Briefly, the protein samples (40 \u03bcg per lane) isolated from the hippocampus were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk for 2 h and incubated overnight at 4\u00b0C with specific first antibodies. The first antibodies in WB assay are shown in Mice were anesthetized with sodium pentobarbital and then transcardially perfused with 20 ml of PBS followed by 60 ml of 4% paraformaldehyde. The brains were removed and cut into consecutive frozen sections and then incubated overnight with CD11b antibody  and anti-glial fibrillary acidic protein (GFAP) . The secondary antibody  was applied to the sections for 40 min at 37\u00b0C. The images were performed by a fluorescent microscope .2 incubator. BV-2 cells were pretreated with LPS (1 \u03bcg) for 1 h and then incubated with MMI-0100 (10\u22125 to 10\u22128 M) for 24 h. In vitro experiments include cell viability assessment (MTT), real-time PCR, and Western blotting. The results of cell stability are shown in BV-2 cells were obtained from cellbank.org.cn. BV-2 cells were cultured in DMEM containing 10% fetal bovine serum at 37\u00b0C in a humidified 5% COTo confirm the penetration of the MMI-0100 in the brain tissue of mice, the fluorescent dye Cy7.5 was used. The specific synthesis method is shown in Two hours after intranasal administration of Cy7.5-MMI-0100 and Cy7.5 alone into the nasal cavity, mice were sacrificed, and perfused with 20 ml of PBS to remove residual blood followed by 60 ml of 4% paraformaldehyde. The frozen brain tissue was cut into 30-\u03bcm-thick sections using a cryotome and observed with an Olympus FluoView FV1000 confocal laser scanning microscope .t test was used to examine whether the DI of each group was significantly different from the chance level (50%). Differences among more than two groups were assessed by one-way ANOVA, followed by Bonferroni's post-hoc test, and p < 0.05 was considered to indicate a statistically significant difference.All data are presented as the mean \u00b1 SEM for two repeats twice of each experiment. Statistical analysis was conducted using SPSS 19.0 (IBM Corp.). A p < 0.01), but A\u03b21\u221242  or LPS  treatment significantly disrupted memory; the average DI of this group  was significantly lower than that of vehicle + vehicle  being significantly higher than the 50% chance level (1\u221242 (200 pmol) or LPS (1 \u03bcg) group  infused into the bilateral hippocampus displayed a good memory performance, compared to the A\u03b21\u221242-induced AD model mice (p < 0.01). However, these pro-inflammatory factors were significantly down-regulated after MMI-0100 application , SH-SY5Y (a human derived neuroblastoma cell line), and U251 (glioma cell lines) cells in vitro. The results showed that MMI-0100 can exhibit anti-inflammatory effects on these four different cells. The detail results are shown in Subsequently, we confirmed the anti-inflammatory response of MMI-0100 in LPS-treated BV-2 cells. As shown in p < 0.01), A\u03b21\u221242  or LPS  treatment significantly disrupted memory, and the average DI of this group  was significantly lower than that of vehicle + vehicle. Intranasal administration of MMI-0100 (25 nmol), 14 days after injection of A\u03b21\u221242 or 3 days after injection of LPS, showed a good memory performance  being significantly higher than the 50% chance level  and astrocytes (GFAP) expression, while the expression of both markers was markedly decreased in the MMI-0100-treated mice. These results suggest that MMI-0100 suppressed the activation of microglia and astrocytes induced by LPS in mouse brain. Meanwhile, qRT-PCR and Western blotting experiments were applied to detect that a series of pro-inflammatory cytokines, including IL-6, IL-1\u03b2, TNF-\u03b1, and iNOS, were dramatically increased in the hippocampus of LPS- or A\u03b2us works , 40. MMIMicroglia, the macrophages and immune defense of the brain in CNS, are activated to secrete several pro-inflammatory cytokines and neurotoxic mediators around the amyloid plaques in AD brain , 6. We t1\u221242 in NOR and OLR tasks. To demonstrate the delivery of MMI-0100 into the mouse brain, ex vivo fluorescence imaging experiments were performed. A strong NIR fluorescent signal coming from Cy7.5-MMI-0100 was observed over the whole brain at 2 h after intranasal injected peptide. Brains from control mice treated with Cy7.5 or saline alone displayed no significant signal. Intranasal MMI-0100 ameliorates the memory deficit induced by LPS or A\u03b21\u221242, and this route of administration has the potential for non-invasive therapeutic intervention for depression.Intranasal infusion allows drugs to rapidly enter the CNS via intracellular neuronal olfactory and extracellular trigeminal associated pathways, bypassing the BBB to effectively interact with their receptors in multiple brain regions , 20, 41.1\u221242 and provides neuroprotection against LPS- or A\u03b21\u221242-induced neuroinflammation in vitro and in vivo, and the mechanisms may be related with down-regulation of MK2 pathways. Meanwhile, intranasal administration of MMI-0100 significantly ameliorates the memory deficit induced by A\u03b21\u221242 or LPS in object and location memory tasks. Near-infrared fluorescent and confocal microscopy experiments were applied to detect the brain uptake and distribution of intranasal MMI-0100. Together, the previous reports and present results suggest that MK2 protein target may represent a novel treatment strategy for neuroinflammation disease. Moreover, intranasal infusion of MMI-0100 bypassed the BBB to rapidly enter the brain, which is a novel promising treatment for the CNS disease.Overall, the present study firstly demonstrated that central injection of MMI-0100 relieves memory impairment induced by LPS or A\u03b2The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.All of the procedures in this experiment were approved by the Ethics Committee of Lanzhou University, China (Permit No: SYXK Gan 2009\u20130005).JJ, ZW, XL, and XC conducted the experiments, performed the analysis, and wrote the manuscript. YN analyzed the data and imaging. YN and XC facilitated the equipment and software to perform the behavior experiments. SL and HX provided peptide drug MMI-0100. CM designed the experiments and contributed to writing and editing the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors.5 transformants colonies. Out of these ~\u20093000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype.We constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~\u200910So far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis. Saccharomyces cerevisiae and its close relative possess ~\u2009150\u2009bp centromere and can bind to a single microtubule. The homologous chromosome separation mainly depends on the mono orientation of the sister kinetochore because of which the paired homolog separates to the opposite pole. On the other side in the MII the sister kinetochore are biorient because of which the sister chromatids separates to the opposite pole. In these organisms, sister kinetochore co-orientation in MI depends mainly on two processes, first on a specialized four protein complex known as monopolin and second the CCP by Spo13, shugoshin (SGO) and protein phosphatase 2A (PP2A) [Transfer of the genetic information from one generation to the other through cell division is critical for survival . In sexuA (PP2A) \u20137.S. pombe the Pcs1 form a complex with Mde4 and both localizes to the core of the centromere like monopolin complex. These two proteins don\u2019t participate in the monoorientation of sister kinetochore but play an important role in the amphitelic kinetochore orientation during MII and in mitotic division [The monopolin complex is composed of the four proteins Mam1, Csm1, Lrs4 and Hrr25. Mam1 is a meiosis specific protein, which express from the pachytene to metaphase I . The Csmdivision .Saccharomyces cerevisiae strains with individual genes deleted and having GFP dots on both the copy of Chromosome III. They observed the non-disjunction and premature separation of the chromosomes in some strains. A similar observation was done in fission yeast where Shugosin was recruited to the centromere by Bub1 [The centromeric cohesin during MI is mainly replaced by Rec8. At the time of anaphase I the Rec8 is cleaved by seperase along the chromosome arm region, but protected in the centromeric region , 16. The by Bub1  and same by Bub1 .Saccharomyces cerevisiae and Moa1 in S. pombe is the only meiosis specific kinetochore protein identified. The meiosis-specific protein Spo13 is also necessary for kinetochore co-orientation. In its absence, the monopolin complex initially associates with kinetochore but cannot be maintained there [Spo13 has also been reported to play role in centromeric cohesion protection , 20. Desed there , 7, 21. ed there , 23. SKMed there . Moreoveed there . Thus, Sed there .To find out the additional proteins responsible for the SKM and CCP, we started with the construction of two strains. In first strain we expressed Mam1 and Cdc5 to attain the forceful kinetochore co-orientation in mitosis. Cdc5 expression helps the Lrs4 and Csm1 to localize in the nucleus so that it can form the monopolin complex with Mam1 even in mitotic cell division. In the second strain we are expressing the Rec8 and Spo13 to increase the centromeric cohesion protection. So in these strains we are forcefully generating the meiosis I condition. In case of mitotic division the cells were found to be sick, utilizing this phenotype, we can screen the specific cDNA library to find out the novel factors. This report shows the simple methods to screen the novel factors from the cDNA library for a specific pathway or process.MAM1 and CDC5 genes from S. cerevisiae was cloned into pESC-URA to form pESC-CDC5-MAM1-URA construct, similarly the pESC-REC8-SPO13-URA construct was made as described in the methodology  and the transformants were grown in SC-URA broth supplemented with 2% raffinose till 0.8 OD600. These samples were induced with the 2% of galactose till 4\u2009h and the samples were prepared for the DAPI staining and the cells were observed under fluorescence microscopy. We observed two types of cells, first was properly segregated having GFP dot with proper DAPI segregated  on chromosomal segregation we first integrated a tandem array of tetO sequences near the centromere of chromosome V SGY229). These cells also expressed a tetR-GFP fusion, which binds to tetO, to visualize the repeats. We transformed this strain with the 29. TheseTo check the effect of the expression of Rec8 and Spo13 on growth of the cells, the construct were integrated in CRY1 and spotting was done on galactose as well as on dextrose plate (as described in method). We observed that the growth on the dextrose plate  which had compromised APC activity.Similarly, we checked the effect of Mam1 and Cdc5 expression on the growth of the strains Fig. b. Straincdc16\u2013123, cdc16\u20131, cdc20\u20131). We first cloned CDC5 and CDC5-MAM1 along with the gal promoter in the pRS405-LEU vector. We integrated pRS405, Cdc5 (using pRS405-CDC5-Leu) and Cdc5-Mam1 (using pRS405-CDC5-MAM1-Leu) independently along with the gal promoter in cdc16\u2013123, cdc16\u20131, cdc20\u20131 to make SGY4129, SGY4130, SGY4131, SGY4132, SGY4133, SGY4134, SGY4135, SGY4136 and SGY4137 strains respectively. The transformants were checked for the expression of Cdc5 and Mam1 in these strains (data not shown). Then we did the frogging on galactose as well as on dextrose plates at 23\u2009\u00b0C  were very slow at 23\u2009\u00b0C  and cdc16\u2013123 transformed with pRS405-leu, pRS405-CDC5-Leu, pRS405-CDC5-MAM1-Leu  grew normally at 23\u2009\u00b0C, 25\u2009\u00b0C, 30\u2009\u00b0C  was slow growing as compared to SGY4132 (cdc16\u20131 transformed with pRS405-Leu), but SGY4134 strain (cdc16\u20131 transformed with pRS405-CDC5-MAM1-Leu) was unable to grow  of SGY4132, SGY4133 and SGY4134 strains respectively and were transformed with pRS316-gal empty vector. In every transformation equal amount of chemical was used and lastly equal volume of cells was spread from SGY4132, SGY4133 and SGY4134 strains on SC-leu-ura plate supplemented with galactose and found no colony in strain SGY4134  and SGY4119 was transformed with empty vector pRS316-GAL (used to prepare library). After transformation both the samples were spread on SC-leu-ura plate supplemented with either galactose or dextrose at three different temperatures 25\u2009\u00b0C, 30\u2009\u00b0C, 37\u2009\u00b0C simultaneously. Plates were observed on the regular interval. We observed that the appearance of the colonies on galactose plate was delayed as compared to the dextrose plate. The transformants of the dextrose plate grew normally while transformants on galactose plate appeared after 45\u2009h of incubation which get prominent after 65\u2009h at 30\u2009\u00b0C . The equal volume of the transformed cells was spread in each experiment so that the number of cells remains constant on each plate. The transformants were selected on SC-leu-ura medium supplemented with galactose or dextrose and colonies were counted. We found that the survivability on the galactose plate of SGY4119 was very less as compared to controls Fig. c. In casSalI and ClaI and send for the sequencing to identify the gene cloned in the vector. This is the simple method to identify novel factors in any organism using cDNA library. Similarly, we can use the SGY4134 to identify the additional factors working in sister kinetochore mono orientation.Further, we analyzed that there were two types of colonies, in the first type of colony, the phenotype was flat and color was little whitish and look like a sick colony. We checked the cell under microscope and found that the cells were elongated as we mentioned earlier  has been identified with similar function . But, thcdc16\u20131, cdc20\u20131 and cdc20\u2013123 were a kind gift from the Prof. M. Jayaram, University of Taxus. cdc16\u20131, cdc20\u20131 and cdc20\u2013123 strains were modified by integration.For gene integration, promoter shuffling, C-terminal and N-terminal tagging of proteins, PCR-based approach was adopted using appropriate plasmid borne cassettes obtained from Euroscarf. C-terminal and N-terminal protein fusions were verified both by PCR and Western blotting  were transformed with these construct and expression of the cloned proteins was checked after galactose induction, we first grow the cells in SC broth lacking uracil for overnight in 5\u2009ml tube . Secondary inoculation was done in 50\u2009ml flask and initial OD600 was set 0.2 and grown in same condition. The cells were grown till 0.8 OD600, induction was done with 2% galactose in same condition. Protein extraction was done by NaOH method [H method . ProteinTo analyze the defect in chromosomal segregation in cells, first cells were grown in SC medium supplement with 2% raffinose and induction was done for 4\u2009h using 2% galactose as described earlier for protein extraction. CEN-V GFP dots were analyzed in cells that were fixed in 5% formaldehyde for 10\u2009min, washed twice and stored in 0.1\u2009M phosphate buffer pH\u20097.4. Before the microscopic analysis samples were fixed with 50% EtOH then wash with 0.1\u2009M phosphate buffer and suspended in 1\u2009\u03bcg/ml DAPI solution. Images were acquired using z-stack (at every 0.25\u2009\u03bcm) multichannel image acquisition tool of Zeiss Axiovision software and Zeiss Axio Observer.Z1 microscope. Approximately 100 cells were counted per samples for statistical analysis.600 was maintained at ~\u20090.2 and then grows till ~\u20091.0 OD600. Equal cell from each sample were taken and different dilution was made from 1 to 10\u2212\u20095 and spotting was done on the synthetic complete medium or dropout medium agar plates having galactose or dextrose as a carbon source separately.To analyze the growth defect, the strains were first grown in synthetic complete medium devoid of uracil (raffinose was used as a carbon source) for overnight at 30\u2009\u00b0C and 200\u2009rpm shaking condition. Then secondary inoculation was done and the initial ODAdditional file 1:Figure S1. Characterization of pRS316-GAL-cDNA library (A) Image of some of the clones of pRS316-GAL-cDNA library digested with SalI and NotI. Clone efficiency and average insert size were calculated. Out of these two values the number of colonies required for the screening was also calculated. (B) Transformation efficiency of the library by transforming yeast strain (CRY1) with 850\u2009ng of cDNA library and transformants ware diluted for 1000 times and spread on the SC-ura plate."}
+{"text": "Our calculations showed that the band-gap tunability of the monolayer Sb was much more sensitive to hydrostatic pressure than that of the monolayer BP and MoS2. Furthermore, the monolayer Sb was predicted to change from an indirect band-gap semiconductor to a conductor and to transform into a double-layer nanostructure above a critical pressure value ranging from 3 to 5 GPa. This finding opens an opportunity for nanoelectronic, flexible electronics and optoelectronic devices as well as sensors with the capabilities of deep band-gap tunability and semiconductor-to-metal transition by applying mechanical pressure.In this paper, the band-gap tunability of three monolayer semiconductors under hydrostatic pressure was intensively investigated based on first-principle simulations with a focus on monolayer antimony (Sb) as a semiconductor nanomaterial. As the benchmark study, monolayer black phosphorus (BP) and monolayer molybdenum disulfide (MoS Recently, two-dimensional 2D) nanomaterials have exceptional properties for optoelectronic nanodevices ,2,3,4,5.D nanomat2) has been the most extensively studied in 2D forms because of its stability at room temperature in air [Besides graphene, atomically thin 2D forms of layered transition metal dichalcogenides (TMDs) have recently attracted remarkable scientific and technical interest ,12,13,14e in air , alternae in air ,16, and e in air ,18. In ce in air ,20,21,22e in air . It is ae in air . It shoue in air .2 increases to a direct band-gap of 1.8 eV in single-layer form. Moreover, monolayer BP shows great mobility and in-plane anisotropy, which makes BP a potential material in the optoelectronic and energy device field. In contrast, antimony (Sb) in bulk is a typical metalloid element with metallic luster. Therefore, single-layer semiconductors as novel nanomaterials deserve to be deeply investigated, especially for novel electronic and optoelectronic device applications.There are many interesting layer-dependent properties in 2D nanomaterials ,18,19, wRecently, the band-gap engineering of 2D semiconductors has attracted more research interest by means of chemical functionalization ,27,28, i2, BP, and Sb, based on first-principle calculations, focusing on the band-gap tunability of single-layer semiconductors under hydrostatic pressure. In this paper, the band-gap changes of the monolayer semiconductors of Sb, BP, and MoS2 under hydrostatic pressure were systematically investigated for exploring the band-gap tunability. We found that the band-gap value of the monolayer Sb could be well controlled by applying hydrostatic pressure. Its pressure sensitivity and band-gap tunability were more effective than that of the monolayer MoS2 and BP. Moreover, the electrical structures of the monolayer Sb were also studied under different hydrostatic pressures. The breakage of bonds and the phase transition were predicted to occur above a critical hydrostatic pressure ranging from 3 to 5 GPa. This finding opens a new opportunity for device applications of monolayer Sb to nanoelectronics, MEMS, and sensors.This paper investigated the band-gaps of three single-layer semiconductors, i.e., MoS2 and BP with buckled honeycombs under ambient conditions [2, BP, and Sb are shown in 2 were provided by the Cambridge Crystallographic Data Center (CCDC). The lattice parameters of the Sb monolayer were calculated, and a = b = 4.141 \u00c5 and d = 3.0 \u00c5 were obtained after relaxation. When performing first-principle simulations for monolayer materials, a vacuum layer in the z-direction between two monolayers is commonly used for isolating self-interaction and interlayer effects. A vacuum layer thickness ranging from 15 to 20 \u00c5 is usually adopted in the literature [The atomic structure of monolayer Sb is similar to that of monolayer MoSnditions ,35. The terature ,36.In this benchmark study, all first-principle calculations were carried out by both the Vienna Abinitio Simulation Package   and the Furthermore, in the VASP simulation, the ultrasoft pseudopotential (USPP) is applied and the generalized gradient approximation (GGA) with the parameterized Perdew\u2013Burke\u2013Ernzerh (PBE) functional is adopted as the exchange correlation function. For the USPP, to reduce the plane wave basis set and cut-off radius, the closely interacting electrons in the atomic core region are removed by generalized orthogonal conditions. Because only the close interaction electron orbitals in the atomic core region need high cut-off energy, with less sacrifice of calculation accuracy, the calculation efficiency is greatly improved when the USPP is used, and it is easier to find geometric stable states under pressure conditions. Kohn\u2013Sham schemes based on GGA functionals can describe the total energy and related properties of crystals and molecules accurately but underestimate the band-gap. In CASTEP, GGA with Heyd\u2013Scuseria\u2013Ernzerh screened hybrid functional (HSE06) is applied as the exchange correlation function, which includes an empirical scissors correction based on a screened Coulomb potential . The empAs explained in above section, there are large differences of the PBE and HSE06 calculated band-gaps reported in the literature ,40. TherMoreover, in the simulation model, the plane wave energy cut-off was set to 550 eV and the K-space was divided by a 15 \u00d7 15 \u00d7 1 mesh along the directions of a, b, and c, respectively. The conjugate gradient algorithm was used for ion iteration and ion coordinates during the process of geometry optimization. The cell volume and cell size were not constrained.Band-gap is a fundamental physical parameter for semiconductors, which is not only related to bonding properties and crystal structures, but also to photon and electron transport properties.2 under hydrostatic pressure loadings.A band-gap benchmark study of the monolayer Sb was performed using different Hamiltonians. The calculated results are listed in 2 with a comparison of the experimental (EXP) data available from literature [2, respectively. The deviation around 5% is reasonably close to the experimental results. At this time, the experimental data for the monolayer Sb are not available (NA). However, its band-gap can be predicted around the value of 1.775 eV based on our benchmark calculations. In contrast, the relative deviation of VASP-GGA-HSE06 (V-H) with the experimental data from [2, respectively, which indicates the V-H can be a reasonable model for band-gap calculations.VASP: Vienna Abinitio Simulation Package; CASTEP: Cambridge Sequential Total Energy Package; LDA: local density approximation; GGA: generalized gradient approximation; PBE: Perdew-Burke-Ernzerh functional; HSE06: Heyd-Scuseria-Ernzerh screened hybrid functionalFurthermore, the first-principle simulation results are listed in terature ,41. As sterature ,41 was cata from ,41 was c2 were significantly changed by applying different values of hydrostatic pressure, based on our first-principle calculations. 2, BP, and Sb at 0, 1, 2, 3, and 4 GPa, respectively. Both MoS2 and BP show direct band-gaps, located at the high symmetry points K and \u0393, respectively, at 0 GPa pressure. However, the monolayer Sb show indirect band-gaps. The global conduction band minimum (CBM) is located at points between the high symmetry points \u0393 and X, while the global valence band maximum (VBM) is located at the high symmetry point \u0393. When the applied pressure was further increased to 2 GPa, the locations of the CBM of MoS2 and the VBM of BP changed. It is also shown that the band-gap eventually decreased to 0 eV at 4 GPa hydrostatic pressure for BP and Sb, but not MoS2, which requires much higher pressure for tuning the band-gap.The band-gaps of the monolayer semiconductors of Sb, BP, and MoS2 under different hydrostatic pressures. The values of band-gaps calculated by V-P and C-H under different pressures ranging from 0 to 8 GPa are consistently shown in Further investigation was performed by applying different pressures from 0 to 8 GPa for 12 case studies based on VASP-GGA-PBE (V-P), VASP-GGA-HSE06 (V-H), and CASTEP-GGA-HSE06 (C-H). The extensive calculation results are summarized in 2 did not change monotonically with applied pressures. When the applied pressure was increased, the direct band-gap initially increased, up to the maximum value. The responding pressure values, however, were dependent on the computational functionals, as shown in 2 initially increased from 1.83 eV to a maximum value around 2 eV at 2 GPa pressure. It then decreased with the increase in applied pressures above 2 GPa. Moreover, the critical hydrostatic pressure around 2 GPa of the MoS2 band-gap for the direct\u2013indirect transition of the band structure is very much consistent with the literature, reported as 1.9 GHz from the experimental data [2 were obtained at each pressure from 2 to 8 GPa based on V-P, V-H, and C-H, as depicted in However, the band-gap value of the monolayer MoStal data . ConsistThe band-gap values of the monolayer semiconductors of Sb and BP consistently decreased linearly with the increase in applied hydrostatic pressure. The band-gap values of BP decreased with the increase in hydrostatic pressures from 0 to 8 GPa, as shown in 2. Specifically, the band-gap of Sb can be tuned to zero at low pressure around 3 GPa, estimated from the average value, which is experimentally achievable for the nanostructure of the monolayer Sb.Therefore, the monolayer Sb can be deeply tuned by applying much lower pressure, as compared to the monolayer BP and MoS2 was reported by applying high pressure up to 40 GPa so that the 0.4 eV band-gap was reached from the first-principle calculations [2 is a compound semiconductor with much stiffer mechanical properties, as shown in The bulk MoSulations . This ca2, BP, and Sb for investigating the dynamical stability of the nanostructures. It is clearly shown that before critical pressure, the in-plane lattice parameters of a and b, as well as the out-of-plan lattice parameter d, were all stable with small displacements. However, after a critical pressure was reached, the in-plane lattice parameters of a changed relatively significantly and the out-of-plan lattice parameter d remained stable for the monolayer BP. Instead, after critical pressure of approximately 3 GPa, the out-of-plan lattice parameter d and the in-plane lattice parameters of a and b significantly changed for the monolayer Sb. Combined with analysis from In contrast, we investigated the lattice displacements of the monolayer MoSFurther investigation of the charge densities showed that the monolayer Sb can be completely transformed from a semiconductor to a metal. The 3D diagrams of the charge density distribution of the monolayer Sb under different pressures were prepared using VESTA, as shown in 2 were intensively investigated by applying different pressures from 0 to 8 GPa for case studies, based on the first-principle calculations of VASP-GGA-PBE, VASP-GGA-HSE06, and CASTEP-GGA-HSE06 functionals in this paper. This benchmark study indicates that the monolayer Sb exhibits semiconductor properties with an indirect band-gap and an estimated value of 1.77 eV. Moreover, the band-gap values of the monolayer semiconductors of Sb and BP decreased linearly with the increase in the applied hydrostatic pressures. However, the band-gap values of the monolayer MoS2 did not change monotonically with the applied pressures. A critical hydrostatic pressure around 2 GPa for the direct\u2013indirect transition of the band structure of the monolayer MoS2 is very much consistent with the literature, reported as 1.9 GHz from the experiment data [In summary, the band-gap characteristics of the two-dimensional monolayer semiconductors of Sb, BP, and MoSent data .2. Specifically, the band-gap of Sb can be tuned to zero at a low pressure around 4 GPa, as estimated from our calculations, which is experimentally achievable for the nanostructure of the monolayer Sb. The band-gap tunability of the monolayer semiconductors of Sb, BP, and MoS2 under hydrostatic pressure opens new opportunities for potential applications to nanoelectronic, flexible electronics and optoelectronic devices, as well as sensors.Further investigation of the charge densities showed that the monolayer Sb can be completely transformed from a semiconductor to a metal at a critical pressure ranging from 3 to 5 GPa or 4 GPa, as estimated from our calculations. Therefore, the monolayer Sb can be deeply tuned by applying a much lower pressure, compared to BP and MoS"}
+{"text": "Spontaneous cystic artery-gallbladder fistula is an extremely rare condition described in the medical literature. We have found two articles in the literature describing fistula formation between the cystic artery and gall bladder. In this report, we present a unique case of a cystic artery-gall bladder fistula that resulted in massive gastrointestinal bleeding through cystic artery erosion and was adequately managed with coil embolization. Haemobilia is a rare cause of upper gastrointestinal bleeding usually secondary to an iatrogenic or trauma-induced abnormal cystic artery and biliary tract connection. Spontaneous cystic artery-gallbladder fistula has rarely been discussed in the medical literature . In thisA 62-year-old\u00a0female with a pertinent past medical history of HIV on antiretroviral therapy, dyslipidemia and glaucoma initially presented to the emergency department for evaluation of abdominal pain and bright red blood per rectum.\u00a0The abdominal pain had started two days\u00a0before the presentation; it was described to be generalized and associated with bloating. She also endorsed having\u00a0three bowel movements with bright red blood per rectum at home. She\u00a0had undergone outpatient colonoscopy screening the previous year, which had been reported to be normal. She denied any history of adenoma,\u00a0polyps, and personal or family history of gastrointestinal-related cancers. She only had a pertinent surgical history of appendectomy. Her drug history was significant for the daily consumption of non-steroidal anti-inflammatory drugs once\u00a0in the past two years for chronic right hip pain.Initially, in the emergency department, the patient remained hemodynamically stable. The liver function tests showed mildly elevated mixed pattern with alkaline phosphatase of 313 IU/L , aspartate aminotransferase of 92 Units/L , and alanine aminotransferase of 65 Units/L . Given her history of abdominal pain, a CT scan was obtained, which demonstrated porcelain gall bladder with suspicion of a fistulous tract between her gallbladder, colon, and duodenum , and proton pump inhibitors. She was evaluated by general surgery and gastroenterology and was planned for esophagogastroduodenoscopy (EGD) and colonoscopy the next day. Ultrasound of the abdomen was additionally obtained to visualize the hepatic structures, which showed mild hepatomegaly with suspected stable 2.5-cm gall stone and negative Murphy\u2019s sign, ruling out cholecystitis. The common bile duct was noted to be normal with 4 millimeters in size . In the second portion of the duodenum, a bright red blood clot was noted, which was not dislodged upon washing. As bleeding was noted to be dripping from the ampulla, there was high suspicion for\u00a0haemobilia likely due to hepatic artery fistula into the gall bladder. Intervention radiology was immediately consulted and, upon reviewing the CT scan again, there was high suspicion\u00a0for cystic artery pseudoaneurysm. However, when the patient emergently underwent a hepatic angiogram, cystic artery fistula with the gallbladder was confirmed. Subsequently, cystic artery coil embolization was performed to control the bleeding vessel. The patient was transferred back to the intensive care unit for further monitoring.After reviewing the imaging studies and extensive discussion with surgical, radiology, and gastrointestinal teams, it was concluded that the diagnosis of the gallbladder-duodenal fistula was misleading due to the thickened gallbladder wall. In order to further improvise on the anatomical structural findings, MRI of the abdomen with and without contrast was obtained, confirming no evidence of any fistulous tract between gall bladder and duodenum Figure . Gall blCurrently, she is recovering at home and will continue to follow up with us for possible elective cholecystectomy for finding the porcelain gall bladder, which may represent the underlying malignancy.Spontaneous fistula formation between the cystic artery and gall bladder has rarely been reported in the medical literature. As seen in our patient, it may result in a rare cause of haemobilia -3. HaemoIdentifying vascular-biliary fistula can pose a diagnostic challenge. In our clinical case, the initial CT scan of the abdomen falsely suggested possible fistula formation between gall bladder, colon, and the duodenum. However, on EGD, active bleeding from the ampulla of Vater was clearly noted in the duodenum. No abnormal tracts suggestive of biliary-enteric fistula were visualized during the endoscopic procedure. A prompt angiogram was performed by the intervention radiology, which effectively identified the bleeding vessel to be a cystic artery and the patient underwent coil embolization. Although aneurysm and pseudoaneurysm of the cystic artery are considered important differentials leading to such vascular-biliary fistula, no such abnormality was noted in the angiogram. Therefore, the cystic artery was coiled, which successfully controlled the bleeding ,10.The exact mechanism of the spontaneous cystic artery and biliary tract fistula is not properly defined. However, it can be suggested that the inflammatory process following the stone formation in the biliary tract and mechanical pressure of the stone may have resulted in adhesion formation with the anatomical structures in proximity, promoting fistula formation .As the suspicion for cystic artery erosion resulting in active bleeding in the gastrointestinal tract through the cystic artery-gallbladder fistula was considered very high, cystic artery coil embolization was performed. The bleeding was successfully stopped, resulting in the stabilization of the patient. The MRI studies performed later confirmed the suspicion for cystic artery-gall bladder fistula by ruling out any suspicious abnormality in the biliary tract. As limited information is available in medical literature for the treatment of spontaneous cystic artery-gallbladder fistula, we believe our case with coil embolization will provide a unique example in the management of such a clinical entity . Given tA spontaneous cystic artery-gall bladder fistula is a unique clinical condition and hence its diagnosis is challenging. It may cause massive gastrointestinal bleeding as observed in our clinical scenario. It should be considered as a differential for haemobilia. From our clinical scenario, it can be suggested that minimally invasive treatment with coil embolization can constitute the best strategy for management, thereby securing the hemodynamic stability of the patient. It may also be used as a bridge to surgical resection of the gall bladder if deemed necessary. Further review of medical literature is warranted to\u00a0understand the abnormal fistulous formation between\u00a0vasculature and gall bladder."}
+{"text": "Clinical evaluation of Lyme Borreliosis (LB) is the starting point for its diagnosis. The patient's medical history and clinical symptoms are fundamental for disease recognition. The heterogeneity in clinical manifestations of LB can be related to different causes, including the different strains of Borrelia, possible co-infection with other tick transmitted pathogens, and its interactions with the human host. This review aims at describing the heterogeneous symptoms of Lyme Borreliosis, as well as offering a practical approach for recognition of the disease, both in terms of clinical features and diagnostic/research tools. The genus Borrelia includes three Groups: Lyme Borreliosis (LB), Reptil Associated (REP), and Relapsing Fever (RF) Group .Borrelia burgdorferi sensu lato complex. The main tick vector for Borrelia species in Europe is the Ixodes ricinus (Ixodes scapularis and Ixodes pacificus (Ixodes persulcatus. These ticks are possible vectors of Lyme Borreliosis (LB) as well as other pathogens, including viruses, intracellular bacteria, and Protozoa which can co-infect humans (LB co-infections)  is an anthropozoonosis, caused by different genospecies of the  ricinus , in Ameracificus \u20135, whileacificus  and Russacificus  it is thections) , 9. Thered to LB , 10. In ssettiae , 5, B. lsitaniae , 7, B. sielmanii , and B. laisiana . Specifilaisiana , 13. Thepical LB ] and fivariensis . The hetBorrelia mayonii which has been reported to cause high spirochetemia , tinea corporis (mini EM), and erythema necroticans migrans.Serological testing is not recommended because of their poor sensitivity in the early stages of LB. In order to achieve the best outcome for patients, antibiotic treatment should be started without delay.Secondary EM is characterized by multiple erythematous lesions, which do not develop round the site of the tick bite. It can consist of a few or several plaques that can be located throughout the body . The lesBorrelia afzelii and more rarely by B. garinii and B. bissettii , \u03b22-Microglobulin, and serological tests for Ehrlichia (Anaplasma)  . HistoloDifferential diagnosis includes cutaneous marginal zone lymphoma PCMZL, , which cPCR for Borrelia on tissue's DNA  can target OspA as reported by Cerroni , but alsACA is the pathognomonic symptom of late LB. Patients, at presentation, should be asked whether they remember being bitten by a tick several months or even years before and whether they ever had an EM. Since the clinical appearance of ACA is not distinctive, it is of key importance to be generally alerted of the possibility of ACA in patients with bluish-red discoloration of a limb with or without swelling and/or atrophy, especially where LB is endemic , 37.Unilateral acrocyanosis is present in the initial phases. This feature is followed by atrophy of the upper and/or lower limbs in an asymmetric manner, which, due to thinning and consequent greater transparency of the skin, allows the vessels of the dermis to be more visible. This condition leads over time to thinning of the most involved limb . ACA Fi is usualIn addition to ACA, in some cases, other atrophic-sclerodermic manifestations may be related to LB , 40.Serology by chemiluminescence is usually very high in VlsE IgG; in Western-Blot, p93 (p83/100) and DbpA are generally observed.Borrelia afzelii .Skin biopsy for histological examination and PCR for Borrelia are also possible for research purposes. Isolation of Borrelia in BSK medium from skin lesion can result in the growth of Other possible skin manifestations that have been associated with LB are: urticaria , purpuraImportant information to be obtained from patients includes: the geographical area where the patient lives (if endemic or not for LB), if, in the previous weeks or months the patient has been in wooded areas, if he/she has traveled or has been camping, or has spent time in public parks and gardens or if he/she owns any pets. Requested information should also include the date of the onset of symptoms, recollection of a tick bite and/or of a circular erythema as well as the location and the duration of the skin lesion. In the case of a positive, response, the patient should be asked if he/she was previously treated with antibiotics, what type of antibiotics, and what the duration of treatment was. Other clinical manifestations can be fever, lymphadenopathy, balance disorders, dizziness, and photophobia .Arthritis occurs after 4 days to 2 years  from EM \u201349. In aIn the early phase, the patient presents mono- or oligoarticular migrant arthralgia at the level of the large joints. The first affected joint is often near the site of the EM or the tick bite. However, sometimes other large or small joints, such as the temporomandibular joint (TMA), are also affected . Over tiThe articular involvement in the late phase has different clinical features compared to the typical migrant myo-arthralgia of early LB. The clinical symptomatology is not easy to distinguish from arthritis due to other causes. The disorder can become chronic or intermittent, with attacks lasting from a couple of weeks to a few months, which can be followed by resolution of symptoms. The intensity of the attacks decreases over time. Hyperpyrexia is not usually present, but a general sense of fatigue is common.Swelling of the joints with marked functional impotence is often present. Affected knees, for instance, may have very large effusions  . If thosMuscular system involvement includes myalgia, muscle weakness, and myositis  with difTo confirm diagnosis, it is useful to perform a serological ELISA test followed by a Western Blot. In case the patient reports having headaches and/or a fever, tests for TBE, Ehrlichia (Anaplasma), Rickettsia, and Bartonella coinfections are suggested. A rheumatologic examination can be also requested.B. burgdorferi s.l. are present in high titers in patients with Lyme arthritis, while a negative IgG serology rules out the diagnosis . Facial paralysis is bilateral in 25% , 60. ParAmong children in Europe, the most common manifestations are facial nerve palsy (about 55%) and lymphocytic meningitis (about 30%) .Meningopolyneuritis (Garin-Bujadoux-Bannwarth) with radicular pain and sometimes paresis of extremities or the abdominal wall , 63, neuPseudo tumor cerebri associated with LB was first described in 1985 . SubsequInfection of the central nervous system is observed in 2\u20134% of Lyme neuroborreliosis, typically in the late or chronic stage of the disease . EncephaNeuroborreliosis can be associated with speech disorders, recent cognitive, and affective disorders , psychiaBorrelia miyamotoi, which can be transmitted by the same tick as LB, should be considered  , and posIn most patients, examination of the cerebrospinal fluid (CSF) reveals lymphocytic pleocytosis, damage to the blood-CSF-barrier, and an intrathecal synthesis of immunoglobulin IgM, IgG, and sometimes IgA ; the proDuring paralysis of the facial nerve, the CSF often presents lymphocytic pleocytosis even in the absence of signs and symptoms of meningitis .After the onset of neurological symptoms, for a short time, intrathecal synthesis may not be detectable and CSF pleocytosis may be absent especially in children with isolated paralysis of the seventh cranial nerve . The proThe use of chemokine (C\u2013X\u2013C motif) ligand 13 (CXCL13), a B-cell attracting chemokine, was debated for the laboratory diagnosis of acute Lyme neuroborreliosis in CSF . CXCL13 Borrelia garinii is mainly related to typical early Lyme Neuroborreliosis  while Borrelia valaisiana causes neurologic Lyme manifestations less frequently  .In addition to dyspnea, chest pain, or irregular heartbeat, typical symptoms include syncope episodes . On physIf heart involvement in LB is suspected, a cardiological examination is suggested. The following investigations should be addressed: 12-channel ECG and 24-h ECG Holter , chest X-ray ; echocardiography ; cardiac MRI, and in selected cases myocardial biopsy for histological examination and cultural isolation of Borrelia . ElectroOcular manifestations can be linked to a direct involvement of the eye or can be secondary to Neuroborreliosis. Ocular involvement, is possible at every stage of LB and they can be summarized as follows:Follicular conjunctivitis often self-limited, and,Photophobia.They can appear in the first stages of LB.In the early disseminated phase, these manifestations are possible:Macular edema,Uveitis and Iridocyclitis,Optic Neuritis and Neuroretinitis,Retinal Vasculitis and Choroiditis,Branch Retinal Vein Occlusion (BRVO) ,White Dot Syndrome ,Stromal Keratitis and Episcleritis.Intermediate uveitis is the most common uveitis in LB. Posterior uveitis is mostly associated with chorioretinal involvement .Keratitis is characteristic of the second and third stages of LB and may either be interstitial or ulcerative. Episcleritis and scleritis are rare and can be observed mainly in the late phase of LB .Regarding ocular manifestations due to Neuroborreliosis, they include:Myositis of Extraocular Muscles,Facial Palsy and other Cranial Nerve Palsies ,Horner's syndrome .Coinfections should be suspected in the following cases , 104:\u2713 in the presence of fever and headache,\u2713 in patients diagnosed with LB, who do not clinically improve or,\u2713 whose symptoms have changed  after adequate antibiotic treatment,\u2713 when patients have leukopenia and neutropenia, persistent after treatment, or high ESR,\u2713 when patients present purple, persistent skin lesions, even the same purpuric Erythema migrans (in our experience).In these cases tests for Rickettsia, Anaplasma , BartoneThe spirochetes may persist in affected organs even months to years after the initial infection, causing a chronic form of illness. Therefore, antimicrobial agents have been found to have a role in all stages of the disease .When patients come to the Lyme Disease Center, because they have been found to be positive for anti-Borrelia antibodies, it is necessary to request an accurate medical history including the geographical area where the patient lives, recollection of a tick bite, and if applicable, the recollection of a circular rash, its possible location, and its duration. This collection of information should be followed by an accurate examination for the presence of LB related symptoms. Medical history should also include any previous antibiotic treatment.In the absence of any reported tick bite or EM and related clinical manifestations, if the serological test results are positive in IgG antibodies it is recommended to perform a WB, whereas positive IgM may not be specific, and serology should be repeated after 6 months.When the skin, the myo-articular system, and/or the nervous, cardiac or ocular systems are involved, specific investigations must be carried out, as indicated in the two previous paragraphs.These patients should also be subjected to immunological testing, as Borrelia antigens can induce autoimmune diseases in predisposed subjects (Trigger Factor).In some cases, Borrelia induces the production of antibodies against certain surface antigens, which cross-react with specific sequences of organism structures (antigenic camouflage). It is known, in fact, that there can be cross-reactivity between OspA and the human leukocyte function antigen (LFA) , 111, asA thorough diagnostic examination should be based on the clinical picture, the organs involved, the serological pattern, and the tests that have been already performed.The persistence of symptoms related to LB can be observed in untreated patients as well as in patients who have undergone treatment but continue to present symptoms. Untreated patients can develop persistent signs and symptoms, which usually involve the joints and less commonly the nervous system . PatientSome published authors of medical research recognize mainly two clinical scenarios: the first characterized by typical symptoms of post-Lyme disease when symptoms persist for <6 months, and post-treatment Lyme disease syndrome or chronic Lyme disease if symptoms are debilitating and persist after treatment .In the International Lyme and associated diseases society (ILADS) guidelines, \u201cchronic Lyme disease\u201d is described as a multisystem illness with persistent symptoms , 120, inBorrelia burgdorferi is likely due to the development of biologically less active permanent forms (Spheroblasts and round shapes) and of biofilm  and Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) have several clinical features in common, including fatigue, musculoskeletal pain, and cognitive difficulties. The Canadian Clinical Criteria for CFS/ME diagnosis include the following symptoms: Fatigue > 6 months, limited physical activity, unrefreshing sleep, impaired thinking and speech, vertigo, post-exertional fatigue, stress induced by exertion, reduced concentration, orthostatic intolerance, food intolerance .Immunologic mechanisms have been suspected to play a role in both PTLDS and CFS/ME.In CFS/ME patients, serum Activin B levels were significantly elevated compared with control subjects. Elevated Activin B levels together with normal Activin A levels identified patients with the diagnostic symptoms of CFS/ME , 147.It has also been hypothesized that there is an immunosignature specific to CFS/ME and that this could aid the diagnosis. Scientists were in fact able to identify a 256-peptide signature that separates CFS/ME samples from healthy controls .Borrelia burgdorferi; however there was no significant difference in the prevalence of anti-neural antibody reactivity between CFS/ME patients and healthy controls  are classified as probably not affected by Lyme disease. These patients should be considered positive only if, after 1 month, serology tests demonstrate serum conversion.In some cases a rapid test response is required, ELISA or CLIA . ClinicaBorrelia burgdorferi s.l. complex. Test systems comprise different techniques including the Enzyme-linked immunosorbent assay (ELISA), the Enzyme-Immunoassay (EIA), the Enzyme-Linked Fluorescence Assay (ELFA), the Chemoluminescence Immunoassay (CLIA), Luminex, Fluoro-Immunoassay (FIA), and Western Blots/Immunoblots. Some tests use antigens obtained from native Borrelia bacteria, whilst others use manufacturing methods to prepare recombinant antigens. In some assays a mixture of both are used.Several commercial products are available for detecting IgG and/or IgM antibodies against 2 terminals, and one internal variable region. The invariable, internal areas are masked and protected by the \u201cin vivo\u201d external variable regions. Due to the continuous modifications of its external antigenically variable component, Borrelia is able to escape the immune system.The European and North American guidelines indicate that the diagnosis of LB is currently based on a two-tier serology at all stages of the infection, except when erythema migrans is present . The twoBorrelia burgdorferi has been reported to quantitatively vary after antibiotic treatment  peptide of reatment \u2013159, altof Borrelia burgdorferi s.l. complex. The test kit manufacturers clearly define the interpretation for positive, negative, and equivocal samples.Immunoblot (western blot) is generally used to confirm positivity and can characterize the immune responses to specific proteins The European Union Concerted Action on Lyme Borreliosis/EUCALB has conducted a multicenter study for the standardization of the interpretative criteria of immunoblot results in Europe. Although a set of eight bands were identified as significant in each participant laboratory, no single rule was formulated for use across Europe . The senhttps://www.arminlabs.com/en/tests/tickplex).In recent years, other commercially available serological tests have been developed for Borrelia detection. Among them, the TickPlex assay is an ELISA-based test, which also contains a new antigen for round bodies/persister forms of Borrelia. This assay has been reported to be useful in different stages of LB and the upgraded test also allows to simultaneously determine IgM and IgG antibodies of several tick-transmitted bacterial and viral pathogens , the Barbour-Stoenner-Kelly II (BSK-II) medium, and the commercially available BSK-H medium , the DNA extraction protocols, the possible use of systems for enrichment of microbial DNA, the PCR targets and PCR approach  influence PCR sensitivity (Borrelia invades the intracellular niche has been suggested (B. burgdorferi (s.l.) spirochetes have been reported. These include looped or ring-shaped forms, blebs, round bodies, and cell wall deficient forms; spirochete colonies or biofilm aggregates have also been described. The above-mentioned morphologies can impact Borrelia detectability by PCR. Biofilm busters to increase Borrelia load have been suggested for more accurate PCR tests  and the enzyme linked immuno-spot (EliSpot) test have been commercialized. They are based on the detection in patients' blood of Borrelia-specific T-lymphocyte, notably the T helper lymphocytes, which are reported to circulate in the blood in detectable numbers only during an active immune response against Borrelia and to persist in a non-florid infection in lymphoid organs  , but alspatients  as well patients . An immupatients , was als peptide . Other a peptide  and the reatment .Written informed consent was obtained as part of the hospital procedures from the individuals and/or minor' legal guardian/next of kin for the publication of any potentially identifiable images included in this article.GT managed the clinical aspect of the review, MR the section dedicated to serology, SB the section related to direct diagnosis go LB. All authors drafted and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Therefore, we aimed to study the effect of TiO2 on the substantia nigra of midbrain.Widely used Titanium dioxide nanoparticles . Then, Motor tests including pole and hanging tests were done to investigate motor disorders. The animal brain was removed for histological purposes. Accordingly, immunohistochemistry was performed to detect tyrosine hydroxylase positive cells, and then toluidine blue staining was done to identify dark neurons in the substantia nigra. Eventually, the total number of these neurons were counted using stereological methods in different groups.40 male BALB/c mice were randomly divided into five groups: three groups received TiO2-50 group increased and also the time recorded for animals to hang on the wire in the hanging test significantly decreased (P<0.05) in comparison with other groups. Also, the average number of tyrosine hydroxylase positive neurons in TiO2-25 and TiO2-50 groups significantly decreased as compared to the TiO2-10 and control groups (P<0.05). The total number of dark neurons in the TiO2-25 and TiO2-50 groups was substantially higher than the TiO2-10, control and normal saline groups (P<0.05).The results showed that the time recorded for mice to turn completely downward on the pole in the TiO2, depending on dose, can cause the destruction of dopaminergic neurons and consequently increase the risk of Parkinson\u2019s disease.Our findings indicated that TiO The dens part contains dopaminergic (DA) neurons that produce neuromelanin, which protects these neurons against oxidative-related damages by inactivation of free radicals . BesidesPD is known as a common motor syndrome of the CNS that is diagnosed with symptoms such as bradykinesia, rigidity, instability in condition, and tremor . Recent 2 nanoparticles, as well as the lack of sufficient investigations on the effects of TiO2 nanoparticles on the SN of the midbrain, the present research was designed to study the neurotoxic effects of these nanoparticles on DA neurons. For this purpose, pole and hanging tests were used to investigate motor disorders. Furthermore, the immunohistochemistry and toluidine blue staining were used to determine the number of tyrosine hydroxylase (TH) positive neurons and dark neurons, respectively.  With regards to the advancements and developments of nanotechnology and the widespread usage of TiOChemical and preparation2 nanoparticles used in this study were characterized with a size of 10 nm, purity of 99%, specific surface area>150 m2/g (Brunauer-Emmett-Teller (BET) equation), and anatase crystalline phase obtained from the Nano Lima Corporation (Iran). Primary antibody (anti TH) was purchased from Abcam, and secondary antibody (goat anti-rabbit IGg) was purchased from Dako.TiO These nanoparticles were prepared immediately before being used as a form of suspension in the normal saline and administrated as a form of gavage.Animalsad libitum access to water and food.Forty male Balb/c mice aged 7\u20138 weeks and weighing 25\u201330 gr were obtained from the animal center of Mashhad University of Medical Science, Mashhad, Iran. During the study, animals were kept in the standard conditions in terms of temperature (20 \u00b1 2 \u00b0C) and 12 hr light/dark cycle, and After adaptation, animals were randomly divided into five equal groups (n=8):2-10 group: received 10 mg/kg of TiO2 nanoparticles by gavage for 45 days (1) TiO 45 days , 22.2-25 group: received 25 mg/kg of TiO2 nanoparticles by gavage for 45 days. 2) TiO2-50 group: received 50 mg/kg of TiO2 nanoparticles by gavage for 45 days (3) TiO 45 days .4) Normal saline group: received 0.5 ml normal saline by gavage for 45 days.5) Control group: without any intervention.Motor testsAt the end of experiments, motor tests were performed to determine the motor disorders for each of the five groups.Pole testMice were placed on top of a pole 55 cm high and 8 mm in diameter. The time spent by all animals to turn downward was recorded entirely. Finally, the average time was compared between the five studied groups. Then the average time spent in each group was compared to the other groups .Hanging testIn this test, animals were placed on a 100 cm wire, 40 cm high, and 2 mm in diameter. Then the hanging wire was rotated to place the animals in a hanging condition. The time that each mice was able to keep itself hanging was carefully recorded. Finally, the average time of each group was compared to the other groupsTissue preparation+) neurons and toluidine blue staining to identify dark neurons in the SN of the midbrain.After motor tests, the animals were anesthetized using chloroform, and their skull was cut along the sagittal suture, and the animal brain for histological studies was removed, fixed in 10% formalin solution, and then the tissues were dehydrated, cleared, and embedded in alcohol, xylene, and paraffin, respectively. 5 mm cuts were serially prepared from the samples. A total of 8 sections with an interval of 10 sections were selected randomly and used for immunohistochemistry to detect TH positive (THImmunohistochemistry\u00b0C for 15 min and followed by incubation with Bovine Serum Albumin (BSA) (0.01 g BSA+ 1000 \u00b5l PBS+ 10 \u00b5l Triton x) at room temperature (20-25 \u00b0C) for 30 min. In the next step, the slides were placed in hydrogen peroxide  in PBS for 15 min to block endogenous peroxidase. After that, incubation with goat serum was made at room temperature for 20 min, and then the samples were incubated with primary antibody  overnight. In the next step, the slides were washed with PBS and exposed to goat anti-rabbit IGg secondary antibody  with 1:100 concentrations for 90 min. All samples were treated with 3, 3\u2019-Diaminobenzidine (DAB) and H2O2 at room temperature for 10 min. After washing the samples with running water, they were counterstained with Haematoxylin, dehydrated in increasing graded alcohols, cleared in xylene solution, and mounted with cover glasses. Eventually, the sections were prepared to study by optical microscopy  for 5 min and antigen retrieval was done at 96/4 croscopy , 25.Toluidine blue stainingFor toluidine blue staining, at first, sections were cleared with xylene, rehydrated with descending alcohols and then stained with 1% toluidine blue solution for 20 sec. Afterward, the tissue slides were washed with distilled water (DW), dehydrated in increasing graded alcohols, cleared in xylene, and mounted with cover glasses. Then, they were prepared for study by optical microscopy , 27.Measurements+ cells and dark neurons were counted by using the counting frame. The number of TH+ cells and dark neurons were separately calculated for each sample by using the following formula:First, with the aid of a light microscope  equipped with a camera attached to a computer, the colored samples of SNpc were photographed with \u00d720 and \u00d740 lenses. Images were transferred to the computer monitor. Then, THIn this formula, \u201cNA\u201d represents the number of neurons in area, \u201c\u2211Q \u00ae\u201d refers to the sum of counted particles in certain sections, \u201ca/f\u201d is related to the area associated with each frame, and \u201c\u2211P\u201d means the sum of frame-associated points hitting the defined space .Statistical analysisP<0.05 was considered significant difference between groups. All data obtained from experiments were analyzed using SPSS 16 software and one-way ANOVA and Tukey\u2019s tests. Assessment of motor skill testsEvaluation of pole test2-50 group compared with TiO2-10 (P<0.01), TiO2-25 (P<0.05), control (P<0.001) and normal saline (P<0.001) groups. While no significant difference was found between the other groups . Also, the mean time taken to hang in the TiO2-50 group revealed a significant decrease in comparison with TiO2-10 (P<0.001) and TiO2-25 groups (P<0.05). This time in TiO2-25 group showed a significant decrease in comparison with control and normal saline groups (P<0.05)  .Histopathological studyImmunohistochemical evaluation+ neurons of the SNpc were visible in studied groups. TH+ neurons were less visible in samples belonging to TiO2-50 and TiO2-25 groups in comparison with control and normal saline groups. While, TH+ neurons were more visible in TiO2-10, control, and normal saline groups . Besides, there was no significant difference in the TiO2-10 group in comparison with control and normal saline groups; while there was a significant decrease in the number of TH+ neurons in TiO2-50 group compared to TiO2-10 (P<0.001) and TiO2-25 (P<0.01) groups. Also, the number of these neurons in the TiO2-25 group was significantly lower than TiO2-10 group (P<0.05) (The results of TH(P<0.05) .Toluidine blue staining2-50 and TiO2-25 groups was more than this number in other TiO2-10, control, and normal saline groups (P<0.001)  .2-25 and TiO2-50 groups significantly increased as compared to TiO2-10 group (P<0.001). In the TiO2-50 group, the number of these neurons was significantly higher than that of the TiO2-25 group (P = 0.003)  .2 nanoparticles on the DA neurons and also formation of dark neurons in the SNpc. Our results showed that the administration of TiO2 nanoparticles with different doses could reduce the number of TH+ neurons and increase the number of dark neurons. Previous in vitro studies have also shown that TiO2 nanoparticles increase oxidative stress, impair mitochondrial function, resulting in reduced survival of PC12 cells (the cells similar to DA neurons) in the growth medium  and also are the primary source of dopamine in the CNS . Althoug In PD, voluntary movements are slowed down because of damage to the direct pathway of the motor loop and also due to damage to the indirect pathway, limbs tremor occurs at the rest time. Environmental factors play a significant role in the development of CNS diseases and nanoparticles can be one of the important issues affecting the CNS due to their specific characteristics  and also worldwide usage , 36.2 nanoparticles reduce the number of TH+ neurons in the SN. Several possibilities exist regarding the mechanisms of neuronal damage of these nanoparticles: for example, it has been confirmed that administration of TiO2 nanoparticles in mice causes a collapse in the brain electrolytes balance by increasing the levels of calcium and sodium and decreasing the amount of magnesium, potassium, and zinc. This collapse is also due to disruption of the operation of sodium-potassium, potassium-magnesium, and calcium pumps (2 does neuronal damage is the formation of neuroinflammation. These nanoparticles increase the level of interleukin 6 and also NF-kB in the brain and result in activation of inflammatory reactions (2 neurotoxic effects is to increase the oxidative stress factors and reduce the antioxidant factors of the cells. By increasing the amount of O2-, H2O2, and MDA, these nanoparticles lead to cellular damage (2 nanoparticles is another important factor. The results of the Wu and Xiu studies in 2016 showed that in the in vitro environment, TiO2 increased the accumulation of \u03b1-Synuclein in PC12 cells (2 nanoparticles and the mechanisms of PD appearance. In the present study, it was also found that TiOum pumps . Becauseum pumps . Anothereactions . On the eactions . Anotherr damage . Additior damage . In addi12 cells . It has 12 cells . Therefo2-50 group significantly increased. In addition, the results of the hanging test indicated a significant decrease in the time until the animal stays on the wire in the TiO2-50 group. Previous studies have also shown that the mice exposed to high doses of TiO2 show signs of brain damage such as tremor and lethargy (2-50 group, the number of DA neurons reduced and as a result, the amount of dopamine intensively decreased and PD symptoms were observed.  The results acquired from behavioral pole test of the present study showed that the time taken until the animal falls from the pole in the TiOlethargy ; considelethargy . The out2 nanoparticles result in the death of neurons by increasing the activity of apoptosis enzymes such as Caspase-3 and Caspase-9, increasing Bax, and decreasing Bcl2 (An essential component for examining pathological changes in the CNS is the dark neuron. The structure and function of the dark neurons will change after the damage, and they will be wrinkled, basophilic and dark blue , 47. Theing Bcl2 , 49.2 nanoparticles have the potential function for damaging the DA neurons of SNpc. Excessive access of these nanoparticles to DA neurons may also increase the risk of PD.According to our investigation, it can be concluded that TiONo conflicts of interest."}
