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+{"text": "In this study we built a complete tetrameric mouse [AChET]4\u2013ColQ atomic structure model, based on the crystal structure of the [WAT]4PRAD complex. The structure was optimized using energy minimization. Block normal mode analysis was done to investigate the low-frequency motions of the complex and to correlate the structure model with the two known crystal structures of AChE tetramer. Significant low-frequency motions among the catalytic domains of the four AChE subunits were observed, while the [WAT]4PRAD part held the complex together. Normal mode involvement analysis revealed that the two lowest frequency modes were primarily involved in the conformational changes leading to the two crystal structures. The first 30 normal modes can account for more than 75% of the conformational changes in both cases. The evidence further supports the idea of a flexible tetramer model for AChE. This model can be used to study the implications of the association of AChE with ColQ.Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine in the neuromuscular junctions and other cholinergic synapses to terminate the neuronal signal. In physiological conditions, AChE exists as tetramers associated with the proline-rich attachment domain (PRAD) of either collagen-like Q subunit (ColQ) or proline-rich membrane-anchoring protein. Crystallographic studies have revealed that different tetramer forms may be present, and it is not clear whether one or both are relevant under physiological conditions. Recently, the crystal structure of the tryptophan amphiphilic tetramerization (WAT) domain of AChE associated with PRAD ([WAT] Acetylcholinesterase (AChE) breaks down acetylcholine in the neuromuscular junction and other cholinergic synapses to terminate neuronal signals. AChE exists as tetramers anchored by structural subunits to the cell membranes in the brain or the basal lamina in the neuromuscular junction. Based on a crystal structure of the tetramerization domain of AChE with a proline-rich attachment domain of the anchoring proteins, a symmetric model of the complex of AChE tetramer with the anchoring protein tail was constructed. Block normal mode analysis revealed the presence of several low-frequency, low-barrier normal modes corresponding to inter-subunit motions. Previous crystal structures of AChE tetramer could be rationalized using these normal modes. These low-frequency modes are due to the presence of a flexible hinge in the structure of AChE. This study paints a picture of a flexible AChE tetramer with different conformational states interconverting easily under physiological conditions, which has important implications on the function of AChE. In particular, AChE is not trapped in the compact tetramer structure, for which access of substrate to two of the active sites is somewhat limited. Rather, the tetramer fluctuates to expose all four of its active sites to ensure rapid removal of acetylcholine. T) with a 40-residue C-terminal \u201ct-peptide\u201d is the only form expressed in the brain and adult muscles of normal adult mammals 4\u2013ColQ complex model and calculated the 100 lowest normal modes. By projecting the conformational changes onto these normal modes, it was found that the two AChE tetramer crystal structures could be rationalized by using these low-frequency normal modes.Here we applied BNMA to the [AChET]4\u2013ColQ complex model has a quasi-4-fold axis as shown in T]4\u2013ColQ complex. Reaction-rate calculations are currently underway using this model.Our [AChET]4\u2013ColQ structure model. The first 100 lowest frequency normal modes were obtained. These normal modes are sufficient to capture all the collective motions, and motions with higher frequency are usually localized to a small domain 4\u2013ColQ complex model, this subdomain makes contact with the C-terminal extension of ColQ, as is evident from the correlation map. In addition, this subdomain participates in the inter-subunit interface with the clockwise neighboring subunit.The correlation map can identify collective motions, which are often important large-scale motions related to the protein's biological function. Due to the large amount of data, only one AChET]4\u2013ColQ complex, they represent two conformational states with large conformational changes to the complex structure. By projecting the conformational changes onto the eigenvector of each normal mode, it is possible to identify the degree to which each mode is involved in the conformational change 4\u2013ColQ complex. The cumulative involvement plot in \u22121 and 1.89 cm\u22121, respectively. In the case of the loose tetramer structure, the most involved modes are the second and fourth lowest frequency modes with involvement coefficients of 0.35 and 0.44, respectively. The vibrational frequencies of these two modes are 1.66 cmAll the seven inter-subunit motions involve a flexible hinge in residues 540 to 545 (D544 is the first residue in the WAT sequence), with T543 at the center of the flexible hinge. These residues have no correlation in motion with either AChE or the WAT/PRAD complex as seen in T]4\u2013ColQ complex that minimize the RMSD to the two crystal structures. The RMSD between the models of the [AChET]4\u2013ColQ complex and the compact tetramer structure decreased from 23.9 \u00c5 to 15.7 \u00c5, while for the loose tetramer structure it went from 28.9 \u00c5 to 17.6 \u00c5. The large RMSD values are due to the large translational and rotational movement required for each AChE subunit to realign between the [AChET]4\u2013ColQ complex model and the crystal structures. As a comparison, the RMSD for the two crystal structures is 34.9 \u00c5, i.e., even larger than the initial RMSD between the [AChET]4\u2013ColQ complex model and the two crystal structures. When the two final models of the [AChET]4\u2013ColQ complex from projection were examined, it was found that they were significantly distorted in respect to the monomeric AChE. However, both models show a similar dimer of dimers as seen in the two crystal structures. The orientations of the active site gorges were originally all parallel to the tetramer plane. After applying projected motions from BNMA, two gorges were pointing downward and the other two slightly upward. It should be noted that this is a rather approximate demonstration using a simplified linear combination of normal mode motions. The application of normal modes for proteins is strictly only valid for very small conformational motions.Using a simplified linear combination of these 100 normal modes, we generated two series of transient models of the [AChET]4\u2013ColQ complex may possess significant flexibility due to the presence of a flexible hinge at the interface of AChE and WAT. Under physiological conditions, there is a distribution of many different conformational states, including the two crystal structures and the symmetric tetramer model we constructed based on the [WAT]4PRAD complex structure. Due to the low barrier uncovered by our analyses, these conformational states can easily interconvert. Experimental findings support such a conformational transition. For example, under conditions that the compact AChE tetramer is grown, all four active site gorges can be occupied by fasciculin 4\u2013ColQ complex model, we added the missing residues 35\u201340 in WAT and extended PRAD to 16 more residues in the N-terminus and 19 more residues in the C-terminus using SYBL . The conformations of these residues were copied from the existing chains, i.e., the area with residues 35\u201340 was the same coiled \u03b1 helix as the rest of the WAT, and all residues in ColQ were in polyproline II conformation. Mouse sequence of ColQ tail was downloaded from the Swiss-Prot database. While the sequence in the [WAT]4PRAD complex crystal structure was from human, the only differences were D35E in the WAT and T3M in the PRAD from human to mouse. SeMet21 was mutated back to Met.The crystal structure of the [WAT]ata Bank . Chains T. The mouse AChE structure was taken from a preoptimized structure based on the PDB entry (see Accession Numbers section) [T]4\u2013ColQ complex model. VMD version 1.82 [T]4\u2013ColQ complex model was further optimized by 5,000 steps of conjugate-gradient energy minimization, using the sander module of Amber8 (first 100 steps were steepest descent search). The solvation effect was mimicked with a distance-dependent dielectric coefficient of 4r. A 20-\u00c5 cutoff was used in evaluating nonbond interactions. The final RMS force in energy minimization was 0.01 kcal/mol/\u00c5.The next step was to connect WAT to AChE to form AChEsection) . Since bsection) , includisection) , which dion 1.82  was usedion 1.82 , using tion 1.82 . The iniion 1.82  worked bT]4\u2013ColQ complex model. The advantage of BNMA is that the real atomic potential can be used and projected to a smaller dimension using blocks of atoms 4PRAD complex (1VZJ), mouse AChE (1MAH), and acetylcholine receptor structure (1OED).The accession numbers for the proteins described in this paper can be found in the SwissProt database ("}
+{"text": "The application of high-throughput genomic tools in nutrition research is a widespread practice. However, it is becoming increasingly clear that the outcome of individual expression studies is insufficient for the comprehensive understanding of such a complex field. Currently, the availability of the large amounts of expression data in public repositories has opened up new challenges on microarray data analyses. We have focused on PPAR\u03b1, a ligand-activated transcription factor functioning as fatty acid sensor controlling the gene expression regulation of a large set of genes in various metabolic organs such as liver, small intestine or heart. The function of PPAR\u03b1 is strictly connected to the function of its target genes and, although many of these have already been identified, major elements of its physiological function remain to be uncovered. To further investigate the function of PPAR\u03b1, we have applied a cross-species meta-analysis approach to integrate sixteen microarray datasets studying high fat diet and PPAR\u03b1 signal perturbations in different organisms.We identified 164 genes (MDEGs) that were differentially expressed in a constant way in response to a high fat diet or to perturbations in PPARs signalling. In particular, we found five genes in yeast which were highly conserved and homologous of PPAR\u03b1 targets in mammals, potential candidates to be used as models for the equivalent mammalian genes. Moreover, a screening of the MDEGs for all known transcription factor binding sites and the comparison with a human genome-wide screening of Peroxisome Proliferating Response Elements (PPRE), enabled us to identify, 20 new potential candidate genes that show, both binding site, both change in expression in