+{"text": "Im Rahmen der aktuellen Diskussion des G\u2011BA zur Einf\u00fchrung neuer Mindestmengenregelungen (MMR) in Deutschland untersucht die vorliegende Studie das Meinungsbild aktiver Thoraxchirurgen zu Mindestmengen (MM) bei der operativen Behandlung des Lungenkarzinoms.Die Auswahl der thoraxchirurgischen Zentren f\u00fcr die Onlinebefragung erfolgte auf Basis des Krankenhausverzeichnisses 2017 (Bundesamt f\u00fcr Statistik), der Lungenkrebszentren (Deutsche Krebsgesellschaft), der zertifizierten Kompetenzzentren Thoraxchirurgie (Deutsche Gesellschaft f\u00fcr Thoraxchirurgie), der Kliniken mit thoraxchirurgischem Schwerpunkt und der deutschen Universit\u00e4tskliniken. Abgefragt wurde der potenzielle Einfluss einer MMR auf die Ergebnisqualit\u00e4t, Versorgungsqualit\u00e4t, \u00f6konomische Aspekte und auf die Versorgungsstruktur. Des Weiteren wurde eine Empfehlung f\u00fcr eine MM gefordert und aktuelle Ausnahmeregelungen bewertet.Es wurden 145 Kliniken (R\u00fccklaufquote 85\u202f%) mit 454 Thoraxchirurgen (R\u00fccklaufquote 54\u202f%) kontaktiert. Bei hoher Akzeptanz von MM zur Verbesserung der Ergebnisqualit\u00e4t erwarten 78,4\u202f% der befragten Operateure eine Zentralisierung der chirurgischen Versorgung, welche jedoch nach Aussage von 70,1\u00a0\u202f% zu keiner Verschlechterung der Versorgung von Lungenkrebspatienten f\u00fchren w\u00fcrde. Etwa 46,1\u202f% der Teilnehmer rechnen mit einer \u00f6konomischeren Versorgung und 83,3\u202f% sprachen sich f\u00fcr die Einf\u00fchrung einer MMR mit einer durchschnittlichen MM von 67 anatomischen Lungenresektionen pro Jahr und pro Zentrum aus.n\u2009=\u200967) liegt etwas unter der Vorgabe f\u00fcr chirurgische Prim\u00e4rf\u00e4lle eines zertifizierten Lungenkrebszentrums.Eine MMR zur chirurgischen Therapie des Lungenkarzinoms findet unter aktiven Thoraxchirurgen eine hohe Akzeptanz. Die geforderte MM ( Der Gemeinsame Bundesausschuss (G-BA) pr\u00fcft derzeit die Einf\u00fchrung einer Mindestmengenregelung (MMR) f\u00fcr Lungenkrebsoperationen in Deutschland, zur potenziellen Verbesserung der Qualit\u00e4t des Behandlungsergebnisses. Die vorliegende Studie untersuchte dabei das aktuelle Meinungsbild aktiver Thoraxchirurgen zu Mindestmengen (MM) bei der operativen Behandlung des Lungenkarzinoms mit Empfehlung einer konkreten MM und Bewertung der aktuell g\u00fcltigen Ausnahmeregelungen.In der Diskussion um die Zukunftsf\u00e4higkeit der Krankenhausversorgung werden zum notwendigen Strukturwandel diverse Ziele diskutiert . MindestDie anatomische Lungenresektion  gilt als Standard bei der onkologisch-gerechten operativen Versorgung eines Lungenkarzinoms , daher wDie Aufarbeitung von Daten aus Deutschland auf Basis der DRG(\u201ediagnosis related group\u201c)-Statistik der Jahre 2005 bis 2015 zeigte nahezu eine Verdopplung der Krankenhausletalit\u00e4t in Zentren mit einem Behandlungsvolumen von weniger als 25 anatomischen Lungenresektionen pro Jahr 5,7\u202f%) bei der Hauptdiagnose Lungenkarzinom im Vergleich zu Zentren mit einem Volumen von mehr als 75 anatomischen Lungenresektionen pro Jahr  mit Festlegung von Reverenz- bzw. Grenzwerten ist komplex und kann nur dann einen Beitrag zur Qualit\u00e4tssicherung liefern, wenn sie auch von den behandelnden Chirurgen anerkannt und umgesetzt werden . Aus dieDie Auswahl der kontaktierten Zentren erfolgte anhand des Krankenhausverzeichnisses 2017 (Bundesamt f\u00fcr Statistik), der assoziierten thoraxchirurgischen Zentren der Lungenkrebszentren nach der Deutschen Krebsgesellschaft (DKG), den Kompetenzzentren f\u00fcr Thoraxchirurgie der Deutschen Gesellschaft f\u00fcr Thoraxchirurgie (DGT) sowie den Schwerpunktzentren f\u00fcr Thoraxchirurgie , den thoraxchirurgischen Abteilungen der deutschen Universit\u00e4tskliniken und durch eine zus\u00e4tzliche manuelle Recherche . Es wurdDer Befragungszeitraum erstreckte sich vom 17.01.2019 bis 26.02.2019. Der Fragenkatalog umfasste insgesamt 15\u00a0Fragen. Im ersten Teil wurde nach dem Ausbildungsstand sowie nach Struktur und Versorgungszahlen am Zentrum des Teilnehmers gefragt. Der zweite Teil besch\u00e4ftigte sich mit allgemeinen Aspekten zum Thema MM, w\u00e4hrend abschlie\u00dfend neben thoraxchirurgisch spezifischen Aspekten auch um eine Empfehlung f\u00fcr Grenzwerte von MM f\u00fcr anatomische Lungenresektionen beim Lungenkarzinom gebeten wurde. Die Umfrage wurde als Onlinesurvey zur Verf\u00fcgung gestellt und \u00fcber die Plattform SoSci Survey  realisiert. F\u00fcr akademische Umfragen ist die Nutzung des Service kostenfrei. Die verschl\u00fcsselte Daten\u00fcbermittlung und -speicherung mit Serverstandort in Deutschland folgte den Vorgaben der Datenschutz-Grundverordnung (DSGVO) in ihrer aktuellen Fassung.2-Test genutzt. Verwendet wurde die Statistiksoftware SPSS . Das Signifikanzniveau wurde auf p\u202f=\u20090,05 definiert.Die statistische Analyse erfolgte deskriptiv. H\u00e4ufigkeit und Verteilung der Antworten wurden absolut und prozentual ausgewertet. Zur Analyse von Gruppenvergleichen wurde der Fischer-Exakt-Test/\u03c7n\u202f=\u2009193) oder befanden sich in der Fachweiterbildung (n\u202f=\u200921). Der Gro\u00dfteil der Teilnehmer stammte aus Zentren mit 6 bis 8 (n\u202f=\u2009144) bzw. 3 bis 5 (n\u202f=\u200940) Fach\u00e4rzten sowie 1 bis 2 (n\u202f=\u2009123) oder 3 bis 5 (n\u202f=\u200955) Weiterbildungsassistenten f\u00fcr Thoraxchirurgie. Etwa 56\u202f% der Teilnehmer stammten aus Lungenkrebszentren der DKG, 19,7\u202f% aus Zentren mit geplanter Zertifizierung zum Lungenkrebszentrum (sog. Transitstatus), 20,2\u202f% der Teilnehmer waren aus Kompetenzzentren f\u00fcr Thoraxchirurgie (DGT) sowie 26\u202f% aus Organkrebszentren (DKG) ohne Lungenkrebszentrumsstatus und/oder onkologischen Zentren der DKG . Keine Zertifizierung am Standort gaben 9\u202f% der Teilnehmer an.Insgesamt 244 von 454 adressierten Teilnehmern aus 122 Zentren nahmen an der Umfrage teil. Dies entspricht einer R\u00fccklaufquote von 53,7\u202f% der Teilnehmer und 84,7\u202f% der Zentren. F\u00fcr die Analyse g\u00fcltige Interviews (>90\u202f% der Fragen beantwortet) leisteten 223 der 244  Teilnehmer. Etwa 96,5\u202f% der Teilnehmer waren Fach\u00e4rzte f\u00fcr Thoraxchirurgie  als auch f\u00fcr den Fachbereich Thoraxchirurgie im Speziellen  der Teilnehmer sahen das Potenzial f\u00fcr eine \u00f6konomischere Versorgung und 78,4\u202f% erwarteten eine Zentralisierung der chirurgischen Versorgung von Patienten mit Lungenkarzinom durch Einf\u00fchrung einer MMR Abb.\u00a0.n\u202f=\u2009170; 83,3\u202f%) sprachen sich f\u00fcr die Einf\u00fchrung von MM in der chirurgischen Behandlung des Lungenkarzinoms aus. Nur 16,7\u202f% der Teilnehmer hielten dies dagegen f\u00fcr nicht sinnvoll. Die Bef\u00fcrworter der Einf\u00fchrung einer MMR gaben im Mittel 67 anatomische Lungenresektionen pro Jahr und pro Zentrum an  mit dem Potenzial einer \u00f6konomischeren chirurgischen Behandlung  von Patienten mit Lungenkarzinom erwartet. Die Empfehlung der Thoraxchirurgen hinsichtlich der H\u00f6he der MM lag bei 67 anatomischen Lungenresektionen pro Jahr. Diese Empfehlung liegt im Vergleich zu den derzeit geltenden MM f\u00fcr andere operative Eingriffe sehr hoch, aber immer noch unterhalb der geforderten MM anatomischer Lungenresektionen pro Jahr zur Zertifizierung zum Lungenkrebszentrum (DKG), die bei \u226575 chirurgischen Prim\u00e4rf\u00e4llen liegt . Seit de8,1\u202f% mitn\u202f\u2265\u200975) eher unwahrscheinlich. Aus gesundheitspolitischen Aspekten stellt sich die Frage, ob eine MM in der H\u00f6he von 25\u00a0Eingriffen nicht ausreicht, um eine \u201eGelegenheitschirurgie\u201c auszuschlie\u00dfen. Aktuell werden ca.\u00a014\u202f% der anatomischen Resektionen beim Lungenkarzinom durch Kliniken mit <25 anatomischen Lungenresektionen pro Jahr durchgef\u00fchrt [Betrachtet man die derzeit bestehenden MM f\u00fcr operative Verfahren, so ist die Einf\u00fchrung einer MM, welche sich an den DKG-Kriterien f\u00fcr Lungenkrebszentren orientiert n\u202f\u2265\u20095 eher unhgef\u00fchrt . Diese EDie European Surgical Association (ESA) betont in einem Konsensuspapier neben dem Krankenhausfallaufkommen auch die Bedeutung des Operationsvolumens des Chirurgen und seine Spezialisierung . FachlicNur wenige Studien fragen nach der Beziehung zwischen Fallaufkommen des Chirurgen und Ergebnissen nach Lungenresektion. Aktuelle Untersuchungen zeigen zwar einen wesentlichen Einfluss chirurgischer Fallzahlen auf die Krankenhaus- bzw. 30-Tage-Letalit\u00e4t , 20 und Die Spezialisierung des Chirurgen wirkt sich ebenfalls positiv auf die Ergebnisse der Lungenresektion aus , 23. DieInternational wird von der Leapfrog-Gruppe in den USA f\u00fcr 11\u00a0Operationen sowohl eine Mindestmenge f\u00fcr die Krankenh\u00e4user als auch f\u00fcr die Operateure gefordert. F\u00fcr Lungenresektionen bei Karzinom betr\u00e4gt das Krankenhausvolumen \u226540\u00a0Eingriffen pro Jahr bei einem Chirurgenvolumen von \u226515\u00a0Eingriffen pro Jahr . Weder iEs sollte ebenfalls diskutiert werden, ob die alleinige Anzahl an Operationen f\u00fcr die Beurteilung der Versorgungsqualit\u00e4t ausreichend ist. Neben der Ergebnisqualit\u00e4t sollten auch die Aspekte der Struktur- und Prozessqualit\u00e4t ber\u00fccksichtigt werden. Um Qualit\u00e4t messen zu k\u00f6nnen, muss zun\u00e4chst der Anspruch an gute Qualit\u00e4t festgelegt werden, insbesondere da durch den G\u2011BA eine neue Ausnahmeregelung f\u00fcr \u201ehohe Qualit\u00e4t\u201c eingef\u00fchrt wurde, die eine Genehmigung zur Durchf\u00fchrung auch unterhalb der MMR zul\u00e4sst. Allerdings m\u00fcssten daf\u00fcr messbare Qualit\u00e4tsindikatoren (QI) zur Bewertung herangezogen werden k\u00f6nnen, welche als Surrogat-Parameter f\u00fcr in der Regel Teilbereiche der gew\u00fcnschten Qualit\u00e4t stehen. Einzelne QI k\u00f6nnen naturgem\u00e4\u00df in der Regel nur Teilaspekte des Qualit\u00e4tsanspruches abbilden. Somit wird die Verwendung mehrerer QI im Sinne von Qualit\u00e4tsindikatorprofilen empfohlen . BewertuMit der Einf\u00fchrung von MMR f\u00fcr Krankenh\u00e4user in Deutschland im Jahr 2004 wurden auch Ausnahmetatbest\u00e4nde (AT) formuliert. Diese erm\u00f6glichen die Abrechnung von MM-Operationen auch unterhalb der MM-Vorgaben. Die AT sind f\u00fcr alle MMR g\u00fcltig und w\u00fcrden somit auch eine potenzielle Regelung f\u00fcr anatomische Resektionen beim Lungenkarzinom betreffen. Die aktuellen AT wurden von den Umfrageteilnehmern der Studie \u00fcberwiegend positiv bewertet. Dies betrifft zum einen die AT f\u00fcr \u201eerstmalige Erbringung einer Leistung\u00a0(\u2026) oder die erneute Erbringung nach mindestens 24-monatiger Unterbrechung\u201c . Auch diF\u00fcr die Umgehung bestehender MMR konnten neben den schon diskutierten AT auch bestandene Abrechnungsmodalit\u00e4ten genannt werden. Durch die Novellierung der MMR durch den G\u2011BA zum 01.01.2018 muss der Krankenhaustr\u00e4ger nun vor Erbringung der Leistung gegen\u00fcber der Krankenkasse darlegen, dass die vorgegebenen MM im folgenden Kalenderjahr erreicht werden k\u00f6nnen. Ist dies nicht m\u00f6glich, ergibt sich kein Verg\u00fctungsanspruch durch die Krankenkassen \u201327.Am 16.08.2018 wurde das Institut f\u00fcr Qualit\u00e4t und Wirtschaftlichkeit im Gesundheitswesen (IQWiG) durch den G\u2011BA mit einer systematischen Literaturrecherche inklusive Evidenzbewertung zum Zusammenhang der Leistungsmenge und der Qualit\u00e4t des Behandlungsergebnisses bei der chirurgischen Behandlung des Lungenkarzinoms beauftragt. Die Ergebnisse der Literaturrecherche des IQWiG (Stand 08.10.2019) wurden am 06.11.2019 ver\u00f6ffentlicht. Es wurden 23\u00a0Beobachtungsstudien in die Analyse eingeschlossen, wobei nur 19\u00a0Studien verwertbare Daten f\u00fcr die Darstellung und Bewertung des Zusammenhangs zwischen der Leistungsmenge und der Qualit\u00e4t des Behandlungsergebnisses bei der chirurgischen Behandlung des Lungenkarzinoms beinhalteten . Acht deDurch die Beauftragung des IQWiG durch den G\u2011BA zur systematischen Literaturrecherche zum Zusammenhang zwischen Leistungsmenge und Qualit\u00e4t des Behandlungsergebnisses wurde ein wesentlicher Schritt zur Diskussion \u00fcber die Einf\u00fchrung einer MMR zur chirurgischen Versorgung des Lungenkarzinoms (Thoraxchirurgie bei Lungenkarzinom) in Deutschland gemacht. Analog zu bereits bestehenden Publikationen wird in der abschlie\u00dfenden Beurteilung des IQWIG \u201evon einer h\u00f6heren Sterblichkeit bei geringerer Leistungsmenge ausgegangen\u201c. Einschr\u00e4nkend wird auf die insgesamt geringe Datenlage/-qualit\u00e4t verwiesen, welche keine Aussagen zu konkret in die Versorgung eingef\u00fchrte Mindestfallzahlen zulassen. Diese aktuelle Umfrage unter Thoraxchirurgen (244 Teilnehmer aus 122 Zentren in Deutschland) zeigt, dass unter den aktiv in die Versorgungssituation des Lungenkarzinoms integrierten Thoraxchirurgen eine hohe Zustimmung  zur Einf\u00fchrung einer MMR mit den aktuell g\u00fcltigen Ausnahmeregelungen besteht. Es wurde von diesen eine durchschnittlich MM von 67 anatomischen Lungenresektionen pro Jahr und Zentrum empfohlen. F\u00fcr die Beurteilung der Qualit\u00e4t einer Versorgungseinrichtung sollte jedoch die Verwendung eines einzelnen Qualit\u00e4tsindikators nicht ausreichend sein. Hier bieten Indikatorprofile aus Zertifizierungsprozessen  eine zus\u00e4tzliche, validere Grundlage. Als Kennzahl bietet die Anzahl der durchgef\u00fchrten anatomischen Lungenresektionen aber ausreichend wissenschaftliche Evidenz und unter aktiven Thoraxchirurgen eine hohe Akzeptanz zur Weiterentwicklung der Versorgung von Lungenkrebspatienten in Deutschland.Die gro\u00dfe Mehrheit  der befragten Thoraxchirurgen bef\u00fcrwortet die Einf\u00fchrung von Mindestmengen (MM) in der chirurgischen Versorgung des Lungenkarzinoms.Die durchschnittliche Empfehlung unter den Bef\u00fcrwortern von MM betrug 67 anatomische Lungenresektionen bei der Diagnose Lungenkarzinom pro Jahr und Zentrum.Es besteht eine hohe Korrelation zwischen der Anzahl an chirurgischen Prim\u00e4rf\u00e4llen am jeweiligen Zentrum der Teilnehmer und der H\u00f6he der empfohlenen MM.Die aktuell g\u00fcltigen Ausnahmeregelungen wurden \u00fcberwiegend positiv bewertet, sodass diese auch bei der chirurgischen Versorgung des Lungenkarzinoms greifen sollten."}
+{"text": "Prognosemodelle zur Intensivbelegung mit COVID-19-Patienten sind in der aktuellen Pandemie wichtig zur strategischen Planung der Patientenallokation und Vermeidung regionaler \u00dcberlastung. Sie werden oft vollst\u00e4ndig an retrospektiven Infektions- und Belegungsdaten trainiert, wodurch die Prognoseunsicherheit exponentiell mit dem Prognosehorizont anwachsen kann.Wir schlagen einen alternativen Modellansatz vor, bei dem das Modell weitgehend unabh\u00e4ngig von den zu simulierenden Belegungsdaten erstellt wird. Die Verteilung der Bettenbelegungen f\u00fcr Patientenkohorten wird direkt aus Belegungsdaten aus \u201eSentinel-Kliniken\u201c berechnet. Durch Kopplung mit Infektionsszenarien wird der Prognosefehler durch den Fehler der Infektionsdynamikszenarien beschr\u00e4nkt. Das Modell erlaubt eine systematische Simulation von beliebigen Infektionsszenarien, die Berechnung von Korridoren f\u00fcr die Bettenauslastung sowie Sensitivit\u00e4tsanalysen im Hinblick auf Schutzma\u00dfnahmen.Das Modell wurde anhand von Klinikdaten und durch Anpassung von nur 2\u00a0Parametern an die Daten in der St\u00e4dteregion Aachen und Deutschland gesamt vorgenommen.Am Beispiel der Simulation der jeweiligen Bettenbelegungen f\u00fcr das Bundesgebiet wird das Belastungsmodell zur Berechnung von Belegungskorridoren demonstriert. Die Belegungskorridore bilden Schranken f\u00fcr die Bettenbelegungen f\u00fcr den Fall, dass die Infektionszahlen spezifische Grenzwerte nicht \u00fcberschreiten. Dar\u00fcber hinaus werden Lockdownszenarien simuliert, die sich an retrospektiven Ereignissen orientieren.Unser Modell zeigt, dass eine deutliche Reduktion der Prognoseunsicherheit in Auslastungsprognosen durch gezielte Kombination von Daten aus unterschiedlichen Quellen m\u00f6glich ist. Es erlaubt eine beliebige Kombination mit Modellen und Szenarien zur Infektionsdynamik und kann damit sowohl zur Belastungsprognose als auch f\u00fcr Sensitivit\u00e4tsanalysen f\u00fcr zu erwartende neuartige Spreading- und Lockdownszenarien eingesetzt werden.www.springermedizin.de zur Verf\u00fcgung. Bitte geben Sie dort den Beitragstitel in die Suche ein, das Zusatzmaterial finden Sie beim Beitrag unter \u201eErg\u00e4nzende Inhalte\u201c.Die Onlineversion dieses Beitrags (10.1007/s00063-021-00791-7) enth\u00e4lt die Simulation der Prognosekorridore der Intensivbettenbelegung f\u00fcr die Bundesl\u00e4nder. Beitrag und Zusatzmaterial stehen Ihnen auf  Die SARS-CoV-2-Pandemie hat im Rahmen der ersten COVID-19-Welle im Fr\u00fchjahr 2020 zu einer Spitzenbelastung der Intensivstationen mit mehr als 3500\u00a0COVID-19-Patienten im April 2020 gef\u00fchrt, die durch den ersten Lockdown bis Anfang Oktober 2020 auf niedriges Niveau zur\u00fcckgef\u00fchrt werden konnten. Der im Oktober einsetzende starke Anstieg der Infektionen f\u00fchrte zu einer zunehmenden Belastung der Intensivmedizin. Trotz der hohen Intensivbettenzahl drohen die Ressourcen im Winter 2020/2021 vollst\u00e4ndig ausgelastet zu werden [Integrierte Prognosemodelle f\u00fcr den Verlauf der Covid19-Infektion und die damit einhergehenden Belastungen f\u00fcr die Krankenversorgung auf Basis von SEIR(Susceptible \u2013 Exposed \u2013 Infected \u2013 Removed \u2013 Modelle)-Modellen \u20136 modellModelle vom SEIR-Typ weisen in einer Pandemiephase weit unterhalb der Herdenimmunit\u00e4t eine intrinsische exponentielle Instabilit\u00e4t des L\u00f6sungsverhaltens auf. Hierdurch resultieren Unsicherheiten im Gesamtmodell, die tendenziell exponentiell mit dem Prognosehorizont wachsen. Au\u00dferdem ist die Verkn\u00fcpfung der Parametrisierung durch ver\u00e4nderte politische Rahmenbedingungen nicht explizit integriert, sondern muss aus Daten retrospektiv gesch\u00e4tzt werden, wodurch ein Zeitverzug resultiert. Netzwerkbasierte Modelle , die dasDaher schlagen wir ein Belastungsmodell vor, bei dem die Dynamik des Infektionsgeschehens explizit entkoppelt wird von der Modellierung der Bettenbelegungen und nur die zu erwartende Dynamik der Bettenbelegung bei vorgegebener Infektionsdynamik simuliert wird. Das Belastungsmodell ist dabei so ausgelegt, dass es die reale Verteilung der Bettenbelegungsdauern explizit erfasst und so auch atypische Verteilungen simulieren kann. Das Modell wird dabei bis auf wenige Parameter nicht mithilfe einer gegebenen Infektionsdynamik und zugeh\u00f6rigen Bettenbelegungen parametrisiert, sondern mithilfe von separat erhobenen Belegungsdaten aus \u201eSentinel\u201c-Kliniken. Daher ist die Identifizierung des Belastungsmodells numerisch robust, d.\u202fh., kleine Ver\u00e4nderungen in den Parametern und den Infektionsdynamiken k\u00f6nnen qua Design keine zeitlich exponentiell anwachsenden Auswirkungen auf die Bettenbelegungen haben. Durch unser Modelldesign wird numerische Robustheit und Prognosesicherheit gewonnen, daf\u00fcr verlangt unser Ansatz eine geeignete Auswahl von \u201eSentinel\u201c-Kliniken mit ausreichender Verf\u00fcgbarkeit von Daten. Die vorliegende Arbeit soll zeigen, dass schon mit Daten von wenigen \u201eSentinel\u201c-Kliniken mit hohen Belegungszahlen eine gute Modellqualit\u00e4t mit gro\u00dfer Generalisierbarkeit auf L\u00e4nder- und Bundesebene erreicht werden kann.1(t),\u00a0\u2026 um(t), aus mit ui(t) als der t\u00e4glichen Infektionszahl der i\u2011ten Altersgruppe im zu simulierenden Gebiet. ui(t) k\u00f6nnen aus Datenquellen, Prognosen oder Simulationen von Szenarien gewonnen werden.Das Belastungsmodell geht von einer gegebenen Infektionsdynamik, uDer Kern des Modells ist die Quantifizierung des erwarteten zuk\u00fcnftigen Bettenbedarfs der Kategorie\u00a0k  zum Zeitpunkt\u00a0t f\u00fcr einen zum Zeitpunkt\u00a0t\u2032 infizierten Patienten der Altersgruppe\u00a0i quantifiziert durch die Belastungsfunktion B, B\u202f=\u20090 f\u00fcr t\u202f<\u202ft\u2032.k(t):Die Gesamtbelastung GB(t) des Gesundheitssystems der Kategorie\u00a0k im zu simulierenden Gebiet zum Zeitpunkt\u00a0t ist dann eine Faltung der Belastungsfunktion B mit den Infektionszahlen ui(t\u2032) der Infektionsdynamik vom angenommenen Szenario ui(t) resultieren in einer Abweichung \u03b4GB(t):Abweichungen \u03b4uDie H\u00f6lder-Ungleichung garantiert, dass die resultierende Abweichung stets innerhalb eines Korridors von der BreiteDie Berechnung der Belastungsfunktion B aus gemessenen Belegungs- und Infektionsdaten mithilfe von Maximum-Likelihood-Verfahren aus der Modellgleichung Gl.\u00a0LoS,k,i) die Verteilung der Aufenthaltsdauer der Patienten in Bettenkategorie\u00a0k ist. S ist die Wahrscheinlichkeitsverteilung f\u00fcr den Zeitverzug zwischen Einweisung und Infektion und R die Wahrscheinlichkeit f\u00fcr einen Bettenbedarf der Kategorie\u00a0k f\u00fcr einen infizierten Patienten der Altersgruppe\u00a0i.Da weder der Zeitpunkt der Infektion\u00a0t\u2032 noch der genaue Zeitpunkt der Aufnahme auf die Intensivstation sowie der Beginn der Beatmung bekannt ist, muss im Modell f\u00fcr jede Bettenkategorie ein unbekannter Zeitverzug aus den Belegungsdaten f\u00fcr jede Bettenkategorie einzeln gesch\u00e4tzt werden. Hierf\u00fcr wird die Belastungsfunktion B approximiert werden durche(k) mit einer Spannbreite von 7\u00a0Tagen angenommen. Da zwischen Infektion, Erfassung der Diagnose und Einweisung ein unbekannter Zeitverzug besteht, der au\u00dferdem zeitlich leicht schwanken kann, wird te(k) aus Belegungs- und Infektionsdaten mithilfe der Gl.\u00a0S\u00a0wurde normalverteilt um trel multipliziert mit einem von der Kategorie\u00a0k abh\u00e4ngigen Gesamtrisikofaktor Rg(k):Das Risiko R wird approximiert als Produkt aus dem relativen Risiko der Altersgruppen Rrel wird aus der Verteilung der Altersgruppen in den \u201eSentinel\u201c-Kliniken und der Altersverteilung der Infektionsdynamiken gesch\u00e4tzt. Das absolute Risiko Rg(k) f\u00fcr jede Bettenkategorie wird mit Gl.\u00a0Rg(k) und te(k) wird ein \u201eleast square fit\u201c mit Kreuzvalidierung  verwendet, die te(k) und Rg(k) werden als Erwartungswert der in jedem Kreuzvalidierungslauf gefitteten Parameter berechnet. Als Modellunsicherheit wird der Fehler im Testset angenommen.F\u00fcr die Sch\u00e4tzung von RIn der vorliegenden Modellversion wurden zur Modellidentifizierung Daten aus den folgenden Quellen verwendet:Dashboard Robert Koch Institut (RKI) . Es wurdt\u00e4gliche Infektionszahlen (RKI) : F\u00fcr dieZur Kompensation der 7\u2011Tage-Periodizit\u00e4t der Infektionsreportings wurden die tagesaktuellen Daten gegl\u00e4ttet. Hierzu wurden die Daten des jeweiligen Tags \u00fcber die letzten 5\u00a0Wochen mit einem kubischen \u201espline\u201c interpoliert und jeder Tag durch den Median der resultierenden 7\u00a0\u201esplines\u201c approximiert.Testintensit\u00e4t (RKI) : Die InfDie Parametersch\u00e4tzung des lokalen Modells f\u00fcr die St\u00e4dteregion Aachen wurde anhand der Daten IG NRW durchgef\u00fchrtF\u00fcr Deutschland wurden Daten des DIVI-Intensivregisters  verwendeLoS,k,i) wurden mithilfe von anonymisierten Patientendaten der Uniklinik RWTH Aachen (N\u202f=\u2009451) sowie des Krankenhauses K\u00f6ln-Merheim  berechnet. Es werden nur Patienten eingeschlossen, die die Behandlung zum Zeitpunkt der Modellierung abgeschlossen haben und ab Mai 2020 aufgenommen wurden.Die Belastungsfunktionen B\u2032 sowie der Risikofunktionen wurden mit 100-fach-Bootstrapping mit jeweils 80\u202f% der Daten durchgef\u00fchrt.Die Sch\u00e4tzungen der Belastungsfunktionen B\u2032(te(k) und Rg(k) f\u00fcr die St\u00e4dteregion wurden die gemeldeten Bettenbelegungen um die Zuweisungen von au\u00dferhalb der St\u00e4dteregion f\u00fcr das UK Aachen als Haupttr\u00e4ger der Zuweisungen reduziert. Die Parameter f\u00fcr Deutschland wurden aus den DIVI-Intensivregisterdaten ohne Korrekturen gesch\u00e4tzt. Es wurde 20-fache Kreuzvalidierung mit jeweils 80/20-Splitting in Trainings- und Testdatensatz verwendet. Als Fehlerfunktion wurde f\u00fcr jede Kategorie der mit der absoluten Belegungszahl gewichtete quadratische relative Fehler sowohl im Training- als auch im Testdatensatz verwendet.Bei der Berechnung der Parameter tAlle Belegungsdaten wurden auf die Altersgruppen des RKI-Dashboard bezogen. Die Verteilung der Patienten des UK\u00a0Aachen seit Mai 2020 mit abgeschlossener Behandlung ist in Tab.\u00a0Die daraus berechneten Belastungsfunktionen\u00a0B\u2032 f\u00fcr die Altersgruppen\u00a04\u20136 sind in Abb.\u00a0rel\u00a0, gesch\u00e4tzt aus den Belegungsdaten des Universit\u00e4tsklinikums Aachen ist in Abb.\u00a0Das relative Risiko RWie erwartet steigt das Risiko innerhalb jeder Bettenkategorie mit dem Alter deutlich an, wobei in der Intensiv- und Beatmungskategorie der Abfall in der Altersgruppe\u00a0\u2265\u202f80 Jahre auff\u00e4llt. Dieser kann jedoch auf die Patientenverf\u00fcgungen zur\u00fcckgef\u00fchrt werden.e(k) und Rg(k) anhand eines Maximum-Likelihood-Sch\u00e4tzers mit Kreuzvalidierung identifiziert. Der relative gewichtete Fehler f\u00fcr die St\u00e4dteregion Aachen zum Zeitpunkt 16.12.2020 liegt zwischen 10\u201320\u202f%  als L\u00f6sung einer dynamischen Folge simuliert und mithilfe des parametrisierten Modells die resultierenden erwarteten Bettenbelegungen in jeder Kategorie simuliert.Als Beispiel sei hier eine Simulation mit einem Lockdownszenario demonstriert, das analog zur Dynamik des Herbstlockdowns in Frankreich simuliert wurde. Hierzu wurde angenommen, dass sich bis zur Aktivierung des Lockdowns die aktuelle Infektionsdynamik in allen Altersgruppen unver\u00e4ndert fortsetzt. Aus den franz\u00f6sischen Infektionsdaten  wurde deAls Beispiel k\u00f6nnen die Intensivbettenbelegungen f\u00fcr den Start des Lockdownszenarios ab dem 16.12. simuliert werden Abb.\u00a0.Ein weiteres Beispiel ist die Simulation der Konsequenzen von \u201eweichen\u201c vs. \u201eharten\u201c Lockdownszenarien am Beispiel des zweiten Lockdowns in \u00d6sterreich. Es wird dabei der Start am 16.12. simuliert mit Parametrisierung des zweiten Lockdowns in \u00d6sterreich sowie 50 und 25\u202f% Effizienz (Abb.\u00a0Das vorgestellte Belastungsmodell erlaubt die Simulation der Bettenbelegung in den Kategorien Krankenhaus, Intensivstation und Intensivstation mit mechanischer Beatmung f\u00fcr beliebige Szenarien der Infektionsdynamik. Der Schwerpunkt des Modells ist die numerisch robuste Simulation der resultierenden Bettenbelegungen ohne exponentielles Wachstum der Unsicherheiten. Dies wird durch die weitgehende Sch\u00e4tzung der Modellparameter aus separaten Daten von \u201eSentinel\u201c-Klinken erreicht. Es erlaubt eine numerisch stabile Simulation von Infektionsszenarien und eignet sich als Simulator bei Optimierungsfragestellungen. Es ist daher nicht als Ersatz, sondern als Erg\u00e4nzung zu den etablierten epidemiologischen Modellen der Infektionsdynamik zu sehen.Trotz der numerischen Stabilit\u00e4t des Modellansatzes bleiben f\u00fcr eine breite Nutzung, insbesondere f\u00fcr die lokale Simulation, offene Herausforderungen zu l\u00f6sen: Die Belastungsfunktion wurde bisher auf Basis von Daten der Uniklinik der RWTH Aachen und des Krankenhauses K\u00f6ln-Merheim berechnet. Auch wenn die Fallzahlen ausreichend erscheinen und beide Kliniken als repr\u00e4sentativ f\u00fcr entsprechende Maximalversorger in Deutschland f\u00fcr COVID19-Patienten gelten k\u00f6nnen, sind f\u00fcr eine robuste Prognose insbesondere in l\u00e4ndlich gepr\u00e4gten Gebieten weitere repr\u00e4sentative Daten erforderlich.Eine weitere Unsicherheit liegt in der Konstanz der Datenqualit\u00e4t, insbesondere der Infektionsdaten, die durch Meldeverzug sowie limitierte Laborkapazit\u00e4ten einen variablen zeitlichen Verzug haben k\u00f6nnen.Des Weiteren enthalten die Simulationen durch die wochenperiodischen Gl\u00e4ttungen einen Zeitverzug, der besonders bei lokalen Simulationen bei lokalen Outbreak-Events zu zeitverz\u00f6gerten Prognosen f\u00fchren kann.Insgesamt konnten wir zeigen, dass eine Modellierungsstrategie, die die Modellierung der Infektionsdynamik von der Modellierung der resultierenden Bettenbelegungen numerisch und datentechnisch entkoppelt, zu einer deutlichen Reduktion der Prognosefehler beitragen kann und f\u00fcr Szenariosimulationen geeignet ist. Eine systematische Weiterentwicklung in Richtung einer breiten, lokalen Nutzung, auch f\u00fcr zuk\u00fcnftige Infektionslagen, erfordert jedoch die systematische Verf\u00fcgbarkeit entsprechender Versorgungsdaten aus einem breiteren Panel von \u201eSentinel\u201c-Kliniken und unterstreicht damit die Bedeutung einer landesweiten Koordination der Datenverf\u00fcgbarkeit f\u00fcr Forschung und Versorgung."}
+{"text": "Foeniculum vulgare Mill. (1.01), whereas the Aloaceae family (0.86) had the highest FUV. Acorus calamus L. (80%) had the highest FL percentage. The leaves were identified as the most used plant part and decoction was the dominant mode of a medicinal preparation. Out of the plants and their uses documented in our study, 26.7% of the medicinal plants and 71.8% of the uses were novel. In conclusion, the diversity of medicinal plant uses in the Ngadisari village could contribute to the development of new plant-based drugs and improve the collective revenue of the local society.The people of Tengger, Indonesia have used plants as traditional medicine for a long time. However, this local knowledge has not been well documented until recently. Our study aims to understand the utilization of plants in traditional medicine by the people of Tengger, who inhabit the Ngadisari village, Sukapura District, Probolinggo Regency, Indonesia. We conducted semi-structured and structured interviews with a total of 52 informants that represented 10% of the total family units in the village. The parameters observed in this study include species use value (SUV), family use value (FUV), plant part use (PPU), and the relative frequency of citation that was calculated based on fidelity level (FL). We successfully identified 30 species belonging to 28 genera and 20 families that have been used as a traditional medicine to treat 20 diseases. We clustered all the diseases into seven distinct categories. Among the recorded plant families, Poaceae and Zingiberaceae were the most abundant. Plant species within those families were used to treat internal medical diseases, respiratory-nose, ear, oral/dental, and throat problems. The plant species with the highest SUV was  Documenor tribe .Indonesia has around 40,000 different plant species, of which approximately 6,000 are used for traditional healing processes . BTSNP iThe Tengger people use plants from the BTSNP for traditional ceremonies , as well2. Like other regions in Indonesia, Ngadisari village has only two seasons; dry and rainy. The rainy season spans the months of November-May, whereas the dry season spans June-October. The present study was conducted from 2018\u20132019. Most inhabitants belonged to the Tengger tribe and rely on agriculture. According to the Indonesian Statistics Bureau (BPS) data, the Ngadisari village has a population of 1,543 inhabitants or about 507 family units (households) [Allium fistulosum L.), potato (Solanum tuberosum L.), cabbage (Brassica oleracea L.), carrot (Daucus carota L.) and corn (Zea mays L.) are examples of plants that contribute to the income of these communities [This study was carried out in Ngadisari village, which belongs administratively to the Sukapura district in Probolinggo Region of the Republic of Indonesia. It is located at 7\u00b0 55\u2019 18\u201d S 112\u00b0 57\u2019 21\u201d E around the Bromo Tengger Semeru National Park (BTSNP) . Ngadisaseholds) . The popThis study was authorized (SK No. 091650/IT2.VII/HK.00.02/2018) by the Institute of Research and Community Service (LPPM) of the Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia. Verbal informed consent was obtained from each informant before conducting the interview process.Data collection was obtained through semi-structured and structured interviews with informants who knew or used plants as medicine. This technique is commonly used in ethnobotanical studies . InterviTaxonomical identification was conducted to verify the samples that were raised during the interviews. An herbarium was also prepared to obtain dry specimens supporting the taxonomical identification. However, the herbarium method was used only for unknown species. Photo documentation and herbarium of medicinal plants were then identified by Christin Risbandini under the laboratory of plant bioscience and biotechnology, Institut Teknologi Sepuluh Nopember, Indonesia using key dichotomy and some references , 19.Diseases that commonly occur in the Indonesian region were grouped into seven categories including gastrointestinal disorders (GI) ; dermatological diseases (DO) ; urogenital and gynecological problems (UGP) ; skeletomuscular disorders (SD); internal medical diseases (IM) ; respiratory-nose, ear, oral/dental, throat problems (RT) ; and others (OT) (motion sickness).et al. [et al. [The relative frequency of citation was calculated using the fidelity level (FL) formula according to Friedman et al.  and Oued [et al. . FL is tNp is the number of informants who mentioned or claimed the use of plant species for a particular healing process/medicinal treatment. N is the total number of informants who cited the plant species for various kinds of medicinal treatment.Where UVis) for a particular medicinal species divided by the total number of informants (Ni). The SUV was calculated according to Hoffman and Gallaher [SUV signifies the value of a medicinal plant species used by the people from Ngadisari village. It is calculated as the sum of the informant species use values  that are used as medicinal bioresources. The PPV is calculated according to Gomez-Beloz  as folloRU(plant part) and \u2211RU represent the sum of the cited plant parts and the total number of cited uses for a given plant, respectively.Where \u2211Mandevilla sanderi (Hemsl.) Woodson, Jatropha curcas L., Cymbopogon nardus (L.) Rendle), Microsorum buergerianum (Miq.) Ching., Paederia foetida L., Solanum muricatum Ait., Zingiber zerumbet (L.) Sm., and Senna alata (L.) Roxb. Also, different medicinal uses for known plants (71.8%) were observed in the present study, compared to the study conducted by Batoro [The people of Tengger receive their knowledge of traditional medicine from their ancestors. This knowledge is inherited and subsequently preserved across generations . We founy Batoro  (Table 1Adas (Foeniculum vulgare Mill.) has been used to treat urticaria/hives (DD), cough (RT), and to overcome motion sickness (OT). Betelvine (Piper betle L.) has also been used for more than one disease including leucorrhoea (UGP), hives or urticaria (DD), and worm disease (GI). This demonstrated that the use value of these species is quite high compared with that of other medicinal plants [The highest number of species in one category was found in the category of IM with 12 species, followed by nine species in RT and six species in DD . We alsol plants .Foeniculum vulgare Mill. (1.01), Aloe vera (L.) Burm. f. (0.86), Acorus calamus L. (0.8), Apium graveolens L. (0.76), and Allium fistulosum L. (0.71). Previous studies also demonstrated that fennel is frequently used as medicinal plants in Indonesia [F. vulgare is categorized as a plant used to treat dermatological problems (DO). People of Tengger inhabiting Ngadisari village use F. vulgare to treat urticaria, hives, or itching. Our results are also in accordance with other studies that revealed F. vulgare as a traditional medicine for people suffering from itching or other dermatitis problems [Species use value demonstrates the value of a medicinal plant species used by the people from Ngadisari village. Our results revealed that the SUV of the reported plants varied from 0.01 to 1.01 . Five spndonesia , 25 and ndonesia . Our datproblems .Aloe vera (L.) Burm. f., Acorus calamus L., Apium graveolens L., and Allium fistulosum L. have been reported in previous ethnobotanical studies. For example, Reimers et al. [et al. [Aloe vera to treat hair problems. Meanwhile, the use of Acorus calamus L. to treat fever has been reported by Rajput et al. [A. graveolens and A. fistulosum L. have been used by traditional Chinese and Indonesian people to reduce blood pressure and cough, respectively [et al. [Similar medicinal uses of s et al.  and Sale [et al.  reportedt et al. . Also, Aectively , 31. Fin [et al. , where tJamu\u2014an Indonesian traditional medicine [et al. [In total, 30 medicinal plant species have been recorded in our study. All belong to 20 different families, with Poaceae and Zingiberaceae being dominant in the study area (each consisting of four species) followed by Apiaceae (three species). The remaining families were represented by one or two species . Poaceaemedicine . Sharifi [et al.  also desA. vera or locally named as crocodile\u2019s tongues has been frequently used in some regions such as Southern Africa [The total number of species within a given family has been calculated to obtain their FUV. Our results showed that Aloaceae had a high FUV (0.86), followed by Acoraceae (0.80), Piperaceae (0.69), and Euphorbiaceae (0.65). Other families represented low FUV (< 0.60) . High van Africa , Asia [3n Africa , Nigerian Africa , and Indn Africa  to treatA. calamus is also cited by other ethnobotanical studies around the world including China [Jatropha curcas). The Tengger people use this Barbados nut species to treat mouth ulcers and hyperuricemia. Our data supports other studies; for example, Abdelgadir and Staden [et al. [J. curcas is potentially used to treat hyperuricemia.ng China , India [ng China , Nepal [ng China  to treatng China . Reporteng China . Finallyd Staden  reported [et al.  also repet al. [et al. [A. calamus demonstrated the highest FL for fever (80%), followed by A. graveolens (76.92%) and A. fistulosum (71.15) for treating hypertension and cough, respectively. Based on a previous study, plants with a high percentage of FL are more frequently used as bio-pharmacological resources [According to Imran et al. , the fid [et al.  reportedesources  and shouesources  bioassayesources , 50.S. muricatum, Z. zerumbet, C. nardus, C. domestica, P. guajava, G. mangostana, P. foetida, S. alata, K. galanga, C. sativum, Brassica sp. Low fidelity levels might also explain the low abundance of plant species in this region. Furthermore, it might also indicate that there is little information about the use of this medicinal plant among the people of Tengger in the Ngadisari village. Even though some plants possess low FL, these species should not be abandoned to preserve traditional knowledge of the society in treating some diseases as reported by Chaachouay et al. [Some species have low percentage of FL (1.92%) related to various diseases . Exampley et al. .According to Hoffman and Gallaher , calculaJ. curcas leaves and stem have been used to treat hyperuricemia and mouth ulcer, respectively.Leaves are the major plant components commonly reported to be used as herbal medicine materials in Indonesia , 53 and People from the Ngadisari village use many methods to prepare plant parts before using them as herbal medicine. The decoction is considered the main mode of preparation (40.9%), followed by pounding (15.9%) and burning (13.6%). Meanwhile, eating raw (9.1%) and smearing (6.8%) contribute and of the total mode of preparation in the present study. Other miscellaneous modes of preparations constitute the remaining 13.6% . Some otA. graveolens are eaten raw to reducing hypertension symptoms and leaves of A. vulgaris are applied directly by clogging into the nose to stop nosebleeds. All this local knowledge is preserved and applied by the people of Tengger in Ngadisari village. This practice is common in other regions in Indonesia such as in Madura and Bali [Some plants can be prepared without any processing. For example, leaves of and Bali , 66.in vitro plant tissue culture could also be used as an alternative way to conserve medicinal plants documented in this study. Finally, the information we obtained could enable the local communities to develop, market, and profit from dried herbal products, which then substantially improving the collective revenue of the local society.Our results highlighted the use of medicinal plants by people from the Ngadisari village, Indonesia. A total of 30 medicinal plant species were recorded in the present study. They belong to 20 different families, where Poaceae and Zingiberaceae were the most representative families. A high number of plant species were used for treating internal medical diseases, respiratory-nose, ear, oral/dental, and throat problems. Leaves were the most popular plant part used and decoction was the most common method of preparation. These findings indicated potential roles of medicinal plants used in the Ngadisari village. Furthermore, our study characterized the cultural values of the people of the Ngadisari village. The species use, family use values and fidelity levels presented here may be used to further support plant conservation and pharmacological studies for new drug discovery. Out of all the plants we reported, approximately 26.7% were novel medicinal plants. In addition, 71.8% of the plant uses we documented of medicinal species were also novel. Some highly cited species recorded in our study warrant further biochemical analyses to evaluate their bioactive substances. Moreover,"}
+{"text": "Sleep quality and duration play a pivotal role in maintaining physical and mental health. In turn, sleep shortage, deprivation and disorders are per evidence the risk factors and facilitators of a broad spectrum of disorders, amongst others including depression, stroke, chronic inflammation, cancers, immune defence insufficiency and individual predisposition to infection diseases with poor outcomes, for example, related to the COVID-19 pandemic. Keeping in mind that COVID-19-related global infection distribution is neither the first nor the last pandemic severely affecting societies around the globe to the costs of human lives accompanied with enormous economic burden, lessons by predictive, preventive and personalised (3P) medical approach are essential to learn and to follow being better prepared to defend against global pandemics. To this end, under extreme conditions such as the current COVID-19 pandemic, the reciprocal interrelationship between the sleep quality and individual outcomes becomes evident, namely, at the levels of disease predisposition, severe versus mild disease progression, development of disease complications, poor outcomes and related mortality for both -\u00a0population and healthcare givers. The latter is the prominent example clearly demonstrating the causality of severe outcomes, when the long-lasting work overload and shift work rhythm evidently lead to the sleep shortage and/or deprivation that in turn causes immune response insufficiency and strong predisposition to the acute infection with complications. This article highlights and provides an in-depth analysis of the concerted risk factors related to the sleep disturbances under the COVID-19 pandemic followed by the evidence-based recommendations in the framework of predictive, preventive and personalised medical approach. Keeping in mind that COVID-19-related global infection distribution is neither the first nor the last pandemic severely affecting societies around the globe to the costs of human lives accompanied with enormous economic burden, lessons by predictive, preventive and personalised medical PPPM/3PM) approach are essential to learn and to follow being better prepared to defend against global pandemics. To this end, 3PM knowledge towards the COVID-19 pandemic is regularly updated in the literature . Unique PM approaThis article focused on the comprehensive sleep quality impacts, follows the series of publications presenting valuable 3PM lessons towards the COVID-19 relevant knowledge. Sleep quality plays a pivotal role in maintaining physical and mental health. In turn, sleep shortage, deprivation as well as sleep disorders are per evidence the risk factors and facilitators of a broad spectrum of disorders, among others including depression, stroke, chronic inflammation, cancer, immune defence insufficiency and individual predisposition to infection diseases with poor outcomes. Therefore, sleep deficits are highly relevant for the COVID-19 distribution and outcomes.Sleep quality and duration have been shown to play a pivotal role in immune health. Accumulated research data clearly indicate that the immune defence is supported by sleep, whereas sleep deprivation, e.g. caused by insomnia and circadian disruption, can severely impair the immune system functionality serving therefore, as a reliable predictor , 7. DuriIn this context, the relationship between sleep quality and proper immune defence functionality becomes particularly relevant for the COVID-19 pandemic outcomes. Indeed, tiredness occurs as one of the common symptoms in the early stages of COVID-19 infection, and as the diseases progresses, insomnia may emerge as an additional problem, with disturbed sleep persisting beyond the acute stage of the disease. In terms of possible long-term effects of COVID-19 that exceed the typical recovery period, bundled under the term long COVID-19 or post-acute sequelae of SARS-CoV-2 infection, effects on sleep and fatigue are apparent, with sleep problems and fatigue becoming typical symptoms of long COVID-19 6\u00a0months after infection. In the context of long COVID-19, Huang et al. reported in a sample of N\u2009=\u20091655 that 437 individuals (26%) showed sleep problems and fatigue or muscle weakness for 1038 individuals (63%) .Among its multi-faceted functions, sleep has been shown to regulate glucose metabolism and weight gain, both of which are acknowledged risk factors of diabetes, obesity and sleep apnoea, which in turn have been associated with a higher predisposition to disease on viral infections  and to sConsidering the importance of vaccination as the primary strategy to combat viral pandemics, research data provide clear evidence towards an important role of the sleep quality in the level of vaccination efficacy . It has While there are no specific antiviral therapeutic agents available for the COVID-19 yet , 25, sleA retrospective analysis performed by Columbia University indicated improved survival rates for the intubated ICU patients, if they were treated with melatonin . To thisHealthcare givers, among the shift workers in general, are particularly susceptible to impaired sleep, due to rotating working hours and night work. Shift work, especially night work, induces a disruption of the circadian rhythm compromising the neuroimmune-endocrine homeostasis including a reduction in natural killer cells activity that has been well documented for nurses \u201337. SleeAn epidemiological survey conducted in December 2019 in Wuhan revealed the highest prevalence of the poor sleep quality in 18.2% of the sample (N\u2009=\u20097236) specifically for healthcare givers demonstrating compared to all other professional occupations . In partBased on the above presented facts, a reciprocal interrelationship has been hypothesised between the sleep quality and individual outcomes for both COVID-19 diseased and medical staff involved in the care. If being valid, this concept opens great prospects for implementing risk assessment, patient stratification, individualised prediction and targeted prevention to persons at high risk among professional and in the population linked to the well-justified educational measures.Systematic literature search has been conducted utilising electronic data available in PubMed and Google Scholar.Literature search investigated effects of COVID-19 on a sleep quality in hospital populations, separately considering both \u2014 the medical staff and affected patient cohorts.Studies dedicated to the sleep quality of healthcare givers were identified by following combinations of the keywords, \u201cCOVID-19\u201d, \u201csleep quality\u201d, \u201cmedical staff\u201d, \u201csleep disturbances\u201d and \u201cinsomnia\u201d. The review was limited to published research articles written in English. Only original research articles about or literature reviews of the effects of sleep on medical staff with reported sleep assessments as outcome variables were included.Studies dedicated to the sleep quality of the affected patients were identified by following combination of the keywords, \u201cCOVID-19\u201d, \u201cpatients\u201d, \u201csleep\u201d, \u201csleep quality\u201d and \u201cICU\u201d. The review was limited to published research articles written in English. Only original research articles or literature reviews on the effects of COVID-19 on patients\u2019 sleep were included. Furthermore, only studies measuring sleep status were included.Both search approaches are summarised in Figs. Potentially related to early awakening by medical staff and disrupted sleepDue to sedative administrationDue to mechanical ventilation of affected COVID-19 patients .Table Poor sleep quality was consequently associated with the lower levels of lymphocytes in the blood of COVID-19 patients . To thisFurther, an evident gender difference has been observed: female patients suffered more frequently from poorer psychiatric outcomes, such as depression and anxiety, compared to the male patients , 53.To improve sleep quality in COVID-19 patients, Liu et al. suggest implementing progressive muscle relaxation, for which anxiety-relieving effects were also reported .Table In agreement with earlier demonstrated sex differences in the sleep quality , 82, femData collected clearly demonstrate that particularly frontline healthcare givers reported worse sleep outcome variables, such as insomnia symptoms or significantly decreased sleep quality accompanied by increased levels of stress and burnout as well as increased appearance of psychiatric symptoms such as depression and anxiety. Indeed, under the pandemic condition, the frontline healthcare givers are facing sudden outbreak, which given the severity of the pandemic leads to the work overloads and extended working time frame with insufficient time to recover and consequent chronic exposure to stress, psychological distress  and incrSleep deprivation in shift workers is associated with a higher incidence and severity of respiratory infections compared to non-shift workers , 44, sugHowever, stress response and stress-related predisposition to pathologies have been demonstrated as being highly individual. Contextually, for working in shifts, an application of predictive diagnostic and patient stratification approaches followed by the targeted preventive measures is strongly recommended utilising corresponding tools and recommendations developed for a population screening and identification of individuals in sub-optimal health conditions , 85 and Contextually, perceived social and psychological support is considered as the preventive strategy to mild poor sleep quality and psychiatric symptoms in healthcare givers treating the COVID-19-affected patients , 77, 78.Analysed data clearly demonstrate that insomnia symptoms persist in big portion of the COVID-19 patient cohort with the monitored prevalence which significantly exceeds the level of insomnia in the general population. Several underlying causes have been proposed. Poor sleep quality was further associated with the decreased immune system functionality in the COVID-19 patients , suggestAs the generalised anti-COVID-19 prevention, the evidence-based periodontal healthcare is strongly recommended .To this end, periodontopathic microflora is implicated in systemic inflammation and pneumonia development, in severe cases leading to sepsis and death. Diagnosed periodontitis is associated with high risk of admission to intensive care units and increased COVID-19-related death . To thisTaking into account multi-faceted anti-inflammatory and anti-mitochondriopathic effects of melatonin, melatonin treatment is strongly recommended as an essential pillar of the anti-COVID-19 protection for the shift workers, COVID-19-affected individuals and patients suffering from chronic non-communicable disorders under the viral pandemic conditions \u201391."}