the condition studied. Lastly, we found a non random localization of the differentially expressed genes in the genome.in silico analysis filtered by evolutionary conservation. The analysis enabled us to indicate potential gene candidates that could fill in the gaps with regards to the signalling of PPAR\u03b1 and, moreover, the non-random localization of the differentially expressed genes in the genome, suggest that epigenetic mechanisms are of importance in the regulation of the transcription operated by PPAR\u03b1.The results presented are potentially of great interest to resume the currently available expression data, exploiting the power of  The availability of public gene expression repositories, such as GEO  and Arracis-element of promoter of a target gene.Lipids serve as membrane constituents and supply and storage of energy. The related fatty acids are precursors of a wide range of bioactive molecules. They have the ability to regulate a wide variety of cellular processes through the induction of changes in gene expression. Indeed, fatty acids and some oxygenated derivates are ligands able to activate a class of transcription factors, called peroxisome-proliferator-activated receptors (PPARs), key regulators in energy storage and metabolism. PPARs  belong to a superfamily of nuclear hormone receptors that share a common action mechanism: the formation of heterodimers with the nuclear receptor RXR and the consequent binding to Homo sapiens, Mus musculus, Rattus norvegicus and Saccharomyces cerevisiae) is based on the known evolutionary conserved regulatory mechanism responsive to fatty acid-rich diet. In S. cerevisiae the homologous of mammalian PPAR\u03b1-RXR is Pip2p-Oaf1p [PPAR\u03b1 is expressed primarily in metabolic tissues  but elevated levels are also present in the digestive  and cardiopulmonary  systems, and plays a central role in almost all aspects of fatty acid catabolism in particular in the liver. Recent studies demonstrate that the role of PPAR\u03b1 is not limited only to metabolism but it also acts in many processes like inflammation , immunit2p-Oaf1p . These t2p-Oaf1p . Taking In this study we provide a list of homolog genes subjected to comparable stimuli, showing significant changes in expression levels after changes in PPAR\u03b1 activity. Thus, taking advantage of all datasets available on PPAR signaling and high fat diet, our study offers a comprehensive overview of the key pathways and cellular processes regulated by PPAR\u03b1. We identified a series of chromosomal regions in the mouse genome specifically enriched by PPAR\u03b1 related genes suggesting common regulatory mechanisms. Finally we provide a useful method and interesting information to identify new target genes integrating the results of our work on gene expression with those previously obtained by Lemay et Hwang on PPRE sequence .Array Express  and GEO Saccharomyces cerevisiae dataset performed in our laboratory. On the whole, we included in the analysis 202 hybridizations  experiments on rat, mouse and human hepatocytes, where PPAR\u03b1 signaling is activated treating cells with WY14643, (ii) experiments in which PPAR\u03b1 signaling is completely inhibited using mouse PPAR\u03b1 knockout, (iii) experiments in which the organism under study were fed with high fat diet, comprehensive of the S.cerevisiae with expression data of several knockout yeast for transcription factor involved in oleate response, and two mouse datasets with experiments belonging at three category of experimental design mentioned above.Additional datasets were selected to test the results of our work. /i, where p is the p-value of the gene, n the total number of genes and i is the number of genes at or better than p. Statistical analyses was performed with R software http://www.r-project.org.Gene expression of Affymetrix datasets were quantified and separately normalized using echnique  and. Entechnique  was usedweb tool . In ordeweb tool , a moderUnfortunately, not all the datasets contained sufficient numbers of biological replicates as required for powerful inference. Fold change cut-off, filtered by variance coefficient, was used to select DEGs in those datasets with less than 3 replicates per gene .p-value cutoff of 0.1. Similarity structure on metabolic enrichment characteristics across datasets was performed using cluster analysis. A Boolean matrix with pathways in rows and datasets in columns was generated, where matrix cells equal to 1 identified significant enrichment of a given pathway in a given dataset, and 0 otherwise [Enrichment analysis on metabolic pathways was calculated for each dataset using Fisher exact test based on hypergeometric distribution with a therwise ,22. Usintherwise  a hierarMus musculus HomologeneID.Homologene database  was usedp-value (see previous paragraph for details), we decided to adopt as meta-analysis procedure the vote counting approach proposed by Rhodes and colleagues [j of the S total number of datasets with j = 2...S. The idea was to compare the observed number of significant genes shared by at least j studies (observed gene enrichment) with the number of significant genes shared by at least j studies obtained by chance (random gene enrichment). The number j was defined through a permutational approach. Permutational steps were the following: i) Q-values of each dataset were randomly permutated so that genes in each signature  changed randomly, but the number of genes in each signature remained the same, ii) the number of genes differentially expressed common to at least j datasets was calculated for j ranging from 2 to the total number of datasets, iii) step i) and ii) were repeated 1000 times, iv) average and empirical confidence intervals (at confidence level 95%) of the number of random gene enrichment for each j (across the 1000 simulations) were calculated. Then, we compared the observed number of genes shared by at least j studies with the confidence interval obtained through the permutational approach and choose those js showing a significant difference between observed and random number of gene enrichment. Finally among these js we selected the minimum j such that the ratio between the expected and observed number of genes shared was less than 10%. In our analysis the number of j leading to 4% of false positives was found to be equal to 6 [Given the presence of some datasets without gene lleagues . The votj identified through the permutational approach described above. The virtual reference set for the hypergeometric distribution was obtained by selecting all the genes common in at least j platforms. In our analysis the reference set contained 15,463 genes. This new general approach, specifically adapted for meta-analysis enrichment analysis, showed several advantages, accurate results, faster and easier execution.An enrichment analysis, similar to that described in the previous paragraph, was applied on MDEGs. In this case, using hypergeometric distribution, the enrichment test set was represented by the MDEG list, while the reference set should be virtually generated according to the number A gene ontology network was drawn and analysed by means of the BINGO plug-in  of CytosS. cerevisiae and one for mouse data. The differentially expressed genes of each dataset are filtered by the species specific MDEGs and matrix with genes in row and dataset in column were build. Using TM4 [The validation set was divided by organism, we performed one validation with sing TM4  a hierarp-value < 0.05 were used as significant cut-off thresholds.Over-represented putative transcription factor binding sites have been detected for the lists of differentially expressed genes with oPOSSUM web tool . The defFollowing the approach proposed by Vogel et al.  we searcOur work aims at developing methodological and computational procedures for the study of metabolic pathways and conserved regulatory mechanisms underlying the fundamental biological response to high fat diet, with additional goal to suggest novel candidate genes belonging to PPAR\u03b1 signaling.We used a cross-species meta-analysis approach for the integration, at the gene level, of sixteen transcriptional datasets from different organisms  and experimental platforms . The datasets included into the analysis were focused on either genetic or dietary perturbations.First, we evaluated the presence of possible trend in clustering due to organism, platform, tissue and experimental design variability characterising the sixteen datasets selected for the analysis. After inferential and enrichment analysis on each single dataset, functional similarities among studies have been performed through cluster analysis (see Methods for details). We expected that if some biases would be present in our analysis, datasets should be grouped according to the sources of variability. Figure Analyzing the data intra and inter datasets, the meta-analysis approach allows us to find the most frequently deregulated genes in the tested condition.Comparing the MDEG list with the DEG lists of datasets analyzed individually, we observe that the meta-analysis led us to identify a smaller number of total genes but biologically more strongly correlated to the studied condition, probably reducing the false positive genes and recovering true positive genes eliminated by a strict statistic at a single study level. A cross-species meta-analysis provides an added value to find conserved genes and for this reason is more reliable.Meta-analysis approach identified 164 differentially expressed genes shared by at least 6 datasets (MDEGs)  and argininesuccinate lyase (Asl) were included in MDEGs ,43.Finally, Kersten et al. demonstrated that PPAR\u03b1 regulates carbohydrate metabolisms in particular by acting on gluconeogenesis  and goveAll this evidence demonstrates that PPAR\u03b1, despite its primary role in regulating the metabolism of fatty acids, acts as a master regulator of the rate of utilization of the various energy substrates in relation to food availability, and the identified network of biological process provides full details of all these aspects.In addition to biological processes network, the cellular component network Figure  highlighin silico predicted PPRE.The results of pathway enrichment analysis are shown in Additional File Cross-species analysis allows deciphering molecular complexity through evolutionary constrains. As expected mammalian organisms share the largest amount of genes Figure , howeverS. cerevisiae adapts to oleic acid as a sole carbon source inducing transcriptional modulation of both peroxisomal and mitochondrial function [Looking at functions and processes linked to the yeast MDEGs, we observed that 7 out 41 genes,  were directly involved in fatty acid metabolism and transport. CRC1, POT1, YAT1, FOX2 and SPS19 are targets of the transcription induced by Oaf1-Pip2 in yeast . The 41 function . In addifunction , AAT2 hofunction ,43, ARG4function , FOX2 hofunction ,49 and Yfunction ,38. InteM. musculus. The resultant tree of experiments, obtained clustering by similarity of expression the selected 164 mouse MDEGs, perfectly split the 4 experiments in 2 groups. In the first group we find experiment studying activation of PPAR\u03b1 signaling, in the second group experiment in which there is not PPAR\u03b1.  