+{"text": "During the COVID-19 pandemic, the need to establish the prevalence of sleep dysfunction and psychological distress, identify predisposing and protective factors, and explore effective management strategies remains an important priority. Evidence to date suggests that a considerable proportion of COVID-19 patients experience significant sleep disturbances (estimated to afflict up to 50\u201375%) as well as psychological distress such as depression, anxiety, and traumatic stress. Duration of hospitalization, pre-existing mental health concerns, lower absolute lymphocyte count, and increased neutrophil-to-lymphocyte ratio have been all associated with a greater risk of sleep dysfunction in infected and hospitalized patients. Furthermore, in this review, we discuss the link between sleep deprivation, susceptibility to viral infections, and psychosocial wellbeing in relevance to COVID-19 and summarize the existing evidence regarding the presence and role of sleep apnea in infected individuals. Finally, we highlight the importance of suitable interventions in order to prevent and manage sleep dysfunction and avoid long-term physical and psychological implications. Future research should aim to provide high-quality information including in high risk, underserved, or difficult to reach populations and on the long-term consequences and effectiveness of applied interventions. At the end of December 2019, a series of cases of a novel coronavirus (SARS-CoV-2), causing respiratory infections in humans were described in Wuhan, China. The virus spread rapidly worldwide causing the coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) declared COVID-19 a \u2018Public Health Emergency of International Concern\u2019 on the 30 January 2020 and a \u2018pandemic\u2019 on the 11 March 2020 [Previous studies have shown that infectious disease outbreaks have been associated with disturbed sleep and psychological distress, i.e., traumatic stress, depression, and anxiety ,5,6. ConMoreover, some patients who have been infected with SARS-CoV-2may display prolonged new, recurring, or ongoing symptoms for more than four weeks after infection, sometimes even after an initial symptom recovery . These hTherefore, the consequences of poor sleep in COVID-19 sufferers are multiple, varied, and far reaching. Here, we aim to summarize the prevalence of sleep dysfunction in the face of COVID-19, identify the risk factors for its development, and explore ways in which we can minimize its impact. In addition, we discuss the links between psychosocial stress, sleep deprivation, and susceptibility to viral infections in relevance to COVID-19 as well as the presence and role of obstructive sleep apnea (OSA). Sleep is an important factor for human wellbeing in order to maintain daily functions , while lIn all groups studied, sleep problems were positively associated with psychological distress, such as depression and anxiety. Country of residence and lockdown period significantly influenced the development of sleep difficulties. However, the meta-regression analysis in this study indicated that factors such as age, gender, country, and marital status did not contribute to the prevalence of sleep problems. Furthermore, COVID-19 patients displayed the highest prevalence of sleep problems with HCPs having the second place , especiaAnother recently conducted meta-analysis on this subject  also conThe definition of insomnia varied among the published studies during the pandemic; most referred to insomnia in its broader presentation, as insomnia disorder or insomnia symptoms, with limited information about its onset and maintenance  . Being aThe instrument used to estimate sleep problems in most of the studies (50%) was the Pittsburgh Sleep Quality Index (PSQI) . The meaHowever, the pandemic is ongoing, and the results of different studies cannot be generalized. Nevertheless, it remains important to evaluate the impact of COVID-19 on sleep in different ethnicities, different age groups, and different strata of society, including those with limited access to health care services as well as the longer-term consequences . FurtherSARS-CoV2\u2032s most frequent severe clinical finding is pneumonia, leading to acute respiratory distress syndrome (ARDS). However, mounting evidence has shown that it can affect the nervous system, causing neurological symptoms from the early phases of the disease . Sleep iIn a prospective study evaluating the neurologic disorders in hospitalized COVID-19 patients in New York, patients with new neurological complications during hospitalization were matched to COVID-19 patients without neurological complications and were followed up for 6 months . Almost In fact, there is evidence to support a link between coronavirus infections and various nervous system manifestations. Neuroinflammation has been also noted with COVID-19 but appears to play a role in other neuropsychiatric diseases, several of which are characterized by immune-inflammatory states, and their treatments may have anti-inflammatory properties and effects. The underlying mechanism of the psychiatric, neuropsychiatric, and sleep consequences of COVID-19 are multifactorial and may include the social isolation, the severity of viral infection, immunological reactions, different treatments applied as corticosteroids, the Intensive Care Unit (ICU) stay, and the social stigma .Sleep strongly affects the immune system, and there is evidence that sleep deprivation negatively impacts immune responses ,40, possThese findings are suggestive of a potential close relationship between poor sleep quality during hospitalization and worse clinical outcomes in COVID-19 patients. This is further supported by evidence indicating that sleep deprivation has a substantial effect on immune cell number, function, and cytokine production. Lack of sleep may promote inflammatory factors release and impair human immunity , and botHigh levels of psychological distress and poor mental health have been reported since the start of the COVID-19 pandemic in various populations ,50,51. IThis bi-directional relationship is likely multifactorial. Thus, a high prevalence of sleep problems could be partly explained by psychological distress and sleep-related factors due to quarantine and lockdown as delayed bedtime and sleep onset as well as fear of COVID-19 and illness . Fear ofIn addition, another systematic review and meta-analysis that aimed to assess the prevalence of depression, anxiety, and sleep disturbances of COVID-19 patients found that 45% of COVID-19 patients experienced depression, 47% experienced anxiety, and 34% experienced sleep disturbances . AnotherObesity, hypertension, diabetes, dyslipidemia, cardiovascular, and pulmonary disease have been found to be associated with severe outcomes from COVID-19 infection ,60. One This finding could be partly explained by the fact that OSA patients are often overweight or obese with multi-morbidity such as hypertension, diabetes, and cardiovascular disease, which all adversely affect COVID-19 related severity mortality outcomes ,60. AddiOSA also impairs sleep quality, causing sleep deprivation due to frequent awakenings from respiratory events. In turn, sleep deprivation has been shown to affect the immune system, increasing interleukins  and tumor necrosis factor-a (TNF-a), promoting inflammation ,73 and pTelemedicine may be used for the evaluation and follow up in order to minimize hospital visits and limit patients\u2019 exposure to the virus. Patients diagnosed with OSA with suspected COVID-19 infection should be closely monitored for early detection and treatment to prevent severe outcomes. On the other hand, OSA diagnosis should be considered in COVID-19 patients with characteristic clinical findings. Various societies have issued guidelines regarding the safe function of sleep laboratories for the diagnosis and treatment of sleep disorders ,80,81,82For the treatment of OSA patients during the COVID-19 pandemic, home positive airway pressure (PAP) should be applied using telemonitoring. General protective measures for patients and staff are important: disposable probes and non-leaky full facemask with a filter to the tubing is suggested. Additionally, during titration, humidifiers are not recommended . All theEvidently, there is a need for appropriate and tailored management strategies and interventions across different populations including the general public and high-risk groups such as HCPs and COVID-19 patients including improved sleep hygiene, identification of various risk factors at individual, interpersonal, institutional, and community levels, and early and accurate recognition of sleep dysfunction and psychological distress . SpecialMedication strategies such as the administration of melatonin or sedating psychotropic medications have been also suggested in order to improve the sleep quality of COVID-19 sufferers ,83,84. SThe prophylactic administration of melatonin and melatonin receptor agonist, Ramelteon, improved the sleep of ICU patients, decreasing the prevalence of delirium and ICU length of stay, as it may have alleviated ARDS due to its anti-inflammatory and immune-enhancing effects . MelatonAdditionally, a selective serotonergic agent fluvoxamine showed encouraging preliminary results in limiting disease progression in mild to moderate COVID-19 illness in a randomized trial , as it aInsomnia should initially be treated according to the European Sleep Research Society and the American Academy of Sleep Medicine guidelines with cognitive behavioral therapy (CBT) ,92. MediMore than one year into the pandemic and despite the implicit limitations in the quality of available studies, the evidence points to the magnitude of effect of COVID-19 on the sleep and psychological wellbeing of the general population and high-risk groups ,98. Slee"}
+{"text": "Enterococcus durans isolate A8-1 from a fecal sample of a healthy Chinese infant, we determined the tolerance to low pH, survival in bile salts and NaCl, adhesion ability, biofilm formation, antimicrobial activity, toxin gene distribution, hemolysis, gelatinase activity, antibiotic resistance, and virulence to Galleria mellonella and interpreted the characters by genome resequencing. Phenotypically, E. durans A8-1 survived at pH 5.0 in 7.0% NaCl and 3% bile salt under aerobic and anaerobic condition. The bacterium had higher adhesion ability toward mucin, collagen, and Bovine Serum Albumin (BSA) in vitro and showed high hydrophobicity , auto-aggregation activity (51.7%), and could co-aggregate (66.2%) with Salmonella typhimurium. It had adhesion capability to intestinal epithelial Caco-2 cells (38.74%) with moderate biofilm production and antimicrobial activity against several Gram-positive pathogenic bacteria. A8-1 can antagonize the adhesion of S. typhimurium ATCC14028 on Caco-2 cells to protect the integrity of the cell membrane by detection of lactate dehydrogenase (LDH) and AKP activities. A8-1 also helps the cell relieve the inflammation induced by lipopolysaccharide by reducing the expression of cytokine IL-8 (P = 0.002) and TNF-\u03b1 (P > 0.05), and increasing the IL-10 (P < 0.001). For the safety evaluation, A8-1 showed no hemolytic activity, no gelatinase activity, and had only asa1 positive in the seven detected virulence genes in polymerase chain reaction (PCR), whereas it was not predicted in the genome sequence. It was susceptible to benzylpenicillin, ampicillin, ciprofloxacin, levofloxacin, moxifloxacin, tigecycline, nitrofurantoin, linezolid, vancomycin, erythromycin, and quinupristin/dalofopine except clindamycin, which was verified by the predicted lasA, lmrB, lmrC, and lmrD genes contributing to the clindamycin resistance. The virulence test of G. mellonella showed that it had toxicity lower than 10% at 1 \u00d7 107 CFU. According to the results of these evaluated attributes, E. durans strain A8-1 could be a promising probiotic candidate for applications.To evaluate the probiotic characteristics and safety of  Enterococcus, as one of the indigenous bacteria in the intestine, belongs to the class of facultative anaerobic lactic acid bacteria (LAB). Enterococcus spp. is distributed widely and can be separated from the environment, food, and human and animal gastrointestinal tract and has strong resistance to harsh stress and can survive at different conditions  and veterinary medicine    , and theaecalis) .Enterococcus has both probiotic character and potential pathogenicity, and some strains can cause important infections and diseases, such as endocarditis; bacteremia; and urinary, intra-abdominal, pelvic infections, and central nervous system infections .Enterococcus durans A8-1, Enterococcus faecalis ATCC29212, Lactobacillus rhamnosus GG BL379, and Bifidobacterium infantis CICC6069 were inoculated into de Man, Rogosa and Sharpe (MRS) medium  and incubated aerobically with constant temperature shaker at 37\u00b0C for 18\u201324 h. About the streak-plating growth, A8-1 and BL379 were cultured on MRS agar using MRS broth with 15 g/L agar for 18 h. For the anaerobic culture, the bacterial cells were inoculated on the same medium and incubated in an anaerobic chamber  with a modified atmosphere of 82% N2, 15% CO2, and 3% H2 without shaking. For the growth of Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa PA01, P. aeruginosa ATCC27853, Enterococcus hormaechei ATCC700323, Salmonella typhimurium ATCC14028, and Escherichia coli ATCC35218, nutrient broth was used .2.Caco-2 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s minimal essential medium  supplemented with 10% fetal bovine serum  without antibody. The cells were kept at 37\u00b0C in an atmosphere containing 5% COOne gram of stool sample was mixed with 0.9% sterile saline solution to a final volume of 10 mL, and 0.1 mL of this dilution was spread on the MRS agar plate (MRS broth with 15 g/L agar) and cultured anaerobically at 37\u00b0C for 48 h. After incubation, colonies were randomly selected from each sample and subcultured on MRS plates for further analysis. Single colonies were picked out for Gram staining and microscopic observation and catalase, oxidase production, and nitrate reduction tests .For further confirmation, the 16S rRNA gene sequence (1.4 kb) was amplified, and sequenced by Sangon Biotech  Co., Ltd. Primers used were 16S-27F: 5\u2032 AGA GTT TGA TCC TGG CTC AG 3\u2032, 16S-1492R: 5\u2032 GGT ACC TTG TTA CGA CTT 3\u2032. The polymerase chain reaction (PCR) amplification conditions were as follows: initial denaturation for 5 min at 94\u00b0C, 35 cycles of denaturation for 30 s at 94\u00b0C, annealing at 60\u00b0C for 30 s, extension at 72\u00b0C for 60 s, and a final elongation step of 5 min at 72\u00b0C. Multiple alignments with sequences of closest similarity were analyzed using CLUSTAL W, and a phylogenetic tree was constructed by using the neighbor-joining method.600 = 0.1 at fresh MRS with different pH 3.0, 4.0, and 5.0; bile salts  and NaCl . The negative control was MRS blank medium at pH 6.5. Three replicates were set for each medium. Growth was monitored by optical density at 600 nm every 30 min at 37\u00b0C for 21 h in a microtiter plate reader   and resuspended in ODGermany) . MaximalGermany) .E. faecalis ATCC29212, S. aureus ATCC25923, P. aeruginosa PA01, P. aeruginosa ATCC27853, E. hormaechei ATCC700323, S. typhimurium ATCC14028, and E. coli ATCC35218. A8-1 was inoculated into MRS broth and incubated at 37\u00b0C for 24 h. The cells were removed by centrifugation at 9,710 \u00d7 g for 2 min at 4\u00b0C. The supernatants were filter sterilized and added to 96-well plates at 0, 25, 50, 100, 150, and 200 \u03bcL, made up to 200 \u03bcL with fresh medium. Finally, each test indicator bacteria was added to the well at the concentration of OD600 = 0.1. Growth of test indicator bacteria was monitored every 30 min by optical density at 600 nm with an automatic microplate reader for 12 h at 37\u00b0C (The minimum inhibitory concentration (MIC) method was used to determine the antibacterial activity of A8-1. The indicator bacteria were as follows:  at 37\u00b0C .600 of 0.8 (A0). Then, 1 mL xylene and 1 mL chloroform were added separately to 3 mL of A8-1 cell suspension and mixed thoroughly. Then, the water and xylene phases were separated for 30 min at room temperature. The aqueous phase was removed, and the new OD600 was measured (A1). The cell surface hydrophobicity (%) was calculated using the following formula: Hydrophobicity (%) = [(A0\u2212A1)/A0] \u00d7 100%. The strain was classified into low (0\u201329%), moderate (30\u201359%), and high hydrophobicity (60\u2013100%).The hydrophobicity, auto-aggregation, and co-aggregation assays were performed according to 600 about 0.6 (A0). Bacterial cell suspensions were vortexed for 10 s and subsequently incubated at room temperature for 5 h, and the new OD600 was measured (At). The auto-aggregation percentage was determined using the following equation:For auto-aggregation, A8-1 was incubated overnight and washed in PBS twice and then resuspended in PBS with ODE. durans A8-1 and S. typhimurium ATCC14028 were incubated overnight separately and washed in PBS twice and then resuspended in PBS with OD600 about 0.8. Equal volumes (2 mL) of A8-1 and S. typhimurium ATCC14028 were mixed and incubated at room temperature without agitation for 5 h. Control tubes contained 2 mL of the suspension of each bacterial cells. The OD600 of the mixtures and controls were measured after incubation. The percentage of co-aggregation was calculated using the following formula: Co\u2212aggregation (%) = [(Ax + Ay)/2\u2212A(x + y)]/(Ax + Ay) \u00d7 100%, where Ax and Ay refer to the OD600 of the A8-1 and S. typhimurium ATCC14028 cell suspension, respectively, Ax + y represents the absorbance of the mixed bacterial suspension tested after 5 h.For co-aggregation, L. rhamnosus GG BL379 was used as a positive control; its adhesion rate was set as 100%, and the relative adhesion of A8-1 cell to BL379 was calculated.Strain binding to different substrates was evaluated as reported previously . Mucin (Adhesion (%) = (fluorescence intensity of A8-1)/(the free cFDA-labeled BL379) \u00d7 100.7 CFU/mL. Caco-2 cells cultured by high-glucose DMEM were seeded in 96-well plates and incubated at 37\u00b0C. The 200 \u03bcL of A8-1 suspension was added to each well containing Caco-2 cells and then incubated for 2 h. Caco-2 cells with DMEM was set as control. The Caco-2 cells were collected and washed three times by PBS to remove the unadhered A8-1. Then, trypsin was added to Caco-2 cells to lyse the adherent A8-1. Finally, the mixture of each well was cultured on an MRS solid plate to count the adhered bacteria (The A8-1 was incubated overnight and washed in PBS twice and then resuspended in 1 mL DMEM medium (without antibiotic) with a final concentration of 10bacteria .The adhesion (%) = [(CFU/mL) adhered bacteria/(CFU/mL) added bacteria] \u00d7 100.VITEK 2 Compact with AST-GP67  was used to access the antimicrobial susceptibility of the A8-1 to 15 clinical antibiotics, which included penicillin , ampicillin , high-level gentamicin (synergistic) , high-level streptomycin , ciprofloxacin , levofloxacin , moxifloxacin , erythromycin , clindamycin , quinupristin/dalofopine , linezolid , vancomycin , tetracycline , tigecycline , and nitrofurantoin . According to the MIC obtained, the results were judged according to Clinical Laboratory Standard Institute criteria (CLSI M100 S28)  and the Hemolytic activity of A8-1 was evaluated as described previously . A8-1 waOvernight cultured A8-1 was inoculated into gelatin medium  and incubated at 37\u00b0C for 48 h. Then, the tube was placed at 4\u00b0C for 1 h and it was observed whether there is liquefaction immediately. If the bacteria could produce gelatinase, there was liquid in the tube.600 = 1.0. The 50 \u03bcL of bacterial suspension was added to 150 \u03bcL of fresh MRS broth and incubated in 96-well plates at 37\u00b0C for 24 h. Also, 200 \u03bcL of MRS broth without bacteria was set as negative control. After 24 h, the culture medium was poured out. The wells were washed with sterile PBS three times to remove free-floating planktonic bacteria and then dried at room temperature. The biofilm was then fixed with methanol and stained with crystal violet. The control hole was rinsed with sterile water three times until it turned colorless. Then, 200 \u03bcL ethanol was added into each well, and optical density of stained adherent cells was measured at 595 nm by microplate reader. According to the cutoff OD (ODC), the biofilm-producing ability was determined as follows: OD \u2264 ODC set as non-biofilm-producer (0), ODC < OD \u2264 2ODC set as weak biofilm producer (+), 2ODC < OD \u2264 4ODC set as moderate biofilm producer (++), and OD > 4ODC set as strong biofilm producer (+++).Quantitative assessment of biofilm formation was evaluated as shown previously . OverniggelE (gelatinase), cylA (cytolysin), hyl , asa1/agg (aggregation substance), esp , efaA (endocarditis antigen), and ace/acm (collagen adhesion)  . The priGalleria mellonella) larvae were arranged  were maintained on woodchips in the dark at 15\u00b0C until being used. The overnight cultures of E. durans A8-1, E. faecalis ATCC29212, and B. infantis CICC6069 suspension were adjusted with concentrations of 1 \u00d7 106, 1 \u00d7 107, and 1 \u00d7 108 CFU/mL. Ten randomly selected larvae were used in each group. Each larva was inoculated the bacterial suspension via the rear left proleg using a 10 \u03bcL Hamilton animal syringe. The MRS medium was injected into the larvae and set as negative control. The treated G. mellonella larvae incubated at 37\u00b0C for 3 days, and the survival rate of the G. mellonella were recorded every 12 h.For the virulence activity assay, tests in wax moth (arranged . The G. 5 cells per well) and cultivated to a single layer. For the treatment, there were three treatment groups, S-A8-1 (competition group), A8-1 + S (exclusion group), and S + A8-1 (replacement group). S-A8-1, 1 \u00d7 107 CFU/mL S. typhimurium ATCC14028 and 1 \u00d7 107 CFU/mL A8-1 culture were added into the cell wells at the same time and incubated for 2 h; A8-1 + S, 1 \u00d7 107 CFU/mL A8-1 culture were added into the cell wells and incubated for 2 h and then 1 \u00d7 107 CFU/mL S. typhimurium ATCC14028 was added and incubated for another 2 h; S + A8-1, 1 \u00d7 107 CFU/mL S. typhimurium ATCC14028 culture was added into the cell wells and incubated for 2 h and then 1 \u00d7 107 CFU/mL A8-1 added and incubated for another 2 h. Finally, 1 \u00d7 107 CFU/mL S. typhimurium ATCC14028 incubating solely with Caco-2 cells for 2 h was set as control. After incubation, the cell wells were washed three times by PBS to remove the unadhered S. typhimurium ATCC14028 cells. The amount of adhered S. typhimurium ATCC14028 to the Caco-2 cells were counted by bismuth sulfite agar plate /the counted adhered S. typhimurium ATCC14028 in control (CFU/mL).Inhibition rate (%) = the counted adhered S. typhimurium ATCC14028, A8-1, A8-1 + S, S + A8-1) and one control. After incubation, the supernatant of cell culture was collected after centrifuging at 1,500 rpm for 10 min at 4\u00b0C. The activity of extracellular alkaline phosphatase (AKPase) was assayed in the collected supernatant using a kit  as described (Caco-2 cells were cultured and treated as mentioned into four treatment groups (escribed . The AKPescribed .5 cells per well). After treatment, cells were washed twice with PBS and incubated with 5 mg/mL MTT working solution for 4 h at 37\u00b0C. Then, the supernatant was removed, and the culture was resuspended in 150 \u03bcL of DMSO to dissolve MTT formazan crystals, followed by mixing on a shaker for 15 min. The absorbance was measured at 570 nm using a microplate reader. The effect of A8-1 culture and Lipopolysaccharides (LPS) on cell viability was assessed as the percentage of viable cells in each treatment group relative to untreated control cells, which were arbitrarily assigned a viability of 100%.It was carried out as described previously by 5 cell/well and incubated until the cells grew to monolayer. There were two treatment groups, A8-1 + LPS and LPS + A8-1. For the A8-1 + LPS group, 2.5 \u00d7 106 CFU A8-1 cells were added into Caco-2 cells and incubated for 6 h; the wells were washed three times by PBS, and 1 mL fresh DMEM was supplied and then 10 \u03bcg LPS was added and incubated for another 6 h. For the LPS + A8-1 group, 10 \u03bcg LPS was added into Caco-2 cells and incubated for 6 h; the wells were washed three times by PBS, and 1 mL fresh DMEM was supplied and then 2.5 \u00d7 106 CFU A8-1 cells were added and incubated for another 6 h. The different treated Caco-2 cells and cell culture supernatant were collected at 6 and 12 h, separately. The contents of IL-8, IL-10, and TNF-\u03b1 in the supernatant were detected by an ELISA kit (Sigma-Aldrich). Cell RNA extraction and relative mRNA expression of IL-8, IL-10, and TNF-\u03b1 were determined according to the instructions of corresponding kits (Sigma-Aldrich). GAPDH was selected as the internal reference gene, and the relative mRNA expression levels of IL-8, IL-10, and TNF-\u03b1 were calculated according to the 2\u2013\u0394\u0394CT method. The untreated Caco-2 cells were used as control, and all tests were performed in triplicate.Quantification of cytokine levels in cell culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative PCR (qPCR) . Caco-2 \u00ae 4413021). The DNA concentration and purity was quantified with the NanoDrop2000. It was sequenced on the Illumina HiSeq\u21222000 platform at Gene de novo Biotechnology Co., Ltd. . The reads were de novo assembled by SOAPdenovo version 2.04, 1 The predicted genes of E. durans A8-1 were compared with the comprehensive antibiotic resistance database (CARD)2 (3 (4 (5 (6 (7 to search for the secondary metabolite biosynthetic gene clusters.Whole-genome DNA of A8-1 was extracted by a kit (Applied Biosystems (CARD)2  and viruARD)2 (3  for iden)2 , STAMP10, GraphPad Prism 7, and SPSS V20.0  were used to perform statistical analyses. Data were presented as means \u00b1 SEM. +), catalase (negative) and oxidase production (negative), nitrate reduction test (negative), a final strain of A8-1 was confirmed by 16S rRNA sequence analysis . The maximum biomass of A8-1 in a 0.5, 1, and 2% bile salt environment were higher than that of the control group (600 of A8-1 was close to the control at pH 5.0 (P < 0.01) and 1.75% NaCl   . Under a > 0.05) .L. rhamnosus GG BL379 (positive control), A8-1 showed higher adhesion to mucin (P < 0.01), BSA (P < 0.01), and collagen. The adhesion ability of A8-1 to mucin, collagen, and BSA was 5.2, 1.6, and 5.6 times higher than L. rhamnosus GG BL379. For the adhesion to Caco-2 cells, the adhesion of A8-1 was 38.47%, which is higher than L. rhamnosus GG BL379  determined in our study.Compared with S. typhimurium with a co-aggregation percentage of 66.2 \u00b1 2.9%.For the surface adhesion ability, A8-1 showed a high hydrophobicity of 79.2 \u00b1 3.1% in chloroform and moderate hydrophobicity of 49.2 \u00b1 4.4% in xylene; it had 51.7 \u00b1 4.5% for the auto-aggregation after 5 h of incubation and was able to co-aggregate with P. aeruginosa PA01 and E. coli ATCC35218, the MIC was 25 \u03bcL supernatant, and for the S. aureus ATCC25923, P. aeruginosa ATCC27853, E. hormaechei ATCC700323, S. typhimurium ATCC14028, the MIC was 50 \u03bcL supernatant.The fermentation supernatant of A8-1 showed different antibacterial activity against the indicator strains for P = 0.005) and was increased in the S. typhimurium ATCC14028 group , whereas AKP activity was changed with a similar trend to LDH but no statistical difference. In the A8-1 + S group, activities of LDH and AKP in the Caco-2 cell supernatant were all significantly decreased compared with that of the Salmonella group . Even in the S + A8-1 group, LDH activity was still significantly lower than that of the Salmonella group . Those results show that strain A8-1 could protect the integrity of the Caco-2 cell membrane in pretreatment and inhibit the damage of Salmonella to Caco-2 cells.The enzyme activities of LDH and AKP in the supernatant of cell culture were selected as indicators for the integrity of the Caco-2 cell membrane . CompareS. typhimurium ATCC14028 to Caco-2 cells. It is found that, in S-A8-1 (competition group), A8-1 can reduce the adhesion of S. typhimurium to cells without statistical difference . In A8-1 + S (exclusion group), the adhesion of S. typhimurium to Caco-2 cells was significantly reduced  possibly because A8-1 could inhibit the growth of S. typhimurium and occupied the binding sites on the surface of the Caco-2 cells. However, there was no statistical difference for the adhesion to Caco-2 cells between S + A8-1 (replacement group) and the S. typhimurium group  (S. typhimurium to Caco-2 cell.The plate counting method was used to explore the antagonism of A8-1 against the adhesion of = 0.592) . Those r5, 2.5 \u00d7 105, and 1 \u00d7 106 CFU/mL could all significantly increase the Caco-2 cell viability  except the 2.5 \u00d7 106 CFU/mL group . So 2.5 \u00d7 106 CFU/mL of A8-1 cells was selected for the following assays.Results of cell viability treated by A8-1 are shown in P = 0.002); the IL-10 were significantly increased in both A8-1 + LPS and LPS + A8-1 groups . TNF-\u03b1 increased in both intervention groups, but there was no significant difference compared with LPS group . For the relative expression of mRNA, the IL-8 in A8-1 + LPS and LPS + A8-1 groups were significantly decreased ; IL-10 were significantly increased in both A8-1 + LPS and LPS + A8-1 groups . TNF-\u03b1 expression was decreased in both groups without statistical difference . Those results showed that A8-1 could reduce the secretion of IL-8 and increase the secretion of IL-10 and TNF-\u03b1 in response to LPS stimulation in Caco-2 cells.The 10 \u03bcg/mL LPS was added to the medium and induced the inflammation of Caco-2 cells, and the anti-inflammation ability of A8-1 was detected by IL-8, IL-10, and TNF-\u03b1 through ELISA and qPCR . CompareE. durans A8-1 to antibiotics was determined by measuring MICs, and the results were compared to the cutoff values for Enterococcus species as defined by EFSA and CLSI. E. durans A8-1 was found to be resistant only to clindamycin but was susceptible to penicillin, ampicillin, high-level gentamicin (synergistic), high-level streptomycin, ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, quinupristin/dalofopine, linezolid, vancomycin, tetracycline, tigecycline, and nitrofurantoin  Test, and there were no statistical differences in E. durans A8-1 and B. infantis CICC6096 within 107 and 108CFU/mL groups (P > 0.05). Apparently, A8-1 could be considered safe , 85 cell colonization\u2013related genes, 268 binding ability genes, and four antioxidant genes .The circular chromosome of NA genes . Genome ahp, A8-1_2612, A8-1_2613) and NADH peroxidase  that were found in the genome. For the polysaccharide biosynthesis\u2013related genes, there were eight genes located in the genome, including A8-1_ 0155 (polysaccharide biosynthesis glycosyltransferase), A8-1_0235 (polysaccharide core biosynthesis protein RfaS), A8-1_0854 (polysaccharide transport system ATP-binding protein), A8-1_1612 (sugar transferase), A8-1_1617 (polysaccharide cholinephosphotransferase), A8-1_1621 (polysaccharide core biosynthesis protein RfaS), A8-1_1639 (polysaccharide chain length determining protein CapA), and A8-1_1764 (polysaccharide biosynthesis protein). In addition, based on the previous studies, we also screened for a set of genes involved in imparting important probiotic functions as described in The probiotic-related genes in the genome were also analyzed. The cholylglycine hydrolase (EC 3.5.1.24) gene responsible for bile salt hydrolysis action was identified in one copy within the A8-1 genome (A8-1_2053). Fibronectin/fibrinogen-binding protein (A8-1_2247) and collagen-binding protein (A8-1_0314) were found in the genome allowing them to bind the GI tract, suggesting an important role in adhesion and colonization in intestinal mucosal surfaces. Also, the resistance to hydrogen peroxide is imparted by genes alkyl hydroperoxide reductase (AAC (6\u2032)-IIH and AAC (6\u2032)-IID; three \u03b2-lactam resistance genes mecC, mecB, and mecA; and one fluoroquinolone resistance gene mfd were predicted in CARD. Regarding the possibility of acquired resistance by horizontal gene transfer (HGT), there was no detection of any acquired antibiotic resistance genes. In addition, efaA/scbA was found in virulence gene prediction ; however, the similarity score was only 50.1%. Meanwhile, the asa1 gene detected by PCR was not found in the genome sequencing results, which may explain the non-toxic activity to the G. mellonella larvae. In addition to the above genes, there predicated some genes responsible for the secondary metabolites, such as bsh , which may be related to the bile salt tolerance and survival in the intestinal tract for A8-1; cap8E , cap8E , cas4J  genes related to capsular polysaccharide synthesis were also found in the genome, which may contribute to protect bacteria itself and resist the phagocytosis of host cells; fliN , which is involved in synthesis of flagellin, which could be recognized by Toll like receptor TLR5, and activate innate immunity, upregulate the expression of tight junction protein Occludin and mucin and protect the intestinal barrier.The drug-resistance and virulence genes were annotated by database of CARD and VFDB. Two aminoglycoside resistance\u2013related genes Enterococcus spp. are mainly from the gut of human and animal and can be detected in fecal samples, which are more competitive than isolates from other environments and deserve more attention for probiotic screening  and two G + indicator bacteria . Moreover, bacteriocin showed a narrow antibacterial spectrum against the same related strains. In this study, the growth of the indicator bacteria was used to determine the antibacterial ability of Enterococcus isolates, but the specific types and production of antibacterial active substances were not discussed. To further analyze the bacteriostatic mechanism of A8-1, the eight specific polysaccharide biosynthesis\u2013related genes, which were annotated in the genome sequence of A8-1, deserve more attention.The first step for good probiotics to exert probiotics in the host is to adhere to the cell surface, which is also the basis for probiotics to show the barrier protection function. Also, for probiotic enterococci, it is an important factor in colonization and competitive exclusion of enteropathogens . The strll model . The adhll model . Similarnsidered . Metabolnsidered . A8-1 haP > 0.05). A8-1 may have the potential to inhibit inflammatory response. When inflammation occurs, A8-1 can reduce the expression of cytoinflammatory factors to reduce the inflammatory response of cells to LPS. It is worth noting that probiotics have highly diverse effects on the level of immune regulatory cytokines, mainly related to the specificity of strains and cell lines , and aminoglycoside antibiotics are generally the preferred drugs for the treatment of enterococci infection, which may make the strains more resistant to such antibiotics  should be measured, and the cell tight junction protein can be further detected to explain the effect of A8-1 on the maintenance of gut permeability and intestinal barrier function and interpret the mechanism of inflammation suppression through the inflammatory response-related signal pathways. Furthermore, animal models will be established to evaluate the safety and functionality of E. durans that is able to tolerate and survive the simulated gastric and intestinal juices and has the potential to colonize the intestinal epithelial cells. Furthermore, we also showed that it contains no obvious pathogenicity or virulence genes. Taken together, our findings suggest the efficacy of probiotic E. durans A8-1 in exerting an adherence to the cell surface to show the barrier protection function and competitive exclusion of enteropathogens with reductions in the levels of inflammatory cytokines. According to the results of these evaluated attributes, E. durans strain A8-1 could be a promising probiotic candidate for applications.In summary, we have identified a strain of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/BH and YC: conceptualization and writing\u2014review and editing. YZ, JW, and LS: main experiments. LL and JY: genome analysis. RD: data and bacterial curation. BH and YZ: writing\u2014original draft preparation. BH: supervision. BH and RD: funding acquisition. All authors agreed to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Cancer cells alter metabolic processes to sustain their characteristic uncontrolled growth and proliferation. These metabolic alterations include (1) a shift from oxidative phosphorylation to aerobic glycolysis to support the increased need for ATP, (2) increased glutaminolysis for NADPH regeneration, (3) altered flux through the pentose phosphate pathway and the tricarboxylic acid cycle for macromolecule generation, (4) increased lipid uptake, lipogenesis, and cholesterol synthesis, (5) upregulation of one-carbon metabolism for the production of ATP, NADH/NADPH, nucleotides, and glutathione, (6) altered amino acid metabolism, (7) metabolism-based regulation of apoptosis, and (8) the utilization of alternative substrates, such as lactate and acetate. Altered metabolic flux in cancer is controlled by tumor-host cell interactions, key oncogenes, tumor suppressors, and other regulatory molecules, including non-coding RNAs. Changes to metabolic pathways in cancer are dynamic, exhibit plasticity, and are often dependent on the type of tumor and the tumor microenvironment, leading in a shift of thought from the Warburg Effect and the \u201creverse Warburg Effect\u201d to metabolic plasticity. Understanding the complex nature of altered flux through these multiple pathways in cancer cells can support the development of new therapies. Cancer is a complex genetic disease that arises from elaborate changes to the genome. This includes a cumulative collection of gain-of-function mutations that stimulate oncogenes, loss-of-function mutations that inactivate tumor suppressor genes, and mutations that inactivate stability genes involved in proliferative cell division, all of which help facilitate the transformation of a cell to a malignant phenotype. Characteristics typical of cell with a malignant phenotype include an unlimited ability to replicate, avoidance of apoptosis, insensitivity to anti-growth signals, continuous angiogenesis, self-sustained growth signals, tissue invasion, and metastasis . In ordeOtto Warburg\u2019s description of glucose metabolism in cancer cells was made almost a century ago, and it remains a key concept in the field of cancer metabolism. The Warburg Effect states that cancer cells rely on aerobic glycolysis (the conversion of glucose to lactate in the presence of oxygen) for ATP production, as compared to normal cells that rely on oxidative phosphorylation (OXPHOS) . Since WGlucose metabolism is considered one of the most important aspects of cancer cell metabolism as it supplies intermediates and precursors for several other key metabolic pathways, including the generation of amino acids, nucleotides, and lipids . Thus, oHow exactly does a cell shift from OXPHOS to aerobic glycolysis? Over the years it has remained a highly researched area and has been found to involve elaborate mechanisms and molecules. Hypoxic conditions, resulting from inadequate vascularization, act as key initiator in the transition. As hypoxia increases in the tumor microenvironment, the cells employ certain stress responses as a means of survival . These sAll of the aforementioned pathways and molecules have crosstalk with the master regulator and oxygen-sensing transcription factor HIF-1 . Some exRecently, a \u201cmetabolic plasticity\u201d theory of cancer cells has been described, where cells still have fully functional OXPHOS machinery and can switch between OXPHOS and aerobic glycolysis, or even perform them simultaneously . This afThe discovery of heterogeneity in tumors led to a paradigm shift from the Warburg Effect to the \u201creverse Warburg Effect\u201d, where aerobic glycolysis in cancer cells metabolically supports adjacent cancer cells . This alA second key source of energy for cancer cells is the essential amino acid glutamine (Gln). Glutamine is the most consumed amino acid in cancer and the dependence on glutamine for growth is a hallmark of the disease . Cancer To attenuate oxidative damage and produce additional macromolecules, cancer cells increase the process of glutaminolysis, although the levels vary with heterogeneity of tumor, patient, and cancer type . GlutamiReprogramming the cell to perform glutaminolysis is achieved through various oncogenes and is thought to be coordinated with the reprogramming of glucose metabolism  34]. On. On34]. Recently, research has described a shift in glutamine nitrogen metabolism, referred to as a \u201csecond Warburg-like effect\u201d . This efThe pentose phosphate pathway (PPP), also referred to as the hexose monophosphate shunt, is an offshoot pathway of glycolysis that plays an important role in glucose metabolism, and consequently, cancer metabolism . AlthougThe PPP is composed of two phases or branches that undergo reprogramming in cancer through various mechanisms. Commitment of a cell to the PPP is regulated by the first phase of the PPP, the oxidative branch . The firReprogramming of the oxidative branch is primarily achieved through mechanisms involving G6PD, as this enzyme is the \u201cgateway\u201d between glycolysis and the PPP. Hence, a large amount of research has focused on study of regulation of this enzyme . PPP oxiThe second branch of the PPP is the nonoxidative branch and utilizes the product of the oxidative branch, ribulose-5-phosphate (R5P), to generate glycolytic intermediates and nucleotide precursors . This brAdditionally, there is crosstalk between glycolysis and the PPP via the nonoxidative branch, which can greatly impact the regulation of these pathways in regard to cancer. The ability of the nonoxidative branch to adjust flux depending on metabolic needs allows it to act as a \u201cbridge\u201d between phase one of glycolysis and phase one of the PPP and allows cancer cells to exhibit \u201cmetabolic plasticity\u201d. Specifically, the different modes of the PPP influence glycolytic flux and vice versa. Increased glycolysis upregulates intermediates, such as F6P and G3P, which can be used to generate ribonucleotides in the nonoxidative PPP. Inactivation of glycolytic enzymes, such as PFK1, can occur during oxidative stress to increase production of NADPH via the diversion of G6P to the oxidative PPP ,54. CrosAberrant lipid metabolism is one of the most pronounced metabolic alterations in cancer, and it greatly contributes to cancer cell growth and tumorigenesis . Lipids,Cells can acquire lipids in one of two ways, de novo synthesis or uptake . Most liCancer cells activate de novo lipogenesis by upregulating several enzymes involved in the pathway, specifically acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase 1 (SCD1) . These ede novo lipogenesis provides cancer cells with the ability to shunt into different biosynthetic pathways to create lipids with a wide variety of functions that allow them to adapt and respond to their surroundings and ensure continued proliferation. Specifically, increased FA synthesis reduces the number of polyunsaturated FAs (PUFAs) and increases the number of monounsaturated FAs (MUFAs). This helps provide protection from lipid peroxidation as PUFAs are subject to peroxidation in the presence of ROS. Increased FA synthesis in cancer cells also confers protection from ROS, contributes to pro-angiogenic signaling, and provides an escape from immune surveillance , a, a111], The main outputs of 1C metabolism are purines and pyrimidines, which provide the building blocks for DNA synthesis. Nucleotide biosynthesis requires cofactors generated through 1C metabolism pathways . In cancCancer cells upregulate de novo nucleotide biosynthesis through both the aforementioned upregulation of 1C metabolic pathways and downstream pathways. A major mechanism specific to de novo nucleotide biosynthesis includes the upregulation of thymidylate synthase, inosine synthetase, and rate-limiting enzyme phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) via c-Myc expression ,130.SAM is the primary regulator of methylation levels in a cell that is produced from 1C metabolism. It is required for the methylation of DNA, histones, and other substrates . MethylaGlutathione is a tripeptide consisting of cysteine, glycine, and glutamate; thus, its production is regulated by 1C metabolic pathways . GlutathNADH, NADPH, and ATP are important for multiple metabolic and biosynthetic pathways. As such, rapidly proliferating cancer cells display an increased need for these molecules. There are several reactions of 1C metabolism that contribute to the generation of NADH, NADPH, and ATP. This includes the production of NADPH and ATP by MTHFD1 in the folate cycle, NADH production by MTHFD2, and NADPH production by MTHFD2L . SpecifiIn addition to NEAAs that are heavily studied, i.e., serine, glycine, glutamate, and glutamine, the NEAAs alanine, aspartate, asparagine, arginine, cysteine, and proline are emerging as players in the tumor metabolic landscape .Alanine is synthesized by alanine aminotransferases using carbon from pyruvate and nitrogen derived from glutamate. Although alanine contributes to major cancer growth pathways, its role in cancer is still up for debate. Thus far, the alanine biosynthetic pathway has been connected to cancer proliferation and the secretion of alanine by pancreatic stromal cells is used in the TCA cycle .Aspartate is linked to cancer growth and proliferation in a number of ways. Produced from OAA and glutamate-derived nitrogen, aspartate is crucial for the transfer of electrons from the cytosol to the mitochondria via the malate-aspartate shuttle (MAS). In the only irreversible step of the MAS, aspartate is exchanged for cytosolic glutamate and a proton by the aspartate-glutamate carrier (AGC) to provide electrons for OXPHOS. Since the concentration of aspartate in the plasma is low, cancer cells rely on the biosynthesis of aspartate by aspartate aminotransferase in the mitochondria ,134,135.Asparagine is another NEAA necessary for cancer cell growth under certain conditions. Specifically, when glutamine is depleted, asparagine is necessary for protein synthesis through its restoration of glutamine production ,136,137.Cysteine is used by cancer cells as a carbon source, with increased cysteine bioavailability acting as a stimulus for metabolic reprogramming. When there is no limit on cysteine uptake, its contribution to cell growth and proliferation mainly occurs through cysteine catabolism . CysteinMetabolic reliance on cysteine has been observed in several cancer types and concurrently involves the upregulation of cysteine catabolism and cysteine synthesis, as well as upregulation of the expression of cysteine transporters. Specifically, two enzymes involved in cysteine catabolism, cystathionine \u03b2-synthase (CBS) and cystathionine \u03b3-lyase (CSE), are often upregulated. Increased expression of CBS and CSE is linked to enhanced rates of proliferation in cancer cells and is controlled by PI3K/Akt and Wnt pathways, respectively. CBS and CSE also play a role in cysteine synthesis, which occurs through the transsulfuration pathway (TSP) from methionine and serine . TherefoProline is an important component of proteins, especially collagen, as its cyclic shape allows for a variety of protein structures. It is synthesized from glutamate and degraded by proline dehydrogenase. Both the biosynthetic and degradation pathways are regulated by Myc, giving proline context in the field of oncogenic signaling. Catabolism via proline dehydrogenase can promote cancer cell survival and have a tumor-suppressive function, depending on the type of tumor and the conditions of the tumor microenvironment. Proline is also a limiting factor for protein synthesis in some types of tumors . AdditioBranched chain amino acids (BCAAs) include the essential amino acids valine, leucine, and isoleucine and they play an important role in tumor cell growth and proliferation . BCAAs fRegardless of the mechanism preferred, the enzymes involved in the first step of BCAA degradation are upregulated in cancer and they are reversible. This includes cytosolic branched-chain aminotransferase (BCAT1) and mitochondrial branched-chain aminotransferase (BCAT2), which convert BCAAS into branched-chain \u03b1-keto acids (BCKAs) by transferring the amino group to \u03b1-KG to generate glutamate or the reverse reaction . SpecifiIn tumors that favor direct BCAA usage and low BCAA catabolism, such as breast cancer and leukemia, BCAT1 catalyzes the reamination of BCKAs to BCAAs, resulting in the accumulation of BCAAs. High levels of BCCAs then promote tumor growth by activating mTORC1 and the mTOR downstream signaling pathway ,140.Other tumors, such as gliomas, rely on increased BCAA catabolism via BCAT1 for growth. BCAA degradation restricts \u03b1-KG, which diminishes the activity of \u03b1-KG-dependent dioxygenases. Low \u03b1-KG levels attenuate the activity of a specific group of \u03b1-KG-dependent dioxygenases, termed EGLN prolyl hydroxylases. EGLN prolyl hydroxylases block HIF-1 activation by tagging it for proteasomal degradation, and thus, decreased \u03b1-KG levels impair this group of enzymes and cause HIF-1 activation. HIF-1 activation allows cancer cells to survive in hypoxic conditions through the activation of target genes. Additionally, increased BCAA catabolism results higher production of glutamate, which can be used for DNA and protein synthesis to facilitate cancer cell proliferation . Tumors After degradation, BCKAs then undergo decarboxylation via an irreversible reaction by the branched-chain \u03b1-keto acid dehydrogenase (BCKDH) complex, located in the mitochondria. The activity of the BCKDH complex is regulated by pair of enzymes, branched chain keto acid dehydrogenase kinase (BCKDK) and Mg2+ / Mn2+- dependent 1 K protein phosphatase (PPM1K), which are upregulated in many tumors. BCKDK overexpression suppresses BCKA decarboxylation but enhances tumor growth by increasing BCAA levels and activating the MAPK pathway ,146. WheWhile healthy cells typically undergo programmed cell death or apoptosis during regular progression of the cell cycle, cancer cells evade apoptosis to enhance growth, proliferation, and survival under hypoxic conditions. Many of their apoptosis evasion mechanisms are linked to metabolic reprogramming .Glucose metabolism, specifically enhanced glycolysis, is one of the main hallmarks of metabolic reprogramming in cancer. Glucose metabolism is linked to the evasion of apoptosis in several ways. Many of the same signaling molecules involved in the upregulation of glycolysis are also involved in the suppression of apoptosis. For example, the hypoxic tumor microenvironment induces expression of aforementioned HIF-1, which in turn, leads to overexpression of glycolytic enzymes. Upregulation of glycolysis and glucose uptake are linked to resistance to apoptosis as increased glycolysis and glucose uptake prevents oxygen related damage to the cell, and thus, results in decreased apoptosis. HIF-1 also directly induces resistance to apoptosis via suppression of pro-apoptotic B cell lymphoma 2 (BCL-2) family protein BH3 interacting-domain death agonist (BID)  148,149149.Akt, another regulator of glycolysis in cancer cells, blocks apoptosis via suppression of two pro-apoptotic BCL-2 family proteins, p53 upregulated modulator of apoptosis (PUMA)  and glycLipid metabolic reprogramming in cancer is also linked to the regulation of apoptosis, specifically via pathways involving sphingolipid production . CeramidAlthough the role of oncogenes, transcription factors, and other downstream signaling molecules has been widely established in mechanisms of cancer metabolic reprogramming, non-coding (nc) RNAs are emerging as important players. As ncRNAs were initially considered to lack biological function because they do not encode proteins, they play a role in cancer progression by regulating enzymes and pathways involved in the metabolic reprogramming of cancer cells. This regulation primarily occurs through glucose, glutamine, and lipid metabolism and involves two types of ncRNA, long-chain non-coding RNA (lncRNA) and microRNA (miRNA) . Our recent understanding that ncRNAs can affect a cell has also implicated deregulated ncRNA expression in cancer development and progression. Amplification of chromosomal regions that encode for oncogenic ncRNAs are found in cancer . Some ofThe reprogramming of metabolic pathways in cancer involves not only tumor cells themselves but also interactions between tumor cells and host cell populations . The tumNutrient sharing and metabolic symbiosis are common in multiple types of tumors and most significantly involve lactate. Glucose-derived lactate plays a multifaceted role in that it can originate in hypoxic cancer cells and feed nearby tumor cells, originate in fibroblasts and feed tumor cells, or originate in tumor cells and feed mesenchymal stem cells and fibroblasts. It can also act as a signaling molecule and affect immune cell populations, either by polarizing macrophages toward a tumor-associated macrophage fate or inhibiting antitumor T cells . This moNutrient recycling between tumor cells and fibroblasts is also important for tumor growth. The main function of fibroblasts is to produce and secrete extracellular matrix, which increases the diversity of macromolecules surrounding the tumor cells . The macWhile many symbiotic tumor-host cell metabolic interactions contribute to tumor growth, competition between tumor and host cells for nutrients also significantly influences cancer progression . In ordeIt has been suggested that treatment with a ketogenic diet impairs the metabolism of cancer cells and restores immune cell function. High in fat and protein and low in carbohydrates, a ketogenic diet results in less carbohydrate uptake, and consequently, leads to cancer cell starvation and cell death . A ketogIncreased knowledge and understanding of metabolic reprogramming have led to the development of cancer therapies that target various aspects of metabolism. Pharmacological targeting of these pathways has the potential to significantly reduce cancer growth and proliferation. This is evident by the large number of drugs in development that target glucose metabolism, lipid metabolism, one-carbon metabolism, and various growth pathways . With fuMuch progress has been made in the field of cancer metabolism since Warburg\u2019s initial observations about 100 years ago. While cancer cells were previously thought to perform only aerobic glycolysis, it is now evident their growth and proliferation is dependent on the reprogramming of a large number of pathways, such as the TCA cycle, the PPP, lipogenesis, 1C metabolism, and BCAA metabolism, as well as the utilization of alternate substrates, conferring them with metabolic plasticity. Altered flux through metabolic pathways is coordinated by many different genes and regulatory molecules that work together to support enhance growth and proliferation. Crosstalk between many of these pathways is evident, including that between glycolysis and the PPP and the regulation of apoptosis through metabolism. Understanding the complex nature of altered metabolic flux in cancer cells and its context dependent nature is crucial to the development and application of new therapies."}