was not significant, although giving an indication of non randomness. However we are aware that the only presence of a PPRE does not necessarily result in a change in gene expression and vice versa many of the changes in gene expression we discover might be due to a not-direct interaction with PPARa or a combined action of more TFs.Mining the literature we can find that Lemay and Hwang  have alrWith the aim of deeply investigate the presence of other TF binding sites, we accomplished a genome-wide screening of the region flanking the transcription starting site of all mouse MDEGs, searching binding site (TFBS) for all TFs contained in the Jaspar database . The scrWe believe that, despite the previous not significant p-value, the overlapping genes might represent an important set of response activators. This belief is strengthened by the fact that the overlap between the results of the two methods is composed by 5 genes  all already known as target gene of PPARa , by ligand and by the recruitment of HAT complex (Histone Acetylase) [In our opinion, this finding is a further confirmation that the mechanism of transcription regulation operated by PPAR\u03b1 involve epigenetic processes. Indeed in literature we can find molecular and etylase) . Unfortuetylase) . In yeasetylase) . The facin silico analysis suggest that transcription factor induction and chromatin state seem to be the principal factors mediating the response to excess dietary fat. This probably allows PPAR\u03b1 to bind a PPRE and to regulate more than one gene at the same time.At the light of these findings, The proposed computational methods contribute towards the advances in integrative analyses of genomic data that still represent a major, and partially unresolved, computational issue. Through the selected strategy we were able to scan the expression data currently available and to suggest directions and new candidates to be investigated. This demonstrates the utility of the undertaken approach to exploit cross-species analysis and define gene signatures of the evolutionary conserved mechanisms as key elements to decipher the complexity of genome-wide data.DC and EC designed the experiment, performed the analyses, developed the method and wrote the paper, CR developed the method supported the analyses and wrote the paper, EM performed the yeast microarray hybridizations, MR discussed the results designed the experiment and corrected the paper, BVO corrected the paper and discussed the results, MM corrected the paper and discussed the results. All authors have read and approved the final manuscript.The MDEGs list.Click here for fileThe file contain the complete output of BINGO, the KEGG enrichment table, the list of MDEGs know as target of PPAR\u03b1, complete output of oPOSSUM, the table of co-localized genes.Click here for file"}
+{"text": "Objective: To find out the frequency of vitamin D deficiency and its relation with glucose parameters and the incidence of gestational diabetes (GDM).Methodology: Gestational diabetes was diagnosed with 75 gram oral glucose tolerance test. Forty-four pregnant women diagnosed with GDM and 78 non-GDM pregnant women were enrolled as case and control group, respectively in this descriptive study. Vitamin D status was classified as deficiency at \u226420 ng/ml for serum 25(OH)D concentrations.Results: The mean ages were 33.4\u00b15.2 (18-44) years and 29.7\u00b14.1 (21-39) years, mean BMI was 30.6\u00b15.9 kg/m\u00b2 (19.5-46.1) and 25.9\u00b14.4 kg/m2 (16.5-38) in case and control groups, respectively. The frequency of GDM was found 9.38%. The mean serum vitamin D levels in GDM group were significantly lower than in non-GDM subjects (p=0.07). A total of 56.8% of GDM patients were compared with 35.8% of control group which had Vitamin D deficiency and the difference was significant (p= 0.02). There was no significant association between vitamin D levels and fasting glucose, insulin and HbA1c. Vitamin D levels were inversely correlated with clothing style, parathyroid hormone levels, dental problems and muscle cramps.Conclusions: The association of maternal Vitamin D status with the markers of glucose metabolism in pregnancy needs prospective studies. We aimed to investigate the frequency of Vitamin D deficiency in pregnant women with gestational diabetes and the relation of Vitamin D levels with the glucose parameters in the presence of gestational diabetes. A total of 44 pregnant women with gestational diabetes and 78 healthy non-GDM pregnant women who presented at Gynecology and Obstetric Department of Baskent University Hospital Istanbul between 2009 and 2011 were included in this cross-sectional, descriptive and controlled trial. The diagnosis of gestational diabetes was determined by the guidelines proposed by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) with a 75-gr 2 hour oral glucose tolerance test at 24-32 weeks\u2019 gestation. GDM is diagnosed when the fasting serum glucose is 92\u00a0mg/dL or greater, or the 1-hour value is 180\u00a0mg/dL or greater or when the 2-hour value is 153\u00a0mg/dL or greater. The subjects were divided into two groups according to the presence of gestational diabetes as case (group 1) and control (group 2). Serum fasting insulin, 25 (OH) D, creatinine, calcium (Ca), phosphorus (P), magnesium (Mg), alkaline phosphatase (ALP), parathyroid hormone (PTH), thyroid-stimulating hormone (sTSH), albumin, HbA1c, liver transaminases (AST and ALT) were also measured and the body mass indexes (BMI) were calculated in all groups. Serum 25 (OH) D levels \u226420 ng/mL are accepted as the cutoff level for vitamin D deficiency since this level was the highest level to maintain the serum PTH levels below 45 pg/ml. Pregnant women with history of pregestational diabetes, multiple pregnancies, fetal abnormality, chronic renal or liver failure and history of consumption of calcium supplements or vitamin D were excluded from the study. All subjects gave written informed consent and the study was approved by the local ethics committee (KAO9/355). All patients were asked about their consumption of prenatal vitamin/food supplements, dairy products/fish and any drugs that may alter glucose metabolism and antihypertensive medication, family history of diabetes and obesity, the period of daily physical activity and sun exposure. These collected maternal fasting plasma samples were kept frozen at \u221220\u00b0C. Serum glucose was measured by using an enzymatic in vitro test. 25-hydroxy vitamin D3 was analyzed by a human ELISA kit , with interassay coefficient of variation (CV) of 2.6% intraassay CV of 2.3%. Serum Ca, P, Mg, (ALP) and glucose levels were measured using the enzymatic colorimetric method\u00a0 (Roche Integra 800), while\u00a0 serum 25 hydroxy vitamin D 25(OH) D levels were measured during the fall and the winter with a chemiluminescent immunassay method (CMIA)  Normal ranges were between (NA): 15.7-60.3ng/ml (summer), 8.8-46.3ng/ml (winter). The intra-assay variation ranged from 2.6-4%. Insulin levels were analysed with CMIA , NA: 2.6-24.9 \u03bcU/ml. Intra-assay coefficients of variation were 2.3-4.2%. Serum PTH levels were measured with electrochemiluminescent immunassay method (ECLIA) , NA:15-68pg/ml. Intra-assay coefficients of variation was 3-6.5%. Serum calcium (Ca) and phosphorus (P) levels were measured with enzymatic colorimetric assay . Intra and inter-assay coefficients of variation were 0.5-0.6%, and 0.5-0.3% for Ca; 0.5-0.5% and 0.3-0.6% for P. Serum glucose measured with enzymatic colorimetric assay  intra and interassay coefficients of variation were 1.98-0.65% and 0.84-0.93% respectively. HbA1c was detected with turbidimetric assay method  Intra and interassay coefficients of variation were 0.88-0.77% and 1.88-1.45%, respectively. Statistical analysis was performed using SPSS 16.0 for Windows. Student T test, Fisher\u2019s exact test and Spearman corelation test were used for the comparisons of categorical variables and correlations between two variables. Statistical significance was set at p value of less than 0.05. A total of 413 pregnant women were screened during the study period and 44 pregnant women were diagnosed with GDM. Seventy-eight non GDM pregnant women were enrolled as control group. All subjects were matched in their multivitamin use, magnesium levels, family history of diabetes and obesity, sun exposure and daily physical activities. Sociodemographic characteristics and metabolic parameters of the study groups are shown in  There was a negative correlation between serum 25 (OH) D and parathyroid hormon levels  . There w Family history of diabetes and obesity was 73.3% (n=34) and 45.5% (n=20), respectively in pregnants with GDM. These rates were 77.8% (n=35) and 46.7% (n=21) respectively in control. Only 12% of group 1 and 11.5% of group 2 were engaged in regular physical activity of 30 min/day duration, the others were not doing any type of exercise. Dairy products consumed per day were approximately 2.4 portions in all groups.  The mean Vitamin D levels of veiled pregnant women were significantly lower compared to nonveiled  in pregnant women . The main symptoms in Vitamin D deficient pregnant women were dental problems, muscle cramps, weakness, soreness and sweating. Dental problems and muscle cramps were seen more frequent in Vitamin D deficient pregnant women compared to non-vitamin D deficient pregnant women  and 4.1 The frequency of GDM was found among 9.38% women in our study. This rate was consistent with the literature regarding the increasing gestational diabetes prevalance in the world.11,12 However we did not find significant association of low levels of serum 25 (OH) D with elevated risk for GDM even after adjustment for conventional risk factors for diabetes. Data relating vitamin D status to GDM risk are inconsistent and limited. In one cross-sectional study, serum 25(OH) D levels during 24-28 weeks of gestation were lower in GDM women than in nondiabetic pregnant women.9 In another study, maternal serum 25 (OH) D concentrations measured at the time of GDM screening test were significantly and inversely associated with fasting glucose, although the association of Vitamin D with GDM risk was not statistically significant.8 Our findings are also consistent with a study in an Indian population, showing no significant association between 25 (OH) D concentrations (at 30 weeks of gestation) and GDM risk.6 Our study provides data indicating that maternal Vitamin D deficiency (serum 25-[OH] D <20 ng/mL) is prevalent among our pregnant population. This evidence is consistent with low vitamin D levels in either pregnant or non-pregnant individuals in our country as in the rest of the world.13 Significantly reduced risk of type 1 diabetes mellitus development was found when high doses of vitamin D supplementation (up to 2000 IU/d) were given during infancy in a birth-cohort study.14 Deficiency of vitamin D was shown to be associated with fetal and brain growth deficiency and type 1 diabetes in children.5,15-17 Higher risks of preeclampsia and cesarean section ratios were shown in pregnants with low levels of Vitamin D.16,17 Moreover supplementation of vitamin D has been demonstrated to decrease the risk of preeclampsia by 27% in a study.18 Vitamin D deficiency was also associated with increased risk for pregnancy complications and serious health problems in the offspring.19 In the present study, we did not find any significant difference in engaging physical activity between two groups. Outdoor physical activity, since correlated with sun exposure, could be a protective factor for insulin resistance, obesity and GDM.20-22 Vitamin D deficiency can result from a variety of causes including malabsorption problems, obesity, age, skin color, altitude, lack of Vitamin D in breast milk and in diet, the side effects of certain medications and inadequate exposure to sunlight.  