+{"text": "This work can enrich the investigation of RTV, and may provide useful reference for performance evaluation and replacement of RTV in substations.Due to high humidity, the ageing of room temperature vulcanized silicone rubber (RTV) has been a serious problem in Southwestern China. In order to solve the problem of RTV life prediction, the aging classification method was established by analyzing the microtopography of RTV samples in this paper. Besides, the comprehensive analysis of RTV element content, partial element content ratio, and major chemical groups of RTV samples in each aging level were conducted. It is found that as the ageing level increases, the element contents of C, Si, O, Al change accordingly and the ratio of C:Si drops from 2.39 to 1.54, and absorption peaks of the chemical groups of Si-(CH In recent years, due to the excellent hydrophobicity and anti-pollution performance, RTV has been widely used in transformer substations \u20133. HowevDuring the service, silicon rubber endure strong electric field and can possibly be affected by many factors such as humidity, pollution and ultraviolet radiation . After a2 composition can be used to evaluate the surface state of RTV [Outdoor insulation performance of RTV is determined by its surface state, researchers have carried out a lot of work on polymers surface condition monitoring \u20139. Basede of RTV . Xia este of RTV . Alok ste of RTV . Besidese of RTV . It can Besides, the relationship between physical and chemical properties and surface state can reveal the changing mechanism of RTV. Rahmat have done a lot of work to study the physical and chemical properties of polymer material. The hydrophobicity, mechanical test, leakage current test, Fourier infrared spectroscopy (FTIR) and scanning electron microscope (SEM) test are analyzed to study the influence of the multi-stress aging on high temperature vulcanized silicone rubber \u201316. It iIt can be concluded that the study of RTV from on-site operating condition is relatively lacking. In this paper, the RTV samples from high humidity area are studied. Based on the microtopography of RTV surface, a new classification method is proposed. Moreover, the relationships between physical and chemical properties and surface state are established by adopting a variety of detecting methods.In this paper, the RTV samples in the Yibin \u00b1800kV UHV DC converter station are studied. Yibin \u00b1800kV UHV DC converter station is owned by the State Grid Corporation of China, and we have got the authority to test the RTV coatings of the post insulators in this converter station. The converter station is located in Shuanglong Town, Yibin City, China, 104\u00b035\u2032 east longitude and 28\u00b045\u2032 north latitude. The annual average temperature is only 18\u00b0C, the average annual sunshine hours are 976 hours, the annual average air humidity is as high as 80%, and the climate type is mid-subtropical humid monsoon climate.All the samples have been in service for more than 7 years, and they are from high-voltage post insulator and low-voltage reactor post insulators respectively. 20 typical pieces of RTV samples are shown in 3) wrapping on the outside of the long chain.The main molecular structure of RTV used in Yinbin converter station is polydimethylsiloxane (PDMS) . The molSix tests were performed for the RTV samples, including hydrophobicity, surface morphology, surface microtopography, element content, chemical structure and dielectric properties. All the tests were conducted in the condition monitoring of power transmission and transformation equipment research center of Shaanxi Province, and the test instruments are shown in The OCA40Micro static contact angle measuring instrument produced by German Dataphysics Company is used to test the static contact angle of the RTV surface.A video microscope produced by Nanjing Nanpai Technology Co., Ltd. is used to analyze the particle size of the RTV surface.The VEGA 3 SBH scanning electron microscope (SEM) produced by Czech TESCAN is used to characterize the RTV surface microscopic morphology, and the magnification is set to 1000 times.The Octtance Prime energy dispersive spectrum analyzer (EDS) produced by EDAX, USA is used to analyze the surface chemical elements of RTV.-1~400cm-1.The Vertex70 infrared spectrometer (FTIR) produced by German Bruker is used to analyze the surface chemical structure of the RTV sample, and the analysis wavelength is set to 4000cmBroadband dielectric spectroscopy impedance meter produced by Novocontrol, Germany is used to analyze the dielectric constant, dielectric loss and conductivity of RTV sample at 20\u00b0C and 50Hz.The static contact angle method is employed the test the RTV samples . And theIn this section, SEM is used to get the surface micromorphology of RTV samples. The typical SEM image of one single RTV sample is shown in The RTV samples with surface roughness of 10\u201340\u03bcm, 40\u2013100\u03bcm and over 100\u03bcm are primarily designated as ageing level 1, ageing level 2 and ageing level 3, respectively.Level 1: The microscopic morphology of RTV samples in this level with a particle diameter of 10\u201340\u03bcm is shown in Level 2: The microscopic morphology of RTV samples in this level with a particle diameter of 40\u2013100\u03bcm is shown in Level 3: The microscopic morphology of RTV samples in this level with a particle diameter more than 100\u03bcm is shown in To further evaluate the surface state of RTV samples, surface morphological parameters should be acquired. A portable video microscope with the magnification ranging from 1 to 225 was used to test all the 20 samples. And the morphology pictures of RTV samples in each levels are acquired, and they are shown in The typical particle size distribution of According to the observations in this section, comparison of average particle diameter method and SEM surface roughness method, the surface state of the sample less than 100\u03bcm observed by portable microscope is similar to the level 1 observed by SEM. The surface state of samples larger than 100\u03bcm observed by portable microscope is similar to the level 2 and 3 observed by SEM. It shows that the surface state proposed by the portable microscope used are consistent with the three aging classification proposed by SEM. And this method can be used as a supplement to the surface roughness method.In the previous section, 20 pieces of RTV samples are divided into three different ageing levels according to their microscopic morphology (SEM). Multiple testing methods including element content analysis, FTIR analysis and dielectric properties analysis are conducted in this section. The chemical properties of RTV samples with different levels are also given in this section.By analyzing the molecular structure of PDMS, the relative contents of elements C, Si, O, Al are selected as characteristic element. The average relative content of these four elements at three different ageing levels are shown in According to the PMDS molecular formula, the relative ratio of C:Si should be more than 2, which is consistent with the results of EDS analysis. The average content ratio of C:Si of RTV samples with different ageing levels are shown in The main components of this type of RTV formula are PDMS, Aluminum Trihydrate (ATH) and Silica. In FTIR, the aging level of RTV can be evaluated by the absorbance peaks of several chemical groups. The absorbance of different chemical groups are shown in 3)2, Si-O-Si, Si-CH3, C-H in CH3 of RTV samples under different ageing levels are analyzed, as shown in The average absorption peaks of four chemical groups Si- in more detail. According to the particles, holes and cracks on the RTV surface under 1000 times, the RTV aging state is divided into three levels. Other method is the average particle diameter analysis method based on a portable microscope (\u00d750), this method divides the RTV aging state into three levels based on the RTV surface particle diameter.3)2, Si-O-Si, Si-CH3 and C-H in CH3 gradually decrease with increasing roughness level of RTV. Therefore, the surface roughness level is effective to reflect the ageing level of RTV.As the surface roughness and the average particle diameter of RTV increases, atom content of C, O, Si and Al and the ratio of C:Si of RTV coating change significantly. The ratio of C:Si drops to 1.69 at level 2, indicating that the PMDS molecular structure is severely damaged. In addition, the absorption peaks of chemical groups such as Si-Click here for additional data file."}
+{"text": "Characteristics of the translation product are often used in translation process research as predictors for cognitive load, and by extension translation difficulty. In the last decade, user-activity information such as eye-tracking data has been increasingly employed as an experimental tool for that purpose. In this paper, we take a similar approach. We look for significant effects that different predictors may have on three different eye-tracking measures: First Fixation Duration (duration of first fixation on a token), Eye-Key Span (duration between first fixation on a token and the first keystroke contributing to its translation), and Total Reading Time on source tokens (sum of fixations on a token). As predictors we make use of a set of established metrics involving (lexico)semantics and word order, while also investigating the effect of more recent ones concerning syntax, semantics or both. Our results show a, particularly late, positive effect of many of the proposed predictors, suggesting that both fine-grained metrics of syntactic phenomena (such as word reordering) as well as coarse-grained ones (encapsulating both syntactic and semantic information) contribute to translation difficulties. The effect on especially late measures may indicate that the linguistic phenomena that our metrics capture  are resolved in later stages during cognitive processing such as problem-solving and revision. Translation difficulty prediction, which aims to assess the difficulty of a translation task, is a topic of interest within Translation Studies that can benefit both pedagogical and research settings. Advances in translatability could for instance ensure that appropriate text material is used in translation classes, and to create general-purpose machine translation (MT) systems that are trained on a balanced mix of simple and hard texts. On the other hand, it could also help the research fields of Translation Studies and psycholinguistics to select source material of suitable translation difficulty for experiments. Even though a well-established methodology to quantify a source text's translatability does not exist (yet), the problem of translation difficulty has gained some attention over the years.r correlation. Additional sentence-level metrics were introduced in Vanroy et al.  by only making use of the source text, similar to our previous work . Finally, we end with broad conclusions and suggestions for future research in section 6.A lot of work has been done on the relationship between ST and TT, particularly on the concept of literal translation and the  transfer of the source text to the target. We will discuss one specific way how literal translation can be operationalised  translation that is being produced and that this effect decreases over the duration of the translation process. The literal translation hypothesis has received supporting evidence from translation process studies that measure the effects of literality metrics (see below) on process data semantic properties of a source sentence with its translation. As such, these metrics operationalise literality and can be used to measure the impact of literality, but also of divergent structures in general, on the translation process.The literal translation hypothesis and the way that is has been operationalised, is often used in translation process research as predictors for cognitive load during translation  between the source text and a plausible translation. We focus on the latter type of difficulties. The metrics that follow were all suggested in previous work to model the relationship between a source sentence and its translation in different ways. They all rely on word alignment information. Word alignment is the linking of a source word with its translated word(s) so that the relationship between smaller units can be quantified.To be able to investigate the first point of the definition of literal translation of Schaeffer and Carl  \u201cthe worr = 0.8 with the real population , then HCross will be low, and otherwise it will be high. Schaeffer and Carl  find thaWhere HCross is a way to quantify the uncertainty of word reordering, word translation entropy  are suitable translations. It gives us a (limited) insight in the different options that translators can choose from . A high HTra value means that many options are available and that a single, straightforward choice is not necessarily available. As a consequence, a high word translation entropy is expected to have an impact on process data as well: more choices to choose from for a given word in a specific context, is likely to require more time to make a decision. This has been confirmed in a number of studies. Effects of HTra were reported on total production duration . In a way, HSTC encompasses both HTra and HCross discussed above. It is intended as a single metric to measure the (non-)literality of a translation, both (lexico)semantically and syntactically.Recently, a new entropy-based metric has been introduced that incorporates different types of information into a single metric Carl, . It is cCarl  shows thWith the exception of Cross, the above measures are all meant to be calculated involving a relatively high number of translations. The main idea is that a sufficient number of translations approximate all the possible choices that translators are faced with, and that more choices (or less-straightforward ones) lead to a more difficult translation process. Vanroy and colleagues introduced different syntactic metrics that are not reliant on multiple translations and each focus on different aspects of syntactic differences between a source text and its translation , which means that for some aligned structures the cross value of a source word could differ from its aligned target word, because that target word is aligned with other source words as well. In Vanroy et al.  reordering on process data. Similar to word_cross above, sequence cross (or seq_cross) was introduced in Vanroy et al.  between the two, a common metric to measure differences between trees. TED looks for the most optimal way to transform the source tree into the target tree by making use of different operations: match  source item(s) and its corresponding target item(s). In process-based studies, the focus lies on the source text. The translation unit here is considered to be the source item(s) that a translator processes one at a time  the unit of translation remains a much discussed topic, approached from different directions. A distinction can be made based on the focus of the research, i.e., the translation process or its product. In product-emphasised studies, it is generally accepted that the translation unit (TU) is a Rather than investigating a single type of translation unit in process data, Carl and Kay  proposesAlves and Vale , and conImmonen and M\u00e4kisalo  aim to fIt is clear that research is actively involved in the translation unit, but clear-cut definitions do not exist. A translation unit is a variable concept: it differs between participants and tasks, and may or may not necessarily correspond to syntactic units. In this paper, however, we rely on the minimal product-based view that a translation unit is a pair of  source and target items. We investigate both small, word-based units and larger (word group) units.In this section, we first discuss a couple of improvements that were made for the current paper to metrics that we introduced in earlier work. Then, we describe our dataset and the processing that was applied to it, followed by a description of the regression models that were built and the involved variables.In section 2.2, we discussed methods to quantify the relationship between a source sentence and its translation. Methodologically, the current paper makes some small improvements to the metrics that we introduced in Vanroy et al. .word_cross, seq_cross, and ASTrED. The sequence cross value of a group is passed on to all the words belonging to that group. Each word thus has a word_cross value, based on word alignment and its own reordering, and a seq_cross value that is based on the alignment of the word group that it belongs to. These sequence alignments  can greatly reduce the number of alignments and, consequently, the cross values calculated on these groups (seq_cross) can be much smaller than their word_cross equivalent because there are less (group) alignments present in the sentence to cross compared to word alignments.First and foremost, previous work focuses on sentences. In the current study we zoom in on individual words. That means that some metrics were re-implemented so that word-level analyses could be done.seq_cross itself was improved as well. We now consider m-to-n alignments of consecutive items as valid aligned word groups. In other words, Requirement 1c does not apply to these so-called multi-word groups (MWGs), but as an alternative requirement all source words need to be aligned with all target words of the construction. The assumption here is that m-to-n alignments are used for groups of words or phrases that cannot be easily compositionally aligned, such as idioms or free translations of specific concepts. Semantically, however, the source and target side should constitute the same concept or phrase. Note that this does not necessarily mean that from a monolingual perspective these constructions are multi-word expressions or idiomatic expressions: MWGs are purely based on the alignments between the source and target words belonging to the construction. As an example of a MWG, consider the following translation, where \u201cmarine sentinels\u201d \u2014 \u201cwachters van de zee\u201d constitutes a MWG according to our specification and as such only one alignment link will be needed between the two groups rather than the m-to-n word alignments .word_cross value of 8. However, its sequence alignment , has a seq_cross value of 1 because the m-to-n construction that it crosses is considered a valid sequence and only has one alignment link connecting \u201cmarine sentinels\u201d to \u201cwachters van de zee\u201d instead of eight. Example 2 can be visualised as in word_cross, and sequence cross. The groups of words that adhere to the requirements above are boxed in and aligned (solid black lines). Their original word alignments are given in grey dotted lines. If a word does not belong to a multi-word group, it is its own singleton group . Cross and word_cross are calculated on the alignments of the single words, whereas sequence cross uses the alignments between word groups. On the word-level , \u201ccalled\u201d crosses eight alignment links. On the word-group level, however, this is reduced to only one.Note that allowing m-to-n alignments to be groups, also greatly reduces the sequence cross value of other words: because \u201ccalled\u201d is aligned with \u201cgenoemd\u201d it crosses the m-to-n alignment, leading to a large astred_change value of \u201cFALSE\u201d (match) or \u201cTRUE\u201d (no match), indicating whether a specific operation needs to occur on this word.Minimal changes were made to ASTrED as well to accommodate the focus on the word level. For each source word we check whether it was matched (and not changed) or whether an edit operation was necessary to transform this specific node to create the target tree (changed). These operations can only be deletion or substitution because insertion can only happen for target words. Each word, then, has an For our experiments, we use a subset of ROBOT Daems, , a pre-eThe full dataset consists of post-edited and from-scratch translations of eight news articles by ten student translators (P1-P10) and twelve professionals  , Eye-Key Span (EKS), and Total Reading Time on source tokens (TrtS). FFDur, a very early measure, is the time in milliseconds of the first fixation when a source word is first encountered. Eye-Key Span is the time between the first fixation on a source word and the first keystroke that contributes to the translation of that word  as predictors in our regression models. We repeat them below for clarity. These predictors were chosen because there is a lot of variation in the aspects that they model: some are semantic, others are syntactic; some require multiple translations and others do not; some are word-based whereas others make use of word groups. Our experiments compare these different aspects to one another in terms of their effect on the translation process.Cross (section 2.2.1): relative word reordering. We use the absolute value of Cross in our experiments : entropy version of CrossHTra (section 2.2.3): word translation entropyword_cross (section 2.2.5): absolute word reorderingseq_cross : absolute word group reorderingastred_change : compares linguistic structure of source and target sentence while taking word alignment information into accountlmerTest . We use HCross as the base model because it is a syntactic, entropy-based measure. The metrics by Vanroy and colleagues are also syntactic, but not entropy-based, which can lead to an interesting comparison. If convergence was not possible in subsequent models with the new predictors, we excluded predictors one by one until convergence was possible and compared these to a base model with the same predictors\u2014apart from HCross.Prior to model building, for each dependent variable, we excluded data points from the raw data which differed by more than 2.5 standard deviations from the mean for each participant. This resulted in no case in a loss of more than 3%. All models had, as random variables, participant and item . The first model we built always included HCross\u2014whether it was significant or not. We then included word form frequency . 99.6% of the First Fixation Duration after exclusion of outliers on the basis of the raw data were under 500 ms, while 73% of the excluded outliers were over 500 ms . 87.9% of the Total Reading Time after exclusion of outliers on the basis of the raw data were under 5,000 ms. 68.7% of the excluded outliers were over 16,000 ms . 86.7% of the EKS datapoints after exclusion of the outliers on the basis of the raw data were under 500.000 ms, while 85.8% of the excluded outliers were over 500.000 ms (range 91.580\u20132.648.372 ms). In other words, extremely long First Fixations, Total Reading Times and EKS were excluded. This is reasonable practice. The fact that often, model results were different after exclusion of residual outliers suggests that these results were often strongly affected by residual outliers, as will be shown. In the interests of transparency, we report model results before and after of exclusion of residual outliers.2-test, and Akaike's Information Criteria , which reports the variance of the fixed effects only. In all models, skewness was below |1| and kurtosis below |3| after exclusion of residual outliers. Variance inflation factors in all models were below 2.Finally, we compared models in which the critical predictors were significant with each other, again via the anova function. We report results from the \u03c7word_cross, seq_cross, astred_change, absolute Cross, HCross, HTra, HSTC on three eye-tracking measures: First Fixation Duration, Eye-Key Span, and Total Reading Time on source tokens. In the overview tables, the \u201cANOVA (HCross)\u201d column compares each model individually with HCross (\u03c72). This HCross model is always shown first. \u201cANOVA\u201d compares for each model whether it significantly improved over the previous model . \u201cbase\u201d indicates when a model has been used as the first reference model in an ANOVA. When the models are compared, all residual outliers are included. The variance that they account for is given in \u201cR2 (outliers).\u201d Separate models are also built that exclude for each model its respective residual outliers. These results are reported in \u201cR2 (no outliers).\u201d In each table only those predictors are included that had a significant effect (with or without outliers) on the dependent variable. Significance of the specific predictor under scrutiny are given in the p columns. The individual significance levels of secondary fixed effects  were not reported but in all cases they were significant (p <0.05). The BIC and AIC columns are given for transparency to indicate the absolute goodness-of-fit of the models (lower is better), as discussed in section 3.3.In this section, we present the effects of the predictors word_cross and HSTC have a significant effect. HCross performs best in terms of BIC/AIC as well as R2 when outliers are included. Neither word_cross nor HSTC perform better according to the ANOVA. However, when outliers are removed, only word_cross has still a significant effect suggesting that outliers were driving the effects in HCross and HSTC in the first place. Only very little variance is explained in these settings.word_cross and HSTC are given in The effect plots for the base model HCross, seq_cross only converged when the word ID (the index of the word in the sentence) was excluded as a predictor . Therefore, a separate HCross model was built (HCross+) that similarly contains the source text ID (the index of the word in the text) and word frequency, but not the word ID. With these fixed effects, seq_cross performs significantly better than HCross according to the ANOVA but it is also evident from their respective BIC/AIC values. On top of that, HCross does not have a significant effect in this context. For that reason, the HCross+ model was not included in the second ANOVA. All results with respect to EKS are given in Eye-Key Span is considered a late measure, assuming that the translator has fixated a word long enough to at least start producing a translation for it. It does imply, however, that initial problems have been resolved when the production of a word starts (but revision may still happen at a later stage). Many predictors show a significant effect. However, astred_change, HTra and HSTC. The base model HCross . seq_cross and HSTC, word group based metrics, are the best performing models according to their BIC/AIC, with HSTC coming out on top. Their effect is highly significant (p <0.01). Absolute Cross is the third best fitting model followed by HTra and finally word_cross. The fixed effects in the HTra model explains the most variance in Total Reading Time, however. Note that HCross did not have a significant effect. Therefore, it was not part of the second ANOVA comparison. In that case, the word_cross model was the reference model (because it has the highest BIC/AIC), although it was just marginally significant in the first place.Similar to Eye-Key Span, Total Reading Time , is affected by many predictors . The basword_cross (the base model for the ANOVA comparison), seq_cross and HSTC are visualised in The effects of word_cross was significant both with and without outliers but again, the variance explained was very small.In our experiments, we see very little effect of our predictors on the early measure of First Fixation Duration (FFDur) and those that are significant only explain variance by a minimal amount. Furthermore, both HCross and HSTC (both entropy measures) lose their significance when their residual outliers are removed. The effect of HTra and absolute Cross on FFDur as reported in Schaeffer et al.  could noseq_cross can be observed but the explained variance is low as is the significance of the predictor. This effect is only present when the word ID predictor is dropped. Because of that, a fair comparison cannot be made with the other predictors by themselves for this dependent variable. Except for word_cross, which is not significant, all other predictors show a positive significant effect. Especially the measures involving semantic information perform well , closely followed by structural changes between ST and TT (astred_change). Absolute Cross is further behind, with a considerable gap in BIC/AIC between astred_change. It is also not significant without the outliers. The same is true for HCross. Therefore, we can cautiously confirm the results reported in Schaeffer and Carl  and performs significantly better than absolute Cross, although the difference in R2 is minimal. All predictors explain more variance in TrtS than any predictor could in EKS. The reason for this may lie in late, conscious processes. Even after a translation is being generated (EKS is the time from the first fixation on a word until the first keystroke that contributes to its translation), additional fixations on a word may indicate control and revision processes that are active. The implication could be that more divergent source and target structure (in terms of the significant predictors) require longer control and/or revision processes but this needs further investigation. Surprisingly, the significant positive effect of astred_change did not continue in TrtS. This could be related to the aforementioned control processes: syntactic divergent structures may have a significant impact on the problem-solving process right before a translation can be produced , but as soon as that problem is resolved, such structural issues are not likely to cause issues during later fixations on the word .In Total Reading Time, similar effects can be observed with respect to the semantic measures . Interesting, however, is that both absolute Cross and r et al.  concerniseq_cross and HSTC involve word groups, it is tempting to attribute their significant effects on late processes, especially TrtS, to a gradual increase of the cognitive unit of translation . However, because absolute Cross is word-based, the suggestion would be that the unit of translation increases in a compounding manner. In other words: in later stages of the translation process, both individual words and (surrounding or involved) word groups are important to the translator. During later processes, a translator may be trying to incorporate or resolve larger units while still taking into account the properties associated with the single word. As mentioned before, a lot of research exists on translation units  and insertions and deletions (partly what astred_change models) need more specific attention from the translator. But it does not explain why semantic measures such as HTra and HSTC only have a late effect; the variance in FFDur that is explained by the fixed effects (with HSTC) is very small and HSTC does not have a significant effect when residual outliers are excluded. It may be the case that TL features are activated during first contact but that they simply do not pose a problem yet. Another likely explanation is that more data (in terms of the number of data points) is needed to show consistent, early effects.Why we found more effects in late measures  compared to early eye-tracking measures is not easy to explain. One possibility is that our metrics especially model language properties that need conscious decisions. Whereas early measures are often indicative of automatic processes, later measures hint toward conscious decision-making and problem solving, which cannot be resolved automatically Kiraly, . This exr = 0.8 when approximately ten translations are available for a given text (we have between nine and eleven). It is hard to tell then whether entropy-based metrics based on more translations would lead to a greater effect on the process data.Conclusions concerning entropy are difficult to make because a variety of factors are involved. HTra and HSTC both have a semantic component, whereas HCross and HSTC contain syntactic information. HSTC involves word groups, whereas HTra and HCross are metrics on the word level. A single statement on the effect of entropy cannot be made. What we can indefinitely say, though, is that more translations could change the picture. Carl  shows thseq_cross, which is based on word-group reordering, has a particularly strong significant late effect which poses interesting questions about the cognitive unit of translation and how that unit might change during the translation process.Although strong conclusions are hard to draw because of the size of our dataset, our results indicate that particularly late process measures are affected by the predictors. The reason for this may lie in the conscious processes that occur in such late stages, like problem-solving and revision. In addition, we find that HSTC, an entropy-based metric that incorporates both word group translation and reordering probabilities, is the best-fit predictor across the board. This is perhaps unsurprising, exactly because it entails both syntax and lexicosemantic information while also being based on all available translations. In terms of metrics that are not based on probabilities, absolute Cross has a consistent significant effect in the late measures. In this paper we investigated the effect of a number of predictors that each model different parts of the relationship between a source text and its translation(s). Although our results are promising, \u201cit is dangerous to make sweeping generalisations about translation processes\u201d  for a given source word, which in turn can be used in a translatability measuring system which predicts difficulties for a given source text without access to a translation.Specifically for the PreDicT project, it is very promising to see that metrics that do not rely on multiple translations also show an effect. Ultimately we wish to predict the difficulty of a given source text, and these results indicate that such singular metrics have predictive power as well. Technically speaking, that is very important: it is much easier to find parallel corpora with one translation than with multiple translation. Such large parallel corpora can be used to train a machine learning model to predict these relevant features  the translation unit, the distinction between (lexico)semantic and syntactic predictors (and their relevance in the time course of the translation process), and whether or not entropy-based measures are a necessity in predicting cognitive effort.The dataset Daems,  analysedBV, MS, and LM discussed and agreed upon the experimental design of the study. Data (pre)processing  was done by BV. MS performed all of the statistical experiments in R. The results were discussed and agreed upon by BV, MS, and LM. BV wrote the first draft of the article with the exception of the description of the creation of statistical models, which was written by MS and revised by BV for consistency with the rest of the text. MS and LM made suggestions for improvement, which BV took into consideration in subsequent revisions. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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": "Recent publications have presented research showing that WD repeat domain 4 (WDR4) plays a significant role in various kinds of malignant tumours. However, the expression profile of WDR4 is still unspecified, as is its significance in the analysis of human pan-cancer. We conducted an in-depth analysis of three aspects of WDR4 expression patterns from 33 types of cancer and determined the value of WDR4 for prognostic prediction and carcinoma drug resistance prediction. WDR4 was expressed in different cancer cell lines at inconsistent levels. Aberrant expression of WDR4 has been observed in various malignant cancers and is significantly implicated in overall survival outcomes. The expression level of WDR4 is also strongly associated with tumour immunity, such as immune scores and tumour-infiltrating immune cells. The level of WDR4 is related to microsatellite instability and tumour mutation burden in several types of malignancy, and validation studies implied that WDR4-associated terms and pathways are involved in malignancy. We explored the expression level of WDR4 across 33 types of cancer and showed that WDR4 plays a significant role during cancer development. More crucially, WDR4 is associated with immune infiltration, which suggests that WDR4 could be an immunotherapy target in cancers. In summary, our research showed that WDR4 plays a vital role in tumorigenesis and has the potential for to be targeted with treatments. Over the recent years, the incidence of carcinomas has increased at a substantial rate worldwide, which is primarily attributable to lifestyle and improvements in healthcare methods for detecting tumours. Malignant tumours are one of the principal causes of mortality in both developing and developed nations, with limited therapeutic success achieved worldwide , 2. CurrWD repeat domain 4 (WDR4) is a member of the WD-repeat protein family, which is associated with a variety of cellular developments, including cell cycle evolution, signal transduction, gene regulation, and apoptosis \u201317. WDR4In our study, we aimed to investigate the expression of WDR4 and its prognostic significance in human tumours using data from the TCGA. The involvement of WDR4 in tumour infiltration, microsatellite instability (MSI), and tumour mutational burden (TMB) was analyzed in various types of cancer. Gene set enrichment analysis (GSEA) was performed to explore the principal mechanisms. The results of this study provide information regarding the role of WDR4 in tumours, reveal the relationship between WDR4 and tumour-immune interactions, and clarify the potential underlying mechanisms.https://tcga-data.nci.nih.gov/tcga/). Our research comprises more than 20,000 initial tumour samples and corresponding non-carcinoma samples for 33 types of carcinoma. The data from the Cancer Cell Line Encyclopedia (CCLE) project were downloaded from the website http://www.broadinstitute.org/ccle. For this study, only open-access data were used, which excluded the requirement of authorization from the Ethics Committee.The patient datasets and processes were taken from The Cancer Genome Atlas (TCGA), a cornerstone of cancer genomics projects.  analysis was implemented to evaluate the association between overall survival (OS) outcomes among TCGA carcinoma patients and the WDR4 expression level by the log-rank test, and a survival-associated forest plot was generated.WDR4 gene expression data was obtained from the TCGA to compare the expression levels among the carcinomatous and adjacent normal samples. A univariate Cox model was applied to evaluate the association between patient survival outcomes and WDR4 expression levels. A https://cistrome.shinyapps.io/timer/) is a complete method for the systematic study of the immune infiltration level of several types of carcinoma [The tumour immunity estimation resource , analysed the TMB scores, and determined the association between TMB and WDR4 expression levels. MSI is considered an extensive polymorphism of the microsatellite sequences resulting from DNA polymerase slippage. Recently, it was hypothesized that cancer patients with elevated MSI benefit from immunotherapy, and MSI has been used as a marker of genetic uncertainty in malignancy. We analysed the MSI score of every patient and then performed a correlation analysis between WDR4 expression levels and the MSI score.TMB represents the number of alterations in a specific cancer genome. Many studies have revealed the importance of TMB as a predictive biomarker for patient checkpoint inhibitor sensitivity . We acquhttp://www.broadinstitute.org/gsea/index.jsp). When the number of random sample arrangements was 100 and the significance threshold was P < 0.05, R software (http://r-project.org/) and Bioconductor (http://bioconductor.org/) were applied to visualize our results.Additional gene set enhancement investigation was similarly achieved utilizing GSEA  software v2.2.1 , BLCA (P < .001), BRCA (P < .001), KICH (P < .001), LGG (P < .001), LIHC (P < .001), LUAD (P < .001), MESO (P < .001), SARC (P < .001), SKCM (P < .001) and UVM (P < .001); however, WDR4 expression appeared to be a protective factor in READ (P < .001) as shown in P < .001), KIRC (P = .043), LGG (P < .001), LIHC (P < .001), LUAD (P < .001), MESO (P < .001), READ (P < .001), SARC (P < .001) and UVM (P < .001), as illustrated in In the OS outcome study, Cox regression revealed that higher expression of WDR4 is a risk factor in ACC (P < .001), BLCA (P < .001), BRCA (P < .001), KICH (P < .001), KIRC (P < .001), LGG (P < .001), LIHC (P < .001), LUAD (P < .001), MESO (P < .001), SARC (P < .001), SKCM (P < .001) and UVM (P < .001), as illustrated in P < .001), BRCA (P < .001), KICH (P = .01), LGG (P < .001), LIHC (P < .001), LUAD (P < .001), MESO (P < .001), SARC (P < .001) and UVM (P < .001), as illustrated in The Cox regression analysis of DSS indicated that high WDR4 expression is a risk factor in ACC (P = .048), BRCA (P = .032), KIRP (P < .001), LIHC (P = .002), and SARC (P = .0014), as illustrated in P = .024), BRCA (P = .01), KIRP (P = .0011), LGG (P < .001), LIHC (P < .001) and SARC (P < .001), as illustrated in The Cox regression analysis of DFI indicated that higher WDR4 expression was a risk factor in ACC (P < .001), BLCA (P < .001), BRCA (P < .001), KIHC (P < .001), KIRP (P < .001), LGG (P < .001), LIHC (P < .001), LUAD (P < .001), MESO (P < .001), PAAD (P < .001), PCPG (P < .001), PRAD (P < .001), SARC (P < .001) and UVM (P < .001), as illustrated in P < .001), KICH (P < .001), KIRP (P = .0011), LGG (P < .001), LIHC (P < .001), MESO (P = .004) and SARC (P < .001), as illustrated in The Cox regression analysis of PFI revealed that higher WDR4 expression is a risk factor in ACC (Tumour-infiltrating lymphocytes (TILs) can be independent predictors of SLN (sentinel lymph node) involvement and cancer survival outcomes. We observed an association between WRD4 expression levels and the levels of immune infiltration in numerous carcinoma types using TIMER analysis. It was discovered that the WDR4 levels were significantly correlated with the infiltration levels of CD4+ T and CD8+ T cells in 21 types of carcinoma, B cells in 14 types, neutrophils in 15 types, macrophages in 14 types, and dendritic cells in 19 types. As WDR4 expression had prognostic value in BRCA, KIRC, and LIHC, the correlation between WDR4 expression levels and the degree of immune infiltration in BRCA, KIRC, and LIHC is shown in To investigate the relationship between WDR4 expression and various infiltrating immune cells, the associations between WDR4 expression and immune markers in a range of immunocytes were studied, as shown in WDR4 expression was typically correlated with the stromal score in LUCS, BRCA, and TGCT, the Est_ImmuneScore in LUCS, SKCM, and THCA, and the ESTIMATE score in LUSC, TGCT, and SKCM .As shown in P = .0032), STAD (P < .001), SKCM (P = .044), SARC (P = .024), PRAD (P = .002), LUAD (P = .011), LGG (P < .001), LAML (P = .049) and BRCA (P < .001) but negatively associated with THYM (P = .037) and THCA (P < .001), as illustrated in P = .0087), STAD (P = .016), SARC (P < .001), LUSC (P < .001), LUAD (P = .026), LIHC (P = .011), KIRP (P = .045), KIRC (P < .001), HNSC (P < .001), CESC (P < .001) and BRCA (P = .0011) but negatively associated with READ (P = .01) and COAD (P = .03), as illustrated in We found that WDR4 expression was positively associated with TMB in UCEC  are independent predictors of SLN involvement and cancer patient survival outcomes , 27. FurGenetic alterations are the main cause of malignancy. Specific gene mutations can predictors of patient prognosis and of the patient\u2019s response to treatment. The adaptive immune system recognizes and identifies cancers due to nonself-neoantigens associated with somatic mutations. The TMB level influences the production of immunogenic peptides, therefore influencing the response to immune checkpoint inhibitors \u201336. In tOur thorough pan-cancer investigation illustrated the role of WDR4 in tumour cells and tissues. Furthermore, we discovered that WDR4 levels can serve as a valuable prognostic biomarker for some types of tumour. According to the findings presented in the current report, the WDR4 level is associated with cancer immunity. Similarly, our latest integrated omics-based workflow could be adopted to identify novel targets for carcinoma treatment."}
+{"text": "With a high rate of attrition and burnout of teachers as a global concern, teacher resilience has become a trendy topic in the research of their professional development as one of the pillars of positive psychology (positive character traits). However, the literature reveals that little research has been done on the mid-career teachers in the Chinese context, especially on how resilience may be nurtured, sustained, or eroded over time. Focusing on a mid-career EFL female teacher (the author) in China as a case study, this longitudinal self-reflective study employs a narrative inquiry to investigate the challenges that the experienced teacher was encountered with and to depict her trajectories of resilience-building by fleshing out the interaction between challenges, resources, and coping strategies in her three different scenarios. \u201cHard data,\u201d such as teaching journals, reflective field notes, and messages with students were collected and analyzed inductively by using thematic analysis, and \u201csoft data,\u201d like memory was also referred to. The findings unfolded challenges confronting the experienced teacher peculiar to the Chinese context and charted a detailed bumpy journey of resilience building in three phases, accompanied by her growing emotional, intellectual, and psychological capacities. Implications are drawn out for teacher resilience building, school leaders, and policymakers. A body of research has proved that teaching has become one of the most demanding and stressful professions in any field in the 21st century . High woAn overview of previous studies reveals that almost all the research on teacher resilience were conducted in Western contexts . Few stuIt should be noted that this study is exploratory and interpretive, so the generalizability of the findings will be limited . By provResilience is a relative, multi-faceted, and developmental construct . StudiesAn overview of the previous studies on teacher resilience mainly involves three aspects: risk factors, protective factors, and coping strategies. Risk factors, stressors, or challenges are a prerequisite for initiating resilience building . StudiesWhile resilience presumes the existence of risk factors, the resilience-building relies heavily on resources (or protective factors) and strategies with which one can \u201cbounce back.\u201d As for personal resources, important factors identified are mainly related to motivation and emotion, such as efficacy, a sense of moral purpose, agency, stroke, credibility, and optimism . AccordiThe previous studies contribute a lot to our understanding of the multi-dimensional construct, yet teacher resilience, to a large extent, is also fluctuating and relevant to different scenarios and career phases , 2013. UWhat challenges has the mid-career college EFL teacher been confronted with in the Chinese context?What resilience-building processes has the mid-career college EFL teacher undergone in coping with the challenges?Narrative inquiry is adopted as the research methodology. Narrative, as an \u201calternative paradigm for social research\u201d and complementary to experiment, survey, and other methods, appeals more to \u201clikeness\u201d and sense-making by providing a rich description of individuals\u2019 experiences , p.1. InAs a form of qualitative research, it also features being iterative, emergent, and interpretive in terms of data collection, data analysis, and data interpretation . It is tAs a self-narrative inquiry case study, in this research, the participant is the author \u2013 LN (the author\u2019s acronym), a mid-career female EF teacher in a Double First-Class University in central China. Based on purposeful sampling, the author meets two selection criteria: (1) she has taught in the frontline for 11years and stepped into a mid-term career phase, with shifting challenges from personal lives and occupational reforms; (2) in spite of adversities, she showed resilience and made above-average achievements in teaching, researching, and managing roles. Her excellence in teaching was measured by evaluation from students and colleagues, and her awards in several high-rank national teaching contests; her achievements in research was judged by research projects she participated in and articles she published; and her managing quality as a deputy director of the College English Department was evaluated by her leaders and colleagues.To ensure the participant was qualified to be selected as a resilienct teacher, the resilience scale of In the VITAE study, In order to discover the shifting challenges encountered by the author, and rebuild the bumpy journey of her resilience building, data were collected from the source between her fifth and eleventh teaching years in four sections: (1) Teaching journals were produced by LN after class at the end of each unit, and diaries concerning challenges and resilience-building, some of which were written in her notebook, and others were uploaded online in her blog. (2) Class observations and field notes were written in her notebook or shared in the \u201cmoment\u201d of WeChat . (3) Chat records with colleagues and students referred to the texts and pictures in WeChat and emails between LN and them, which were mainly in digital format. (4) Research materials included reports and papers in LN\u2019s teaching studies. Overall, the various types of collected data included teaching journals, diaries, class observations, field notes, chat records, and research materials, which helped to enhance the trustworthiness of the study . BesidesTo improve the rigor and credibility of data analysis, the coding process was done inductively through Nvivo 12, a computer-assisted qualitative data analysis software CAQDAS; , which pIn this section, the major themes extracted from the data regarding the challenges confronting the participant are presented first, namely individual challenges, classroom challenges, institutional challenges, and challenges from persistent national reforms. Then, the themes concerning the resilience-building process are stated on terms of challenges, resources, and response and strategies in the three phases: (1) homeostasis-breaking and changes-resisting, (2) reflecting, recognizing, and rebuilding, and (3) assimilating and adapting. To chart a detailed map of the participant\u2019s uneven resilience-building journey, the nuance and intricacies of her physical, emotional, and cognitive responses and strategies are fleshed out chronologically in her self-narration. Meanwhile, excerpts and commentaries from her story are listed as pieces of evidence of the themes, and theories and prior studies are referred to help the discussion of the findings.In order to fully convey the \u201clikeness\u201d and verisimilitude of the reality experienced by the participant, the first person \u201cI\u201d will be used, and the genre of narrative writing will be used in this part.At the age of 30, in my 6th year being an EFL teacher, my orderly life was disrupted by a baby cry. I became a mother. However, the pleasure and curiosity of being a new mother were soon diluted by subsequent challenges.In the first half year, every night I was woken up three to six times to feed and care the crying baby, and I was left exhausted and grumpy the next morning (Excerpt from diaries).The problem was compounded when my maternity leave ended at the 8th month, which soon made me a juggler caught between being a mother and a teacher. I often felt like being split up, despite the help from my mother and husband, enormous amount of time and energy was required to take care of the baby, so I had to burn the night oil to prepare lessons and grade papers for the next day. What I expected then was this dilemma would be relieved as the baby grew up. However, the reality proved that being a mother indicated an eternal change for female teachers, constant roles shifting and working hard to strike a balance between the two demanding sides.At different career phases, teachers face different types of stressors and conditions of work and life . Unlike \u201cIs there any volunteer who would like to share your opinions about this topic?\u201d This was the third time I asked or even begged for students to do the oral task in the class. Still, dead silence. Either because of being embarrassed or being bored, they lowered their heads and buried themselves in smartphone flicking .Engaging college students in oral tasks has always been tough, and individual factors including personality, motivation, inadequate oral English ability, anxiety, contextual factors like topics, learning tasks, classroom environment, and kinesthetic, auditory, and visual modalities have been identified accounting for students\u2019 reticence and willingness to communicate WTC; . Further\u201cWith a phone, knowledge is available to me anytime anywhere\u201d (Excerpt from chat records). Equipped with the \u201cknowledge reservoir,\u201d students now are more like a \u201csupervisor\u201d in the class, since my pronunciation, spelling, and content would often be checked online by students, and my mistakes sometimes would be kindly reminded or left unknown to me, which sometimes made me feel like an imposter caught on the spot. As one of my colleagues said, \u201cWe teachers are edged out gradually from the center by information technology\u201d (Excerpt from chat records). The blended teaching mode mandated by our department since the Covid-19 outbreak meant 20% percent of course tasks were put online and the offline instruction was reduced proportionally. Coupled with this attention loss, the internet directly connects students and learning resources . The heavy workload often made me feel like a \u201cteaching machine,\u201d shuttling from the campus to my apartment day and night, so I had no time to do research for my professional development. Lying exhausted on the bed after work, I often wondered when this machine mode would be over.In the first 5years of my teaching career, every week I had at least 20 teaching hours in the class, which was high above the average workload. In the autumn midterm, I even had In my 8th year, the dream day came. My teaching workload was reduced to 12 hours a week. However, the relief and pleasure brought by the lightened work did not last long. We had a new dean! Unlike the former one who only cared about whether we finished the workload or not, she was very ambitious and demanded all the faculty to aim high in terms of teaching and researching. Inspired by Positive Psychology, she initiated to break the status quo of \u201cbeing settled\u201d and establish an uplifting and even competing culture in our department. We were even classified as \u201ccombustible\u201d or \u201cnon-combustible\u201d according to our degree of motivation and commitment. For the young and middle-aged teachers like me, teaching and research targets were set up, and incentives were also taken to push us forward.Coupled with the demanding dean, our university updated its evaluation and promotion system with higher requirements since it was listed as the Double First-Class University, a project initiated by Chinese authorities for developing a number of world-class universities and disciplines and enhancing Chinese higher education power and international competitiveness. According to the new criteria, in order to be promoted as an associate professor, I need to achieve highly both in terms of teaching and research. For teaching, I had to win at least first prize in the English teaching competitions held provincially or nationally. Although in recent years opportunities to join in the contests are increasing for teachers, it is still very hard for us to win prizes since there are more than 100,000 EFL teachers in China, and many of them are eager to win prizes to accomplish the evaluation requirement. Within our department, there are about 40 young and mid-aged teachers like me who are striving for senior professional titles, and winning prizes looms like a must-do for us, and it is no exception to me. Whenever news about teaching contests is released, dozens of teachers would sign up for it, which seems like a fierce battle without smoke, and this competing atmosphere puts a lot of teachers under great pressure as I learned in private talks with them. The performativity agendas, initiatives, and limited resources may bring stress and anxiety to teachers and even make some teachers marginalized in the institute .Besides the requirement for prizes in teaching contests, at least three high-quality papers published in core journals and one provincial research project are required for professional advancement. Ironically, the new promotion regulation still placed our department as a public English teaching institution while we were assessed in the same way as other teachers from research institutions. The unfair treatment experienced by the EFL teachers in professional evaluation and promotion was also found in other studies .This is more challenging for me in three aspects. For a start, I have not received systematic and disciplined training on academic research in my college life, so writing papers and applying for research projects were so intimidating that I tossed and turned in bed without knowing where to start. Meanwhile, the heavy workload in my first 5years deprived me of time and energy to read academic journals and do research, which in turn left me with a weak foundation of academic ability and a bleak prospect for professional development. Furthermore, the big imbalance between the limited number of paper publishing outlets and the high demand of publishing made ordinary EFL teachers like me stuck in a dilemma with no way out.\u201cI tried so hard, but I do not think I can ever catch up with the shifting education trend.