There are studies showing that prenatal vitamins including 400 IU of Vitamin D are insufficient to supplement the low levels of 25(OH)D in pregnant women.23-25 Although intervention trials assessing the role of Vitamin D supplementation on glucose metabolism among pregnant an/or non-pregnant individuals have inconsistent results and, the dose of supplementation and the duration of follow up period were not standardised in these studies, there is a growing evidence for potential benefits of high-dose Vitamin D supplementation during pregnancy. Our study has several limitations. First, serum 25 (OH) D concentrations determined in late trimester may not show the status of maternal vitamin D during the whole pregnancy period and hence to identify the association of GDM development and vitamin D status. Secondly, the BMI were not matched in our study. Longitudinal studies with serial measurements of maternal plasma 25(OH) D concentrations, and monitoring the development of gestational diabetes in BMI matched pregnant women in relation with these measurements are required to explore the pathophysiology. Our study has contributed to data regarding the increasing prevalance of gestational diabetes and the growing evidence of Vitamin D deficiency in pregnant population. Although there is no consensus for routine Vitamin D screening in pregnancy, being alert about the Vitamin\u00a0D deficiency and its symptoms and optimising when necessary by replacement and/or through taking lifestyle measures may be recommended to prevent the birth of Vitamin D-deficient infants from vitamin D-deficient mothers. The lifestyle measures can include increasing the sun exposure and outdoor physical activity which also contribute to weight control and thus improving glucose homeostasis and/or favourable outcomes in pregnancy. Besides the safety of Vitamin D supplementation has already been known. Further studies are needed to determine the optimal levels and doses of Vitamin D in pregnancy to reduce the health risks for both mother and fetus. Investigating and diagnosing the factors that may negatively affect the development of gestational diabetes as early as possible is very important."}
+{"text": "Diagnosis of Parkinson\u2019 disease (PD) carries a high misdiagnosis rate due to failure to recognize atypical parkinsonian disorders (APD). Usually by the time of diagnosis greater than 60% of the neurons in the substantia nigra are dead. Therefore, early detection would be beneficial so that therapeutic intervention may be initiated early in the disease process. We used splice variant-specific microarrays to identify mRNAs whose expression is altered in peripheral blood of early-stage PD patients compared to healthy and neurodegenerative disease controls. Quantitative polymerase chain reaction assays were used to validate splice variant transcripts in independent sample sets. Here we report a PD signature used to classify blinded samples with 90% sensitivity and 94% specificity and an APD signature that resulted in a diagnosis with 95% sensitivity and 94% specificity. This study provides the first discriminant functions with coherent diagnostic signatures for PD and APD. Analysis of the PD biomarkers identified a regulatory network with nodes centered on the transcription factors HNF4A and TNF, which have been implicated in insulin regulation. Parkinson\u2019s disease (PD) is the second most common neurodegenerative disease. Approximately 95% of cases of PD are idiopathic most likely caused by environmental factors and genetic susceptibility. Unfortunately by the time of diagnosis most of the dopaminergic neurons in the substantia nigra are dead. Diagnosis of PD is based on classical motor symptoms including resting tremor, rigidity, bradykinesia and postural instability. Despite clinical criteria for PD there remains a high rate of misdiagnosis with atypical parkinsonian disorders (APD), such as progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) Messenger RNA (mRNA) transcripts are excellent candidates for diagnostic biomarkers since very small quantities may be amplified and quantified by quantitative polymerase chain reaction (qPCR). Numerous studies have examined changes in global gene expression in postmortem brains of PD patients and these have been analyzed in a meta-genome-wide expression study (GWES) It is clear from the above studies that the loss of nigrostriatal dopamine neurons in PD correlates with changes in splicing within the brain. Brain tissue, however, is not a useful source for PD biomarkers and RNA is often degraded in post-mortem tissue. In contrast, blood biomarkers are useful because they are non-invasive. In this regard, it is clear that the immune system responds to changes in dopamine In an earlier study, total mRNA abundance in whole blood was assayed using standard microarrays in order to identify transcripts associated with risk of PD Here, we identify and validate biosignatures for PD and APD composed of splice variant biomarkers in blood whose expression is altered in patients compared to controls. Network analysis of the PD biomarkers reveals a network centered on the transcription factors HNF4A and TNF, which have been implicated in insulin regulation.Samples used in this study came from early stage PD patients  and age-matched healthy HC, MSA and PSP controls who were enrolled in the Prognostic Biomarker Study (#NCT00653783) In order to identify a splice variant-specific profile associated with PD we probed cDNA prepared from RNA extracted from whole blood of participants with 257,319 probes on microarrays designed to monitor splicing events . In a thPrincipal components analysis (PCA) on the microarray data and cluster analysis of the PCR data showed that PD patients can be separated from controls using the 13 biomarkers . In ordeSamples from HC were compared to those from PD, MSA and PSP patients. The results showed that macf1, mpp1, pkm2, and slc14a1-l are expressed differentially in healthy individuals compared to diseased participants . In ordeTo assess the prediction accuracy of the PD markers in the classification of the training set, we carried out a LOOCV . The prePD is the discriminant score value  as PD and those above as non-PD  as APD and those below as PD  and 39 healthy HC, 17 MSA and 17 PSP age-matched controls were enrolled in the Prognostic Biomarker Study (#NCT00653783). The parent cohort for the PD patients from the PRECEPT, PostCEPT and LAB-PD studies has previously been described Whole blood (20 ml) was collected in the morning between 8 am and 12 pm during the baseline visit using the PAXgene Blood RNA system . The tube was inverted 8\u201310 times and incubated at room temperature of 24 h. The blood samples were frozen at \u221220\u00b0C until processed for total RNA isolation. Samples from PD patients were processed in parallel with those of controls. RNA was extracted using the PAXgene blood RNA kit according to the manufacturer\u2019s protocol followed by DNase I digestion. RNA quality was determined using the RNA 6000 NanoChip kit and an Agilent 2100 Bioanalyzer . Samples with RNA integrity values >7.0 and absorbance 260/280 between 1.7 and 2.4 were used.Amplified and labeled cDNA was prepared using the NuGEN WT-Ovation\u2122 Pico RNA Amplification System and the FL-Ovation\u2122 cDNA Biotin Module V2 . ExonHit Therapeutics, Inc  prepared cDNA from total RNA and the DNA/RNA heteroduplex was amplified by SPIA\u2122  Data analysis was performed with Partek GS 6.5 software . The microarray results comply with MIAME guidelines. An analysis of variance (ANOVA) was performed to compare each group of study participants. 10,563 probes were identified that produced a signal above background that was at least 2-fold changed . The following three analytical steps were used to identify markers that distinguish PD from controls .Identification of splice variants that are differentially expressed in PD patients compared to each control group. A Venn diagram tool was used to identify splice events that were differentially expressed in the PD patients compared to each control group . The union of the splice events identified by this comparison included 61 splice variants .Identification of splice variants that are differentially expressed in PD patients compared to non-PD controls. Another ANOVA was performed to compare PD to the controls, without distinguishing between the type of control. Seventeen splice variants were identified, of which 12 were new candidates .Identification of splice variants with optimized accuracy of prediction. We applied a two-level nested LOOCV. An \u201couter\u201d 39-fold cross-validation was performed to estimate prediction error of the classifier while a nested, \u201cinner\u201d, 38-fold cross-validation was performed to select the best performing classifier. We assessed K-Nearest Neighbor classification models with the number of neighbors 3, 5, 7 and 9 and number of variables from 1 to 12 using an ANOVA. The best performing classification model contained 12 splice events .http://genome.ucsc.edu/). Splice variant-specific primers were designed using Primer Express software  such that one of the primers spanned the splice junction. The High Capacity RNA transcription kit  was used to reverse transcribe 1\u00b5g of total RNA according to the manufacturer\u2019s protocol. The sequence of the primers and the number of cycles used to amplify the products is presented in Candidate risk markers were prioritized based on the role they may play in PD etiology or progression based on information from pathway and disease analysis using Ingenuity  software. The sequence of the splice variants was retrieved from the UCSC genome browser (To assess the correlation between expression of splice variants with binary diagnostic categories (PD vs. controls) we calculated the Pearson correlation coefficient for all splice events represented on the microarrays. To assess the prediction accuracy of the set of 13 risk markers we performed LOOCV analysis on the microarray data for these splice events. A Student t test and one-way ANOVA and tukey-kramer post-hoc analysis was used to compare groups in the analysis of the qPCR data using GraphPad Prism . Discriminant analysis was performed with Partek GS 6.5  and JMP 9.0  software.Figure S1Regions of the risk markers amplified by PCR. Boxes represent exons. Green boxes represent the variant region of the mRNA. Forward arrows represent forward primers and reverse arrows represent reverse primers. Broken arrows indicate that the primer was designed to span the splice junction.(TIF)Click here for additional data file.Figure S2Relative mRNA quantification graphs of the risk markers. (A) Student\u2019s t test and tukey-kramer post hoc analysis was used to compare PD patients with controls . Fold change values relative to a calibrator are displayed with error bars indicating SEM. (B) Student\u2019s t test and tukey-kramer post-hoc analysis was used to compare PD with APD patients. Fold change values relative to a calibrator are displayed with error bars indicating SEM. Gapdh mRNA was used as a control. *p<0.01, **p<0.005, ***p<0.001 and ****p<0.0001. PD is Parkinson\u2019s disease and C is control.(TIF)Click here for additional data file.Figure S3Frequency distribution of the Pearson correlation coefficient for binary diagnostic categories (PD vs. controls). Locations of Pearson correlation coefficient (r) values for PD risk markers are shown with arrowheads. These values are either below 10% percentile (\u22120.2111) or above 90% percentile (0.2045).(TIF)Click here for additional data file.Figure S4Representative relative mRNA quantification graphs of risk markers from a training set of samples. Splice variants that were expressed differentially in HC compared to disease participants. A student t test was used to compare groups. *p<0.01, **p<0.005 and ****p<0.0001. Fold change values relative to a calibrator are displayed with error bars indicating SEM. Gapdh mRNA was used as a control. HC is healthy control, PD is Parkinson\u2019s disease, MSA is multiple system atrophy, and PSP is progressive supranuclear palsy.(TIF)Click here for additional data file.Figure S5Principle components and heat map analysis of the APD biomarkers. (A) Heat map of the data from the qPCR assay of the biomarkers analyzed using the \u0394\u0394Ct method. Each column in the heat map corresponds to a study participant. Each row represents the relative level of abundance of a single splice variant. Each splice variant is denoted by the name of the mRNA. Color scales representing splice variant expression with red representing high abundance relative to the mean abundance; blue representing low abundance relative to the mean abundance; and gray representing no significant change in abundance level between the sample and control. PD patients are indicated by green and APD patients are indicated by yellow. (B) Principle components analysis of 52 samples (training set).(TIF)Click here for additional data file.Table S1Criteria used for inclusion/exclusion of study participants and for clinical diagnosis.(DOC)Click here for additional data file.Table S2Biomarker information. Identifying information for the mRNA sequences that result from an alternative splicing event that are used to diagnose the presence of Parkinson\u2019s disease in a human patient and the specific forward and reverse primers and cycle number used for amplification. 1Numbers represent exons; > < represents between exons; < > represents skipped exon; 2Event and identifier numbers are located at http://www.ncbi.nlm.nih.gov/andhttp://genome.ucsc.edu/cgi-bin/hgGateway.(DOC)Click here for additional data file.Table S3Raw and standardized canonical discriminant function coefficients for the PD biomarkers. Discriminant analysis was performed with Statistica 8.0 and JMP 9.0 software.(DOC)Click here for additional data file.Table S4Discriminant analysis results using the PD biomarkers. Chi Square Test with successive roots removed. Analysis was performed with Statistica 8.0 software.(DOC)Click here for additional data file.Table S5Linear discriminant analysis performed on gene expression data from 124 participants. Sensitivity and specificity values are displayed for the three classification groups.(DOC)Click here for additional data file.Table S6Standardized and raw canonical coefficients for canonical variables in the discriminant function using the APD biomarkers.(DOC)Click here for additional data file.Table S7Discriminant function analysis summary for the APD biomarkers.(DOC)Click here for additional data file.Table S8Summary of chi-square distribution and canonical correlation of the APD biomarkers.(DOC)Click here for additional data file.Table S9Linear discriminant analysis performed on gene expression data from 85 patients. Sensitivity and specificity values are displayed for each classification group.(DOC)Click here for additional data file."}
+{"text": "Negative results are an important building block in the development of scientific thought, primarily because most likely the vast majority of data is negative, i.e., there is not a favorable outcome. Only very limited data is positive, and that is what tends to get published, albeit alongside a sub-set of negative results to emphasize the positive nature of the positive results. Yet, not all negative results get published. Part of the problem lies with a traditional mind-set and rigid publishing frame-work that tends to view negative results in a negative light, or that only tends to reward scientists primarily for presenting positive findings. This opinion piece indicates that in addition to a deficient mind-set, there are also severe limitations in the availability of publishing channels where negative results could get published. There are, as I see it, two crises in science. The first relates to trust, in part, due to weaknesses and failures of the traditional peer review system . The secNegative results are extremely important in science because they indicate what doesn\u2019t work. Such valuable clues thus form the basis of new hypothesis testing and new experiments that could then allow a focus on a narrower set of variables, or options. The existence of negative results is an essential building block for science. Dr. Haiko Sprott defines a negative result as \u201ca scientist is not able to show \u2026 a positive effect of the experiment\u201d. . SandercP value [Many, if not most, studies that show a set of data tend to present \u2013 most likely as an inherent human bias \u2013 the positive, successful results, in relation to negative results, either to show that the results are themselves positive, or to indicate that the negative ones were not successful. Thus, the intrinsic nature of many scientific papers already incorporates negative (or not so positive) results into its framework. Consequently, there are few outlets to publish purely negative results Table\u00a0 simply bP value  inhibitsP value  correctlSo why then, apart from the negative psychological perception of negative results, are more negative results not published? There may also be two additional underlying factors, but ultimately these might be reflecting the negative aspects of positive psychology , which iConsequently, the number of journals that can be found that deal exclusively with negative results is extremely limited Table\u00a0, most ofCuriously, the Committee on Publication Ethics (COPE) has one mandatory clause in its code of conduct for COPE member journal editors related to negative results that states: \u201c14.3. Studies reporting negative results should not be excluded\u201d. Thus, the importance of negative results is recognized, but they are simply not woven into the publishing psyche of scientists and editors or into the publishing fabric of most STM publishers. Thus, the mind set of editors, and their receptiveness towards negative results, needs an overhaul .Most likely such a mental frame-work underlying the selection against negative in favor of positive ones will not only require a structural change, but also a mental or psychological one, including better training of editors to recognize the importance of negative results, and to distinguish negative results from bad science. Scientists also need to be taught to better appreciate the importance of their negative results, although this appreciation can only evolve when there are suitable and sufficient channels for them to express/publish their negative results. Providing additional and expansive ideas, through the public presentation of negative results, also aids in expanding the discussion, provides new vistas and perspectives, and assists those who wish to conduct similar experiments, with valuable experimental signs of what not to do, aptly summarized by Pfeffer and Olsen : \u201cit onlsenso lato, bad science. The inflation of positive results, simply because there is a lack of negative ones, which have not been duly reported, may inflate the \u201cpositive\u201d nature of some studies, and in some cases, these may turn out to be unreproducible. This was demonstrated in the Bayer HealthCare and Amgen cases, the latter not being able to replicate as much as 89\u00a0% of its published findings in prominent cancer journals, leading the National Institutes of Health (NIH) to contemplate the implementation of rules to validate positive results, and to counter the lack of incentives to publish negative results [That said, the reader is duly warned that negative results should not be equated with bad science, lack of scientific rigor, or with poor experimental design. Negative results focus exclusively on those results that did not support a hypothesis, or prove a desired \u201cpositive\u201d outcome, and should never be equated with,  results . Thus, r"}