\u201d (Excerpt from chat records).My colleague Li\u2019s complaint echoed my concern and stress. Actually, whether we teachers welcome it or not, reform and change have been the norm in Chinese foreign language education. The development of Chinese foreign language education has been closely intertwined with Chinese social, economic, and cultural development, and its original aspiration has been and will always be to bring out talents to serve Chinese development and prosperity . Under t(Excerpt from chat records). My colleague\u2019s instruction soon paid off. More and more young teachers in my department began to try the Rain Classroom out in their classes, while I remained reluctant to dabble in it. Deep down inside, for one thing, I held doubt about the effectiveness of technology to enhance the interaction in the class. For another, I feared my authority and centeredness would be challenged by those fad technology. Actually, my doubt and fear are echoed and researched in the studies. Reforms, especially those managed poorly often disturb the relative stability of teachers\u2019 work, sometimes their beliefs and practices and self-efficacy  courses and upgrade our curricular system by developing ESP and EAP courses to meet the needs of the country as well as students to cultivate compound talents with specialty and capability (Data from field notes). This pragmatic orientation in foreign language teaching not only brings out rounds of curriculum reforms, but also implies teachers need to update their knowledge and teaching competencies constantly, which will inevitably arouse a sense of insecurity among them .The challenge to me was enormous and intimidating. Since I started to work, I had been teaching the College English Listening and Speaking Course for more than a decade, and I had no experience of developing a new course. What new course should I choose? And what procedures should I follow to set it up? Those questions haunted me day and night as I fumbled to find a new direction for my professional development.Resilience is a dynamic developmental process and presupposes the presence of threat to the status quo, and thus a positive response to conditions of significant adversity , 2020. IAccording to the division of three scenarios in teachers\u2019 life , my mid-The watershed moment of my teaching career came with a baby cry. I became a mother at age of 30 in my 6th year. Before it, I was immersed in a \u201crosy color period\u201d when I had a happy married life and I felt established in teaching after the novice struggling periods. To some extent, I thought I had realized my dream as a teacher that I cruised teaching in my way, and my teaching was highly evaluated by students at the end of the term. The baby\u2019s coming soon proved to be a disrupter. The equilibrium between life and work was broken since caring for a baby was so demanding physically and mentally that I was mostly occupied by babysitting and had little time for lessons preparation during the first 2years. The time for instruction preparation was so much edged out that I had to stay up late to finish my teaching preparation and daily chores (Data from diaries). As a result, the hormone fluctuation of being a mother and juggling between life and work often left me exhausted, memory deteriorated, and grumpy. As I was busy with baby caring, my young colleagues had passed examinations for PhD and embarked on a new journey for further learning. The peer pressure soon threw me in panic, and undetectably I had come to a crossroad: settled for the status quo, or learning further for professional development (Data from diaries). According to the resilience model , disruptStanding at the crossroad, despite my seeming composure, my response and tackling strategies were still far from positive. As Although riskers like role-shifting as a mother and road-taken decision brought unprecedented challenge and stress, thanks to my supportive family and caring students, their help and encouragement acted as protectors which empowered me to maintain my commitment to work and life instead of falling apart like some middle-aged female teachers suffering from postpartum depression. From the moment of the baby\u2019s birth, my husband began to fulfill his role to care for me and the baby, changing diapers, cradling the baby to sleep, and doing the housework. Meanwhile, in order to ensure me to have a multi-nutrient diet, my mother always prepared the tailored meal for me after reading the cookbook carefully (Data from diaries). They had tried all means to make sure the baby and I live healthily and comfortably. Moreover, I was also uplifted emotionally by my students, dozens of whom flooded in my house to visit me and the baby with flowers and loving words. Their physical and emotional support tided me over the most critical and transformational period. As one student wrote in an email,\u201cThank you so much for your encouraging words, Linda. I was really impressed by your great devotion and commitment to us. In order to arouse our attention in spoken English, you even pronounce words with exaggerated facial expressions and intonation. Although my oral English is bad, you still listened to me carefully and commented on it positively. I am so touched that I will study harder and I will not let your down.\u201d (Excerpt from emails).This letter made me realize that I can make a difference to them. My commitment to students\u2019 learning and achievement can spark their interest to study even to life. Meanwhile, students\u2019 appreciation and applause to me acted like the best buffer for me from the uncertainty and unpredictability in the teaching career. Their recognition was a great spiritual booster and even a lifesaver whenever I was in stress and burnout, especially when I was doubtful about my ability to keep up with the changing teaching methods. As researchers hold, a teaching job is emotional by nature, and it is not a matter of entirely conveying knowledge and content and using novel teaching methods and techniques . Good teMy \u201costrich\u201d way of escaping the changing reality proved to be a failure. As more and more young colleagues were on their way to a doctorate, the piling peer pressure made me realize I had to confront the \u201ccrossroad\u201d question squarely: be settled for the status quo or seek out a way to break through. This is the typical \u201cto be or not to be\u201d question that the mid-career teachers need to answer, and a key watershed of teachers\u2019 long-term perceived effectiveness. Standing at the crossroads of their professional lives, they have to decide whether to pursue career advancement or to remain in the classroom fulfilling the original \u201ccall to teach\u201d .Unlike 2years ago, I could not take the baby as an excuse to defend my demotivation since he was already in kindergarten now. It was time to take action. As I decided to prepare for the doctorate examination, a new regulation released by my university destroyed my hope. It stipulated that all in-service teachers like me should not pursue further degree education anymore or we would be laid off. Although I was furious at being deprived of education right, I had no power to resist but accept it. The only way left for me to advance in the career path was to promote my professional title by achieving more in teaching and research. It was no easier, but I decided to confront the challenges positively after serious reflection upon my teaching experience, which consisted of no shining moments except some praise from students. The mediocre performance made me obscure and marginalized like a shadow in my department. I had to work hard to prove myself and I believed I could do it.God helped those who helped themselves. I would never forget my shining moment on the podium to be awarded the first prize in the FLTRP Star Teacher Contest.I was seen! I not only received rounds of applause from more than a thousand EFL experts and teachers on the spot, and my prize winning news was also headlined on my university\u2019s homepage. I was also bombarded with congratulatory messages from friends and colleagues. This great honor boosted my confidence unprecedentedly. (Excerpt from diaries).Because this national contest was so challenging, with hundreds of EFL teachers competing for very few prizes that I did not expect I could make it. It proved that my painstaking efforts in reading and writing for the teaching design in the past 3months were paid off, and I had the capacity to achieve in teaching and research as long as I devoted myself wholeheartedly.In the next half year, when asked by students, I decided to take the challenge without hesitation to mentor them in a provincial contest. As \u201cI see you have won prizes in teaching. Why not step off your comfort zone to try a new role, the associate director of the sub-department? What do you think?\u201d (Excerpt from chat records). I felt greatly flattered but also anxious and pressured. Managing the institute of dozens of teachers was totally new and more challenging to me, since I was introverted and clumsy at communication. Should I accept this offer? Would I be able to manage it well? After days of consideration, I decided to accept this promotion, believing it was an incentive for me to reach out to colleagues to improve our collegiality.As I was immersed in joy and contentment, the new dean talked to me, The moment I was assigned the new role, I was tested by its daunting responsibilities, the high-stake ones like clarifying and specifying the teaching content, objectives, and procedures to teachers at the beginning of the term, drafting out examination papers for thousands of students at the end of the term. In order to ensure effectiveness, I sought help and advice from several directors and colleagues. Daily office chores like communicating with colleagues, attending a regular staff meeting, and paper-writing work sometimes made me hard to switch off. Even at home, I was also on \u201cwork mode,\u201d working on weekends to finish teaching and non-teaching work. However, I was glad to find my capability at managing and communicating were highly improved with my positive attitude and strenuous efforts. As According to the resilience model , after tAs I stepped into the 10th year of my teaching career, I felt I was transformed a lot compared with the old me 3years ago when I was so panicked and hesitant at the crossroad. I was equipped with more composure, confidence, and resilience, a reservoir built after I survived the challenges with milestone prizes and honors on the road taken. As resilience was not built in a day, teaching career was inherently filled with shifting changes and trials . As I feHow could I make breakthrough in research proposal applying and paper writing? What new extended course should I develop, and how should I develop it? .Meanwhile, our curricular system must be updated since the credit hours of EGP courses were going to be reduced. In retrospect, these challenges dominated the third phase of my resilience-building journey as riskers as well as activators.Research proposal writing and academic paper writing differed in genre, format, and other aspects, yet they had something in common to me \u2013 important but extremely challenging. As a master in translation studies, the lack of strict and systematic education in EFL teaching and researching made me fumble alone in the dark for a long time. The repeated rejection and failures made me so helpless until I joined an academic research team in our institution. To prepare us with the basics of research in EFL teaching and learning, all research members were assigned reading lists and asked to present in the literature discussion regularly. In the heated discussion, I was often inspired by other members\u2019 insights in literature reading and proposal writing (Data from diaries). More importantly, I was so fortunate to join in an important provincial research project about students\u2019 demotivation led by the team leader. I was asked to help collect data by implementing the questionnaire and semi-structured interviews, process data with SPSS, analyze data, and write part of the research project. Although it was far beyond me, I was fueled and motivated greatly by the trustworthiness and close cooperation in the project team that I determined to seek challenges in this process, working around the clock to read literature about motivation studies, watch online courses about SPSS operating, and learn to write the research report (Data from research reports). After 2years of joint efforts by our project team, the research report of this project was highly evaluated and awarded the first prize by the expert committee in Henan Province. Additionally, the academic paper I wrote on students\u2019 coping strategies of demotivation based on this project was also accepted and published successfully.Overall, what I harvested from this research project was far more than the honor, but growing capabilities in all aspects of academic research from literature reading, data collecting, data processing, and data analyzing, to academic paper writing. Moreover, the supportive help from the research team also provided me a sense of safety, belonging, and warmth on the rough road forward. Teachers can hold more positive views toward research and become research literate by joining research organizations and associations, and receiving research training in their workplace . Meanwhietc., of the new course. One day, several colleagues and I were called into a meeting about the new course.In the same vein, the social network and resources enabled me to make a significant breakthrough in course development. Before that, for a long time, I had been haunted by the daunting task to build a new extended course. Despite racking my brain hard, I still had no clue as to the teaching objectives, content, teaching methods, \u201cWe should set up some new courses to cope with the impending credit hours reduction. I hope you work hard together to make it, and all resources needed will be guaranteed,\u201d the dean said (Excerpt from chat records).Inspired and motivated by the dean\u2019s support, we soon established a course-developing team, working together to bring the dream course to a reality. Thanks to the strong support by our institution, we could not only attend online workshops on curriculum development, visit other universities in person to learn about course building and management, but also invite experts to our institution to guide our work (Data from class observations and field notes). Eventually, with the conflation of the dean\u2019s leadership, the institute\u2019s support, our team\u2019s joint efforts, two extended courses were set up and highly evaluated by students, and even landed the great honor of \u201cthe First-Class Course\u201d in Henan province. The groundbreaking establishment made me realize that we could make it as long as we work hard as a team, and there was something more I can do to contribute to the curriculum reform. In spite of the full consciousness of the strenuous effort required, I still made a bold move that I would develop another new course integrating the traditional Chinese culture and EFL teaching, in congruence with the Education Bureau\u2019s initiative to implement Chinese culture-based teaching. Unlike before, instead of sitting and waiting for others\u2019 push, I motivated myself to reach out for leaders and colleagues for their support and help. The new course team was soon set up, in 8months, and we worked hard together to design and refine the teaching content and teaching activities after hundreds of hours of reading, discussing, and evaluating. Now, the new course is full-fledged to meet students in the next semester.As I look back over this period, the challenges to make a breakthrough in academic research and course development were like grains of sand in the oyster, stimulating me to be more responsive and resilient with the ever-changing contexts. At the same time, I was also backed up with various support and resources from the social web interweaved by the dean, our institute, the research team, and the course community. Especially the dean played a significant role to motivate us and support us to move forward when there seemed to be no way out, serving as \u201cweavers of the fabric of resilience initiatives\u201d , p. 299.There are precious qualities nurtured by me in the third phase that distinguishes it from the former two phases: agency to seek changes, challenges, and a growing mindset, and I am more adept at protective factors or coping strategies . While IOverall, teachers\u2019 capacities to sustain their commitment or resilience are moderated by their professional life phases and mediated by the contexts or \u201cScenarios\u201d in which they live and work . As teacIn this research, narrative inquiry was employed to investigate the challenges confronting the mid-career EFL teacher LN, and to map out the detailed and bumpy journey of her resilience building in the three scenarios with distinctive dominant personal, situated, and professional dimensions. Base on the findings, three important observations can be made.First, the findings confirm the prior studies that the teaching career is physically, emotionally, and intellectually demanding in nature, consisting of severe challenges in certain circumstances as well as uncertain and unpredictable trials in everyday life. Therefore, although these challenges could be disruptors of the equilibrium of teachers\u2019 personal and professional life, they could also turn up as initiators of resilience-building process by activating teachers\u2019 psychological, emotional, and cognitive resources. Additionally, as teachers\u2019 professional career path advances, they will face different concerns, tensions, and influences peculiar to the circumstances, and teachers\u2019 responses and strategies will be influenced by the dimensions dominant in their personal, situated, and professional life.Second, the research proposes that there is a reciprocal relationship between positive emotions and positive character traits like resilience and provides evidence to the broaden-and-build theory of Thirdly, in contrast to the findings of the mainstream studies that resilience was an innate psychological attribute, this research finds that its building was the culmination of collective and collaborative endeavors, which involved a complex web of supports and assets from inside and outside, especially the social resources and networks in the critical career phase. Teachers\u2019 resilience is not static that can be accomplished at one stroke; instead, it involves persistent efforts and long-time devotion, and it is dynamic and can fluctuate in levels depending on teachers\u2019 efforts and capacities differ in shifting scenarios.Accordingly, implications are drawn out for teachers\u2019 resilience building and professional development. For individuals, teachers should have a better understanding of the prospect that the persistent pressure and recurring setbacks on the career path are on a daily basis, and confront them positively to enhance their resilience. For policymakers and teacher educators, attention should be paid to the mid-career teachers whose concerns and pressure had long been neglected, and provide in-service education programs on resilience building to help them well cope with the ever-present challenges. For institutional leaders, support and resources should be provided to mid-career teachers who are at the crossroad of surviving or thriving, to ensure their full commitment to teaching and sustainable professional development.This study can be further improved by addressing its limitations in future research. First, although the thick description of a single case \u2013 such as this one \u2013 can be a good starting point for insightful understanding of understudied groups, future studies may as well take a macro-perspective approach such as the large-scare questionnaire to depict a full picture of teacher resilience by focusing on more mid-career teachers in various teaching contexts and circumstances. Second, the trajectory of the experienced EFL teacher resilience-building was mainly a retrospective and interpretative inquiry into the teacher\u2019s past experiences, so future studies can focus on in-service teacher resilience training programs and look into the effect of the intervention to help teachers sustain passion and commitment to teaching.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Academic and Ethical Committee of Henan University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LX conceptualized, collected the data, drafted the manuscript, and edited its language before submission to Frontiers in Psychology.This original research was sponsored by the School of Teacher Education of Henan University. The Research Project is entitled \u201cA Study on Chinese EFL Teachers\u2019 Emotion Regulation, Resilience and Their Work Engagement Based on Positive Psychology (Grant No. YB-JFZX-22)\u201d; the research was also sponsored by the Research Funds for Social Sciences in Higher Education of Henan Province, entitled \u201cA Study on the Teaching Methods for Chinese Culture Courses in English to Cultivate Students\u2019 Ability of Telling the Chinese Stories Well\u201d (Grant No. 2022-ZZJH-522), and was supported by the Undergraduate Teaching Research Project administered by Henan University, entitled \u201cResearch on the Cultivation of College Students\u2019 Ability of Telling the Chinese Stories Well in the New Era\u201d (Grant No. HDXJJG2020-43).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.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": "In recent years, due to the development of standardized diagnostic protocols associated with an improvement in the associated technology, the diagnosis of pelvic endometriosis using imaging is becoming a reality. In particular, transvaginal ultrasound and magnetic resonance are today the two imaging techniques that can accurately identify the majority of the phenotypes of endometriosis. This review focuses not only on these most common imaging modalities but also on some additional radiological techniques that were proposed for rectosigmoid colon endometriosis, such as double-contrast barium enema, rectal endoscopic ultrasonography, multidetector computed tomography enema, computed tomography colonography and positron emission tomography\u2013computed tomography with 16\u03b1-[18F]fluoro-17\u03b2-estradiol. Endometriosis is a common chronic disease affecting 5\u201310% of women of reproductive age globally . AccordiThe ovary is a common site affected by endometriosis . The intIn recent years, due to the development of standardized diagnostic protocols associated with an improvement in technology, the diagnosis of pelvic endometriosis via imaging is becoming a reality. In particular, transvaginal ultrasound (TVUS) and magnetic resonance imaging (MRI) are today the two imaging techniques that can accurately identify the majority of the previously described phenotypes of endometriosis. This review focuses not only on these most common imaging modalities but also on some additional radiological techniques that were suggested for rectosigmoid colon endometriosis and other specific purposes, such as double-contrast barium enema, rectal endoscopic ultrasonography, multidetector computed tomography enema, computed tomography colonography and positron emission tomography\u2013computed tomography with 16\u03b1-[18F]fluoro-17\u03b2-estradiol.TVUS is surely the first-line technique for the diagnosis of endometriosis . The posThe ultrasound features of ovarian endometriosis vary in pre- and postmenopausal patients ,9. The mAlthough some authors suggest that some findings (presence of thickened pericolic fat) correlate with the presence of superficial endometriosis, no reproducibility study has been published . RobinsoRegarding DE, the consensus from the International Deep Endometriosis Analysis (IDEA) group  proposedThe identification of \u201csoft markers\u201d is an easy-to-learn assessment that can even be performed by less experienced operators . \u201cSoft mAccording to the IDEA consensus , the locRecent studies evaluated the prevalence, diagnosis and clinical features of DE involving the parametrium, considering it as a third compartment in addition to the anterior and posterior ones. According to the #Enzian Classification for endometriosis , the medThe DE in the anterior compartment can include hypoechoic linear or spherical lesions, with or without regular contours, cystic spaces, hyperechoic foci and/or regular contours involving the muscularis (most commonly), (sub)mucosa of the bladder  or the uterovesical space  Figure . Albeit In the rectovaginal space (RVS) the DE nodules are described as lesions below a horizontal plane that passes along the lower margin of the posterior lip of the cervix under the peritoneum . Figure. GuerrieExperienced sonographers can reliably identify silent ureteral involvement due to endometriosis with TVUS . The mosAs for the USL, the lesions appear as nodules with regular or irregular margins and often with hyperechoic points, or as a linear hypoechoic thickening with regular or irregular margins  Figure . In the A nodule on the rectosigmoid colon can appear as an irregular hypoechoic nodule in the anterior wall of the rectosigmoid colon with or without hypoechoic or, rarely, a hyperechoic focus with visible retraction and adhesions  Figure . SensitiAccording to the study of Exacoustos et al. , parametTo further increase the diagnostic accuracy of TVUS, there are some additional techniques that can be performed. The simplest additional technique is the tenderness approach . It is aRegarding the need for bowel preparation before a TVUS examination, Ros et al.  concludeIn addition, office\u2009gel sonovaginography (SVG) appears to be an effective outpatient imaging technique for the prediction of bowel DE, with higher accuracy for the prediction of rectosigmoid compared with anterior rectal DE . SVG canBased on the high diagnostic accuracy reported in the literature, of all classification and staging systems for endometriosis, many are based on ultrasound. In order to achieve the quality of care of a tertiary center obtained by expert examination in regular practices, in particular in the USA, Young et al.  suggesteThree-dimensional TVUS (3D TVUS) can also be used for the evaluation of DE. This technique allows for the possibility of obtaining volumes that can be reconstructed manyfold after a single sweep with an infinite number of viewing planes . In partMRI is commonly used for the diagnosis of endometriosis. However, a consensus on the best descriptors of DE on MRI is lacking. The European Society of Urogenital Radiology (ESUR)  guidelinIn order to obtain optimal visualization of the anterior compartment, it is suggested to perform the MR exam with moderate repletion of the patient\u2019s bladder; this is requested because an overfilled bladder could determine the detrusor contractions with subsequent obliteration of the adjacent recesses, thus compromising the identification of small parietal nodules ,65. WhilIt is possible to perform the MR exam with different magnetic field strengths (1.5 T or 3 T). The difference in adopting a 3T scanner is related to the better spatial resolution that is possible to acquire with this scanner technology. Moreover, high-resolution phased array coils (with 8\u201316 channels) are necessary to optimize the image quality. Currently, it is suggested to not perform an exam for endometriosis detection with a low magnetic field or open MRI because of the resulting sub-optimal image quality .From a technical point of view, the use of a high-resolution acquisition sequence (3 mm or less) in the different spatial planes  in TSE-T2w are fundamental to evaluating DE. The use of the TSE T1w and the TSE T1W fat sat is requested to detect the presence of blood products in the adnexa (for the detection of endometrioma) .Moreover, the spatial orientation  of the acquisition planes is very important because it allows for detecting and exploring more challenging areas, such as the USL, as demonstrated by Bazot et al. .In the MRI assessment of endometriosis, the use of contrast material is debated because in most cases, it does not add any significant information; however, it is suggested that the adoption of CM could help with distinguishing DE from other pelvic inflammatory conditions, in particular, it is useful to differentiate a ruptured endometrial cyst from a tubo-ovarian abscess .Ovarian endometriosis can contain varying different types of tissues, including proteins and fluids, but its pathognomonic characteristic is related to the presence of blood products at different stages of catabolism . UsuallyIn the MR assessment of an endometrioma, another potential diagnosis issue is differentiating endometrioma from a luteal hemorrhagic cyst because both lesions contain blood. In this case, differentiating the stage of the hemorrhage (acute versus chronic) could be useful and this could be obtained in the case of the presence of the T2 dark spot sign that is associated with the presence of chronic hemorrhage and, therefore, can be very helpful in differentiating endometriomas (positive for the T2 dark spot sign) from a luteal hemorrhagic cyst (negative for the T2 dark spot sign due to the acute hemorrhage phase) . There aSmall superficial peritoneal implants are defined as those lesions that are <1 cm in diameter; these are the first grossly recognizable lesions on the surface of pelvic organs or the pelvic peritoneum . NodulesIn the case of nodules of endometriosis involving the bladder, it is possible to find two different patterns: localized involvement with evidence of nodularity in the bladder wall or/and diffuse wall thickening . The chaThe MRI study of the ureter can be difficult  and MRI A widely studied target with MRI in the endometriosis involvement is the USL ; this isAs a topographical analysis, the most frequently involved area is the proximal medial portion of the USL. In a recently published meta-analysis, Medeiros et al.  found a Furthermore, in the case of vaginal involvement, it is possible to identify direct and indirect findings. In the case of a direct finding, a nodule  or the thickening of the parietal wall can be detected; in this case, the signal of the T1W sequence is also related to the presence and amount of hemoglobin in the hemorrhagic foci . As an iThe final localization of the DIE is the rectosigmoid wall, which is characterized by four layers, namely, serosa, outer longitudinal muscularis, inner circular muscularis and mucosa . EndometRegarding parametrial involvement, MRI imaging of retraction of the obliterated umbilical artery toward the abdominal cavity was shown to be associated with the presence of endometriosis in the ipsilateral paracervix . There aIn a meta-analysis, Guerriero et al.  comparedHowever, for the USL, rectovaginal septum and vaginal DE, a new meta-analysis reported that MRI shows better sensitivity than transvaginal ultrasound .Among the recent advances in imaging, the MRI/TVUS fusion technique is a new non-invasive diagnostic tool that could be useful in clinical practice because it may allow for improving the TVUS detection of some abnormalities using MRI and vice versa . There iAlthough not a substitute for the methodologies described above, some additional radiological techniques were proposed for some locations and specific purposes. We reviewed the most common techniques used in some locations of endometriosis, in particular, for the rectosigmoid colon. These techniques are the following: double-contrast barium enema, rectal endoscopic ultrasonography, multidetector computed tomography enema and computed tomography colonography .The double-contrast barium enema (DCBE) was historically the first exam used to diagnose bowel endometriosis  but is cThe limit of this approach is due to the fact that it is a pure luminographic approach and, therefore, endometriosis is suspected when the bowel lumen is narrowed at any level from the sigmoid to the anus ,90. The DCBE offers indirect information because the cause of the lumen narrowing is not visible and requires the use of more advanced cross-sectional imaging that is nowadays becoming the first-line standard in this scenario . IncomplAnother technique described by some authors is rectal endoscopic ultrasonography (REU). REU was traditionally used to stage rectal cancer , but in Another imaging technique that can be used for the detection of endometriosis is the multidetector computerized tomography enema (MDCT-e). This is an MDCT with colonic distension obtained with water. This technique is widely used for the imaging detection and characterization of colorectal cancer , but due2 is pumped in via a flexible tube inserted into the rectum fluoro-17\u03b2-estradiol (FDG PET/CT) was proposed as a feasible technique ,122. In To conclude, with advances in imaging techniques, much has already been done in recent years for the improvement in the diagnosis of endometriosis, both with an increase of the sensitivity and specificity of those techniques and preferentially using a less invasive diagnostic approach (relative to surgery). Certainly, more studies are needed to further expand knowledge in this area."}
+{"text": "The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated \u03b2-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase. Pregnancy is dependent upon the success of various steps including ovulation, fertilization, blastocyst development, endometrial differentiation , embryo implantation, and placental formation. Among them, the differentiation of endometrial stromal cells (ESCs) into decidual cells is essential for the acquisition of endometrial receptivity for implantation and subsequent placentation. During the menstrual cycle, the estrogen-primed proliferative endometrium is decidualized by the action of progesterone P4) and cAMP in the mid-secretory phase. Decidualization is characterized by the transformation of ESCs into secretory decidual cells, which produce insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL) ,3,4. The and cAMPSenescent cells produce various secretory factors including inflammatory cytokines such as interleukins (ILs) and chemokines. This phenotype is called the senescence-associated secretory phenotype (SASP), and SASP factors further induce senescence of surrounding cells. In cancer cells, the SASP stimulates tumor growth and metastasis and triggers an immune recognition and tumor clearance . AlthougIt has been reported that a member of the forkhead family of transcription factors, forkhead box protein O1 (FOXO1), is associated with the expression of decidualization markers such as IGFBP1 and PRL . FOXO1 mThe post-ovulatory increase in P4 in the corpus luteum is involved in decidualization . P4 mostWe and others previously demonstrated that endometrial PGRMC1 expression decreases during the secretory phase of the menstrual cycle and the small interfering RNA (siRNA)-mediated knockdown and inhibition of PGRMC1 promotes ESC decidualization in vitro ,27. HoweP4 (#P0130), dibutyryl-cAMP , and AG-205 (#A1487) were purchased from Sigma-Aldrich . Antibodies against PGRMC1 (D6M5M) and FOXO1 (C29H4) were obtained from Cell Signaling Technology . The antibody against glyceraldehyde-3-phosphate dehydrogenase  was from FUJIFILM Wako Pure Chemical Corporation .n = 3) were younger than 45 years and had regular 28\u201332 day menstrual cycles. Endometrial tissues in the proliferative phase were prepared for ESC culture as described previously [v/v) charcoal-treated fetal bovine serum , 50 U/mL penicillin, 50 mg/mL streptomycin, and 0.25 mg/mL fungizone (Life Technologies). In order to induce decidualization, the ESCs were treated for two days with db-cAMP (0.5 mM) and P4 (1 \u03bcM), abbreviated as D/P.Human endometrial tissue samples were collected from female patients with leiomyoma and who were undergoing surgery at the Tokyo Medical University Hospital. All patients , PGRMC1 siRNA  or FOXO1 siRNA , using Lipofectamine RNAiMAX Transfection Reagent  according to the manufacturer\u2019s instructions . After tProtein lysates were prepared from ESCs with a RIPA buffer  according to the manufacturer\u2019s instructions. Equal amounts of protein (20 \u03bcg) were separated by SDS-PAGE and electrophoretically transferred into polyvinylidene difluoride membranes. The membranes were probed with primary antibodies against PGRMC1 (1:2500) and FOXO1 (1:2500). Immunoreactive bands were detected using enhanced chemiluminescence after incubation with a horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG. The membranes were then treated with a stripping solution and re-probed with an antibody against GAPDH . The relative band intensity was evaluated by the densitometric analysis of digitalized autographic images using ImageJ software  and normalized to that of GAPDH .The total RNA was extracted using the reagent ISOGEN II  according to the manufacturer\u2019s instructions. The RNA (100 ng) was amplified by real-time RT-PCR using a SYBR Green Luna Universal One-step RT-qPCR Kit . Sense (S) and antisense (AS) primers were 5\u2032-AAGGGTGACAGCAACAGCTC-3\u2032 (S) and 5\u2032-TTCTGCACACGAATGAACTTG-3\u2032 (AS) for PGRMC1, 5\u2032-AAGGGTGACAGCAACAGCTC-3\u2032 (S) and 5\u2032-TTCTGCACACGAATGAACTTG-3\u2032 (AS) for FOXO1, 5\u2032-AGACAGCCACTCACCTCTTCAG-3\u2032 (S) and 5\u2032-TTCTGCCAGTGCCTCTTTGCTG-3\u2032 (AS) for IL6, 5\u2032-AAGACATACTCCAAACCTTTCCA-3\u2032 (S) and 5\u2032-CCAGACAGAGCTCTCTTCCA-3\u2032 (AS) for IL8, 5\u2032-AACAGAAGCCAACTGGGTGAATG-3\u2032 (S) and 5\u2032-CTCCAAGAGAAAGCACTTCATTGC-3\u2032 (AS) for IL15, and 5\u2032-AGCCACATCGCTCAGACA-3\u2032 (S) and 5\u2032-GCCCAATACGACCAAATCC-3\u2032 (AS) for GAPDH. The fold change in the expression of each gene was calculated using the \u0394\u0394Ct method with GAPDH as an internal control .w/v) paraformaldehyde for 10 min and then washed twice with PBS. The staining was performed overnight at 37 \u00b0C in PBS (pH 6.0) containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mM MgCl2, and 1 mg/mL X-gal. The fold change in SA-\u03b2-Gal-positive cells was evaluated in five randomly chosen fields and expressed and compared with the control group.SA-\u03b2-Gal staining was performed as previously described . BrieflyESCs treated with AG205 or PGRMC1 siRNA were incubated with D/P for 2 days and detached with trypsin-EDTA. The cells were resuspended in the medium containing Hoechst 33342  and incubated for 45 min at 37 \u00b0C in the dark. The cell suspension was analyzed by flow cytometry . The percentage of cells in the different phases of the cell cycle was calculated with the FlowJo software .g at 4 \u00b0C for 10 min, and the supernatant was collected in order to determine the IL8 concentration. The concentrations of IL6 and IL8 were determined using an ELISA kit (Abcam ab178013 Human IL-6 SimpleStep ELISA kit and LBIS Human IL8 (CXCL8) ELISA Kit) according to the kit instructions, respectively.The culture medium was centrifuged at 10,000\u00d7 p-value of less than 0.05 was considered statistically significant.The data are expressed as the mean \u00b1 standard error of the mean (SEM) from at least three independent experiments. The significance was assessed using the Dunnett multiple comparisons test. A In order to examine the role of PGRMC1 in cellular senescence in decidualizing ESCs, we evaluated the effects of a PGRMC1 inhibitor, AG-205, that blocks PGRMC1 action ,30,31, aIL8 mRNA was downregulated in the D/P-stimulated decidualizing cells . These results imply that IL8 mRNA expression may be increased by PGRMC1 inhibition and knockdown at an earlier timepoint than analyzed in this study. Similarly for another SASP factor, IGFBP7, our preliminary data showed that the expression was increased by the decidualization stimuli and that the treatment of AG205 promoted the D/P-stimulated IGFBP7 expression (data not shown). Furthermore, the expression of p21 and p53 were not changed by AG205 and PGRMC1 knockdown in ESCs (data not shown). These results suggest that enhanced cellular senescence by PGRMC1 inhibition and knockdown may not be mediated by p53 and p21 induction in ESCs.As SASP factors, IL8 and IL6 protein secretion increased with PGRMC1 inhibition, whereas lization . We confIL15 mRNA increased upon decidualization, different responses were detected upon PGRMC1 inhibition and knockdown. This result may be due to the difference in culture time between experiments using AG-205 and PGRMC1 siRNA. PGRMC1 exhibits haem-dependent dimerization on the cell membrane [The IL15 secreted by decidualized cells activates the NK cells in the uterus and induces the elimination of senescent cells by activating the NK cells . Althougmembrane . The PGRmembrane  and cytomembrane ,41 and tmembrane . AG-205 membrane ,43,44,45Previously, we reported that PGRMC1 inhibition and knockdown enhanced db-cAMP-induced decidualization, even in the absence of P4. We therefore speculated that the action of P4 was not necessary for the stimulatory effect of PGRMC1 downregulation on decidualization. The expression of FOXO1 was increased by the activation of cAMP signaling in decidualizing ESCs . AlthougA limitation of this study is that we evaluated PGRMC1 in cultured ESCs but not in the endometrium in vivo. Human endometrial assembloids consisting of gland-like organoids and primary stromal cells were recently established . They caIn the present study, the functional inhibition of PGRMC1 promoted not only decidualization but also decidual senescence in ESCs . In conc"}
+{"text": "TM) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p\u2009<\u20090.05) significantly segregates the habitat ground control (GC) group from flight (FLT) group. Many pathways associated with cellular stress, inflammation, apoptosis, and metabolism were significantly altered by flight conditions. A decrease in the expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Moreover, mRNA expression of many genes related to anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response were significantly downregulated. Here we report that significantly altered immune reactions may be closely associated with spaceflight-induced stress responses and have an impact on the neuronal function.It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38\u2009\u00b1\u20094\u2009h of splashdown, mice were returned to Earth alive. Brain tissues were collected for analysis. A novel digital color-coded barcode counting technology (NanoString Long-duration spaceflights reportedly induce immune dysregulation, which is considered a risk to astronaut safety and mission success3. Studies to date have demonstrated the altered distribution of peripheral leukocytes, a diminished function of specific leukocyte subpopulations, and skewed cytokine profiles in many astronauts3. Many biomarkers associated with neuroinflammation following space radiation and space stressors in rodents and humans have been identified8. However, the precise nature of immune dysregulation during spaceflight is not well understood. Evidence suggests that acute exposure to galactic cosmic rays (GCRs) induce detrimental CNS changes including an increased level of neuroinflammation, neuronal damage and cognitive deficits similar to accelerated aging12. Ionizing radiation has been shown to elicit neuroinflammation through direct activation of microglia cells13 and through the increased infiltration of immune and inflammatory cells through the damaged blood-brain barrier (BBB)14. Study also showed that both microgravity encountered by astronauts in space and simulated microgravity on earth induce changes in brain structure and function15. Microgravity induces a tremendous shift in body fluids, an increase in brain fluid and alterations in tissue perfusion16. Space microgravity modulates the expression of cellular molecules, and alters pro-inflammation cytokine secretion17. Neuroinflammation is also a central pathological feature of several acute and chronic brain diseases, including Alzheimer\u2019s disease (AD), Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis18.The spaceflight environment is characterized mainly by ultraviolet and ionizing radiation, microgravity, and physiological/psychological stressors. These conditions present a significant hazard to spaceflight crews during the course of mission activities. The susceptibility of the central nervous system (CNS) to spaceflight-induced changes can be particularly devastating to the health, mission performance, and quality of life of the spaceflight crew both acutely and chronically. The health risk from spaceflight-induced neuronal damage and potential adverse neurovascular effects are a chief concern, examples are intracranial fluid redistribution results in alteration of brain perfusion, neurovestibular problems, and cognitive alterations in astronauts19, the content of oxidizable unsaturated lipids20 and low levels of anti-oxidant defenses21. Oxidative injury has been implicated as a causative or contributory factor in a number of neurodegenerative conditions, including aging, and ischemic, traumatic damage26. Our preliminary studies have shown that spaceflight and ionizing radiation cause prolonged oxidative stress and endothelial dysfunction that may lead to chronic inflammation and adverse remodeling30. However, our knowledge about the mechanism and consequences of spaceflight condition-induced neuroinflammation is very limited. Because of the complexity of the processes and pathways that lead to neuroinflammation, an in-depth \u201comics\u201d approach will potentially provide insight to understanding the impact of environmental insults on the CNS and the immune function that may have a long-lasting impact.The CNS is sensitive to oxidative injury due to its high oxygen consumptionThe goal of this study was to characterize changes in gene expression using a neuroinflammatory assay panel to investigate inflammation, neuronal function, growth, metabolism, and stress in mouse brain tissue after spaceflight.p\u2009<\u20090.05) by one-way analysis of variance (ANOVA) and Tukey\u2019s HSD (honestly significant difference) test segregates the ground control (GC) group from the flight (FLT) group (Table https://www.ncbi.nlm.nih.gov/gene/?term=NM_001110350), SLC2A5 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_019741), MERTK (https://www.ncbi.nlm.nih.gov/gene/?term=NM_008587), TREM2 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_025864), RIPK1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_009068) are listed in multiple tables as they are found to play various roles.A set of 54 differently expressed genes (DEG) significantly (up Table . Clusterhttps://www.ncbi.nlm.nih.gov/gene/?term=NM_010398) and SOX4 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_009238). The summary is presented in Table https://www.ncbi.nlm.nih.gov/gene/?term=NM_013560), GADD45G (https://www.ncbi.nlm.nih.gov/gene/?term=NM_011817), CCNG2 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_007635), SESN1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001013370), and CDC25A (https://www.ncbi.nlm.nih.gov/gene/?term=NM_007658) which were significantly upregulated. In general, we see an increased cellular stress response, growth arrest, and a possible trend toward more inflammatory-associated cell death.Genes directly involved with neuronal function were universally downregulated and are summarized in Table 31. Higher scores compared to normalized gene expression general means upregulation while lower scores represent downregulation. Figure p\u2009<\u20090.05), while scores for innate immune response and oligodendrocyte function were greatly reduced in the flight group compared to the controls which may indicate downregulation of the pathways (p\u2009<\u20090.05) by one-way ANOVA and Tukey\u2019s HSD test   Fig. .Fig. 5Sp29. Gene expression in the brain that was analyzed after 90 days of simulated microgravity showed many genes related to catalytic and oxidoreductase activities were downregulated32. However, in contrary to our findings, many genes related to stress, immune response, metabolic process, and/or inflammatory response were significantly upregulated in their study. Duration of environmental stress may play a role in different response of gene expression profiles and pathway regulation.Our data showed robust changes in gene expression profiles after spaceflight. Genes supporting neuronal synaptic signaling and migration were significantly downregulated. Decreased expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Downregulation of microglial gene expression that is important for apoptotic cell clearance, phagocytosis, and proliferation were evident. Genes supporting innate immune responses were also downregulated regarding anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response. Expression of genes that are correlated with DNA damage and cellular stress were upregulated. Many of these observed changes are in line with our previous findings in the brains exposed to simulated spaceflight condition. Following combined exposure of simulated microgravity and radiation, pathways involved in neurogenesis, neuroplasticity, the regulation of neuropeptides, neuronal structure, stress, and cellular signaling are significantly alteredhttps://www.ncbi.nlm.nih.gov/gene/?term=NM_022029) coding for a postsynaptic kinase called neurogranin was found to be significantly reduced in expression. When neurogranin binds calmodulin, the concentration of calcium needed to transduce a signal is increased33. Given the downregulation of NRGN (https://www.ncbi.nlm.nih.gov/gene/?term=NM_022029) and possible decrease in availability of neurogranin, neurons may become more sensitive to firing. KCND1(https://www.ncbi.nlm.nih.gov/gene/?term=NM_008423) codes for a potassium voltage-gated channel involved in neurotransmitter release which has been shown to be inhibited by some types of ROS generating treatments36. Environmental stressor-induced ROS may be provoking the down regulation of KCND1(https://www.ncbi.nlm.nih.gov/gene/?term=NM_008423). DLG4 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001109752) is transcribed into a protein associated with glutamatergic receptor signaling while also potentially participating in dendritogenesis37. Its downregulation observed in our assay has the potential to affect glutamatergic neuron signaling, and affect dendritic morphology leading to further dysfunction. NLGN2 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_198862) codes for a molecule which selectively mediates inhibitory synapses38. Its downregulation may contribute to dysfunction at inhibitory neuron synapses. Previous study has shown that inflammation induced loss of inhibitory nerve terminals or a redistribution of presynaptic machinery in inhibitory neurons may increase risk for developing neurological disease and psychiatric illness, including seizures and schizophrenia39.We saw uniform downregulation of gene expression in our mouse brain samples directly related to neuron function. NRGN (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001110350) behaves as a transcriptional repressor involved in cortical neuron differentiation40. In past studies the silencing of this gene in mice has shown decreased memory consolidation41. It is possible that downregulation may have similar, albeit reduced effects on mice in spaceflight. RELN (https://www.ncbi.nlm.nih.gov/gene/?term=NM_011261) codes for an extracellular matrix serine protease that regulates microtubule function and plays a role in the layering of neurons in the cerebral cortex, among other structures42. Its enzymatic activity that is important for cell adhesion and reduced transcription, as seen in our flight mice, could lead to further reduction of nervous system plasticity. SLC2A5 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_019741) in a gene responsible for the GLUT5 (https://www.ncbi.nlm.nih.gov/gene/2700445) fructose transporter that has been found in numerous areas including the BBB, and the hippocampus44. The downregulation of this gene suggests the decreased ability of cells to utilize fructose as a potential energy source.Regarding genes involved in neuron function and plasticity, we continue to see universal downregulation in the flight tissue compared to GC controls. The gene SIN3A  is a gene coding for a cell-surface receptor which binds to colony stimulating factor-1 (CSF1) (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001113529) and interleukin-34 (IL-34) (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001135100)47. CSF1R null mice have been shown to have fewer phagocytic cells and reduced survival48. Given these findings we suggest that flight mice may have a reduced capacity to generate microglial cells in the nervous system. MERTK (https://www.ncbi.nlm.nih.gov/gene/?term=NM_008587) creates a kinase protein in innate immune cells which mediates engulfment of apoptotic cells and acts as a negative regulator of inflammation51. Downregulation of this gene may contribute to immune dysregulation in our flight mice. TREM2 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_031254) codes for a regulator of microglial chemotaxis and phagocytosis of apoptotic53. In previous in vivo studies with microglial lacking TREM2, decreased migration to areas of apoptotic neurons was shown54. NCF1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001286037) codes for a key subunit of the phagocyte NADPH oxidase system56. Downregulation suggests a reduced capacity for microglial cells to generate an effective microbial defense. SLC2A5 codes for the GLUT5 fructose transporter, as described previously43. With reduced activity, microglial cells may have less capacity to utilize fructose as an energy source and may be more prone to suffer from metabolic related dysfunction.Level of gene expression related to neuron support cells, like microglial cells and oligodendrocytes were also seen universally downregulated. Microglia are the resident phagocytes of the innate immune system in the brain and play an important role in cytokine production58. A couple of key genes involved in the oligodendrocyte function have been significantly downregulated in the flight mice. PLP1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_011123) codes for a major myelin protein that plays an important role in its formation and maintenance59. The implication of significantly downregulated PLP1 indicates impaired neuron axonal function with the possibility of long-term neurodegeneration. OPALIN (https://www.ncbi.nlm.nih.gov/gene/?term=NM_153520) plays a role in oligodendrocyte cytoskeletal remolding and morphology. Downregulation of this gene observed in our study may be an indication of decreased activity of oligodendrocytes. These findings are further reinforced by a significantly decreased oligodendrocyte function pathway score  and SOX4 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_009238). H2-T23 expression is essential for immunological protection and regulation60. HT-T23 dependent T-cell inhibitory interactions aid in preventing expansion of autoreactive CD4 T-cells and collateral autoimmune diseases61. Upregulation of this gene may be a protective response to generalized inflammation in the flight mice. SOX4 directly regulates Innate-like \u03b3\u03b4 T-cell (T\u03b3\u03b417), a major source of interleukin-17 (IL-17) (https://www.ncbi.nlm.nih.gov/gene/?term=NM_010552). Increased SOX4 expression may lead to an increase of this cytokine expression in the flight mice62.In terms of gene expression that associated with general immune functions and inflammation, only two genes were found to be significantly upregulated in our flight mice population, H2-T23 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_009068) has been shown to be a promotor of survival, apoptotic and inflammatory signaling pathways64. However, mice deficient in RIPK1 demonstrated loss of inhibition of a necroptosis pathway which promoted the release of necroptotic damage-associated molecular patterns65. Downregulation of RIPK1 observed in our study may lead to a similar reduction of appropriate apoptosis and an increase in necroptosis resulting in undesirable inflammation. Overall, taking these findings in conjunction with a significantly increased cytokine signaling pathway score  is a pore-forming bactericidal molecule of the innate immune system. It has been shown to be required for the activity of ROS and nitric oxide and their antibacterial effects67. OPTN (https://www.ncbi.nlm.nih.gov/gene/?term=NM_181848) codes for a protein that plays a key role in bacterial autophagic clearance. Deficient mice have also been shown to have impaired interferon regulatory factors (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001159393) signaling and reduced response to toll-like receptor stimulation68. In summary, these results imply the impaired antibacterial capabilities and increased viral susceptibility in the flight mouse brain compared to GC group.Immune dysfunction is further illustrated via the downregulation of two genes important for antimicrobial defense. MPEG1  plays an important role in the metabolism of cholesterol and cholesterol-related compounds72. In humans, complete CYP27A1 deficiency leads to nodule formation in the brain which may lead to dementia, cerebellar ataxia, and spinal cord paresis73. This altered cholesterol metabolism closely associates with inflammatory responses involved in the pathogenesis of AD progression74. In deficient mice, a significant increase in cholestanol in the brain is observed75. Downregulation of CYP27A1, as seen in our flight mice, may lead to neurodegeneration and inflammation in more chronic settings or to a milder degree.Cytochrome P450 27A1 (CYP27A1) (https://www.ncbi.nlm.nih.gov/gene/?term=NM_011817) has been found to play a role in activating checkpoints in the cell cycle following exposure of cells to irradiation76. CCNG2 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_007635) contributes to cell cycle arrest during DNA damage and is upregulated in response to diverse stimuli, including hypoxia77. Additionally, expression of CCNG2 is found to be significantly increased in cell cycle-arrested and terminally differentiated cells78. Upregulation of these genes may be due to exposure to irradiation, among other stressors, during spaceflight.Fourteen genes that were significantly altered in the flight mice compared to GCs were found to have direct roles in cellular growth, proliferation, and stress response. Two upregulated genes play an important part in cell cycle arrest during times of cellular stress. GADD45G (https://www.ncbi.nlm.nih.gov/gene/?term=NM_010238) codes for a nuclear kinase involved in regulating the expression of cell cycle genes via binding to multiple E2Fs, a family of transcription factors79. E2F1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_007891) regulates activation of DNA replication and G1/S transition when interacting with BRD280. BRD2 also interacts with BRD3, a chromatin reader with roles in regulating transcription81. BRD3 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001113573) further regulates transcription by promoting the binding of the transcription factors to their targets82. SIN3A (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001110350) codes for a transcription factor which regulates cell cycle progression by repressing gene expression for cell cycle inhibitor40. It has been linked to functional cellular changes in proliferation, cell cycle, and stem cell function in mice models83. Downregulation of these genes involved in the promotion of cellular proliferation may further amplify the upregulation of genes with roles in cell cycle arrest. These changes are similarly reinforced by a significantly increased epigenetic regulation pathway score  is a kinase involved with responses to cellular stress including apoptosis, inflammation, cellular proliferation, and neurodegeneration. A deficiency of MAP2K4 has been shown to result in cellular susceptibility to stress-induced apoptosis and growth inhibition86. However, it has been demonstrated that disruption of both MAP2K4 and MAP2K7 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001042557) genes was required to thoroughly block cellular growth caused by environmental stressors87. MRE11A (https://www.ncbi.nlm.nih.gov/gene/?term=NM_018736), a part of an exonuclease complex central to the cellular DNA damage response was downregulated. The MRE11 complex is essential to vertebrates and defects lead to sensitivity to DNA damage and cell cycle checkpoints deficiency88. Its downregulation may point to a certain level of dysfunction leading to an impaired response to cellular stress leaving the flight mice more vulnerable to DNA damage.Many downregulated genes were known to be involved in cellular stress responses. MAP2K4  levels using 48-h delayed telemetry data from the FLT group. Water and food bar diet specifically designed by NASA were provided ad libitum to FLT and GC groups. All mice received the same access to food and water. NASA-Ames Research Center and KSC Institutional Animal Care and Use Committees approved this flight study. The study has been done in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health.Ten-weeks-old male C57BL/6 mice  at the time of launch, were flown for NASA\u2019s ninth Rodent Research experiment (RR-9) on SpaceX-12 for a 35-day mission and lived in NASA\u2019s Rodent Habitats aboard the International Space Station. All FLT mice were maintained at an ambient temperature of 26\u201328\u2009\u00b0C with a 12-h light/dark cycle during the flight. Habitat GC mice were kept under similar housing conditions, including temperature, humidity and carbon dioxide  from each mouse (n\u2009=\u20095\u20136 per group) was placed in a sterile cryovial, snap frozen in liquid nitrogen and kept at \u221280\u2009\u00b0C prior to use.Within 38\u2009\u00b1\u20094\u2009h of splashdown, the FLT mice were rapidly euthanized in 100% CO\u00ae neuroflammation pathways panel. The panel includes 757 genes covering the core pathways and processes that define neuroimmune interactions and 13 potential housekeeping genes for normalization. RNA samples (100\u2009ng each) on 2 cartridges were used for the Gene Expression Assay with Mouse Neuroinflammation panel performed on the nCounter MAX system , a multi-channel epifluorescence scanner with Nanostring Advanced Analysis Module plugin for QC, normalization, and differential expression analysis (DE). Data files generated from nCounter system were analyzed using nSolver 4.0 software with the Advanced Analysis module for QC , normalization, DE analysis, and gene-set enrichment analysis. Data normalization included 2 steps: Positive control normalization to correct platform-associated variation and Codeset content normalization using the housekeeping (HK) genes to correct variability of input samples. Geometric means of selected HK probes were used to normalize counts of the samples and DEGs were generated from the normalized counts.Isolation of total RNA from the brain tissues was performed using DNA/RNA/miRNA Universal Kit  according to the manufacturer\u2019s instruction. Briefly, tissue homogenization was performed using 1.5\u2009mm beads  on Minilys homogenizer (Bertin Technologies) in RLT Plus buffer. The purity and concentration of the eluted RNA were measured using Nanodrop 2000  and stored at \u221280\u2009\u00b0C until further analysis. RNA samples (20\u2009ng/ul) were then shipped to Nanostring  and gene expression profiling of brain tissues was conducted using the nCounter86 and data are summarized from changes of a gene set within a given pathway into a single score. To further analyze the associated pathways of the DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was performed using biological process database to identify the annotated sets of genes based on the biological processes in which they participate. Significantly differential expression genes were presented in the tables with individual genes using log10  andlog2 fold change compared to the GC group. Pathview module was used to display upregulated genes or downregulated genes overlaid on KEGG pathways. For DEG and pathway analysis, p\u2009<\u20090.05 was considered statistically significant between FLT and GC groups by one-way ANOVA and Tukey\u2019s HSD test.Gene expression profiling data were analyzed using nSolver analysis and the advanced analysis module software. Advanced analysis module software uses open-source R program for pathway scoring and gene-set enrichment analysis. Using the Reactome pathway database annotations, pathway scores are derived by calculating the first principle component of pathway genes\u2019 normalized expressionFurther information on research design is available in the DatasetReporting Summary"}