+{"text": "Studies with positive results are greatly more represented in literature than studies with negative results, producing so-called publication bias. This review aims to discuss occurring problems around negative results and to emphasize the importance of reporting negative results. Underreporting of negative results introduces bias into meta-analysis, which consequently misinforms researchers, doctors and policymakers. More resources are potentially wasted on already disputed research that remains unpublished and therefore unavailable to the scientific community. Ethical obligations need to be considered when reporting results of studies on human subjects as people have exposed themselves to risk with the assurance that the study is performed to benefit others. Some studies disprove the common conception that journal editors preferably publish positive findings, which are considered as more citable. Therefore, all stakeholders, but especially researchers, need to be conscious of disseminating negative and positive findings alike. Studies with successfully proven hypothesis are represented in the literature in a greater amount than studies that \u201cfailed\u201d to prove the hypothesis, delivering so-called negative results. It seems that \u201csuccessful and productive\u201d studies are more interesting, readable and therefore more \u201cvaluable\u201d for publishers, editors and readers. This can be derived from the fact that the positive results are more favourably cited in the scientific and medical literature  recommends that journals should require the registration of clinical trials to be eligible for publication. ICMJE believes that there is an ethical obligation to share data generated by such trials: \u201c\u2026to prevent selective publication and selective reporting of research outcomes, and to prevent unnecessary duplication of research effort\u201d . This, however, can introduce bias in favour of negative results which is counterproductive. Criteria for publishing should be the quality of the study and its power, no matter the outcome of the study. The results obtained from a methodologically well-designed study are trustworthy no matter if they confirm or disprove the null hypothesis.There are a number of journals whose scope is to publish negative results in the respective science fields to compensate for evident publication bias  or incorrectly concluding that results are due to chance when in fact the effect is present . Oberhofer and Lennon give emphasis to beta statistics urging the authors to apply the same criteria for detecting a Type II error as a Type I error. Negative results should be interpreted more rigorously to assert that the lack of difference is not due to chance. They state that if we historically accept the 95% confidence that a null hypothesis can be rejected it should also be applied to beta statistics (The studies with findings suggesting no effect or opposite effect are considered negative results (Biochemia Medica considers all submitted manuscripts irrespective of the positive or negative findings as long as the manuscript is in the scope of the journal and prepared according to the Instructions to authors. The recommendations on the way the results should be reported for all manuscripts regardless of the positive or negative findings are the same and they comply with the recommendations of all leading organizations. Whether the results are negative or positive, it is recommended to report effect size and confidence intervals. Researchers, journal editors and funders need to be conscious of the importance of negative results and report and support disseminating negative and positive findings alike."}
+{"text": "A structural equation model was used for verification with chronic schistosomiasis to investigate the coagulation\u2013anticoagulation system imbalance and to deduce the mechanism of D-dimer (D-D) level elevation in patients with advanced schistosome hepatic disease. We detected the plasma levels of tissue-type fiber plasminogen activator (tPA), urokinase type plasminogen activator (uPA), plasmin-antiplasmin complex (PAP), plasminogen (PLG), antithrombin (AT), plasminogen activator inhibitor 1 (PAI1), D-D, factor VIII: C (FVIII:C), antithrombin-III (AT-III), PLG, protein S (PS), and protein C (PC) in the healthy people as control (69), patients with chronic schistosomiasis (150) or advanced chronic schistosomiasis (90). FVIII, PAP, D-D, tPA, and uPA plasma levels were significantly higher in the chronic group than in the control group and were also significantly higher in the advanced group. However, AT-III, PC, PS, AT, PLG, and PAI1 plasma levels in the advanced and chronic groups were significantly lower than those in the control group. With progression of disease in patients with schistosomiasis japonica, a hypercoagulable state is induced by the coagulation\u2013anticoagulation imbalance, eventually leading to patients with high levels of D-D. Furthermore, we established a structural equation model path of a \u201cchronic schistosomiasis disease stage\u2013(coagulation\u2013anticoagulation\u2013fibrinolysis)\u2013D-D.\u201d By using analysis of moment structures (AMOS), it was shown that the chronic schistosomiasis stage was positively related to factor VIII and had negative correlation with AT-III; a good positive correlation with PAP, tPA, and uPA; and a good negative correlation with PLG and PAI1. In addition, our results show that the path coefficient of anticoagulation\u2013fibrinolysis system to the chronic stage of schistosomiasis or D-D levels was significantly higher than that of the coagulation system. In conclusion, the coagulation and fibrinolysis imbalance in patients with chronic schistosomiasis, especially with advanced schistosomiasis, is due to the progression of disease stages. As one of the major endemic areas, China, especially the southern part, is still facing a threat from Schistosoma japonicum, where the number of patients was estimated to be 184,943 and the snail-ridden areas to be 365,468.00 hm2 (287.28 hm2 of which was newly found). Individuals can be infected with S japonicum by contact with contaminated water through which cercariae invade the host organism and develop into schistosomula. Schistosomula then migrate through the circulation system to settle at the portal-mesenteric vein system for final development and maturation. Eventually, the adult S japonicum mate and lay eggs, which are transferred through blood flow and deposited in the liver. Inside the eggs, myracidium induce a local inflammation response including egg granuloma and liver fibrosis, and this eventually leads to the chronic infectious status of the host. Infected people with repeated infections of mass cercariae in contaminated water or without proper treatment may develop to the late stage of the disease with more severe symptoms including hepatosplenomegaly, liver portal hypertension, and complications such as hypersplenism and esophageal variceal rupture bleeding.Schistosomiasis is a serious infectious disease caused by S japonicum including its parasitism and migration in the vein system and deposition of eggs in the liver tissue causes specific pathologic responses in the host. Theoretically, blood vessel injuries first induce a local inflammatory response and subsequent imbalance of coagulation and fibrinolysis, each of which may interact with the other and eventually lead to the amplified and systemic pathologic response of the host. Systemic coagulation plays an important role in the compensation of parasite immunity as the subsequent response of inflammation against the parasitism of S japonicum.\u20137 However, to maintain the homeostasis of the blood system, over-secreted fibrins in coagulation need to be further degraded through fibrinolysis factors such as plasminogen and plasmin. Previous studies reported abnormal blood coagulation status in patients with schistosomiasis japonica,\u20137 and our former studies also found increased levels of D-D, especially in patients with advanced schistosomiasis. However, few studies comprehensively examined the coagulation and fibrinolysis system together in patients with schistosomiasis japonica. The mechanism of the imbalance of the coagulation and anticoagulation system and the abnormality of the fibrinolysis system in patients with chronic schistosomiasis  remain unclear. Here, in the endemic area of S japonicum in China, we recruited patients both with chronic and advanced schistosomiasis as well as healthy controls to investigate the variation in coagulation and anticoagulation, and fibrinolysis and antifibrinolysis levels at different disease stages in chronic schistosomiasis patients and further verified the occurrence and its mechanism of coagulation and fibrinolysis imbalance in patients with chronic schistosomiasis.The life cycle of adult 22.1Jiangxi Information Management System for Schistosomiasis Treatment and Assistance were randomly selected in Xinjian, Nanchang, Duchang, and Yugan of Jiangxi province, China. Patients with viral hepatitis and tuberculosis were excluded. A total of 240 patients with schistosomiasis japonica, consisting of 150 patients with chronic disease (chronic group) and 90 patients with advanced disease (advanced group), were finally recruited. The control group comprised 69 healthy individuals who visited the First Affiliated Hospital of Nanchang University for routine medical examination. This study was approved by the Human Research Ethics Committee of the First Affiliated Hospital of Nanchang University. All patients received an explanation about the scope of the study, such as objectives, procedures, and potential risks, and signed an informed consent statement before inclusion in the study.Patients infected with schistosomiasis, previously diagnosed, treated, and recorded in the 2.2Jiangxi Information Management System for Schistosomiasis Treatment and Assistance. On the basis of the comprehensive information of the history of clinical diagnosis and treatment, physical examination, and abdominal ultrasonography, the disease stage of the patients with schistosomiasis japonica was determined strictly in accordance with the diagnostic criteria for schistosomiasis.Stool samples from the control and patient groups were taken on 2 consecutive days, and each sample was tested twice by the Kato\u2013Katz method. The mean egg counts were reported. Information on physical examination and history of clinical diagnostics and treatment were accessed from the 2.3g and stored in 0.5 mL aliquots at \u221280\u00b0C.Venous blood samples, in the morning and after fasting, of all the participants were collected under aseptic conditions in vacuum tubes containing 0.562\u200aM EDTA-K2 and promoting coagulating tubes ; the sample capacity was 2 and 3 mL, respectively. Blood plasma and serum were obtained after rapid centrifugation for 10\u200aminutes at 2000\u00d72.4The routine blood test including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), platelet (PLT), and leukocyte differential count was analyzed by Hematology Analyzer . Liver function tests included aspartate and alanine aminotransferases (AST and ALT), total bilirubin (TB), direct bilirubin (DB), total protein (TP), albumin protein (ALB), globulin protein (GLB), albumin/globulin ratio (A/G), glutamyltranspeptidase (GGT), and alkaline phosphatase (ALP) and were analyzed by automated biochemistry analyzer . Liver fibrosis tests included hyaluronic acid (HA), type III procollagen (PCIII), type IV collagen (IV-C), and laminin (LN) and were also analyzed by the chemiluminescence method . Blood coagulation and fibrinolysis tests were performed using a full-automatic coagulation analyzer  for prothrombin time; thrombin time (TT); activated partial thromboplastin time (APTT); fibrinogen (FBG); D-D; and the activities of blood coagulation factor VIII and AT-III, PLG, PS, and PC. All the reagents used were those specified for the instrument. Enzyme-linked immunosorbent assay  was used to detect the level of plasma tPA, urokinase type plasminogen activator (uPA), PAP, PLG, AT, and PAI1. All tests were carried out strictly in accordance with the standard operating procedures (SOPs).2.52.5.1\u03c72 test. Student t test and 1-way ANOVA were used for within-group comparisons for continuous variables. The Mann\u2013Whitney U test was used for nonparametric comparisons. All the above analyses were performed using SPSS version 19.0 for Windows . All reported P values were 2-sided, and P values <0.05 were considered statistically significant.Test data of normal distribution or near normal distribution are expressed as mean and standard deviation (SD), and test data of skewness distribution are expressed as median . Comparisons between groups for gender were performed using the 2.5.2\u03c72test and standard fitting degree and significance test of the pc of the structural equation model to evaluate the quality of the model and the fitting degree of the model to the data. Finally, we determined whether the causal relationship between the parameters and the default model was reasonable. All the above analyses were performed using AMOS version 22.0 for Windows . P values of the \u03c72 test of the structural equation model greater than 0.05 were not considered statistically significant. P values of the pc significance test of less than 0.01 were considered statistically significant.The theoretical model was established according to the variation tendency of the level of coagulation and anticoagulation and fibrinolysis and antifibrinolysis in different disease stages of chronic schistosomiasis. We speculated that the disease stage of chronic schistosomiasis would directly affect the coagulation system, anticoagulation system, fibrinolysis system, and levels of D-D. The coagulation, anticoagulation, and fibrinolysis systems directly affect the levels of D-D for constructing the initial path diagram and the specific measurement indicators. Accordingly, the chronic schistosomiasis disease stage was defined as the observed variable, and according to the staging score, the control group was designated as 1, the chronic group as 2, and the advanced group as 3, representing the severity of the disease. The coagulation system was also defined as the observed variable, and the observation indicator was factor VIII, which represents the level of blood coagulation. The coagulation and fibrinolysis system was defined as the latent variable, and the observed indicators were tPA, uPA, PAL1, PAP, PLG, and ATIII, which represented the fibrinolysis and anticoagulation levels, respectively. D-D was defined as the observed variable, and the observation indicator was the D-D level. The defined variables were used for descriptive analysis and correlation analysis, and the limiting correlation coefficient was \u22121 to 1, which determined whether there was a correlation. The path coefficients were estimated using the maximum likelihood method, and the path diagram with the path coefficient (pc) was obtained. A positive number indicated positive correlation, while a negative number indicated negative correlation; the greater of the absolute values showed greater influence on the upper indicators, and vice versa. We used the 33.1P\u200a<\u200a0.05).As shown in Table 3.2P\u200a>\u200a0.05). But FVIII:C followed an increasing trend and the level of activity of AT-III, PC, and PS decreased in the control group, chronic group, and advanced group successively, with statistically significant differences among the groups (P\u200a<\u200a0.05). The levels of D-D, tPA, uPA, and PAP showed an increasing trend in the control group, chronic group, and advanced group, while the PLG and PAI1 levels showed a downward trend; there were statistically significant differences among the groups (P\u200a<\u200a0.05), as shown in Table With the progression of the stage of disease of schistosomiasis japonica, coagulation duration showed no significant difference in chronic schistosomiasis , which indicated that the model was fit for data and convergence. Parameter estimation showed that the path coefficients had reached a significant level (P\u200a<\u200a0.001), which indicated that the estimated parameters were in a reasonable range ; this resulted in a hypercoagulable state of the coagulation and anticoagulation systems. This condition was conducive to the formation of thrombus and was not related to the number of platelets. The main reason for this condition was related to the pathological basis of chronic schistosomiasis. A large number of eggs were deposited in the mesenteric vessels after schistosome infection; the eggs and antigens stimulated the vascular wall and activated the coagulation system. However, with the progression of disease stages, patients with chronic schistosomiasis developed a hypercoagulable state induced by the lack of anticoagulation activity, especially in advanced schistosomiasis patients with liver damage, which indicated worsening liver function associated with the development of disease. The hypercoagulable state of the body promoted the activation of fibrinolysis system and inhibition of antifibrinolysis activity, eventually leading to high levels of D-D. In this progression, the activation of fibrinolysis system was dominant, and the antifibrinolysis activity was obviously insufficient, which may be related to the intravascular thrombosis in the patients with chronic schistosomiasis.Analysis of the results showed an increasing trend for the levels of coagulation factors and a decreasing trend for anticoagulation factors with no significant difference in coagulation duration with progression of disease stage in patients with chronic schistosomiasis , fitting degree analysis (NFI=0.957), and pc significance test (P\u200a<\u200a0.001) proved that the model had a high degree of convergence, good fit, and reasonable parameters. This showed that the causal relationship of the theoretical model path was reasonable, and that the theoretical model had been well verified in the verification analysis of the structural equation model. The path analysis results of the structural equation model showed that the disease stage of chronic schistosomiasis had a positive correlation to the level of coagulation and was negatively correlated with the level of anticoagulation, and the pc of anticoagulation was dominant. This indicated that in the chronic schistosomiasis patients with progression of disease stages, the coagulation level was upregulated to a certain extent and the anticoagulation level was downregulated to a certain extent and was dominant. This leads to the development of a hypercoagulable state in the body, which is induced by the coagulation\u2013anticoagulation imbalance. At the same time, the disease stage of chronic schistosomiasis had a positive correlation with the level of fibrinolysis and was negatively correlated with the level of antifibrinolysis. This showed that the level of fibrinolysis with the progression of the disease stages in patients with chronic schistosomiasis was increased to a certain extent, and the level of antifibrinolysis was reduced, resulting in the manifestations of abnormal hyperfibrinolysis. The pc of the anticoagulation and fibrinolysis system to the chronic schistosomiasis disease stage or D-D levels was significantly higher than that of the coagulation system, indicating that the anticoagulation and fibrinolysis systems accounted for the main advantage in the progression of coagulation and fibrinolysis imbalance and D-D upregulation. Our results also suggest that the anticoagulation and fibrinolysis status may reflect the staging of chronic schistosomiasis to a certain extent. In the previous study, the portal blood flow resistance might cause terrible thrombosis and collateral circulation in schistosomiasis, known as cavernous transformation. Although the relationship of staging of chronic schistosomiasis and the anticoagulation and fibrinolysis status was recovered in this study, there was still some limitations, for example the lack of age-matched control, replication validation of the model, and the potential bias or imprecision of the study.In this study, the variation tendency of coagulation and anticoagulation, and fibrinolysis and antifibrinolysis factors was detected and analyzed in different stages of patients with chronic schistosomiasis. The results showed that the hypercoagulable state induced by the coagulation\u2013anticoagulation imbalance caused compensatory hyperfibrinolysis eventually leading to the production of high levels of D-D in patients, which increased with the progression of the disease stage of patients with schistosomiasis. On the basis of this finding, we hypothesized and established a theoretical path model of the structural equation model for chronic schistosomiasis stage-(coagulation\u2013anticoagulation\u2013 fibrinolysis)-D-D. According to the analysis of the default model for the primary data of coagulation, anticoagulation, fibrinolysis, and antifibrinolysis in the different stages of patients with chronic schistosomiasis, and the pc estimation by using the maximum likelihood method, the results showed that the disease stage of chronic schistosomiasis and the level of parameters had good correlation. In addition, the In summary, we found that coagulation and fibrinolysis imbalance in patients with chronic schistosomiasis, especially advanced schistosomiasis, was due to the progression of disease stages and coagulation and anticoagulation imbalance caused by the hypercoagulable state and high levels of D-D by using the structural equation model analysis and verification. It was an important clinical significance in clinical treatment of coagulation and fibrinolysis balance in patients with chronic schistosomiasis, especially with advanced schistosomiasis. However, the indicator number of coagulation and fibrinolysis is still limited in the study, and we focused on improving the number of indicators, verified and analyzed more complex paths on the basis of the theoretical model by using confirmatory analysis of the structural equation model."}