+{"text": "Indium tin oxide thin films were deposited by magnetron sputtering on ceramic aluminum nitride substrates and were annealed at temperatures of 500 \u00b0C and 600 \u00b0C. The structural, optical, electrically conductive and gas-sensitive properties of indium tin oxide thin films were studied. The possibility of developing sensors with low nominal resistance and relatively high sensitivity to gases was shown. The resistance of indium tin oxide thin films annealed at 500 \u00b0C in pure dry air did not exceed 350 Ohms and dropped by about 2 times when increasing the annealing temperature to 100 \u00b0C. Indium tin oxide thin films annealed at 500 \u00b0C were characterized by high sensitivity to gases. The maximum responses to 2000 ppm hydrogen, 1000 ppm ammonia and 100 ppm nitrogen dioxide for these films were 2.21 arbitrary units, 2.39 arbitrary units and 2.14 arbitrary units at operating temperatures of 400 \u00b0C, 350 \u00b0C and 350 \u00b0C, respectively. These films were characterized by short response and recovery times. The drift of indium tin oxide thin-film gas-sensitive characteristics during cyclic exposure to reducing gases did not exceed 1%. A qualitative model of the sensory effect is proposed. A low nominal resistance not higher than or within the specified ranges for commercial sensors is necessary to achieve for developing a competitive device. Commercial SnO2-sensitive elements are obtained by thick-film and ceramic technologies   , where ET. The response times of ITO thin films for all reducing gases at T \u2265 350 \u00b0C do not exceed 20 s, and the recovery times do not exceed 100 s. The longest rest and rect are observed under exposure to NO2, which is caused by the large value of the binding energy of this molecule to the surface [The response and recovery times drop exponentially with  surface .annT = 500 \u00b0C are characterized by the highest responses to H2, NH3, CO and NO2. Therefore, only these films will be further considered. 2, NH3, CO and NO2 concentrations for ITO-500 thin films at MAXT = T. The response increases with the concentration according to the power law, S1,2 ~ ngm, where m is the power index. The m values for H2, NH3, CO and NO2 at MAXT = T are compared in ITO thin films annealed at MAXT = T and under cyclic exposure to H2, NH3, CO and NO2. Estimates have shown that the drift of ITO-500 thin-film characteristics is practically absent under exposure to reducing gases. The deviations of Rair, Rg and S1 from the average values do not exceed 1%. Significant deviations of 5% occurred under exposure to NO2. The I\u2013V characteristics of ITO-500 films in pure dry air and when exposed to H2, NH3, CO and NO2 are linear in the voltage range of \u221210\u201310 V 2, where NNO2 is the surface density of chemisorbed NO2. Changes in G when exposed to gases are caused mainly by changes in e\u03c6s. Interactions between target gas molecules and O\u2212 on the ITO surface can be described by the following reactions [For an n-type semiconductor, oxygen chemisorption leads to the upward bending of energy bands ,21,22. T\u00b0C is O\u2212 . For meteactions ,48,49:H22O, N2 and CO2 molecules are desorbed. Reactions (8)\u2013(12) are the simplest possible and only fundamentally explain the observed sensory effect. Many reasonable variants of other reactions on the surface of metal oxide semiconductors have been proposed. Their consideration is not advisable to describe a qualitative model of the sensory effect for the studied ITO films.As a result of Reactions (8)\u2013(11), the conductivity of the semiconductor increases, and reaction products in the form of H2O3 and SnO2 mixed-oxide system was studied in more detail. The tin oxide content in the mixture varied widely. It was found that with an increase in the content of SnO2 from 0 to 10 wt.%, the conductivity of the mixture increases, and the response to H2 decreases. This composition corresponds to the studied ITO thin film. The limit of SnO2 solubility in In2O3 is 10\u201315 wt.%. The electrically conductive and gas-sensitive properties of the In2O3 and SnO2 mixed-oxide system are determined by In2O3 up to these values of SnO2 concentration. The introduction of Sn into the In2O3 matrix leads to a decrease in the surface density of chemisorbed oxygen ions and the responses of the films to H2. With a further increase in the concentration of SnO2, the conductivity decreases, and the response to H2 increases. Thus, in our case, Sn (SnO2) acts as an additive to In2O3 and reduces the resistance of the film. The sensing properties of the studied ITO thin films are determined by In2O3.In Ref. , the sen2 content of 5\u201310%. The resistance Rair and resistivity \u03c1air of ITO thin films in pure air were measured at room temperature. It is worth noting that for most ITO thin films, high responses to gases corresponded to high nominal resistance. As a rule, high-sensitivity ITO films are characterized by a low thickness, less than 90 nm. High responses were shown by ITO films with an island microstructure [Rair) and relatively high sensitivity to gases .tructure . The diatructure . Neverthtructure . The mai2, NH3, CO, NO2 and CH4 was studied in the operating temperature ranges of 150\u2013450 \u00b0C and 150\u2013550 \u00b0C for ITO films annealed at 500 \u00b0C and 600 \u00b0C, respectively. ITO films annealed at 500 \u00b0C were characterized by higher sensitivity to gases. The maximum responses to 2000 ppm H2, 1000 ppm NH3 and 100 ppm NO2 for these films were 2.21, 2.39 and 2.14 at operating temperatures of 400 \u00b0C, 350 \u00b0C and 350 \u00b0C, respectively. An increase in the annealing temperature led to a decrease in the gas sensitivity of the films. At operating temperatures of at least 350 \u00b0C, the films were characterized by short response and recovery times that did not exceed 20 s and 100 s, respectively, under exposure to reducing gases. The drift of the ITO film characteristics under cyclic exposure to reducing gases did not exceed 1%. A qualitative model of the sensory effect was proposed. The gas sensitivity of the ITO films was determined mainly by In2O3. SnO2 acted only as an additive, a source of Sn donor impurities. The possibility of developing sensors with low nominal resistance and relatively high sensitivity to gases was shown.The structural, optical, electrically conductive and gas-sensitive properties of magnetron-sputtered ITO thin films on AlN ceramic substrates annealed in air for 60 min at temperatures of 500 \u00b0C and 600 \u00b0C were investigated. The microrelief of ITO films annealed at 500 \u00b0C was represented by grains with dimensions of 40\u2013100 nm, which form agglomerates up to 350 nm in size. The tin content in these films was 4.10\u20135.15 wt.%, and the band-gap energy was 3.62 eV. An increase in the annealing temperature to 600 \u00b0C led to increases in the size of grains to 80\u2013140 nm, in the tin content to 4.97\u20136.15 wt.%, and in the band-gap energy to 3.65 eV. In pure dry air, the ITO thin-film resistance annealed at 500 \u00b0C does not exceed 350 Ohms, and the resistance of films annealed at 600 \u00b0C dropped by about 2 times. Sensitivity to H"}
+{"text": "Leishmania donovani is an intracellular pathogen that causes visceral leishmaniasis. The strategies employed by Leishmania donovani to modulate the host epigenome in order to overcome the host defense for their persistence has been worked out in this study. We show that L. donovani negatively affects BRG1, a catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, to alter IFN\u03b3 induced host responses. We observed that L. donovani infection downregulates BRG1 expression both at transcript and protein levels in cells stimulated with IFN\u03b3. We also observed a significant decrease in IFN\u03b3 responsive gene, Class II transactivator (CIITA), as well as its downstream genes, MHC-II (HLA-DR and HLA-DM). Also, the occupancy of BRG1 at CIITA promoters I and IV was disrupted. A reversal in CIITA expression and decreased parasite load was observed with BRG1 overexpression, thus, suggesting BRG1 is a potential negative regulator for the survival of intracellular parasites in an early phase of infection. We also observed a decrease in H3 acetylation at the promoters of CIITA, post parasite infection. Silencing of HDAC1, resulted in increased CIITA expression, and further decreased parasite load. Taken together, we suggest that intracellular parasites in an early phase of infection negatively regulates BRG1 by using host HDAC1 for its survival inside the host.Intracellular pathogens manipulate the host cell for their own survival by contributing to modifications of host epigenome, and thus, altering expression of genes involved in the pathogenesis. Both ATP-dependent chromatin remodeling complex and histone modifications has been shown to be involved in the activation of IFN\u03b3 responsive genes.  Leishmania donovani   cell surface receptor protein  . CIITA i protein  which arer cells . IFN\u03b3, aer cells , via JAK pathway . BAF com pathway . Leishmaolecules . L. dono nucleus . Expressrophages . HoweverLeishmania parasite to manipulate the host defense system, in this study, we have analysed the impact of L. donovani infection on host BRG1, and further affecting IFN\u03b3 responsiveness, using THP-1 cells as the model system , to disrupt the host immune system. A study by Zika et\u00a0al., demonstrated that inhibition of histone deacetylases (HDACs) enhanced the expression of MHC class II cell surface receptor\u00a0protein encoded by the\u00a0human leukocyte antigen\u00a0complex\u00a0(HLA complex)  . We in athe host . In the BRG1 rabbit monoclonal antibody  was purchased from Abcam, UK. STAT1\u03b1 rabbit polyclonal antibody  and \u03b2-actin mouse monoclonal antibody  were purchased from Sigma-Aldrich, USA. Anti-mouse IgG, HRP-linked antibody , and anti-rabbit IgG, HRP-linked antibody  was purchased from Cell Signaling Technology, USA. Alexa green 488-conjugated goat anti-rabbit IgG  was purchased from Thermo Fisher Scientific, USA.L. donovani Bob (LdBob/strain/MHOM/SD/62/1SCL2D) (/1SCL2D)  acquired/1SCL2D) .6 cells/ml) were differentiated and infected as previously described  as described in . ExpressT method .6 cells/ml). Further, qPCR was performed using promoter-specific primers  at 4\u00b0C. 80 \u03bcg of total protein was separated on 8% SDS-PAGE by electrophoresis . Immunoblotting was performed as described before  followed by incubation with Alexa green 488-conjugated goat anti-rabbit IgG (1:200). DAPI (1 \u03bcg/ml) was used to stain the host nuclei and parasite kinetoplastid DNA. All antibody incubations were followed by washes with 0.2% Triton X-100. The images were then visualized under a confocal laser scanning microscope (Olympus FluoViewTM FV1000) at 488 nm wavelength.6 cells/ml) were differentiated, followed by parasite infection and IFN\u03b3 stimulation as mentioned above. Subsequently, the cells were transiently transfected with 1.5 \u03bcg of plasmid overexpressing BRG1 (THP-1 cells (10ing BRG1  for 48\u00a0h5 cells/ml) were transiently transfected with 600 pmole (HDAC1 (3065) siRNA \u2013 SMARTpool  using lipofectamine 3000. The cells were incubated with siRNA for 24\u00a0h to allow gene silencing. Subsequently, the cells were washed for infection and stimulated as described earlier. THP-1 cells were harvested after 6\u00a0h and mRNA expression of HDAC1 and CIITA genes was analyzed by qPCR. ON-TARGET plus Control Pool  was used as a negative control. The basal level of HDAC1 and CIITA in uninfected Sc-siRNA transfected cells respectively were used for data normalization and were taken as 1.0. The transfection efficiency was calculated (ThermoFisher) to be >50%, as has also been reported by the manufacturer .The PMA treated differentiated THP-1 cells (1000 pmole  of siGEN5 cells/ml) were infected and stimulated with IFN\u03b3 as mentioned earlier. After 6\u00a0h, for visualization of intracellular parasites, Giemsa staining was performed, and the parasite load was calculated (THP-1 cells (10lculated .P \u2264 0.05 was considered significant . Error bars used in the figures specify standard error of the mean (Cheeseman and Weitzman).GraphPad Prism (version 5.0) software  was used for plotting data. Statistical analysis was measured using ANOVA. Leishmania infection in the host cells at an initial stage of infection. The impact of IFN\u03b3 on parasite load within the infected macrophages was analyzed by visually counting the intracellular amastigotes after Giemsa staining , 6\u00a0h  and 24\u00a0h  in uninfected and stimulated cells as compared to the resting macrophages. This is similar to the upregulation of STAT1\u03b1 observed in the previous studies , 6\u00a0h  and 24\u00a0h  in comparison to the uninfected, stimulated cells  higher in uninfected cells as compared to the resting macrophages (P = 0.0195) on infection as compared to the uninfected, stimulated cells  instead of live parasites. qPCR data clearly demonstrated that HKLD infection did not have any effect on the regulation of BRG1 expression  cells (CIITA promoter IV (pIV) and promoter I (pI) both mediate IFN\u03b3 inducible gene expression, we hypothesized that the occupancy of BRG1 would be reduced on these promoters leading to the decreased expression of CIITA. Therefore, we checked the occupancy of BRG1 on CIITA pIV and also for the first time on pI  cells . Since CCIITA pIV  and pI  in uninfected cells as compared to the resting macrophages. Further, the occupancy of BRG1 decreased significantly on both pIV  and pI  in infected, stimulated cells in comparison to uninfected, stimulated cells (CIITA promoters is in concordance with the CIITA expression pattern (TOP1) was also checked, which served as a negative control. No occupancy of BRG1 at TOP1 promoter region was observed  are downregulated in IFN\u03b3 stimulated host cells post Leishmania infection  and pI  in uninfected cells when compared to the resting macrophages. On infection, the occupancy of STAT1\u03b1 decreased significantly at both the promoters  as compared to the uninfected, stimulated cells. No binding of STAT1\u03b1 at TOP1 promoter region was detected  significantly in infected cells as compared to the uninfected one  was observed in infected and stimulated cells overexpressing BRG1, compared to infected and stimulated cells overexpressing only vector  and pI  in uninfected cells was observed as compared to the resting macrophages  (Enhanced transcription of genes like 3 and H4 . BRG1 is3 and H4 . TherefoLeishmania infection (HDAC1-siRNA or scrambled-siRNA (Sc-siRNA). In THP-1 cells transfected with Sc-siRNA, comparable levels of HDAC1 were observed in uninfected and infected conditions showing that Sc-siRNA has no inhibitory effect on host HDAC1 expression. When THP-1 cells were transfected with HDAC1-siRNA, a decrease in HDAC1 expression was observed in both infected  and uninfected  cells. This data confirms a specific silencing effect of HDAC1-siRNA on the expression of host HDAC1  and uninfected  cells. Thus, suggesting that downregulation of HDAC1 is beneficial for the expression of CIITA.The expression of HDAC1-siRNA on parasite load, visual counting of intracellular amastigotes was done after Giemsa staining (P = 0.04) was significantly downregulated in cells transfected with HDAC1-siRNA as compared to Sc-siRNA. This data demonstrates the positive effect of host HDAC1 on parasite survival.To determine the role of CIITA promoters   and VI-D&P/569/2016-17/TDT/C from Department of Science and Technology, India (www.dst.gov.in). HB was supported by fellowship from CSIR, GR and EM was supported by D.S. Kothari Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.RMa was funded by EMR/2016/004948 from Science and Engineering Research Board, India (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "This study was aimed to investigate whether patients with epilepsy (PWE) have higher depression and anxiety levels than the normal population in low-risk areas for coronavirus disease 2019 (COVID-19) in the northern part of Guizhou Province, China, during the COVID-19 epidemic, to\u00a0evaluate their knowledge on COVID-19, and to\u00a0analyze related factors for the psychological distress of PWE at this special time.The survey was conducted online from February 28, 2020 to March 7, 2020 via\u00a0a questionnaire. PWE from the outpatient clinic of epilepsy of the Affiliated Hospital of Zunyi Medical University, and healthy people matched for age and sex, participated in this study. Mental health was assessed via a generalized anxiety self-rating scale (GAD-7) and the self-rating depression scale (PHQ-9). The knowledge of COVID-19 in both groups was investigated.P\u2009<\u20090.01) and GAD-7 (P\u2009<\u20090.001) were higher in the PWE group than in the healthy group. There was a significant difference in the proportions of respondents with different severities of depression and anxiety, between the two groups, which revealed significantly higher degree of depression and anxiety in PWE than in healthy people . Overwhelming awareness and stressful concerns for the pandemic and female patients with epilepsy were key factors that affect the level of anxiety and depression in PWE. Further, the PWE had less accurate knowledge of COVID-19 than healthy people (P\u2009<\u20090.001). There was no statistically significant difference between the two groups in the knowledge of virus transmission route, incubation period, susceptible population, transmission speed, clinical characteristics, and isolation measures on COVID-19 (P\u2009>\u20090.05). PWE knew less about some of the prevention and control measures of COVID-19 than healthy people.There were no significant differences in the general demographics between the PWE and healthy control groups. The scores of PHQ-9 (During the COVID-19 epidemic, excessive attention to the epidemic and the\u00a0female sex are\u00a0factors associated\u00a0with anxiety and depression in PWE, even in low-risk areas. Epilepsy is a common disorder of the nervous system, affecting approximately 70 million people worldwide . In 2017The occurrence of public health incidents, such as the Ebola virus that is still circulating in Africa, represents a significant threat to public health and increases the risk of emotional problems. Patients recovering from Ebola have higher scores of anxiety and depression than the normal population . Also noAlthough there has been no COVID-19 outbreak in the economically underdeveloped northern part of Guizhou Province in China, sporadic cases still occurred, and local administrative departments have strictly implemented prevention and control measures. Previous studies have confirmed that depression and anxiety are more commonly seen in PWE than in the general population , 24. MeaThis study was a single-center, cross-sectional study. The questionnaire survey was conducted online through Wechat from February 28 to March 7, 2020. The inclusion criteria of the epilepsy group were: (1) having a history of epilepsy for at least 1\u2009year and being followed up by neurologists every month; (2) aged\u00a0>\u200914\u2009years;\u00a0and (3) being treated in the outpatient clinic of epilepsy of the Affiliated Hospital of Zunyi Medical University. The exclusion criteria were: (1) having nervous system diseases such as cerebral hemorrhage or a tendency of cerebral hemorrhage, intracranial tumor, or cerebral infarction; (2) having understanding disorders, obvious aphasia, communication disorders, mental retardation and hereditary mental disorders; or\u00a0(3) having severe organic diseases such as liver and kidney diseases. The inclusion criteria of the healthy group were: (1) over 14\u2009years old; (2) having good physical condition, no chronic disease; and (3) with no communication disorder or mental retardation. The exclusion criteria of the healthy group were: (1) younger than 14\u2009years; and (2) unable to understand the questionnaire content. This study complied with the recommendations of the Ethics Committee of Affiliated Hospital of Zunyi Medical University, and was approved by the Ethics Committee of Affiliated Hospital of Zunyi Medical University.The content of the questionnaire consisted of four parts. The first part collected demographic information, including sex, age, nationality, and educational level. The second part assessed participants\u2019 knowledge on COVID-19 and the associated control measures. The third part contained anxiety and depression scales. The generalized anxiety disorder-7 (GAD-7) is the only proven scale for PWE anxiety . It is aThe Patient Health Questionnaire 9 (PHQ-9) is a screening tool for depression and has high reliability and validity in PWE . AccordiThe fourth part assessed patients\u2019 adherence to antiepileptic drugs and the seizure status in the previous month before initiation of this study.P\u2009<\u20090.05.Statistical analysis was performed using the SPSS 26.0 software. The Chi-square test was used to compare the classification variables between groups. PHQ-9 and GAD-7 scores were skewed, so the rank-sum test was used for comparison between groups. The Mann-Whitney test was used to compare non-normally distributed continuous variables. The difference was considered statistically significant when A total of 410 questionnaires were collected, of which 10 were excluded because the respondents were under 14\u2009years of age. As a result, a total of 400 valid questionnaires from 200 PWE and 200 healthy subjects were analyzed.Table\u00a0P\u2009<\u20090.01) and GAD-7 (P\u2009<\u20090.001) scores in the PWE group were higher than those in the control group (Table\u00a0P\u2009=\u20090) (Table\u00a0The PHQ-9 (up Table\u00a0. In addi0) Table\u00a0.Table 2SP\u2009<\u20090.01) (Table\u00a0P\u2009<\u20090.05). However, there were no significant differences in anxiety and depression scores between AWE of different ages, between AWE who considered life was disturbed and undisturbed, between AWE taking single ASM and multiple ASMs, and between AWE who had epileptic seizures in the past month and who did not (P\u2009>\u20090.05).The PHQ-9 and GAD-7 scores of the females in the epilepsy group were higher than those of the males (1) Table\u00a0. The PWEThe PWE and control groups both had high accuracy in knowledge on the source of infection (confirmed patients and asymptomatic infection), route of transmission (such as respiratory droplets and contact transmission), incubation period, clinical manifestations , isolation measures, treatment and prognosis of COVID-19. The PWE group had a lower rate of correct answer regarding the need for negative results of two nucleic acid tests  for exclusion from suspected cases, compared to the normal group. Both groups had a high rate of correct answer for the use of 75% alcohol and 84 disinfectant to effectively inactivate the virus, and had a high awareness rate of reducing the number of parties and taking personal protective measures . Among the relevant measures for the prevention and control of COVID-19, staying at home to prevent infection, wearing masks when going out, washing hands frequently, seeking medical treatment, actively isolating after contact with high-risk groups, covering the mouth and nose when coughing or sneezing, and paying attention to keeping warm are measures that were recognized by participants of both groups. Further, the PWE group had a lower recognition rate regarding monitoring of body temperature, proper exercise, and disinfection of the home environment. Besides, the normal control group had a very high awareness rate of other personal protection measures, except the home environment disinfection measure Tables\u00a0.Table 5Cn\u2009=\u2009182) had significantly higher anxiety and depression scores than those in the normal group (n\u2009=\u2009190) (P\u2009<\u20090.05, Table\u00a0The patients in the epilepsy group who cared about COVID-19 daily (PWE are likely to suffer mental disorders. The influencing factors for emotional problems in PWE include sex , age, frGiven the severe morbidity and high mortality in epilepsy patients, the ongoing COVID-19 pandemic has placed an unprecedented stress on the patients, and even more serious social and economic impact than natural disasters such as earthquakes and hurricanes .In this study, we described the mental health status of PWE in the northern part of Guizhou Province during the COVID-19 pandemic, in comparison with the healthy group, and analyzed the related risk factors for PWE mental state fluctuations during the COVID-19 pandemic. We found that 38.5% of PWE had depression and 31% of PWE had anxiety, a rate significantly higher than that in the healthy group. Some studies have shown that over 13% of PWE experienced severe mental stress during the COVID-19 pandemic. The daily amount of time PWE spent on getting information of COVID-19 from the\u00a0media was associated with the severity of psychological distress in PWE during seizures . ConsistWe found that PWE who had a daily follow-up on the COVID-19 pandemic were more prone to anxiety and depression. The input of COVID-19 epidemic information from social media, TV news, radio and other means adds to their psychological burden . A meta-Another factor affecting the mental state of PWE during the pandemic was whether their lives are disrupted. The levels of depression and anxiety of PWE who felt their lives were disturbed were significantly higher than those of PWE who felt no disturbance. It is worth noting that the PWE had normal psychological coping and adjustment abilities. The COVID-19 outbreak occurred during the Chinese New Year Festival, which resulted in cancelation of all celebrations, shut-down of transportation, businesses and public entertainments, and blockade of family reunion. All of the restrictions would cause greater psychological distress in PWE.In the other survey, we found a lower proportion of PWE with correct knowledge on COVID-19 than that in the healthy group, which may be due to the knowledge and education of PWE, or because that epilepsy patients paid too much attention to their diseases and were not interested in COVID-19 knowledge. The lack of awareness of COVID-19 may also cause panic, anxiety and depression in PWE. Cross-sectional studies have also confirmed that the lower the level of education, the greater the risk of depression . The reaThis study had some limitations. With a cross-sectional design, the study did not track the psychological status of the two groups of subjects, nor did it track whether new mental disorders developed as the epidemic progressed. In addition, the psychological assessments were based on online surveys and self-report tools. In future studies, the use of clinical interviews will be encouraged to allow for a more comprehensive assessment.During a public health outbreak, healthcare professionals should focus not only on seizure control but also on the mental health of the PWE. The PWE need more psychological guidance, and the government should provide more psychological assistance to PWE while taking preventive and control measures."}
+{"text": "BackgroundThe prevalence of fetal macrosomia varies worldwide. Its trend has increased over the past decades in many developed nations. It is associated with various maternal and fetal complications. The information regarding the frequency of fetal macrosomia among non-diabetic women is limited in resource-limited countries such as Pakistan. Therefore, this study aimed to determine the number of fetal macrosomia cases among non-diabetic women.MethodologyThis was a cross-sectional study conducted in a tertiary care hospital in Peshawar, Pakistan. A total of 119 pregnant women were enrolled in the study. All pregnant women aged 15 to 45 years who had singleton pregnancies with any parity or gravida and a gestational age of \u226537 weeks were included in the study. Pregnant women with underlying chronic systemic disorders such as diabetes mellitus, gestational diabetes mellitus, hypertension, renal or cardiac disorders, and sickle cell anemia were excluded from the study. Women who did not consent to participate and those with a gestational age of \u226542 weeks at the time of delivery were also excluded from our study. Based on a 5.2% prevalence of fetal macrosomia in the general population, the sample size was calculated using the World Health Organization calculator at a confidence interval of 95%, absolute precision of 0.05 with anticipated population proportion, and a 4% margin of error. The required sample size was calculated at 119. The chi-square test was applied. P-values of \u22640.05 were considered significant.Results2. Post-stratification, spontaneous vaginal delivery was the only significant variable with a P-value of <0.05 in our study.Out of 119 participants, fetal macrosomia among non-diabetic women was seen in 10 (8.4%) cases. The mean age of patients in our study was 29.80 \u00b1 4.33 years. The mean gestational age was 36.05 \u00b1 1.31 weeks, whereas the mean body mass index of participants was 29.17 \u00b1 2.36 kg/mConclusionsThe number of fetal macrosomia among non-diabetic women in our study was 10 (8.4%). Because this was a single-center, hospital-based, cross-sectional study,\u00a0we need to conduct large multi-centered randomized controlled studies to identify the actual prevalence of fetal macrosomia in non-diabetic women in our population. Fetal macrosomia is associated with numerous neonatal and maternal complications such as birth asphyxia, clavicle fractures, and brachial plexus injuries in the fetus. Maternal complications include shoulder dystocia, postpartum hemorrhage, infection, large perineal tears, cesarean delivery, and thromboembolic events ,2. MultiFetal macrosomia, defined as a fetus weighing more than 4,000 to 4,500 g , is a global problem, and its prevalence has increased over the past decades in many developed nations . A case-In this study, we hypothesized that the number of fetal macrosomia cases among non-diabetic women is increasing in Pakistan. Information regarding fetal macrosomia among non-diabetic women and its associated complications is limited in developing countries like Pakistan due to the lack of adequate data collection and follow-up. Therefore, this study aimed to determine the number of fetal macrosomia cases among non-diabetic women.This was a single-center, cross-sectional study conducted in a tertiary care hospital  from September 8, 2020, to March 7, 2021. A total of 119 patients were enrolled in the study. The study was approved by the Institutional Research and Ethical Board of Lady Reading Hospital Medical Teaching Institute (protocol number: 274/2020). Written informed consent was obtained from participants included in this study.Inclusion criteriaAll pregnant women aged 15 to 45 years who had singleton pregnancies with any parity and a gestational age of \u226537 weeks were included in the study. The number of times a woman gets pregnant is termed gravida; it can be primigravida (first-time pregnancy) or multigravida (more than one time). The number of times a woman has given birth to a fetus  with a gestational age of 24 weeks or more is termed parity; it can be primiparous (first time) or multiparous (more than one time). The mode of delivery was categorized into spontaneous vaginal delivery (SVD) or cesarean section. Mothers who gave birth without requiring any surgical intervention were termed SVD, whereas those who gave birth by a surgical intervention were termed cesarean section.Exclusion criteriaWe excluded pregnant women with pre-existing DM or GDM diagnosed by the oral glucose tolerance test (OGTT). We also excluded women with associated chronic medical conditions such as hypertension (HTN), renal or cardiac disorders, and sickle cell disease. Women with multiple pregnancies diagnosed by ultrasound and a period of gestation of \u226542 weeks at the time of delivery were excluded. Further, those who did not consent to participate in the study were excluded.2 (overweight) or >30 kg/m2 (obese).Factors examined included maternal age, gestational age of participants, gravida, parity, body mass index (BMI), previous history of macrosomia, and mode of delivery.\u00a0We considered fetal macrosomia in all newborns who had a birth weight of \u22654,000 to 4,500 g. Detailed history, clinical examination, and laboratory investigation were performed to exclude DM. The non-diabetic status of participants was confirmed from the antenatal records. Pregnant women with fasting plasma glucose (FPG) of 5.3 mmol/L, two-hour plasma glucose of 6.4 mmol/L after a meal, or HbA1C of <6.5% were considered non-diabetic. Fasting blood sugar and two-hour post-meal was repeated during their stay in the hospital. Weight and height were assessed in all patients and were graded according to the World Health Organization (WHO) protocol. BMI of pregnant patients was categorized into those with a BMI of 25-30 kg/mThe questionnaire was developed based on a previously published paper. Based on a 5.2% prevalence of fetal macrosomia in the general population, the sample size was calculated using the WHO calculator with a confidence interval of 95%, absolute precision of 0.05 with anticipated population proportion, and a 4% margin of error. The required sample size was calculated to be 119 . The nonAll the information recorded on the questionnaire was analyzed using SPSS version 20.0 . The mean and standard deviation were computed for continuous variables, such as age, weight, height, BMI, and gestational age, whereas frequency and percentages were calculated for categorical variables, such as parity, gravida, mode of delivery, history of macrosomia,\u00a0previous post-term delivery (>37 weeks), and fetal macrosomia.Stratification with respect to age, gestational age, gravida, parity, BMI, mode of delivery, and previous history of macrosomia was then evaluated to determine effect modification. The Chi-square test was applied. P-values of \u22640.05 were considered significant.2. The detailed demographic characteristics of participants are listed in Table In total, 119 participants were included in this study from September 8, 2020, to March 7, 2021. Out of the 119 participants, fetal macrosomia among non-diabetic women was seen in 10 (8.4%) cases. The mean age of patients in our study was 29.80 \u00b1 4.33 years. The mean gestational age was 38.05 \u00b1 1.31 weeks. The mean BMI of participants was 29.17 \u00b1 2.36 kg/mStratification with respect to age, gestational age, gravidity, parity, BMI, mode of delivery, and previous history of macrosomia was performed to determine significant variables Tables , 3. In oIn this study, fetal macrosomia was noted in 10 (8.4%) cases among non-diabetic women. The mode of delivery (SVD) was the most common variable in our study with a p-value of <0.05. To our knowledge, this is the first study from Pakistan to determine the number of fetal macrosomia among non-diabetic women. Formerly, a case-control study was conducted at the University of Kinshasa. In this study, 8,268 deliveries were performed between January 2007 to December 2016. Out of the 8,268 deliveries, 308 cases were diagnosed with macrosomia with a frequency of 3.7% . Two casGDM is a well-known risk factor for fetal macrosomia. The data is scarce with respect to the development of fetal macrosomia among non-diabetic pregnant mothers, especially in developing countries like Pakistan. Neonatal outcomes of macrosomic infants of diabetic and non-diabetic pregnant women were retrospectively reviewed in 2015. The study compared 170 macrosomic fetuses of diabetic mothers with 739 macrosomic fetuses of mothers without diabetes. It concluded that infants of diabetic mothers are mainly delivered by cesarean section, whereas SVD is the common mode of delivery in mothers without diabetes. Out of 170 macrosomic fetuses of diabetic mothers, 35 were delivered by SVD which was then complicated by shoulder dystocia. On the other hand, SVD was the mode of delivery in 70 of non-diabetic mothers out of 739, which was then complicated by shoulder dystocia. The study concluded that both macrosomic babies of diabetic and non-diabetic mothers are at risk of developing morbidity . Second,2 and pregnant non-diabetic and non-obese mothers. At 19, 30, and 36 gestational weeks, Z-scores of the rate of fetal macrosomia  were assessed between the two groups. The study concluded that Z-scores of the rate of fetal macrosomia were significant in the obese group at 30 and 36 weeks, whereas the difference was not significant between the two groups at 19 weeks of gestation. The study also concluded that maternal obesity prior to pregnancy was linked with fetal macrosomia [2. Even though we discovered eight fetal macrosomic fetuses in the obese group, the difference was not statistically significant (p > 0.05), as shown in Table The mean birth weight has increased tremendously in various regions of the world over the last several years. This might be due to the delivery of a greater percentage of large for gestational age neonates. Previously, 20 pregnant women without impaired OGTT were investigated at 36 weeks of gestation. Parameters assessed were glucose production, energy expenditure, and insulin resistance. The study concluded that the weight of the fetus mainly relies on maternal glucose production, which, in turn, depends on the extent of insulin resistance. This confirms that insulin resistance plays an important role between maternal weight and fetal macrosomia among non-diabetic women . Obesitycrosomia . We cateThe prevalence of fetal macrosomia varies worldwide . This miThe main drawback of this study was that it was a single-center, cross-sectional study with a small sample size; therefore, we were unable to assess the incidence of fetal macrosomia among non-diabetic women in Pakistan with difficulty in making causal inferences as well. It was a questionnaire-based study that was inexpensive to conduct, especially in a resource-limited country like Pakistan. Further studies with large sample sizes in different healthcare facilities must be conducted to determine the exact prevalence of fetal macrosomia in Pakistan.The number of fetal macrosomia cases among non-diabetic women in our study was 10 (8.4%). Because this was a single-center, hospital-based, cross-sectional study,\u00a0we need to conduct large multicentered randomized controlled studies to identify the actual prevalence of fetal macrosomia in non-diabetic women in our population.We recommend that awareness programs must be arranged regularly in resource-limited countries to modify dietary and lifestyle attitudes to reduce the occurrence of fetal macrosomia. This will ultimately reduce\u00a0maternal and fetal complications associated with macrosomia."}
+{"text": "To determine the prevalence, risk factors for macrosomia and pregnancy outcome in women with gestational diabetes (GDM).In this prospective observational study, we included the data of 161 pregnant females diagnosed with GDM. The study was conducted from December 1st, 2020 to June 30, 2021, at the Maternity and Children Hospital (MCH) of Hail, Saudi Arabia. The data regarding risk factors of macrosomia was obtained from each patient. The patients were followed till the delivery of the baby. The data regarding the prevalence of fetal macrosomia and its associated outcomes was noted.The prevalence of fetal macrosomia was 19.8%. Maternal obesity (OR 4.87), poorly controlled diabetes (OR 3.3), previous history of good-sized baby (OR 2.30), previous history of congenital abnormalities (OR 7.2) were the significant risk factors of fetal macrosomia. The prevalence of maternal and fetal complications was high among pregnancies complicated by fetal macrosomia. The prevalence of fetal macrosomia and other fetal complications was high in poorly controlled GDM patients in comparison to optimal control GDM patients.Fetal macrosomia is a common complication among GDM patients. Maternal obesity and poorly controlled diabetes are the common modifiable maternal factors contributing to macrosomia. Gestational diabetes mellitus (GDM) is a worldwide health problem, affecting about 5.0% of all pregnant women.4Fetal macrosomia is defined as if the neonatal weight exceeds >4 Kg, it affects about 10% of the total pregnancies. Macrosomia puts mothers at risk of several complications such as emergency C-section, postpartum hemorrhage, and perianal trauma for mothers, while for babies it increases the chances of fractures of clavicle or humerus bones, birth asphyxia, and shoulder dystocia.8In this present study, we determined the prevalence, risk factors for macrosomia and pregnancy outcome in women with gestational diabetes (GDM).We performed this prospective observational study, starting from December 1st, 2020 to June 30, 2021, at the Maternity and Children`s Hospital (MCH) of Hail, Kingdome of Saudi Arabia. The largest (250 bedded) tertiary care maternity care setup that provides services to the women and children of Hail province.. Inclusion criteria were pregnant women of Saudi origin, who booked in this facility, identified as GDM during the antenatal period, and delivered at this hospital were included in the study. Exclusion criteria were pregnant women with pre-gestational diabetes (Type- I and II DM) or others with medical (hypertensive) and obstetrical conditions, women of non-Saudis origin, and cases with missing information were excluded. We started this study after getting ethical approval from the Research Ethics Committee of the University of Ha\u2019il [Nr.20455/5/42].The study questionnaire was sent to the obstetrician working in the hospital for data collection as a google form. Women fulfilling inclusion criteria who delivered during the study period were informed about the purpose of the study and informed consent was taken to include their information. Their information on risk factors and antenatal course of pregnancy was taken from the patient`s health record which was accessed after approval from the hospital. Their delivery details and outcome are recorded at the time of giving birth. All information was entered in forms by the attending physician (a research team member). We included information about the gestational week at which GDM was diagnosed (onset), HbA1c levels during pregnancy, prenatal risks for GDM, antenatal complication, delivery onset, mode of delivery, intrapartum complications, fetal birth weight, birth trauma, APGAR score at birth, and need of Neonatal Intensive Care Unit (NICU) admission.Participating women were screened and diagnosed as GDM according to the NICE recommendations.10For HbA1c, we used values recommended by National Institute for Health and Care Excellence (NICE) guidelines and cut off of 6.1 used. HBA1c value 6.1 or less was considered as normal (well-controlled GDM), while above 6.1 was considered as high (uncontrolled GDM) during pregnancy .11Babies weighing 4-kg and above were considered macrosomia. Study participants were distributed into two groups. First included women who delivered a baby weighing 4kg or above, considered as macrosomia. The second group consisted of the deliveries where fetal birth weight was less than 4 kg, non-macrosomia.The pre-pregnancy BMI was calculated for the women by measuring their height in centimeters and pre-pregnancy weight in kilograms. The BMI was calculated by using the formula, Weight in Kg/Height in (m)2 and was analyzed as a categorical variable .12Other prenatal risk factors included in the study are the history of GDM in previous pregnancies, diabetes in first-degree relatives, previous history of intrauterine fetal demise, stillbirths, delivery of congenitally anomalous fetuses, and delivery of good size baby/babies (weighing 4kg or more) before. All responses were recorded in the category of \u2018Yes\u2019 or \u2018No\u2019.The information on antenatal complications in the mother (recurrent urinary tract infections (UTIs), pregnancy-induced hypertension (PIH), pre-eclampsia, preterm labor, and development of polyhydramnios) and complications in the fetus  were included. Delivery onset was considered natural if labor started spontaneously and induced where the pregnancy was terminated medically (induction of labor) or surgically (cesarean section) because of pregnancy complications. Mode of delivery included spontaneous vaginal delivery (SVD) and cesarean section (C-section).Intrapartum complications reported include maternal  and fetal  admission). Fetal Macrosomia was defined as newborns with birth weight of 4 kilograms or more. Responses for all the variables were recorded in the category of \u2018Yes\u2019 or \u2018No\u2019.We used Statistical Package for Social Sciences  for data analysis. Independent-Sample T-test was used to compare means and standard deviations for general characteristics of the study population e.g. age, parity, gestational age at diagnosis, fasting & 2-hours postprandial blood glucose level, HbA1c, pre-pregnancy BMI, and weight gain during pregnancy. Descriptive analysis was done to find the frequency and percentage values for early and late-onset GDM and macrosomia. The relationship of the value of HbA1c to the development of macrosomia was calculated by using cross-tabulation in descriptive statistics. The relationship of time of onset of GDM and fetal macrosomia with prenatal risk factors, antenatal and intrapartum complications was determined through bivariate analysis. P-value <0.05 was taken statistically significant.A total of 161 women were diagnosed with GDM during the study period, out of which fetal macrosomia was diagnosed in 32 (19.8%) neonates. Regarding baseline characteristics, the mean parity for Macrosomia was (3.69\u00b12.57) seen while those who delivered babies less than 4kg mean parity was (2.75\u00b12.06) (p=0.036). 2 hours PP BGL was higher in the macrosomia group; 11.01\u00b11.1 versus 9.8\u00b11.9 in normal-weight group, this difference was significant for delivery of babies more than 4 kg (p=0.001) .History of GDM in previous pregnancies showed a non-significant association for the development of macrosomia. At the same time the women who provide a history of delivering good size babies before, only 19 (27.53%) had macrosomia in this pregnancy while in 50 (72.46%) neonatal weight was found to be less than 4 kg . History of late IUFD was not found significant for macrosomia . The history of DM in first-degree relatives was also not a significant risk factor  . The birIt\u2019s obvious from the analysis that shoulder dystocia, extended/3rd-degree tear, immediate PPH, and low APGAR score at five minutes had a significant association with birth weight. Odds ratio analysis showed that a low APGAR score at five minutes was significantly associated with macrosomia . However, macrosomia (birthweight \u2265 4kg) was non-significant for birth trauma to the fetus .On comparison of the level of control of GDM, analysis shows that intrauterine reduced fetal movements and uterine fetal demise are significantly associated with HbA1c >6.1 during pregnancy . Similarly, High HbA1c is significantly associated with increased prevalence of fetal macrosomia . However, it was non-significant for intrauterine growth restriction of the fetus . By this analysis, it\u2019s clear that the HbA1c level has high specificity for intrauterine fetal demise and neonatal birth weight .GDM prevalence is on the rise probably due to the increasing prevalence of elderly pregnant females, obesity, and improvement in antenatal care and GDM detection.21The risk factors of macrosomia in GDM women in this study were maternal obesity, poorly controlled diabetes, previous history of macrosomia, and history of congenital abnormalities in the previous baby. We did not find any association of advanced age with macrosomia. A study by Said et al. reported maternal weight >80 Kg, maternal age \u226530 years, previous history of fetal macrosomia, and GDM as significant risk factors of macrosomia.The other fetal complications that occurred in our patients were shoulder dystocia in 10% neonates, low APGAR score in 8.1%, and NICU admission in 12.4% neonates. On comparison of neonatal complications between the macrosomia and non-macrosomia group, the incidence of LOW APGAR score, shoulder dystocia, and reduced fetal movements was significantly high in macrosomia neonates. Regarding maternal complications, the incidence of 3rd-degree tear was higher in the macrosomia group.We also performed the analysis of fetal complications among women with good control and poor GDM control. The incidence of fetal macrosomia was 27.2% in poorly controlled GDM patients and only 10% in good control GDM. The rate of IUFD and reduced fetal movements was also higher in poorly controlled GDM patients.This study has certain limitations, the major limitation is that the number of macrosomia infants was limited, so studies with larger sample sizes are needed to determine the exact prevalence, risk factors, and outcomes of fetal macrosomia among diabetic mothers.Fetal macrosomia is a common complication among GDM patients. Maternal obesity and poorly controlled diabetes are the common modifiable maternal factors contributing to macrosomia. The outcome of macrosomia is poor in poorly controlled GDM patients in comparison to optimal GDM control. So controlling maternal weight and timely management of GDM can help to reduce the prevalence of fetal macrosomia among GDM mothers.NP & NI: Data collection, study design, manuscript writing, and is responsible and accountable for the accuracy or integrity of the work.AB: Data collection, study design, manuscript drafting.TM: Data collection, analysis and manuscript revision.SA: Did review and final manuscript approval."}
+{"text": "Infectious complications are common during extracorporeal membrane oxygenation (ECMO) and may negatively impact outcomes. However, there is considerable variation in the reported rates of incidence, which hampers the use of infections as a quality benchmark for ECMO centers. To assess the contributing role of poor interrater agreement, three independent raters reviewed medical records from all intensive care unit (ICU) patients who received ECMO for >24\u2009h in our tertiary center between October 2019 and October 2021 for suspected episodes of infection, which were rated based on their date of onset and presumed site/diagnosis. To establish a gold standard, any discrepancies were resolved using an expert panel consisting of two intensivists/infectious disease specialists. During 83 ECMO-runs in 77 patients, we observed a total of 62 adjudicated infectious episodes . Among 81 episodes suspected by at least one observer, 66 (81%) were identified by two, and only 44 (54%) by all three raters, resulting in Fleiss\u2019 kappa of 0.10 . However, if raters concurred regarding infection onset, subsequent agreement on infection site was good . In conclusion, adjudication of infectious episodes during ECMO is associated with poor interrater agreement regarding occurrence\u2014but not site\u2014of infection. This finding might partially explain the significant disparities observed in reported infection rates during ECMO, emphasizing the need for caution when interpreting infection data in this particular population due to the potential for inherent measurement error. In addition, nosocomial infections are among the most frequently encountered complications during ECMO-treatment. However, there is substantial variability in the reported occurrence of ECMO-related infections (ERIs), with incidence rates varying between 12 and 75 episodes per 1000\u2009days at risk, and cumulative incidences between 9% and 65%.5 Furthermore, there are significant inconsistencies between studies regarding the reported association between ERIs and adverse patient outcomes .5 Despite these uncertainties, ERI occurrence is currently included as a quality benchmark for ECMO-centers, for example by the Extracorporeal Life Support Organization (ELSO) registry. For these reasons, it is important to explore methodological factors that could affect the robustness of this criterion.Extracorporeal membrane oxygenation (ECMO) is used to provide circulatory and respiratory support in patients with severe hemodynamic shock and/or respiratory failure. ECMO is a highly invasive support modality reserved to specialized intensive care unit (ICU) settings and is associated with a wide range of circulatory complications, including thromboembolism, coagulopathy, major bleeding, limb ischemia, and circuit failure. ECMO-support invariably presents a further inflammatory trigger due to blood contact with non-endothelialized surfaces of the circuit itself. At the same time, fever in these patients may be masked due to effects of the heater-cooler, and radiological signs of infection may be obscured by the presence of concomitant cardiogenic pulmonary edema or the use of extreme lung-protective ventilator strategies causing airspace opacification. For these reasons we suspected the precision in diagnosing infections in an ECMO population to be poor, which would explain the variability in reported ERI occurrence at least in part. To test this hypothesis, we performed an observational study to assess true ERI incidence as well as evaluate its inherent measurement error due to interrater variability.In the general ICU-population, and ECMO-patients in particular, parameters used for diagnosing infections are difficult to interpret. Whereas non-infectious etiologies of systemic inflammation are already quite common in critically ill patients in general,This study was conducted in the mixed ICU of the University Medical Center Utrecht, The Netherlands. Study patients had been previously enrolled in the Prediction of Weanability, Survival, and Functional Outcomes after ECLS (PRECISE-ECLS) cohort (NCT05444764) as well as in the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) biorepository (NCT01905033). The institutional medical ethics committee approved an opt-out method of informed consent for both studies (reference numbers 21-604/C and 10-056). Consecutive adults who had received venoarterial or venovenous ECMO-support for >24\u2009h between October 2019 and October 2021 were selected for inclusion. There were no exclusion criteria.ERI was defined as any new occurrence of a clinically suspected or microbiologically proven infection for which systemic antimicrobial therapy had been initiated in the period ranging from 24\u2009h after initiation up until discontinuation of ECMO-support. Of note, all patients received selective digestive decontamination which included 4\u2013days use of a third-generation cephalosporin. This as well as any other prophylactic or pre-emptive usage of antimicrobials was disregarded for ERI assessment. For the current analysis, two additional reviewers (raters B and C) independently performed a blinded reassessment of infection status for each day on ECMO-support. To this end, medical records as well as radiology and microbiology findings were examined in detail. This yielded three lists of suspected infectious events for each patient, including their date of onset and presumed site.Patient characteristics, details on the ECMO-procedure and clinical outcomes were collected from the PRECISE-ECLS database. As part of the MARS project, all suspected infectious episodes had already been prospectively adjudicated once by review of medical records by a single observer using strict definitions (further denoted as rater A).To establish an unbiased estimate of ERI incidence we used a \u201cgold standard\u201d reference diagnosis. A diagnosed infectious episode was considered true if all three observers had independently concurred on both infection onset and site. If this condition was not met, a consensus diagnosis was made by an expert panel consisting of two experienced intensivists with expertise in the field of ICU-acquired infections (OC and LD). The interpretation of \u03ba was in accordance with Landis and Koch. Additionally, each rater\u2019s concordance with the \u201cgold standard\u201d was determined. All analyses were performed in R version 4.0.3.Agreement between the three raters was first assessed with respect to ERI presence or absence. During this analysis, a 48-h margin of error regarding estimated infection onset date was allowed. For example, two raters would be in agreement if they had dated infection onset on Monday and Tuesday, respectively, yet would disagree if this were Monday and Thursday (as this would likely constitute an entirely different event). Subsequently, for universally recognized ERIs only, agreement on the primary site of infection was assessed across three categories . Interrater agreement was expressed as observed concordance (%) and Cohen\u2019s kappa (\u03ba) using Fleiss\u2019 generalization for multiple raters.During 83 ECMO-runs in 77 patients, we observed 62 infections according to the reference diagnosis . Overall, 40 (48%) runs were complicated by at least a single infection . The ERIAmong 81 episodes suspected by at least a single observer, 66 (81%) were identified by two, and only 44 (54%) by all three raters, resulting in a kappa of 0.10 (95% CI: 0.00\u20130.19); slight agreement . The obs4 A post-hoc qualitative assessment of the 37 discordant cases that had been reviewed by the expert panel revealed that patients who had consistently elevated inflammation markers with only minor fluctuations offered the greatest diagnostic challenge. However, once raters agreed on infection onset, there was good subsequent concordance on the diagnosis (i.e. presumed site of infection), which concurs with previous observations made in a general ICU popuation.To our knowledge, this is the first study to systematically evaluate interrater agreement for the identification of nosocomial infections in patients receiving ECMO-support. Our findings confirm the great diagnostic challenges we had anticipated in this population due to its specific characteristics, including omnipresent systemic inflammation, disrupted thermoregulation, and difficult-to-interpret chest radiographs. For example, most prior studies as well as the ELSO registry report only culture-proven infections,5 whereas in our study we deliberately chose to include also clinically suspected infections, as not all clinically relevant infections in ICU patients can be documented with microbiological evidence. This comprehensive definition probably increased apparent ERI incidence rates (i.e. 62 infections per 1000\u2009days at risk in our study versus a range of 12\u201375 reported in literature5) and may have introduced some subjectivity. However, a restricted focus on culture-proven infections only represents an oversimplification of the diagnostic complexities encountered in an ECMO-population and would have created a significant underestimation of real-world ERI incidence.Apart from differences in population characteristics, the large variability in reported incidence rates may stem from a strong reliance of ERI diagnosis on local hospital protocols  and the definitions used to classify infections.Although risk stratification was not a primary aim of this study, we observed several patient- and circuit-specific factors that were associated with an increased ERI occurrence, including longer hospitalization prior to ECMO initiation and veno-venous configuration . None of12 This likely contributed to lower rates of Gram-negative pulmonary infections in this cohort. However, challenges regarding the correct identification of infections are universal and we feel that our diagnostic protocols are in line with common clinical practice. Unfortunately, due to limited sample size, we were unable to perform subgroup analyses into specific factors that may have contributed to the observed poor interrater agreement.Our study has certain limitations, such as a potential lack of generalizability due to specific ERI definitions used by us and variability in local diagnostic practices. For instance, despite it being often debated in literature, the use of selected decontamination of the digestive tract in mechanically ventilated patients has become standard of care in the Netherlands.In conclusion, diagnostic adjudication in ECMO patients is associated with poor interrater agreement regarding the occurrence\u2014but not site\u2014of infection. The resulting diagnostic error warrants caution when interpreting infection epidemiology data in this population, precluding their use as a performance benchmark."}
+{"text": "This study aimed to compare the burden and trends of stroke attributed to dietary risk factors in the Belt and Road (\u201cB&R\u201d) countries from 1990 to 2019.The 2019 Global Burden of Disease (GBD) Study was used to gather information on the burden of stroke attributable to dietary risk factors. Numbers and age-standardized rates (ASRs) of deaths, disability-adjusted life years (DALYs) were determined in 1990 and 2019 among the \u201cB&R\u201d countries. The average annual percent change (AAPC) was used to analyze the temporal trends of diet-induced stroke DALYs from 1990 to 2019 and in the final decade (2010\u20132019) by Joinpoint regression analysis.In 2019, the absolute number of stroke deaths and DALYs attributable to dietary risk factors were 671,872 cases  and 1.67 million cases (95% UI 1.15\u20132.24) in China. We found geographical differences in mortality and DALYs of diet-attributable stroke among member countries, with Bulgaria, Hungary and Serbia being the three highest countries in 1990, Bulgaria, North Macedonia and Montenegro in Central Asia in 2019. The ASRs of diet-induced stroke mortality and DALYs were generally declining in most member states from 1990 to 2019, however, the corresponding metrics in Mongolia remained high. The fastest decline in ASR of mortality and DALYs for diet-induced stroke was seen in Estonia, Eastern Europe, with AAPC values of \u22127.09%  and\u2009\u2212\u20096.62% , respectively. We noted a substantial downward trend in ASR of mortality and DALYs from diet-induced stroke changes in the final decade (2010\u20132019) for most member states. The ASR of DALYs for diet-induced stroke decreased greater in females than in males. For those aged 50\u201374, the DALYs for stroke due to dietary risk factors in all other member countries of the \u201cB&R\u201d showed a decreasing trend, except for the Philippines, which rose  and Turkmenistan, which remained stable .The burden of diet-induced stroke varies substantially across \u201cB&R\u201d countries and threaten public health, relevant evidence-based policies and interventions should be adopted to address the future burden of stroke in \u201cB&R\u201d countries through extensive collaboration. Stroke is a global public health problem that imposes a heavy disease and financial burden on individuals and society. According to the 2019 Global Burden of Disease, Injury and Risk Factor Study (GBD 2019), stroke was the second-leading cause of death  and third-leading cause of disability  worldwide . AdditioAs we all know, health issues are no longer the responsibility of individual countries with the rapid pace of globalization. In 2013, Chinese government initiates the Belt and Road Initiative (BRI) to accelerate infrastructure, trade development and business partnerships among 66 countries in Asia, Europe, South America and Africa , 5. AlthRecent epidemiological evidence has found that the large burden of stroke can be attributed to several modifiable factors, such as obesity, hypertension, diabetes, a sedentary lifestyle, or unhealthy diet . Diet isThe GBD 2019 framework, through extensive collection of data sources and statistical modeling, allows for comparable assessment of stroke burden in terms of mortality and DALYs. At present, none of the existing studies on stroke mortality and DALYs attributable to modifiable dietary risk factors had explored differences and the changing trend of DALYs stratified by gender, age and diet-specific risk factors among 66 countries from the BRI. Therefore, this study was conducted to compare the burden and trends of diet-induced stroke from 1990 to 2019 in the \u201cB&R\u201d countries, and to provide the basis for generating prevention and control strategies of stroke for building a healthy \u201cB&R.\u201dhttp://ghdx.healthdata.org/gbd-2019/code/nonfatal-12. DALYs is a composite indicator to assess the disease burden of disability and premature death, which is obtained by summing YLL and YLD.In this study, data on annual diet-induced stroke deaths, DALYs, and respective age-standardized rates (ASR) by gender, age and specific diet risk factor in the \u201cB&R\u201d countries from 1990 to 2019 were extracted from the GBD 2019 database.The composition of \u201cB&R\u201d countries is mainly based on the GBD classification of global regions and international political and economic organizations . BRI incZ-test to check whether the slope for each trend segment is significantly different from prior segment  were reported, and 95% UI was calculated by drawing 1,000 times from each number of posterior distributions, using the 2.5th and 97.5th ordering of the uncertainty distribution . If both the AAPC estimate and the lower limit of 95% UI were positive, ASR of mortality and DALYs showed an upward trend. Conversely, if both the AAPC estimate and the upper limit of the 95% UI were negative, then ASR of mortality and DALYs exhibited a decreasing trend (setting 3% as the cut-off point and\u2009\u2265\u20093% as a larger decrease). Other than that, ASR was considered to be stable . We found no statistically significant differences in AAPC for ASR of diet-induced stroke mortality and DALYs in Kuwait, Mongolia and Turkmenistan in Central Asia and North Africa and Middle East for 1990\u20132019. In addition, we noted a substantial downward trend in ASR of mortality and DALYs from diet-induced stroke changes in the last decade (2010\u20132019) for most member states. The ASR of mortality and DALYs due to diet-related stroke in Mongolia remained stable over the full 30\u2009years, yet showed a decreasing trend in the last decade. Trends in age-standardized mortality rates and DALYs for diet-induced stroke in 2010\u20132019 were not statistically significantly different in Ukraine in Eastern Europe, and Yemen in North Africa and Middle East. See The temporal trend of ASR of mortality and DALYs due to diet-induced stroke for 1990\u20132019 and 2010\u20132019 in \u201cB&R\u201d countries was displayed in p <\u20090.001, respectively). The ASR of DALYs for diet-induced stroke decreased more in females than in males. For males, the AAPCs in Mongolia, Tajikistan, Pakistan and Lebanon were stable between 1990 and 2019, while the change trend of DALYs was stable for female in Azerbaijan and Egypt (p\u2009\u2265\u20090.05) (\u2009\u2265\u20090.05) .The AAPC of age-standardized rates for DALYs due to stroke, attributable to specific dietary risk factors for 1990\u20132019 in the \u201cB&R\u201d member countries was displayed in Based on latest data from the GBD Study 2019, we explored the impact of dietary risks on stroke deaths and DALYs in the member states of the \u201cB&R\u201d over past three decade (1990\u20132019) and the final decade (2010\u20132019). The results showed geographical differences in mortality and DALYs of diet-attributable stroke among member countries, the age-standardized mortality and DALYs were generally declining in most member states. The decreases in ASRs of diet-related stroke burden may be attributed to decreases in the improvement of living standard, increased awareness of self-health, improved screening programs, the diagnosis of patients in the early stages of disease and better access to effective therapy \u201323. BulgFindings from this study found that the fastest decline in ASR of mortality and DALYs for diet-induced stroke was seen in Estonia, Eastern Europe. Estonia has established detailed stroke registration and management system since the 1970s, and the results of the third population-based stroke register in 2005 showed a decrease in stroke incidence and 28\u2009days case fatality rates compared to the previous decade , 28. FurMeanwhile, our study found that the temporal trends of diet-induced stroke DALYs varied considerably by sex, age and specific dietary risk factors. Females displayed lower negative AAPCs than males in most member counties, suggesting a higher downturn in ASR in females. Gender is a key risk factor for cardiovascular diseases, with biological sex  and gender  influencing differences in disease susceptibility and pathology between men and women . Thus, tEvidence suggested that the main dietary risk factors for deaths and DALYs were low in whole grains, high in sodium, low in fruits and vegetables globally and in many countries . Wang etBased on the broadest epidemiological dataset available to date, this study analyzes estimates of stroke mortality and DALYs attributable to dietary risk factors from 1990 to 2019, and the corresponding changes in the last decade for the first time. Meanwhile temporal trend in diet-induced stroke DALYs were also explored by sex, age, and specific dietary factors. The main strength is the data collection of \u201cB&R\u201d member countries using the same methods and modeling used in the GBD study. Several limitations exist in this study. Firstly, dietary risks from GBD dataset were not strictly categorized, for example vegetables, fruits and whole grains are all rich in fiber, to some extent they overlap with the fiber group. Also, we did not consider potential interactions or synergistic effects between different dietary risk factors. Secondly, underreporting or misclassification of stroke cases existed in each member country due to different diagnostic criteria, definition and measurement of dietary risk are not the same around the \u201cB&R\u201d countries. Thirdly, given the diversity of whole grain products, it is quite difficult to accurately measure intake, which can lead to measurement errors. Fourthly, while certain confounders  were taken into account in the GBD framework, other variables, such as socioeconomic status and access to healthcare, could still be sources of bias. Finally, our study is based on a secondary analysis of GBD, thus GBD all limitations also apply to our study, which is why age groups were not mutually exclusive in this analysis.This study compared stroke mortality and DALYs attributable to dietary risk factors from 1990 to 2019, and the corresponding changes in the last decade, and explored the temporal trend of ASR for diet-induced stroke DALYs stratified by gender, age and specific dietary risk factors in \u201cB&R\u201d countries in the past three decades. We found geographical differences in mortality and DALYs for diet-induced stroke among member countries, with a general downward trend in these indicators from 1990 to 2019 in most member countries. A substantial downward trend in ASR of mortality and DALYs from diet-induced stroke changes in the final decade. Notably, the AAPCs of age-standardized stroke mortality and DALYs attributable to dietary risk factors significantly increased in Philippines. The ASR of DALYs for diet-induced stroke decreased more in females than in males. Therefore, prioritization of public health interventions among \u201cB&R\u201d member countries should be evidence-based and data-driven to address the risks and challenges posed by diet-induced stroke through enhanced health collaboration and resource sharing.The original contributions presented in the study are included in the article/YZ and XL conceived and designed the study. YZ, XL, JW, JZ, and YQ analyzed the data. ZL, JY, WX, HL, CM, XX, and WZ provided advice and consultation. YZ wrote the manuscript. All authors read and approved the submitted manuscript.This work was supported by the Basic Research Program of Shanxi Province (Free exploration) project (20210302123216 to YZ), the Science and Technology Innovation Project of Higher Education Institutions in Shanxi Province (2021L221 to YZ), the Special Disease Construction Project of Pudong Health and Family Planning Commission of Shanghai (Grant No. PWZzb2022-20 to ZL), Discipline Construction Project of Pudong Health and Family Planning Commission of Shanghai (Grant No. PWYts2021-02 to ZL), and the General Program of Health Bureau of the Shanghai (202150015 to ZL). All authors had full access to all data in the study and had responsibility for the decision to submit for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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": "Low physical activity (LPA) is linked to the risk of stroke, but the disease burden of stroke attributable to LPA needs to be understood to develop effective preventive strategies. We aim to assess spatiotemporal trends in the global burden of stroke attributable to LPA from 1990 to 2019.Based on the Global Burden of Disease, Injuries, and Risk Factors Study, our research examined deaths, the Disability-Adjusted Life Years (DALYs), the Age-Standardized Mortality Rate (ASMR), the Age-Standardized DALY Rate (ASDR), and the Estimated Annual Percentage Change (EAPC) for stroke attributable to LPA.P\u2009<\u20090.001) and the Socio-Demographic Index (SDI) across different countries in 2019 , respectively, and such patterns were similar to what ASDR and the ASDR-related EAPC had; the Human Development Index (HDI) in 2019 was associated with the ASMR-related EAPC  and the ASDR-related EAPC across different countries , respectively.Deaths and DALYs were on the rise worldwide from 1990 to 2019, with increases of 72.72% for the former and 67.41% for the latter; ASMR and ASDR decreased, with the ASMR-related EAPC of -1.61 (95% CI:-1.71\u2013-1.5) and ASDR-related EAPC of -1.35 (95% CI:-1.43\u2013-1.27); females had more numbers of deaths and DALYs, and the majorities of deaths and DALYs were shared by those aged\u2009\u2265\u200970. The highest burden rates were shared by North Africa, the Middle East, and Tropical Latin America; the ASMR-related EAPC was associated with the ASMR in 1990 (R\u2009=\u2009-0.26, deaths and DALYs of stroke attributable to LPA were on the rise, although their age-standardized rates presented downward over the past three decades; the burden of stroke attributable to LPA showed upward trends especially in those aged\u2009\u2265\u200970 and females in the regions of East Asia, North Africa, and the Middle East, which need more attention to the effects of physical activity on health interventions.Globally,  Physical activity is linked to stroke [the limitation of sedentary activities [worldwide has been occurring since 2001 [to control the risk of unhealthy behavioral risk factors.Stroke, one of the leading causes of mortality and morbidity, has become a public health problem in the world, according to the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) in 2019 . The esto stroke \u20137 and ito stroke \u201311. The tivities , which snce 2001 , and a pwill concentrate in some developing countries with large populations, such as Brazil, Russia, India, and China (BRIC), where the populations aged\u2009>\u200960\u00a0years are predicted to at least double by 2050 [Nevertheless, the current estimates of the global burden of stroke attributable to LPA and its temporal trends are still incompletely understood. Accurate and up-to-date estimates of this burden are important in planning research and the resulting evidence-based strategies for stroke prevention and management.Recent evidence reveals that unhealthy behavioral risk factors, such as a sedentary lifestyle or LPA, tend to increase with development .\u00a0Thus, i by 2050 , 16; LPA by 2050 , the disto estimate spatiotemporal trends in the deaths and DALY of stroke attributable to LPA globally, by regions, HDIs, SDIs, countries, gender, and age groups from 1990 to 2019 for guiding allocations of sports resources, bettering the strategies in the promotion on physical activity, and lowering the disease burden of stroke.GBD provides an opportunity to incorporate newly available datasets, enhance method performance and standardization, and be in response to changes in scientific knowledge. Here, we aimed http://ghdx.healthdata.org/gbd-results-tool) of the Institute for Health Metrics and Evaluation (IHME) at the University of Washington, the World Health Organization (WHO), and members of the Global GBD Collaborative Group. GBD recorded 369 diseases and 87 risk factors in 1990\u20132019 and listed 204 countries and territories and 21 regions, with newcomers of the Cook Islands, Monaco, San Marino, Nauru, Niue, Palau, Saint Kitts and Nevis, Tokelau, and Tuvalu; thus, all the World Health Organization (WHO) members were given in GBD 2019 [Study data were taken from the Global Health Data Exchange  were included in terms of the frequency, duration, and intensity of each physical activity lasting at least ten minutes. The Metabolic Equivalent (MET), a ratio of the working metabolic rate to the resting metabolic rate, was introduced. Based on total MET-minutes per week, physical activity was categorized into inactivity (<\u2009600 MET-minutes per week), low activity , moderate activity , and high activity . Theoretically, the minimum exposure to physical inactivity was 3,000\u20134,500 MET-minutes per week ; thus, LDetails of the GBD 2019 eligibility criteria, the literature search strategy, and data extraction are described in detail elsewhere. In brief,\u00a0stroke was defined by the WHO criteria and was estimated based on the International Statistical Classification of Diseases and Related Health Problems (ICD), GBD 2019, and the Cause List Mapped to the ICD codes of the I60\u2013I62, I62.9\u2013I64, I64.1, I65\u2013I69.998, Z82.3, and G45\u201346.8 [Stroke attributable to LPA was based on defining stroke by combining the distribution of exposure to risk-related LPA [G45\u201346.8  as follothe aggregated countries\u2019 data and used analytic tools on the GBD website. Disease burdens were assessed with a range of indicators, including deaths, DALY, and the Age-Standardized Rates  and the Age-Standardized DALY Rate (ASDR)). ASR was calculated with a no-weighted mean of GBD year\u2019s age-specific proportional distributions for national locations with populations greater than 5 million in GBD year to update the world population age standard. In brief, ASR was generated from several parameters, including a summing up age-standardized rate , a number (or the weight) of persons (wi) in the same age subgroup i (a reference of the standard population), and a dividend of summing up the standard population weight [We collected n weight :\\documenAccording to this formula, we presented\u00a0ASMR per 100,000 person-years, and ASDR per 100,000 people with the direct method of standardization and the WHO\u2019s standard population. The Estimated Annual Percentage Change (EAPC), which was widely used to describe a trend of ASR, was calculated as follows:Where y is equal to the natural logarithm of ASR and x corresponds to the calendar year.http://ghdx.healthdata.org) is a composite index of per capita income, educational attainment, and total fertility rate of all areas, identifying the socio-demographic development status of countries or other geographic regions. It is calculated as the geometric mean on a scale of 0 to 1, by which all countries are divided into five groups: low (0\u20130.455), low-middle (0.455\u20130.608), middle (0.608\u20130.690), middle-high (0.690\u20130.805), and high (0.805\u20131)  [and can be downloaded from the United Nations Development Program (http://hdr.undp.org/en/data).EAPC and its 95% confidence interval (CI) were estimated using a linear regression model. ASRs would be upward, downward, and stable if EAPCs with the 95% CI were\u2009>\u20090,\u2009<\u20090, and\u2009=\u20090. The sociodemographic index (SDI) from the GHDx (.805\u20131)  . Data fr.805\u20131)  . The Humwere quantified as the disease burdens at global, regional, and national levels. The regions and the territories were also divided into 21 groups by region and 15 groups by age. All values for each metric were represented as the Uncertainty Intervals (UIs) using the 25th and the 975th ordered 1,000 draw values of the posterior distribution. Relationships between SDIs and ASRs from 1990 to 2019 were evaluated. With the social development and health outcomes by SDI in 2019 and the baseline level of disease burden by the ASR in 1990, the HDI in 2019 could reflect a national condition in all health sources. To explore the influential factors for EAPC, the Pearson correlation analysis was used to assess the association between EAPC and SDI in 2019, ASR in 1990, and HDI in 2019, respectively [P\u2009<\u20090.05.Deaths, ASMR, DALY, and ASDR ectively . All anaGlobally, deaths of stroke attributable to LPA were on the rise in 1990\u20132019, with an increase of 72.72%, wherein males showed a higher growth (93.55%) than females (63.16%), but females had more death numbers  than males  in 2019  in 1990 to 2.08 per 100,000 persons (95% UI: 0.41\u20135.33) in 2019, with an ASMR-related EAPC of -1.61 (95% CI: -1.71\u2013-1.5); the ASDRs of stroke attributable to LPA also experienced a decline from 43.55 per 100,000 persons (95% UI: 7.56\u2013117.17) in 1990 to 31.16 per 100,000 persons (95% UI: 5.69\u201382.02) in 2019, with an ASDR-related EAPC of -1.35 (95% CI: -1.43\u2013-1.27). Among 21 regions in 1990\u20132019, the EAPCs in most regions were\u2009<\u20090, while the five highest ASMR- and ASDR-related EAPCs were shared by Southern Sub-Saharan Africa , Eastern Sub-Saharan Africa , Southeast Asia , Central Sub-Saharan Africa , and Central Asia  for ASMR and Southern Sub-Saharan Africa , Southeast Asia , Eastern Sub-Saharan Africa , North Africa and Middle East , and Oceania for ASDR, respectively  or with increases of 177.85% for deaths and 145.38% for DALYs in 1990\u20132019; Western Europe had the most deaths  in 1990. North Africa and Middle East had the highest ASMR in 2019 ) and the second highest ASMR in 1990 ), respectively; Tropical Latin America had the highest ASMR in 1990 ). Among 21 regions, North Africa and Middle East had the highest ASDR in 2019 ) and the second highest ASDR in 1990 ), respectively; Tropical Latin America had the highest ASDR ) in 1990  and ASMR in 1990 , respectively  and a significant negative association of the ASDR-related EAPC with HDI across different countries in 2019 , respectively.Figure\u00a0In this study, we estimated spatiotemporal trends in deaths and DALYs of stroke attributable to LPA at the global, regional, and national levels. We found increases of 1.73-fold for deaths and 1.67-fold for DALYs but a decline of ASMR and ASDR for stroke attributable to LPA worldwide; additionally, the global burdens of stroke attributable to LPA varied considerably, with the higher rates in North Africa and the Middle East, Tropical Latin America, and Eastern Europe. Our results also showed an M-shaped association of SDI with the burden of stroke attributable to LPA in 2019 , a decline and a significant association of the ASMR-related EAPC with HDIs across different countries in 2019, and a significant negative association of the ASDR-related EAPC with HDIs across different countries in 2019, respectively; the ASMR and ASDR in 1990\u20132019 showed decline in most countries and territories such as Estonia, Portugal, and Austria, besides the increases in the others like Azerbaijan, Tajikistan, and Lesotho.experienced a decline in the risk of stroke among colorectal cancer survivors in Korea [Physical activity was associated with a decreased risk of stroke in previous studies. Moderate to vigorous exercise in Korea . Similarin Korea ; moderatin Korea . Differein Korea ; longer in Korea ; low phyin Korea , which iin Korea . The patin Korea , 31, andin Korea .we observed the different global burdens by gender, with more deaths and higher levels of DALY, ASMR, and ASDR in females. A potential reason might be that females do fewer physical activities [Unlike males being more susceptible to stroke , 34, we tivities , 36. Nevin the elderly than the young, wherein those aged\u2009\u2265\u200970 shared the majority of deaths and DALYs, which mostly support those elderly with high levels of LPA. Accompanying life expectancy increasing continuously [The global burden of stroke attributable to LPA was more severe inuously , the glowherein the high ASMR and DALY attributable to LPA especially in the countries from the Middle Eastern region and also in the tropical regions , and\u00a0it could be linked to\u00a0the more sedentary lifestyle or high-temperature environment which is less conducive to outdoor physical activities [other possible reasons are fewer demands of physical activities but more changes in recreational and cultural values accompanying with the economic development among those in lower SDI [a potential explanation for those high SDIs with decreasing trends might be high-income countries focusing earlier on promoting physical activity [Burdens of stroke attributable to LPA from 1990 to 2019 varied across the world, with increasing trends in deaths and DALYs in high-middle, middle, low-middle, and low SDI, ns Figs.\u00a0B, and\u00a0itower SDI ; additioactivity . A stablinformation on stroke subtypes was incomplete, and data quality was not easy to guarantee in low- and middle-income countries. Second, a series of indicators were screened and their corresponding trends in burdens of stroke attributable to LPA worldwide were assessed, other risk factors, such as population growth and aging, were incompletely taken into account. Third, due to analytic data from self-reported questionnaires and lack of sufficient validity evidence of the participants\u2019 recall and social desirability, some uncertainties of under or overestimated\u00a0self-report limited the estimates of physical activity and changes in weekly MET-minutes over time,\u00a0accelerometer-measured tool or updated questionnaire should be implemented into these physical activity-related studies in the future.This study included some limitations. First, more attention should be paid to the effects of physical activity on health intervention, and patients with stroke attributable to LPA, especially those aged\u2009\u2265\u200970 and females, should energetically be cared about in the regions of East Asia, North Africa, and the Middle East. Our findings should also help with the development and monitoring of the effectiveness of stroke prevention and management and rehabilitation strategies in different countries and among different populations.We found increases in deaths and DALYs of stroke attributable to LPA worldwide from 1990 to 2019, with different deaths, DALYs, ASDRs, and ASMRs by gender and region. Thus,"}
+{"text": "Heavy menstrual bleeding (HMB) is a common clinical condition affecting adolescent and adult women and compromising their quality of life. Primary hemostasis disorders, affecting platelet plug formation, can be the underlying cause of HMB. They comprise a heterogeneous group of diseases with Von Willebrand disease (VWD) being the most commonly diagnosed; other disorders in this group that have been linked to HMB include (a) Glanzmann thrombasthenia, (b) Bernard\u2013Soulier syndrome, (c) Hermansky\u2013Pudlak syndrome, (d) immune thrombocytopenia (ITP), and (e) Ehlers\u2013Danlos syndromes (EDS) and hypermobility spectrum disorders (HSD). Diagnosing these diseases can be challenging, as the basic laboratory investigations can be within the normal range. Thus, identification of specific clinical features and a thorough hematologic workup can be very important, providing the correct diagnosis. Proper diagnosis of the underlying disorder is important, as management may vary accordingly. Although disease-specific management guidelines exist for some of these disorders such as VWD and ITP, due to the rarity of most primary hemostasis disorders, the best approach for the management of HMB in these women remains elusive. The goal of this study was to create an informative, comprehensive review of the primary hemostasis disorders that have been linked to HMB. This study provides a summary of the basic published information regarding epidemiology, pathophysiology, clinical phenotype, diagnosis, and treatment of HMB in those diseases and serves as a reference guide for further reading. Abnormal uterine bleeding (AUB) is a common debilitating condition for women of reproductive age, having a significant impact on their physical and mental health . It is eFurther platelet aggregation is accomplished by release of adenosine 5\u2032-diphosphate (ADP), serotonin and thromboxane A2 (TxA2), the latter being synthesized from arachidonic acid formed by platelet membrane phospholipids. The final primary hemostatic step is a change in platelet morphology, which now expresses various phospholipids in their membrane surface, necessary for coagulation factor assembly and initiation of secondary hemostasis . As explVon Willebrand disease (VWD) is the most common inherited bleeding disorder, with an estimated prevalence of 0.6\u20131.3% , and it VWD is caused by decreased activity or function of the Von Willebrand factor, secondary to either a quantitative or qualitative defect. VWF is a multimeric glycoprotein which is synthesized and stored in endothelial cells and megakaryocytes and cleared by macrophages in the liver and spleen. It plays a critical role in primary and secondary hemostasis; thus, defective activity can lead to bleeding manifestations. In primary hemostasis, following endothelial damage, VWF binds to exposed extracellular matrix proteins of the endothelial cells and mediates adhesion of platelets, which bind VWF via GPIb receptor. In secondary hemostasis, VWF acts as a chaperone to factor VIII (FVIII), thus preventing premature clearance and degradation ,16,17.Historically, in the middle of the 20th century, VWD had been characterized as \u201cpseudo-hemophilia\u201d due to the low FVIII coagulation activity (FVIII:C) which was a cause of diagnostic confusion between hemophilia and VWD. VWF antigen testing became available through immunoprecipitation in the mid-1970s, thus making the diagnosis possible ,19.VWD is subdivided into three categories, according to the International Society on Thrombosis and Hemostasis (ISTH) classification. Type 1 and Type 3 are quantitative defects, with Type 1 characterized by partial deficiency of VWF and Type 3 by absolute deficiency. Type 1C is a subcategory of Type 1 in which the partial deficiency of VWF is due to increased clearance. Type 2 on the contrary, is a group of qualitative defects comprising of the following subgroups: Type 2A which is due to a defect in multimerization, and types 2B, 2N and 2M caused by abnormal ligand binding; increased binding to GPIb receptor, defective binding to FVIII and defective binding with normal multimers, accordingly ,15.VWD follows an autosomal inheritance pattern with low penetrance and variable expressivity. Types 2B and 2M, as well as the majority of type 1 and 2A cases, are autosomal dominant. Type 3 and Type 2N subtypes on the other hand, exhibit an autosomal recessive inheritance pattern. A single case of uniparental disomy has been reported in type 3 VWD . The relThe clinical picture of patients with VWD varies according to the level of residual VWF activity, the category of disease, as well as age and sex. In adults, the most common symptoms are hematomas, menorrhagia, epistaxis, and bleeding from minor wounds. The majority of patients (60 to 80%) experience bleeding following surgery or dental extractions. A well-recognized, potentially life-threatening situation for these patients is gastrointestinal bleeding from angiodysplasia, more often in older patients with qualitative VWF defects. Intraarticular hemorrhage may be the first symptom in patients with type 2N or type 3 VWD. HMB is the most commonly reported symptom among women of reproductive age with VWD. Some of the published research regarding the prevalence of HMB among women with HMB can be found in Standardized bleeding assessment tools (BATs) can be used to identify specific bleeding phenotypes; however, they have certain restrictions, and it is currently suggested that their use should be limited to patients with low suspicion of VWD and that high-suspicion patients should proceed directly to a laboratory evaluation. The recommendation for the general population is that VWD testing should be reserved for patients with personal or family history of bleeding ,29.Heavy menstrual bleeding since menarche.\u25a0Postpartum hemorrhage,\u25a0Surgery-related bleeding, and\u25a0Bleeding associated with dental work.One of the following conditions:\u25a0Epistaxis, one to two times per month,\u25a0Frequent gum bleeding, and\u25a0Family history of bleeding symptoms.Two or more of the following conditions:Initial evaluation of women suspected to have VWD includes detailed medical and bleeding history and physical examination. A popular test to screen for women who should undergo further laboratory test was developed by Kouides et al. and includes the following .Heavy mWomen with a positive test result should undergo further laboratory testing. The diagnosis of Von Willebrand disease is based on measurements of Von Willebrand factor antigen, the level of Von Willebrand factor-dependent platelet adhesion, and the coagulant activity of factor VIII. Regarding, the VWF-dependent platelet adhesion, the 2021 guidelines by the American Society of Hematology (ASH), the International Society on Thrombosis and Hemostasis (ISTH), the National Hemophilia Foundation (NHF), and the World Federation of Hemophilia (WFH), suggest that newer assays measuring the platelet-binding activity of VWF  should be preferred over the VWF ristocetin cofactor assay (VWF:RCo) for the diagnosis of VWD. However, this suggestion is based on low-certainty evidence . The diaTherapeutic and prophylactic management of patients with VWD is a very challenging topic which is subject to long-standing and ongoing research. As it is outside of the scope of this research to provide a comprehensive review of this topic, we will focus on the management of heavy menstrual bleeding in these patients. Considering that the prevalence of symptomatic VWD is estimated to be around 1 in 1000 (making it a rare diagnosis) it is difficult to conduct large randomized controlled trials with a sufficient number of participants. Brignardello-Petersen et al., in a summary of three Systematic Reviews addressing the first-line management of menstrual bleeding in women with VWD, which included published research up to October 2019, managed to identify only 1 randomized clinical trial (RCT), 1 observational study and 10 case series, which met their eligibility criteria, and the evidence provided by them was assessed as very low to moderate. Furthermore, the RCT included women with heavy menstrual bleeding due to other hemostatic abnormalities of, and not only VWD. Up-to-date, there is no high-quality evidence to guide the management of women with AUB and VWD. Very recently, the results of a phase 3 randomized controlled clinical trial by Ragni et al., comparing the efficacy of Tranexamic acid (TxA) and recombinant VWF for the treatment of HMB were published. A summary of the evidence provided by the research of Brignardello-Petersen et al., regarding the use of the four most commonly used therapeutic interventions for treating HMB; desmopressin (DDAVP), TxA and hormonal therapy including Oral Contraceptive therapy and Levonorgestrel-Releasing Intrauterine System (LNG-IUS) , along wAlthough the aforementioned studies provide limited and moderate- to low-quality evidence regarding the relative efficacy of the recommended treatments for treating HMB in women with VWF, to our knowledge, there are currently no published results of high-quality comparative research to guide treatment. Furthermore, the 2021 ASH Recommendations are in line with the results of the, suggesting either hormonal therapy\u2014Combined Hormonal Contraceptives or LNG-IUS\u2014or tranexamic acid over desmopressin for women with VWD and HMB who do not wish to conceive, and tranexamic acid over desmopressin for those who wish to . The resGlanzmann thrombasthenia (GT) is a rare genetic bleeding disorder, inherited via an autosomal recessive pattern. Although the exact number of GT cases is unknown, the prevalence is estimated to be 1 in 1,000,000 people. Studies suggest that affected women seem to slightly prevail against men (58% vs. 42%). GT patients can be found all around the world; however, consanguinity seems to be a cause of higher prevalence in populations such as Iraqi Jews, some Arab populations, and French Gypsies . In IranThe disease is caused by a defect in the platelet membrane glycoprotein IIb/IIIa, also called integrin \u03b1IIb\u03b23 (ITG aII\u03b23). This glycoprotein acts as a large heterodimeric cell transmembrane receptor with the major function of mediating platelet aggregation and firm adhesion of platelets to the subendothelial matrix. Mutations of its genes lead to a quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP)IIb/IIIa resulting in impaired platelet aggregation. The platelet plug formation is hindered and thus a bleeding tendency occurs .As it exhibits an autosomal recessive pattern of inheritance, bleeding manifestations in GT seem to be limited to homozygous patients, with the heterozygotes being mostly asymptomatic. Bleeding diathesis manifests soon after birth, although some cases of GT are diagnosed later in life . The pheAll the studies published up-to-date agree on the high frequency of HMB in women and girls with GT. However, the exact number varies significantly among them. Early reports by George et al. recorded a prevalence of 98.2% among female patients (54/55), while the GTR, a prospective observational, international registry that included patients from 15 countries worldwide, estimated a prevalence of 73.6% (67/91) among post-puberal women ,38,39. HThe full blood count (FBC) usually shows no abnormalities, with the platelet count being at the lower end of the normal range. Prothrombin time (PT) and activated partial thromboplastin time (aPTT), as well as the blood film are also usually normal in GT. However, the bleeding time is prolonged. The LTA (light transmission aggregometry) for GT is characteristic, with platelet aggregation failing to occur with all agonists other than ristocetin . Flow cytometry confirms the defect in the number or function of GPIIb/III receptors. Type 1 is indicated by absence of GPIIb/IIIa, type 2 by reduction in number and in type 3 GPIIb/IIIa may be expressed but is not functional. The platelet function assay (PFA) is prolonged among patients with GT. Finally, genetic analysis confirms the mutations accountable for the disease ,42.(1)Preventative measures, such as abstinence from bleeding triggers (such as NSAIDS);(2)Topical measures, such as packing in case of epistaxis;(3)Antifibrinolytics, mainly used in surgeries and HMB;(4)DDAVP;(5)Recombinant Factor VIIa (rFVIIa);(6)Female hormones for gynecologic complications;(7)Surgical interventions (such as dilatation and curettage);(8)Red Blood Cell transfusions;(9)Platelet transfusions . PlateleGeneral measures for the treatment and prevention of bleeding episodes in patients with GT include the following:(A)Depo-medroxyprogesterone acetate (DMPA) ,47,(B)HPO axis suppression (leuprolide and goserelin) ,48, and(C)LNG-IUS .There is no consensus about the treatment of HMB in females with GT due to the rarity of the disease. Most of our knowledge derives from case reports and case series. As mentioned above, HMB is particularly common in menarche and severe enough to usually require transfusions. When it comes to managing episodes of HMB, besides administering blood products, hemostasis is usually achieved via intravenous infusion of high-dose conjugated estrogen for 1\u20132 days followed by high doses of oral combined estrogen and progestin, after which combined oral contraceptives (COCs) often need to be used for 2\u20133 months . After tThe rFVIIa has also been used successfully for this cause ,50. WhenBernard\u2013Soulier syndrome (BSS) is a rare platelet membrane receptor defect. According to studies from North America, Europe and Japan, the prevalence of the disease has been estimated as less than 1 in 1,000,000 people, although misdiagnosing and underreporting might lead to an underestimation of the prevalence. The syndrome is usually transmitted in an autosomal recessive pattern with only rare exceptions of autosomal dominant forms . In famiThe bleeding tendency characterizing BSS patients is attributed to mutations of the Gp1b-IX-V gene complex. This membrane protein complex facilitates two major platelet functions (a) binding of platelets to VWF and thus adhesion to the subendothelial space and (b) the ability of thrombin at low concentrations to activate platelets. When those functions are hindered, due to a dysfunctional complex, clot formation is hampered and thus a bleeding diathesis occurs, which can often be severe. Furthermore, the complex is thought to play a vital role in the process of normal platelet generation in the bone marrow, as indicated by the low platelet count and pathologically big size of platelets in Bernald Soulier patients ,42.The bleeding tendency of BSS patients is usually apparent during the first years of life. Common clinical manifestations include (a) recurrent epistaxis, which is usually the most common cause of severe bleeding, (b) gingival bleeding and (c) trauma-induced hemorrhage. However, there are great variations in the clinical profile among BSS patients, as well as in the severity of the symptoms which can worsen or become alleviated after childhood. Pregnancy can pose severe risks on BSS patients or progress uneventfully ,42. SomeHeavy menstrual bleeding can be an important clinical manifestation in the course of the disease. According to a systematic review by Marieke C. Punt et al. that included data from 4 cohort studies, overall, 25% of women with Bernard\u2013Soulier syndrome 13/52) have a present or past history of HMB . In a te have a pWhen a hemostatic disease is suspected, the FBC reveals in most cases a moderate to mild thrombocytopenia which is disproportionate to the bleeding symptoms, thus aiming to differentiate it from immune thrombocytopenia. The initial diagnostic evaluation also includes a blood film examination that can show giant platelets. The bleeding time is prolonged but aPTT and PT are normal. In a person with clinical bleeding manifestations, the combination of those facts can predispose the specialist to the potential diagnosis of BSS. It is important to note though that platelets in BSS patients can be larger than the automatic counter cut-off and therefore in order to avoid underestimation of their number, an optical/manual method is preferred ,53. PlatTreatment in general in BSS shares a lot of similarities with GT and follows the general rules mentioned in the GT section . In partHermansky\u2013Pudlak syndrome (HPS) is a congenital qualitative platelet disorder, characterized by platelets with low amounts of dense granules, making it a delta granule storage pool disorder. It is transmitted via an autosomal recessive pattern and has an estimated worldwide prevalence of about 1 in 100,000 to 1 in 1,000,000, although much more common in Northwest Puerto Rico and the Swiss Valois, where the prevalence reaches 1 in 1800 people .To date, there have been recognized at least 10 different genes responsible for this genetically heterogenous syndrome. Mutations in HPS1 gene are the most frequently reported. Those genes encode proteins responsible for synthesis or function of lysosome-related organelles, such as melanosomes and platelet dense granules. The aftermath of their genetic mutations is the disruption in production or trafficking of those organelles, resulting in the impairment of their related functions in hemostasis and pigmentation .Some of its clinical manifestations are oculocutaneous albinism (OCA), meaning a reduction in retinal, skin and hair pigmentation, variable bleeding predisposition, visual acuity impairment, nystagmus, strabismus, as well as pulmonary fibrosis, granulomatous colitis, renal impairment, and immunodeficiency. The disease\u2019s clinical course and prognosis are characterized by great heterogeneity. Ocular and skin findings commonly lead to early childhood diagnosis, with the skin and hair color ranging from pale white to olive and iris color tending to be blue, green, or brown. However, it should be taken into consideration that sometimes OCA is not easy to recognize, and suspicion arises only after comparing unaffected family members. Bleeding manifestations of HPS also vary and include excessive bruising, epistaxis, gingival bleeding, or other forms of mucosal bleeding, surgical or dental bleeding, menorrhagia and postpartum hemorrhage. Restrictive lung disease typically becomes clinically apparent in the early 30s and granulomatous colitis is severe in about 15% of affected individuals ,57. Hermansky\u2013Pudlak syndrome is a rare disease and therefore our knowledge is limited. A systematic review of case reports and case series was conducted in 2020 by Deborah Obeng-Tuudah et al. about HPS and its obstetric and gynecological complications. According to its findings, HMB is the most common bleeding manifestation in women affected with the syndrome, reported in 53% (8/15) . It is iSuspicion of a delta storage pool disorder is based on the patient\u2019s general features (such as OCA), family history, and a suggestive bleeding phenotype. Initial laboratory testing includes the bleeding time that is usually prolonged and a FBC that is within normal limits as far as the platelet count is concerned. After more common causes have been excluded, platelet aggregometry testing can be performed, which is usually abnormal in HPS. Typically, this consists of decreased aggregation response to arachidonic acid, collagen, and thrombin, but not ristocetin, as well as a deficient second-wave aggregation in response to epinephrine or ADP. Platelet light transmission electron microscopy provides a definitive diagnostic feature of delta storage pool disorders, a significant deficiency in platelet dense granules. As a final step of the diagnostic work-up, molecular genetic analysis allows specific diagnosis of the subtype of Hermansky\u2013Pudlak syndrome, which is important for better management .HPS is a rare disease and therefore there is no standard treatment for patients affected with HMB. Reports of HMB in HPS patients and their therapeutic interventions are scarce. In the following tables  we have 9/L) and increased bleeding risk in the absence of other causes associated with thrombocytopenia [Immune thrombocytopenia, formerly known as Idiopathic thrombocytopenic purpura (ITP), is an autoimmune disorder characterized by reduced platelet counts , ITP is more often a chronic disease in adults with an insidious onset requiring multiple therapeutic approaches . BleedinIn a 2022 cohort study and review of the literature by van Dijk et al., the frequency of HMB among women with ITP ranged from 6% to 55% among the eight included studies, with the wide interval probably reflecting-other than differences in the studied population-also the different assessment methods used and the varying timing in the assessment of the patients; at diagnosis vs. during the course of disease .Despite our knowledge of the pathophysiology of ITP, the diagnosis is still one of exclusion. Usually, the only abnormal lab finding is a low platelet count, while PT and aPTT are most often within the normal limits .9/L and observation for those with a count >30 \u00d7 109/L. It should be noted that in the studies assessed by the expert panel there were none directly comparing corticosteroid use to observation in this patient population. The experts suggest that in those who are eligible for corticosteroid therapy, a short course (\u22646 weeks) of oral prednizone or dexamethazone should be administered. In adults with ITP for \u22653 months who are corticosteroid-dependent or unresponsive to corticosteroids the panel recommends, by order of preference, a Thrombopoietin receptor agonist-specifically either eltrombopag or romiplostim-rituximab or splenectomy. However, it is mentioned that when the choice is made, several factors should be taken into account, including \u201cduration of ITP, frequency of bleeding episodes requiring hospitalization or rescue medication, comorbidities, age of the patient, medication adherence, medical and social support networks, patient values and preferences, cost, and availability\u201d. Furthermore, it should be noted that all the above-mentioned recommendations are based on evidence, which was evaluated as low to very low certainty by the panel [Explicit guidelines for the management of adult patients with immune thrombocytopenia have been published by the American Society of Hematology in 2019 . In adulhe panel ,64.The 2019 ASH Guidelines refer to the general management of all patients with ITP. However, for women with episodes of heavy menstrual bleeding, symptom-specific strategies, common to the disorders mentioned previously, may be needed. Available therapeutic options for HMB are hormonal therapy, endometrial ablation and hysterectomy. However, some of these treatments impair fertility permanently, thus, for women with ITP who wish to retain fertility, hormonal therapies, such as oral contraceptive pills (OCP) or intrauterine devices (IUD), combined with antifibrinolytic therapy are preferably used. These management options may successfully manage HMB ,70,71. IAmong the management techniques, LNG-IUD showed a benefit in reducing menstrual bleeding and improving quality of life assessed by the MMAS score; however, it is important to note that the study population is small, and the results cannot be safely generalized.Joint hypermobility (JH) is not a diagnosis, but a descriptor that defines an excess in the range of motion in one or more joints considering age-, sex-, and race-specific mobility. Generalized joint hypermobility (GJH) is defined as the simultaneous presence of JH in the axial skeleton and the four limbs . The BeiGJH can be symptomatic and a part of certain syndromes although it is also present in the general population in an asymptomatic form. Joint hypermobility syndrome (JHS) was originally conceived and described as a connective tissue disorder combining GJH and one or more systemic symptomatic complications, making it a part of a spectrum of diseases that include Ehlers\u2013Danlos syndromes . This syHowever, the 2017 International Classification of the Ehlers-Danlos syndromes (EDS) replaced previous terms for symptomatic and syndromic joint hypermobility (or joint hypermobility syndrome) with hypermobile EDS (hEDS). It also introduced the term hypermobility spectrum disorders (HSD) for the great range of patients facing joint hypermobility with symptomatic systemic complications, but who do not meet the diagnostic criteria for hypermobile EDS. The 2017 Diagnostic Criteria are applied to diagnose this spectrum of conditions, as indicated in The clinical pictures of hypermobile EDS and HSD are variable. Common manifestations include joint hypermobility, chronic joint pains, recurrent dislocations, subluxations and skin hyperextensibility. However, it is of utmost importance to note that EDS as well as HSD include a wide range of symptoms affecting almost every system, in multiple possible combinations. Some of their extra-articular manifestations include anxiety disorders, depression, chronic fatigue, orthostatic intolerance, functional gastrointestinal disorders, pelvic and bladder dysfunction, pelvic organ prolapse, as well as easy bruising, excessive bleeding in surgeries, and heavy menstrual bleeding ,75.Joint hypermobility as defined by the Beighton score is not rare at all, but the exact prevalence depends on the BS score cut-off value and the population studied. It has been recorded in up to 38.5% of young women and 25.4% of young men when a Beighton score greater than 4 is considered positive . More reTo date, there is very little published research dedicated to the gynecologic symptoms of females with GJH, and this is an area in need of further investigation. However, available data do suggest abnormal uterine bleeding, and other gynecological symptoms are common manifestations of these disorders. In a cohort study including 386 women with hypermobility type Ehlers-Danlos syndrome, 76% of them had a history of menorrhagia/HMB . In anotThe causes of the hemostatic problems in patients with EDS and HSD are yet to be clarified, but have long been hypothesized to be primary hemostatic disorders. Collagen structure is altered in many EDS patients, although genes responsible for hEDS have not been discovered yet. Deformed collagen leads to weakened vessel walls, as well as, defective interaction with platelets and Von Willebrand factor, thus hindering platelet plug formation. hEDS has also been related to mast cell disorders which independently can lead to a bleeding tendency . InteresResearch in the field of therapeutic interventions for the management of HMB in this disease category is scarce. Most therapeutic attempts follow the general recommendations of HMB treatment, although early referral of adolescents to specialist care is recommended, aiding towards a more targeted approach. In the same study mentioned above by Kendel et al. with the 30 patients with GJH and HMB, most of the patients, 68% (20/30), required escalation of their initial therapeutic approach and only 32% (10/30) achieved adequate control of their HMB with the initial treatment selections, indicating GJH as a risk factor of difficult management. The treatment options that eventually lead to control of HMB are indicated on HMB is a common gynecological problem affecting women of reproductive age and compromising their quality of life. When HMB is diagnosed, the C category according to the PALM-COEIN system represents a major group of hematologic clinical entities that have to be included in the differential diagnosis. VWD is the most common disorder in the C category and the most studied disease of those, as a cause of HMB. When the basic hematologic workup has been completed, usually the attention is directed towards other causes outside the C category. However, primary hemostasis disorders can be a cause of HMB even without affecting PT, aPTT and sometimes with normal CBC. Under those circumstances, the clinician must suspect and further investigate the possibilities of hematologic diseases. This review aimed at summarizing the basic findings regarding primary hemostasis disorders and their association with HMB. It includes basic information regarding their epidemiology, pathophysiology, clinical picture, and treatment in a succinct way, in order to act as a reference guide for the clinician. It includes VWD, as well as other causes that could potentially be missed because of their rarity. It acts as a reminder that diagnostic workup can have more steps even when most common diseases have been excluded and emphasizes the importance of a thorough physical examination and a clinical acuity, as indicated by HSD and EDS, platelet disorders and ITP. A simple and easy to use clinical tool such as the Beighton score could be used by both primary care providers and gynecology specialists, in order to pick out those cases that could otherwise be easily missed due to their perceived rarity and their negative laboratory workup. After that point, a specialist hematologist consultation is of great importance in order to reach an accurate diagnosis. This review also aims to provide a disease-specific summary of evidence about each clinical entity mentioned, in order to guide towards a more targeted approach. As a literature and not a systematic review it provides a non-exhaustive view of the topic based on the published literature, and it should not be used as a sole resource to guide diagnostic decisions or therapeutic interventions, but rather as an informative source and a reference for further reading. Further research is needed in order to clarify a possible connection between the remaining rare primary hemostasis disorders and HMB, as well as to gather more solid data about the diseases mentioned and their clinical and therapeutic approach."}
+{"text": "The main drivers of COVID-19 disease severity and the impact of COVID-19 on long-term health after recovery are yet to be fully understood. Medical imaging studies investigating COVID-19 to date have mostly been limited to small datasets and post-hoc analyses of severe cases. The UK Biobank recruited recovered SARS-CoV-2 positive individuals (n = 967) and matched controls (n = 913) who were extensively imaged prior to the pandemic and underwent follow-up scanning. In this study, we investigated longitudinal changes in body composition, as well as the associations of pre-pandemic image-derived phenotypes with COVID-19 severity. Our longitudinal analysis, in a population of mostly mild cases, associated a decrease in lung volume with SARS-CoV-2 positivity. We also observed that increased visceral adipose tissue and liver fat, and reduced muscle volume, prior to COVID-19, were associated with COVID-19 disease severity. Finally, we trained a machine classifier with demographic, anthropometric and imaging traits, and showed that visceral fat, liver fat and muscle volume have prognostic value for COVID-19 disease severity beyond the standard demographic and anthropometric measurements. This combination of image-derived phenotypes from abdominal MRI scans and ensemble learning to predict risk may have future clinical utility in identifying populations at-risk for a severe COVID-19 outcome. COVID-19, the disease caused by the virus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), impacts human health and has debilitating effects in both the short- and longer term . The comWhilst there are few imaging studies that prospectively delineate the impact of COVID-19 on organ health, there are multiple case reports and imaging studies using computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound to assess patients post-infection, with some showing consistent changes characteristic of the response to COVID-19 infection. These include cardiac changes in inflammatory edema, fibrosis, impaired ventricular function, and mass , with acThe UK Biobank (UKBB) is a prospective cohort study of half a million adults in the UK. Beginning in 2014, the UKBB implemented an extensive standardized imaging protocol covering the abdomen, heart and brain . To inveThe aim of the current study is to assess the impact of SARS-CoV-2 infection on abdominal organ health and body composition in 967 cases and 913 controls. Moreover, we assessed the association between severity of COVID-19 disease and multi-organ image-derived phenotypes from the baseline imaging visit. The findings from this study are relevant for determining long-term outcomes and future health needs for both populations and individuals recovering from COVID-19, as well as shedding light on possible factors for disease severity.https://biobank.ndph.ox.ac.uk/showcase/showcase/docs/casecontrol_covidimaging.pdf.Approximately 45,000 UKBB participants have attended a baseline MRI scanning session  prior to the appearance of SARS-CoV-2 in the UK. Starting from February 2021, a total of 1,880 participants who attended the first imaging visit were recruited to a new re-imaging study aiming to determine the impact of COVID-19. Individuals with a confirmed COVID-19 diagnosis  were invited to take part in the study, together with a matched control group. The control group was selected based on negative results or no history of positive results from the aforementioned sources. Cases and controls were paired based on sex, ethnicity, age (\u00b1 6 months), imaging assessment centre, and date of initial baseline scan (\u00b1 6 months). Due to small numbers of non-white participants, ethnic matching was based on a classification of white vs non-white. In line with UK travel restrictions at the time, participants were restricted to those living within 60 km of the UKBB scanning centers at Stockport, Newcastle, and Reading. A detailed description is given here: https://www.ukbiobank.ac.uk).Participant data from the UKBB cohort was obtained as previously described  through During visual quality control of the segmentations, no catastrophic failures were observed and we did not perform any manual editing. We defined a catastrophic failure to be when segmented organs had major components missing or components that did not belong to the structure added (i.e. over or under segmentation). This could be due to underlying data errors such as fat-water swaps, or where the neural network did or did not highlight part of the organ even though the underlying image intensities are free of ambiguities. We included a total of twelve IDPs in this study: volumes of abdominal subcutaneous adipose tissue (ASAT), visceral adipose tissue (VAT), liver, lungs, iliopsoas muscles, kidneys, pancreas, spleen, as well as proton density fat fraction (PDFF) measures of liver and pancreas fat content, and organ iron concentration of the liver and pancreas.Full details regarding the UKBB MRI abdominal protocol have previously been reported . The datggplot2 v3.3.5 package. Descriptive statistics are provided as mean, standard deviation, and range for continuous traits, and as mean, standard deviation, and 95% confidence interval for binary traits. Differences between groups were assessed for statistical significance using a Chi-squared goodness-of-fit test for binary traits, and Student\u2019s t-test for continuous traits.All summary statistics, hypothesis tests, models were performed using the R3.6.3 software environment for statistical computing and graphics . VisualiIn our analyses, we assessed the longitudinal effects of SARS-CoV-2 infection as well as the severity of COVID-19 based on pre-pandemic imaging alone.\u0394age = (agerescan\u2014agebaseline). To capture quadratic effects of age, \u0394age2 was calculated as the square of the difference of the age at rescanning and the age at baseline: \u0394age2= (agerescan\u2014agebaseline)2. The following model was selected to associate changes in abdominal IDPs with COVID-19 diagnosis:Associations between changes in imaging phenotypes and COVID-19 diagnosis required calculating the age difference between the two imaging visits: X describes fixed effects: sex, ethnicity, height, BMI, smoking status, alcohol consumption, and Townsend deprivation index. Baseline and rescan IDP values were standardized. During model selection, we initially included an interaction term between COVID positivity and sex but did not observe any significant interactions (p>0.05), so the interaction term was dropped from the final model above.We determined COVID-19 severity based upon hospitalization with International Classification of Diseases 10th Revision (ICD-10) diagnosis codes U071.1 or U071.2, plus a positive test status from one of the available sources. We also filtered severe cases by pneumonia diagnosis (J128.2) and by placement on a ventilator (Z99.11). For the severity analyses of first imaging visit data only, a second category of COVID-19 severe outcomes also included death with a recorded cause of death as COVID-19 (U07.1). Follow-up analysis of pulmonary diseases was conducted using hospital billing codes J00-J99. The severity analysis included 140 additional SARS-CoV-2 positive samples beyond the matched case/control design in the first analysis, of participants who had attended the first imaging visit but were not recruited as part of the re-imaging study.As a logistic regression model, COVID-19 severity was regressed on standardized baseline IDP values and fixed effect covariates:X included rescan imaging age, imaging age squared, sex, ethnicity, Townsend deprivation index, height, BMI, smoking status, alcohol consumption, and baseline imaging center location. Baseline imaging center was encoded as a categorical variable. During model selection we tested whether baseline IDP association with severity differed by sex but the interaction term was not significant for any of the baseline IDPs (p>0.05). We modeled severity as hospitalized and non-hospitalized outcomes; severe  and non-severe outcomes; and death vs hospitalization. For multiple test correction of independent traits, a Bonferroni adjustment was used to determine a significance threshold, where alpha was set at 0.05. For dependent traits, false discovery rate (FDR) was estimated from p-values using the Benjamini-Hochberg method Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0This study performed a large-scale analysis for abdominal imaging associates body composition with COVID-19 severity. This is timely needed study with comprehensive analysis and decent writing.Does the author perform a human involved QA process for image processing results. I can imagine some segmentation results are failed.It would be helpful to illustrate the rationale of using image processing pipeline [37].Why so many factors have large confidence intervals in Figure 2 and 3?It seems the trend of results is not consistent between Figure 2 and 3 for statistical significance.From Figure 4a, the BMI is a much more impactful feature compared with Age. But in Figure 4b, their importance are almost the same. That is somehow contradictory.Why the visceral fat and liver fat are not used in the Figure 4a?It seems the top predictors are all fat related features, which would have strong interaction.Reviewer #2:\u00a0In this study the authors analyzed data of UK Biobank recruited recovered SARS-CoV-2 positive individuals (n=967) and matched controls (n=913) who were extensively imaged prior to the pandemic and underwent follow-up scanning. They specifically investigated longitudinal changes in body composition, as well as the associations of pre-pandemic image-derived phenotypes with COVID-19 severity. The authors found a decrease in lung volume with SARS-CoV-2 positivity. They also observed that increased visceral adipose tissue and liver fat, and reduced muscle volume, prior to COVID-19, were associated with COVID-19 disease severity. In addition, when training a machine classifier with demographic, anthropometric and imaging traits, they found that visceral fat, liver fat and muscle volume have prognostic value for COVID-19 disease severity beyond the standard demographic and anthropometric measurements. The authors have much expertise in this field of research and the topic addressed by this study is important.Specific comments:1. Introduction: When the authors address post-acute sequelae of COVID-19 they should also discuss the findings of very important studies that investigated this question :259-264; Nat Med. 2022 Mar;28(3):583-590; Lancet Diabetes Endocrinol. 2022 Mar 21:S2213-8587(22)00044-4. doi: 10.1016/S2213-8587(22)00044-4).2. Introduction: The authors should also discuss how the COVID-19 pandemic may generally impact on the cardiometabolic risk and on obesity :135-149).3. Impaired cardiometabolic health and obesity are thought to promote SARS-CoV-2 vaccine-breakthrough infections :75-76). Do the authors have data to address this important point? Otherwise, they should at least carefully discuss this point.4. When the authors address the relationship of obesity with severity of COVID-19, they should also discuss the important findings from the QResearch database of general practices in England, UK study :350-359), which carefully investigated the relationship of obesity, under the aspects of different comorbidities and age, with the severity of COVID-19.5. On what basis were specifically the 1,880 participants, who attended the first imaging visit, selected and recruited to the new re-imaging study? It appears as if these 1,880 participants are the cases and controls. So, what was the initial number of subjects with a positive SARS-CoV-2 test result that was asked to attended the second imaging visit?6. The authors assessed longitudinal effects of SARS-CoV-2 infection, as well as the severity of COVID-19 based on pre-pandemic imaging alone. Does this mean that from the 1,955 participants mentioned in the result section, data of those wo died and of those who refused to participate in the second imaging study, were analyzed for cross-sectional relationships?7. Table 1: For smoking, alcohol consumption and selected diseases reported, the authors indicate that the percentage of the subjects is reported. However, from the data of that table the number cannot be percentages.8. Table 1: If the percentage of subjects having NAFLD amounts to 70-75, why was the percentage of patients with type 2 diabetes so low (2-3 percent). Considering the prevalence of NAFLD in patients with type 2 diabetes :284-296), these numbers should be different.9. Unexpectedly, the authors only observed a significant decrease in lung volume, but no change in any other examined image-derived phenotypes. May it be possible that most of the participants, who attended the second imaging study, had relatively mild COVID-19 and patients with a more severe course of the disease refused to participate in the second exam?********** <font color=\"black\"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #2:\u00a0Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 19 Jul 20221. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: We will update your Data Availability statement to reflect the information you provide in your cover letter.https://www.ukbiobank.ac.uk and we have included this in the data availability statement.Our research was conducted using UK Biobank data. Under the standard UK Biobank data sharing agreement, researchers are unable to directly share data they obtain from the biobank with others. However, all of the the the data generated and used in this paper are available directly from the UK Biobank upon request at Additional Editor Comments (if provided):The paper has been evaluated by two expert reviewers. Both reviewers recognize the strengths of the study but they have also identified a number of major and minor issues that need to be addressed before the manuscript can be considered for publication. In addition to the points raised by the Reviewers, I would ask the authors to more clearly describe 1) in subsection \"Predictive modeling\" whether the data was split on the subject level in the cross-validation and 2) Throughout the manuscript what data exactly was used as input in these models .We would like to thank the editor for this comment, we have addressed this point in the methods and the paper has clearly benefited from this added clarity. We have split the methods into subsections, clearly differentiating the longitudinal analysis and the baseline-only analyses. The predictive modeling was performed on the baseline data only, as we tried to predict disease severity from image-derived body composition before the pandemic. The data were not paired at subject level in the predictive analysis as only one time point was used, and therefore could not be split.-----------------------------------------Reviewer #1: This study performed a large-scale analysis for abdominal imaging associates body composition with COVID-19 severity. This is timely needed study with comprehensive analysis and decent writing.Does the author perform a human involved QA process for image processing results. I can imagine some segmentation results are failed.This is a good point to raise, quality control with big data has to be very stringent, and we did indeed perform extensive QA by visually checking the fifty largest and fifty smallest volumes, as well as another 50 random for each structure. The QA protocol is outlined in [37], however we have added some additional details into the manuscript to explain this more clearly. It would be helpful to illustrate the rationale of using image processing pipeline [37].We thank the reviewer for pointing this out. The preprocessing steps, segmentation models for individual organs and the quantitative analysis of PDFF (proton density fat fraction) and R2* have been applied to approximately 38,000 scans from the UK Biobank in Liu et al. (2021). We have added text in the \u201cImage-derived phenotypes\u201d section that provides the rationale. Why so many factors have large confidence intervals in Figure 2 and 3?The variables affected by large confidence intervals in Figure 3  are self-reported, and therefore have more variability compared to quantitative values; there could also be a bias in answering those questions, given their negative connotation. For Figure 3, the confidence intervals are also quite large for sex as well as ethnicity and imaging centre. We believe this is due to the uneven distribution of severe cases as for example there are about three times more severe cases in men in that particular dataset.It seems the trend of results is not consistent between Figure 2 and 3 for statistical significance.We thank the reviewer for this remark, the plots are quite similar, but the outcome variable is different. Figure 2 summarises the changes between the two timepoints between cases and controls, whereas Figure 3 is about investigating the associations between image-derived phenotypes (IDPs) and severity. We have made this more explicit in the text and captions.From Figure 4a, the BMI is a much more impactful feature compared with Age. But in Figure 4b, their importance are almost the same. That is somehow contradictory.Thank you for raising this point.. Figure 4A are the ROC curves for the three different models  whereas figure 4B is the importance of the random forest features for the last model only. We have changed the caption to reflect this.Why the visceral fat and liver fat are not used in the Figure 4a?Thank you for this question, which falls into the same category as the one above. We did use visceral fat and liver fat in figure 4A, the green line represents the model with Age+Sex+BMI+IDPs, where IDPs are the image-derived phenotypes . We have amended the figure caption accordingly to remove any ambiguity.It seems the top predictors are all fat related features, which would have strong interaction.Yes, we agree that several of the key variables are likely to correlate with each other. We included a regularisation term in the regression model, using the LASSO (Least Absolute Shrinkage and Selection Operator), to perform variable selection and produce a more parsimonious and interpretable result.Reviewer #2: In this study the authors analyzed data of UK Biobank recruited recovered SARS-CoV-2 positive individuals (n=967) and matched controls (n=913) who were extensively imaged prior to the pandemic and underwent follow-up scanning. They specifically investigated longitudinal changes in body composition, as well as the associations of pre-pandemic image-derived phenotypes with COVID-19 severity. The authors found a decrease in lung volume with SARS-CoV-2 positivity. They also observed that increased visceral adipose tissue and liver fat, and reduced muscle volume, prior to COVID-19, were associated with COVID-19 disease severity. In addition, when training a machine classifier with demographic, anthropometric and imaging traits, they found that visceral fat, liver fat and muscle volume have prognostic value for COVID-19 disease severity beyond the standard demographic and anthropometric measurements. The authors have much expertise in this field of research and the topic addressed by this study is important.Specific comments:1. Introduction: When the authors address post-acute sequelae of COVID-19 they should also discuss the findings of very important studies that investigated this question :259-264; Nat Med. 2022 Mar;28(3):583-590; Lancet Diabetes Endocrinol. 2022 Mar 21:S2213-8587(22)00044-4. doi: 10.1016/S2213-8587(22)00044-4).We thank the reviewer for highlighting these papers. Given the extraordinary number of relevant papers that have been published since the pandemic began it was not possible to include all of the references we would have liked. At the time of writing we focussed on the most organ specific publications most directly relevant to this manuscript. We however have expanded our Introduction to include the suggested texts and the paper clearly benefits from this addition.2. Introduction: The authors should also discuss how the COVID-19 pandemic may generally impact on the cardiometabolic risk and on obesity :135-149).We have expanded the part of the discussion where we describe how chronic comorbidities such as obesity, impaired metabolic health, and diabetes are known to be at higher risk of severe disease and poorer prognosis following COVID-19 infection to include this excellent review by Stefan et al.3. Impaired cardiometabolic health and obesity are thought to promote SARS-CoV-2 vaccine-breakthrough infections :75-76). Do the authors have data to address this important point? Otherwise, they should at least carefully discuss this point.The referee raises an important point. Unfortunately we do not have any specific data available to us within this cohort regarding vaccine-breakthrough infections however we have included a reference to this in the introduction. 4. When the authors address the relationship of obesity with severity of COVID-19, they should also discuss the important findings from the QResearch database of general practices in England, UK study :350-359), which carefully investigated the relationship of obesity, under the aspects of different comorbidities and age, with the severity of COVID-19.We have added this reference to the text. 5. On what basis were specifically the 1,880 participants, who attended the first imaging visit, selected and recruited to the new re-imaging study? It appears as if these 1,880 participants are the cases and controls. So, what was the initial number of subjects with a positive SARS-CoV-2 test result that was asked to attended the second imaging visit? https://biobank.ndph.ox.ac.uk/showcase/showcase/docs/casecontrol_covidimaging.pdf, but in brief participants needed to: have already attended pre-pandemia imaging assessment at one of the three imaging sites, live within 60km of the clinic (COVID travel restrictions were still in place during the rescanning study) and had high-quality UK Biobank MRI images available from their first imaging visit. These criteria are in our section \u201cstudy design\u201d on page 6. At the time of preparing this manuscript, a total of 1,880 suitable datasets were available for the study.The UK Biobank rescanning of participants for the COVID study initially aimed to recruit 2,000 participants, the inclusion criteria are listed in detail here: 6. The authors assessed longitudinal effects of SARS-CoV-2 infection, as well as the severity of COVID-19 based on pre-pandemic imaging alone. Does this mean that from the 1,955 participants mentioned in the result section, data of those wo died and of those who refused to participate in the second imaging study, were analyzed for cross-sectional relationships?Data from individuals from only one imaging time point at baseline was used only in the severity analysis (Tables 3 and 4)7. Table 1: For smoking, alcohol consumption and selected diseases reported, the authors indicate that the percentage of the subjects is reported. However, from the data of that table the number cannot be percentages.We have re-made the table to give percentages rather than fractions for categorical variables for easier reading.8. Table 1: If the percentage of subjects having NAFLD amounts to 70-75, why was the percentage of patients with type 2 diabetes so low (2-3 percent). Considering the prevalence of NAFLD in patients with type 2 diabetes :284-296), these numbers should be different.Thanks to the reviewer. We identified a bug in ascertainment of NAFLD cases, which we have now corrected. In fact, there is only a single participant with NAFLD diagnosed via tertiary care records . We have therefore removed this from our analysis as we are not powered to detect differences in incidence or disease severity. To address the poor ascertainment of diabetes from tertiary care records, we have updated our analysis to use an expanded definition of diabetes (UK Biobank Fields 130708 and 130706 for T2D and T1D respectively) which incorporates primary care and self-report as well as tertiary care records. This affects table 1, table 3. We have removed supplementary table 2 as it was the same as T3.9. Unexpectedly, the authors only observed a significant decrease in lung volume, but no change in any other examined image-derived phenotypes. May it be possible that most of the participants, who attended the second imaging study, had relatively mild COVID-19 and patients with a more severe course of the disease refused to participate in the second exam?The referee raises an interesting point, and we too had expected to observe more significant changes in other organs given some reports in the literature, although many of these reports are from subjects with very severe disease. The majority of the UKBB subjects invited for a second scan and who had tested positive for COVID-19, had relatively mild disease (96% mild vs 4% severe). We do not have any information whether subjects with more severe disease were less likely to attend for follow-up scanning, but were reassured that the response rates in the COVID and control groups for repeat UKBB scanning were very similar, with a 58% response rate from participants who had COVID-19 and a 57% response rate from participants who have not been infected. It is however a valid point that there may have been some self-filtering where participants who had very severe COVID-19 (or long-COVID effects) did not want to undergo a second scanning exam; we have added this as a caveat to the study.Regarding the funding, we have removed the separate entry we initially had in the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.AttachmentCOVIDpaperresponse_2.pdfSubmitted filename: Click here for additional data file. 29 Sep 2022 <div> PONE-D-22-06006R1 </div>  <div> Abdominal Imaging Associates Body Composition with COVID-19 Severity </div>  <div> PLOS ONEDear Dr. Basty,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. 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Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Ivana IsgumAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):The authors have performed a careful revision and addressed the comments raised in the review. Nevertheless, Reviewer 3 identified a few issues that I agree with. I would ask the authors to address the first two comments and to consider changing the table for easier interpretation of the results.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0All comments have been addressedReviewer #2:\u00a0All comments have been addressedReviewer #3:\u00a0(No Response)********** <font color=\"black\"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0No********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The quality of the paper has been substantially improved from this round of review. All my concerns have been addressed.Reviewer #2:\u00a0The authors have very well addressed the comments and provide very interesting information about the impact of SARS-CoV-2 infection on abdominal organ health, body composition and lung volume.Reviewer #3:\u00a0This is the first time I have read this paper, not having been part of the previous review round(s). The paper is well-structured and the text is mostly clear, although it is quite verbose. I have primarily focused on the descriptions of the experimental setting, the statistics and the presentation of the results. My specific comments are listed below.- In the Longitudinal analysis section, the authors state \u201c(delta age)^2 was calculated as the difference of the squared age at rescanning and the squared age at baseline\u201d. This is notably different from the formula (age_rescan \u2013 age_baseline)^2, from this same section. By the procedure in the text, (delta age)^2 for ages 60 and 62 would be 62*62-60*60=244, whereas by the procedure described in the formula, (delta age)^2 would be 4. It is unclear which of these results the authors have actually used.- In the section \u201cImage-derived phenotypes\u201d, the authors describe a data validation procedure: visually inspecting a subset of 150 scans (primarily outliers in terms of volume) to ensure the used pipeline is robust. I may have missed it, but I do not believe they describe how possible mistakes were handled. Were all 150 validated scans perfect? If not, were they manually corrected before being used in the analysis presented in this work? This should be clear in the manuscript.- The tables are not pleasant to parse. Many depicted values have more significant figures than would be relevant, resulting in visual clutter. Currently, they seem like tables meant to be copy-pasted into a computer program, rather than tables meant to be readable by humans. I urge the authors to revisit all tables to improve readability. For example, the FDR column in Table 1 can be deleted as it does not add any information here and the ASAT row in Table 2 should be rounded to milliliters.********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoYes:\u00a0Norbert StefanReviewer #2:\u00a0Reviewer #3:\u00a0No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 12 Oct 2022Journal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice. We have double checked each article individually in the reference list. No articles have been retracted. However, several that were at the preprint stage have since been published and we have updated the references accordingly: 34. Scalsky RJ, Chen Y-J, Desai K, O\u2019Connell JR, Perry JA, Hong CC. Baseline cardiometabolic profiles and SARS-CoV-2 infection in the UK Biobank. PLoS One. 2021;16: e0248602.37. Douaud G, Lee S, Alfaro-Almagro F, Arthofer C, Wang C, McCarthy P, et al. SARS-CoV-2 is associated with changes in brain structure in UK Biobank. Nature. 2022;604: 697\u2013707. 38. Sattar N, Ho FK, Gill JM, Ghouri N, Gray SR, Celis-Morales CA, et al. BMI and future risk for COVID-19 infection and death across sex, age and ethnicity: Preliminary findings from UK biobank. Diabetes Metab Syndr. 2020;14: 1149\u20131151. 41. Willette AA, Willette SA, Wang Q, Pappas C, Klinedinst BS, Le S, et al. Using machine learning to predict COVID-19 infection and severity risk among 4510 aged adults: a UK Biobank cohort study. Sci Rep. 2022;12: 7736.Additional Editor Comments (if provided):The authors have performed a careful revision and addressed the comments raised in the review. Nevertheless, Reviewer 3 identified a few issues that I agree with. I would ask the authors to address the first two comments and to consider changing the table for easier interpretation of the results.We thank the editor for the additional comments and finding an additional reviewer.Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The quality of the paper has been substantially improved from this round of review. All my concerns have been addressed.We thank reviewer #1 for taking the time to review our manuscript and revisions.Reviewer #2: The authors have very well addressed the comments and provide very interesting information about the impact of SARS-CoV-2 infection on abdominal organ health, body composition and lung volume.We thank reviewer #2 for taking the time to review our manuscript and revisions.Reviewer #3: This is the first time I have read this paper, not having been part of the previous review round(s). The paper is well-structured and the text is mostly clear, although it is quite verbose. I have primarily focused on the descriptions of the experimental setting, the statistics and the presentation of the results. My specific comments are listed below.We thank reviewer #3 for taking the time to review our manuscript, and agree with the comments intended to make the paper easier to read.- In the Longitudinal analysis section, the authors state \u201c(delta age)^2 was calculated as the difference of the squared age at rescanning and the squared age at baseline\u201d. This is notably different from the formula (age_rescan \u2013 age_baseline)^2, from this same section. By the procedure in the text, (delta age)^2 for ages 60 and 62 would be 62*62-60*60=244, whereas by the procedure described in the formula, (delta age)^2 would be 4. It is unclear which of these results the authors have actually used.Thank-you for pointing out this typo. The formula was incorrect and has been modified to match the text.- In the section \u201cImage-derived phenotypes\u201d, the authors describe a data validation procedure: visually inspecting a subset of 150 scans (primarily outliers in terms of volume) to ensure the used pipeline is robust. I may have missed it, but I do not believe they describe how possible mistakes were handled. Were all 150 validated scans perfect? If not, were they manually corrected before being used in the analysis presented in this work? This should be clear in the manuscript.We have added text clarifying that no mistakes were found during the manual visual inspection of the data and it was therefore non necessary to undertake any corrective manual editing. - The tables are not pleasant to parse. Many depicted values have more significant figures than would be relevant, resulting in visual clutter. Currently, they seem like tables meant to be copy-pasted into a computer program, rather than tables meant to be readable by humans. I urge the authors to revisit all tables to improve readability. For example, the FDR column in Table 1 can be deleted as it does not add any information here and the ASAT row in Table 2 should be rounded to milliliters.We agree with the reviewer that the tables benefit from decluttering and have rounded volumes to millilitres. We removed the FDR column from Table 1, and removed the FDR and Statistic columns from Table 3. Asterisks have been added to p-values that are statistically significant in both tables, with additional text provided for explanation. 10 Jan 2023 <div> PONE-D-22-06006R2 </div>  <div> Abdominal Imaging Associates Body Composition with COVID-19 Severity </div>  <div> PLOS ONEDear Dr. Basty,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. </div>  <div> #reviewer 3 </div>  <div>  <span style=\"color: rgb; font-family: verdana, geneva, arial, helvetica, sans-serif; font-size: 11.2px; background-color: rgb;\"> I commend the authors for their work in improving the manuscript. However, I am quite surprised to hear that there were no errors at all in any of the visually inspected segmentations. Somewhat suspect is that the rendered lung segmentation in figure 1 seems to contain a disconnected left bronchus, which would generally be considered a segmentation error. The authors should define what was considered acceptable and what was considered an error during this quality control step. </span> ============================== <span style=\"color: rgb; font-family: verdana, geneva, arial, helvetica, sans-serif; font-size: 11.2px; background-color: rgb;\"> The tables look a lot better now. A final (minor) suggestion would be to change any p-values larger than 0.10 to decimal notation in Table 3 to make it easier to differentiate large from small numbers at a glance . </span>  </div>  <div> Please ensure that your decision is justified on PLOS ONE\u2019s\u00a0publication criteria\u00a0and not, for example, on novelty or perceived impact.For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data.\u00a0==============================plosone@plos.org. 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Ying-Mei FengAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0(No Response)********** <font color=\"black\"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0Yes********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0Yes********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0No********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0Yes********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0I commend the authors for their work in improving the manuscript. However, I am quite surprised to hear that there were no errors at all in any of the visually inspected segmentations. Somewhat suspect is that the rendered lung segmentation in figure 1 seems to contain a disconnected left bronchus, which would generally be considered a segmentation error. The authors should define what was considered acceptable and what was considered an error during this quality control step.The tables look a lot better now. A final (minor) suggestion would be to change any p-values larger than 0.10 to decimal notation in Table 3 to make it easier to differentiate large from small numbers at a glance .********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #3:\u00a0No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 24 Jan 2023#reviewer 3I commend the authors for their work in improving the manuscript. However, I am quite surprised to hear that there were no errors at all in any of the visually inspected segmentations. Somewhat suspect is that the rendered lung segmentation in figure 1 seems to contain a disconnected left bronchus, which would generally be considered a segmentation error. The authors should define what was considered acceptable and what was considered an error during this quality control step.We thank the Reviewer for taking the time to review the latest iteration of our paper. We agree that the wording of the statement is too generous and we have amended this statement that was in the text:We found no errors in the visual inspections and did not perform any manual editing.To as follows:During visual quality control of the segmentations, no catastrophic failures were observed and we did not perform any manual editing. We defined a catastrophic failure to be when segmented organs had major components missing or components that did not belong to the structure added (i.e. over or under segmentation). This could be due to underlying data errors such as fat-water swaps, or where the neural network did or did not highlight part of the organ even though the underlying image intensities are free of ambiguities. The tables look a lot better now. A final (minor) suggestion would be to change any p-values larger than 0.10 to decimal notation in Table 3 to make it easier to differentiate large from small numbers at a glance .Please ensure that your decision is justified on PLOS ONE\u2019s publication criteria and not, for example, on novelty or perceived impact.Thank you for this comment which improves the readability of the tables. We have amended the tables as suggested. 12 Mar 2023Abdominal Imaging Associates Body Composition with COVID-19 SeverityPONE-D-22-06006R3Dear Dr. Basty,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #3:\u00a0All comments have been addressed********** <font color=\"black\"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0(No Response)********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0(No Response)********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0(No Response)********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0(No Response)Reviewer #3:\u00a0(No Response)********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0I have recommended acceptance in the last round of review. Please do not send it to me again since that was my final decision.Reviewer #3:\u00a0(No Response)********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #3:\u00a0No********** 4 Apr 2023PONE-D-22-06006R3 Abdominal Imaging Associates Body Composition with COVID-19 Severity Dear Dr. Basty:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Jun Mori Academic EditorPLOS ONE"}
+{"text": "At the initial assessment of hand coverage with the hydro-alcoholic gel, the mean palm coverage was 82.2%, mean back coverage was 66.8%, and overall mean hand coverage was 74.5%. Then, two measurements were performed in June (t1) and December 2022 (t2). The third measurement showed an increase in hand coverage, with a mean palm coverage of 97.2%, a mean back coverage of 91.8%, and a mean hand coverage of 94.5% (p < 0.05). Moreover, the mean coverage of the hand-back was lower than that of the hand-palm at all times (p < 0.001). The automated scanner provided evidence supporting its effectiveness in enhancing hand hygiene among hospital employees. These findings have motivated researchers to conduct long-term studies, given the potential effects on HAI prevention\u2014including their impact on HAI trends.Hand hygiene plays a pivotal role in preventing Healthcare-Associated Infections (HAIs). Nevertheless, the quality of hand disinfection techniques remains suboptimal, and the reliability of assessment methodologies is notably lacking. This study aimed to evaluate hand disinfection techniques using an automated system in an Italian research hospital. Hospital employees underwent automated hand-disinfection technique assessment, according to the World Health Organization\u2019s six-step protocol, at a basal time and two further times. Detection was carried out with a scanner that evaluated the effective hand disinfectant coverage through a fluorescent dye present in the hydro-alcoholic gel rub. The hand-hygiene technique of 222 employees was analyzed by HandInScan The most common transmission vectors of healthcare-associated pathogens are the hands of healthcare employees (HCW); thus, hand hygiene represents the single most effective means of reducing healthcare\u2013associated infections (HAI), with relevance for patient care safety and overall hospitalization costs ,2,3,4.HAIs represent the most frequent adverse event associated with patient care, contributing to significant morbidity, mortality, and financial burden in patients and healthcare systems due to direct and indirect costs\u2014thus further representing a central indicator of health service quality . IndirecSince the World Health Organization (WHO) recommendations on hand hygiene created in 2009, a five-dimensional model was proposed to support successful infection prevention and control (IPC): (1) Alcohol-Based Hand Rub (ABHR) at the point of care for the HCW, (2) ABHR assessment and performance feedback, (3) continuous training and education, (4) widespread reminders in the workplace, and (5) institutional safety and a supportive climate ,33,34. RThe IRCCS Fondazione Istituto Neurologico Nazionale C. Mondino (Mondino Foundation), Pavia, Italy, is an Italian research hospital dedicated to the care of patients and research in the field of neuroscience and neurological and neuropsychiatric disorders. The Mondino Foundation is equipped with an Hospital-acquired Infection Control Committee (HICC), a multidisciplinary team dedicated to the IPC function.This study aimed to evaluate Mondino Foundation employees\u2019 hand disinfection technique with an automated system in the context of a broad, multimodal HAI prevention project .This study was conducted at the Mondino Foundation, an Italian research hospital with 131 beds: 60 for neurological disorders, 39 for neurological rehabilitation, 22 for neuropsychiatry, and 10 technical rooms for day-hospital treatment. In 2022, there were around 3500 (2800 adult) hospitalizations and 35,000  patient days.In 2022, the Mondino Foundation\u2019s HICC collaborated with the hospital\u2019s health direction and with the occupational health service to improve IPC activities in the hospital in a comprehensive way. The HICC planned an automated assessment of hand-disinfection technique and performance feedback, integrated with the WHO\u2019s aforementioned five dimensional model. Disinfection protocols were revised, together with protocols on infection control and isolation, the correct use of PPE, and the management of urinary and vascular catheters. In addition, during the first half of the year, nurses were trained in urinary and vascular catheter management to prevent urinary tract and bloodstream infections. In the second half of the year, a workshop on the prevention and management of HAIs and sepsis was held. Point-of-care ABHR was provided by hydro-alcoholic gel dispensers  located at the entrances to outpatient areas and inpatient wards, along the corridors of inpatient rooms, at the hospital entrance, in common and administrative areas, and in each outpatient room. The area supervisors were responsible for refilling the dispensers once they were empty. Posters on correct and frequent hand washing/rubbing and WHO Six Moments for hand hygiene were posted by each dispenser and in each toilet for visitors and staff . The HICIn 2022, the average number of employees in the hospital was approximately 300, including 80 (26.7%) physicians (P), 90 (30%) nurses (N), 80 (26.7%) healthcare assistants (H), and 50 (16.6%) administrative employees (A). Participants were split according to their roles across the three-time intervals, as stated in \u00ae scanner .The evaluation was conducted on the coverage of the disinfected hand area; 2022 was used as the reference year, with an initial measurement in November 2021 (t0) and two additional measurements in June 2022 (t1) and December 2022 (t2).The threshold for passing the test with an excellent technique was to achieve an overall disinfected hand area of 95% ,35. The p-values less than 0.05 were considered to be significant in this study.When comparing the indicators across the four groups of professionals, an analysis of variance was carried out; \u00ae scanner. Overall, 33% (n. 73) were in the administrative department, 27% (n. 60) were nurses, 26% (n. 56) were physicians, and 14% (n. 32) were healthcare assistants. The assessments were conducted and reported at three different time intervals  assessment of hand disinfection coverage with hydro-alcoholic gel in all the participants, the mean palm coverage was 82.2% (SD = 27.4), the mean back coverage was 66.8% (30.5), and the overall mean hand coverage was 74.5%  and t2 (December 2022) showed an increase in hand coverage, together with a reduction in the standard deviation. Specifically, the mean palm coverage was 90.1% (SD = 17.0) at t1 and 97.2% (SD = 5.5) at t2 and the mean back coverage was 72.0% (SD = 27.2) at t1 and 91.8% (SD = 13.5) at t2. The mean hand coverage was 81.0% (SD = 20.7) at t1 and 94.5% (SD = 9.1) at t2.Considering the threshold target of \u226595%, it was exceeded only at t2 for palm coverage . Values close to the threshold (coverage \u2265 95%) were observed for the mean overall hand coverage of all employee categories at t2 . Excluding the results for administrative employees, the values for HCW were above the threshold at t2 . On the contrary, no statistical significance was observed between the different roles of the hospital employees .The increase in the average value for hand palm, hand back, and mean overall hand disinfection coverage for the three times showed statistical significance (p < 0.001).In addition, the mean coverage of the hand back was at all times lower than the hand palm, and the average increase in hand back coverage was higher than for the hand palm\u2014both with high statistical significance  as determined by the baseline measurement. Suzuki et al., in a Japanese hospital with 440 beds and 445 enrolled employees, reported a mean palm coverage of 97.8% for all hand sizes. They also reported 97.5% for the left and right hands using the WHO six-step technique, and 97.9% for both hands using the A6Sw/oI (adapted six-step without interlock) technique. The overall mean dorsal coverage varied from 93.7% to 83.0%. Coverage of more than 90% was obtained for the dorsum of both hands and both techniques performed by small hands, whereas the coverage for large hands was below 86% . From thp < 0.001), and the average increase for the hand back was higher than the hand palm, with high statistical significance (p < 0.001). In addition, there was a statistically significant increase in the average value for the hand palm, hand back, and mean overall hand coverage in the three time intervals (p < 0.05).The subsequent measurements of our study showed a gradual and progressive overall increase, reaching the desired target in specific worker categories . AlthougAlthough there was no distinction between left-handed and right-handed individuals, and the difference was not statistically significant, the first measurement revealed a greater coverage of the left palm and right back. This suggested that right-handed individuals were more likely to pick up the gel with the left palm and rub it on the right back. The overall low initial values could be explained by the ineffectiveness of the technique compared to the WHO\u2019s six-step protocol. Graphical and written feedback and, consequently, the improved technique, would have contributed to the increase in hand coverage as a whole.There was no significant difference between the mean hand coverage at t0, t1, and t2 for hospital employees based on their roles, showing a similar increase over time. This suggested a non-significant difference in hand disinfection technique between healthcare and non-healthcare employees, supporting the efficacy of this hand coverage detection method with direct feedback.p < 0.01, nurses p = 0.03, assistant nurses p = 0.03). In the same study evaluating compliance, the lowest rate was among physicians (53.97%) [Concerning the effects of training and employees\u2019 role in the technique, the increase over the threshold target for physicians\u2014who are typically described as frequently resistant to disinfection practices in terms of compliance and coverage \u2014was of p(53.97%) . Conside(53.97%) . Accordi(53.97%) , there w(53.97%) .Considering the overall data collected and analyzed by HICC, the 2021 hydro-alcoholic gel consumption was 317 L/1000 day, with a \u221210% deviation compared to the WHO minimum standard, whereas in 2022 it was 588.5 L/1000 day, with a +62% deviation compared to the WHO minimum standard. In 2022, HAIs were estimated to occur in 3.6% (3.9% only adult) of all hospitalizations, a decrease from the 5.9% (7.4% of adults) in 2021.The high percentage of coverage, with a significant overall increase and the attainment of the target by the health professionals, along with the increase in gel consumption, could demonstrate staff commitment and support the reported decrease of HAI rates .The incremental trend observed across all disciplines, including administrative ones, suggests that ongoing, integrated training plays a central role in enhancing compliance with proper hand disinfection technique; however, specific basic training is only a partial factor in technique.The application of the scanner test training tool was simple and effective. In addition to being more stimulated by comparison with coemployees, HCWs typically recalled previous measurements and missed areas and corrected them.The study presented some limitations: The sample partially varied over the three measurements, as the scanner was rented for a few days per year. Thus, the staff shifts did not guarantee the participation of all the cohort members. Nonetheless, the sample was representative of the approximately 300 employees of the Mondino Foundation, owing to inter-professional diffusion and ongoing training. Moreover, considering the differences found in left-to-right and palm-to-back coverage, this could be of further interest to distinguish right-handed and left-handed hospital workers.Based on the collected data, the automated and integrated assessment method for hand-disinfection technique proved to be effective in our environment. The hand-disinfection technique scanner was conducted within a multimodal IPC project, along with education/training and adequate consumption of hydro-alcoholic gel, resulting in a decrease in HAI\u2014providing the earliest evidence of efficacy. These findings support the notion that a multi-method approach to the prevention of HAIs is preferable. Long-term efficacy must be monitored, enhancing the roles of all stakeholders\u2014including visitors, patients, and citizens\u2014for the safe management of the healthcare ecosystem."}
+{"text": "Hand hygiene is the most important technique for controlling nosocomial infections. While quantitative evaluations based on the amount of hand sanitizer used and qualitative evaluations such as compliance rates based on direct observation are commonly used to evaluate the effectiveness of this education, more objective evaluation indicators are needed. Here, we evaluated the effectiveness of hand hygiene education by scoring hand hygiene techniques using an artificial intelligence (AI)-based analysis tool.Hand disinfection (proper spreading of fluorescent paint) and hand washing (proper rinsing of fluorescent paint) techniques were scored before and after a hand hygiene education video training using the AI-based analysis tool SCORE!\u24c7.Images were taken with an iPhone before and after hand disinfection and washing. The image data were divided into 72 parts, including the wrists and the backs of the right and left palms. Hand hygiene techniques for each part were evaluated using AI. The effectiveness of the training was also evaluated by job categories.The pre- and post-education scores of 935 participants  increased for hand disinfection (P < 0.01) and decreased for hand washing (P < 0.01). The scores for hand disinfection increased for all occupations, while those for hand washing decreased for all occupations except nursing assistants, indicating that hand disinfection was highly effective in education but hand washing was less effective. In a comparison by job categories, there was a large difference in scores between the job categories in terms of the highest and lowest scores for both hand disinfection and hand washing. In the site-specific evaluation, the back of the hand scored higher in hand disinfection, with no significant difference between left and right hands. On the other hand, the palm and left hand scored higher in hand washing, differing from the hand disinfection results.Evaluating hand disinfection scores using \u201cSCORE!\u201d allows for the assessment of even minor changes that may be difficult to detect visually and provides information on individual techniques that may require improvement.Hiroshi Kakeya, MD, PhD, Asahi Kasei Pharma Corporation: Honoraria|Merck Sharp & Dohme: Honoraria|Sumitomo Pharma Co., Ltd.: Honoraria"}