+{"text": "Lolium rigidum, showing different response patterns, were characterized as being glyphosate resistant (GR). The wide range of values in fresh weight reduction, survival, shikimic acid and EPSPS enzyme activity indicates a different or a combination resistance mechanism. The Line-3 population resulted in minimum reduction of fresh weight and survival values with respect to the glyphosate-susceptible (GS) population, showing 16.05- and 17.90-fold higher values, respectively. There were significant differences in the 14C-glyphosate translocation between GR and GS populations. Moreover, there were differences among the nine GR populations, but they exhibited a reduction in the remaining glyphosate translocation in the treated leaf. The EPSPS gene sequence revealed a Pro-106-Ser substitution in four populations, which could be characterized as being GR with non-target-site and target-site resistance mechanisms. This complexity of several resistance mechanisms makes it necessary to develop long-term integrated weed management strategies to limit further resistance dispersal.Herbicides are the most effective tools for controlling almost 99% of weeds. However, herbicide resistance is a primary concern in modern agriculture. The characterization in new areas and elucidation of the mechanisms of resistance are of vital importance in maintaining the sustainability of herbicides, including glyphosate. Nine populations of  Herbicides are the most effective weed control tools, and control approximately 99% of weeds2. Herbicide resistance is due to the evolution of weed adaptation following selection pressure from repeated herbicide applications and is undoubtedly a major concern in modern agriculture3. Glyphosate is one of the most widely used herbicides, although it also belongs to a class of herbicides with cases of resistance reported in many weed species4. To date, the number of glyphosate-resistant weeds has grown to 37 species worldwide5. Although glyphosate resistance has reached a peak level, it has been controlled by the adoption of best management practices. Glyphosate is still considered a very efficient herbicide, especially under an herbicide-rotation regime where herbicides with different modes of action are used for reducing the glyphosate selection pressure6.Non-controlled weeds cause approximately 34% loss in crop yields worldwide7. Resistance has been documented for the modes of action of all major known herbicides; however, no new modes of action have been commercialized since the 1980\u2019s8. Glyphosate-resistant weed survival is due to different types of resistance mechanisms classified as target site (TSR) and non-target site resistance (NTSR)10. The TSR mechanism is endowed by alterations in the gene encoding the herbicide target protein, causing an increased expression of the target protein or structural changes in the herbicide binding site12. The TSR mechanisms include a target-site mutation (Pro-106 substitution), which was first detected in Eleusine indica13 and which, over the years, has been identified in other weeds16, genomic EPSPS duplication17, and overexpression of EPSPS18. Moreover, a double mutation in E. indica and Bidens pilosa was found in the Thr-102-Ile position followed by Pro-106-Ser11. While a single target-site mutation in the EPSPS gene seems to confer low levels of resistance to glyphosate in the order of two- to fourfold, the double mutation greatly increases resistance levels12.This globally important herbicide (glyphosate) inhibits the nuclear-encoded enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (EC 2.5.1.19), which catalyses the reaction of shikimate-3-phosphate and phosphoenolpyruvate (PEP) to form 5-enolpyruvylshikimate-3-phosphate, which is an important step in the biosynthesis of aromatic amino acids in plants19. NTSR has been described as the most common mechanism of resistance to glyphosate20. The NTSR mechanism can confer unpredictable resistance to herbicides following different modes of action22. Similar to TSR, several glyphosate-resistant weeds have been characterized, with NTSR being the mechanism involved in the resistance28.The NTSR mechanism resulted from reduced absorption and/or translocation, increased vacuolar sequestration, and metabolism to non-toxic compounds, causing a lesser glyphosate transport via the phloem to the EPSPSLolium spp. is an important genus of grass weeds worldwide, with three major species: Lolium rigidum, L. multiflorum, and L. perenne29. These species are principally weeds in perennial crop spheres such as olive groves, vineyards or orchards, and cereal crops. In France and Spain, these grass weeds are widely distributed among the major crops15. L. rigidum is one of the most difficult to control, has the tendency to evolve resistance to herbicides and is reportedly resistant to eleven different herbicide action sites5. This situation reduces the options of diversification with alternative herbicides for an optimal management of this grass weed.L. rigidum populations from France and Spain as well as the determination of their resistance potential to glyphosate, and b) the evaluation of the mechanisms involved in their resistance to glyphosate.The main objectives of the present study were as follows: a) the screening of different \u22121). Almost the GR populations with their 50 plants treated did not exhibit any symptoms of damage caused by the herbicide application (0 score in these populations), but only the Wheat-A population showed a little damage, in which 6 to 50 plants were controlled  to glyphosate and, survived the recommended field dose  used in this study correspondedto L. rigidum . Therefore, each population was compared with France-C to obtain the Resistance Index (RI).The nine GR populations found in the screening assay were studied by means of dose-response experiments. Moreover, two GS populations were studied, which exhibited GR50 and LD50 values with respect to France-C, showing 16.05- and 17.90-fold higher resistance. The Huerta, Sabiote, Bailen, and Wheat-B populations showed lower LD50 values with respect to Line-3, but their values were higher than 2160\u2009g ae ha\u22121 . The France-D, Alamo, France-A, and Wheat-A populations showed LD50 values of approximately 2047.38 and 1138.63\u2009g ae ha\u22121 .Diverse results were observed in different populations Fig.\u00a0. The LinThese results could be divided into two resistance levels. The first one was characterized by higher GR-populations and comprised Line-3, Huerta, Sabiote, Bailen, and Wheat-B. The second one is comprised France-D, Alamo, France-A and Wheat-A, which have lower resistance levels than the above but higher resistance levels than the field dose Table\u00a0. The dif\u22121 fresh weight, 30.13-fold higher with respect to Line-3, the population with the highest GR rate.Figure\u00a014C-glyphosate recovery indifferent populations was approximately 90\u201395%. Different 14C-glyphosate absorption and translocation values were observed at 96\u2009h after treatment (HAT), and the maximum amounts were observed at that time  for the GR and GS populations. The Line-3, Huerta, Sabiote, and Bailen populations exhibited the highest levels of EPSPS enzyme activity, showing 44.97-, 31.97-, 24.62-, and 18.34-fold higher activity with respect to France-C , Huerta (P\u2009=\u20090.7958), and Sabiote (P\u2009=\u20090.7770) with respect to Bailen . The partial EPSPS gene sequence in the Line-3, Huerta, Sabiote, and Bailen populations revealed a single nucleotide substitution of CCA to TCA at codon 106, which was an amino acid substitution from proline to serine . Some populations exhibited both NTSR and TSR mechanisms12.In the present study, both NTSR and TSR mechanisms were studied in order to collect the required information from all the range of mechanisms. In many studies, only one type of mechanism was studied, and usually the TSR mechanism34. These situations could result in a possible resistance in a few years, but certainly they can not be considered to be GR cases20. The populations characterized as being GR could be initially due to these events and, numerous applications for successive years, to increases in the recommended field dose, without the use of herbicides with different mode of action31.It has to be noted that some populations characterized as GS originated from fields that the farmers had previously marked as potentially GR , which was due to an additional NTSR mechanism (reduced glyphosate translocation). Therefore, these populations  were characterized as being GR with NTSR and TSR mechanisms involved. The presence of the latter in these populations makes it difficult to determine the contribution of each mechanism47. For example, Wheat-B and France-D had the NTSR mechanism, exhibiting similar resistance levels to Bailen, in which these two mechanisms were involved. This situation, with both mechanisms was reported in E. colona16, Parthenium hysterophorus20, L. rigidum29, and A. tuberculatus47. Accumulation of these mechanisms could be due to cross-pollination, selection by glyphosate, and environmental conditions34.As evidenced by our results and those obtained in other studies, the TSR mechanism is associated with a smaller amount of shikimic acid and higher levels of EPSPS activityL. rigidum populations exhibited a non-target-site mechanism with reduced translocation. Moreover, four populations had target-site alterations, with Pro-106-Ser substitution in the EPSPS protein. The occurrence of both resistance mechanisms results in higher resistance levels. Several populations and their dispersal into new areas should be further studied in order to evaluate their resistance in the field crops. This action could help farmers; control not only glyphosate-resistance but also multiple-resistance in weeds of high agronomic importance31. Our results would help in establishing a long-term management strategy against herbicide resistance in Lolium spp. As previously stated by Powles48, a major lesson evident from more than three decades of glyphosate use to control billions of plants worldwide is that, where diversity in weed management systems is maintained, weed control by glyphosate can be sustainable. Increased awareness of weed resistance by farmers, extended field monitoring, understanding of the involved mechanisms and integrated weed management strategies are crucial to delay or prevent resistance.In summary, all GR 2 from the coordinate GPS of each population , or susceptible (GS) to glyphosate. Around 20 plants with mature seeds were randomly collected in 50\u2009m\u22122 s\u22121 photosynthetic photon flux, and 80% relative humidity. The populations were transplanted into pots containing sand/ peat in a 1:2 (v/v) ratio and placed in a greenhouse at 28/18\u2009\u00b0C (day/night) with a 16\u2009h photoperiod.All the mature seeds were germinated in Petri dishes with filter paper moistened with distilled water and they were placed in a growth chamber at 28/18\u2009\u00b0C (day/night) with a photoperiod of 16\u2009h, 850 \u00b5mol m\u22121 (3\u2009L\u2009ha\u22121 [recommended field dose]) of glyphosate . Glyphosate was applied at the 3\u20134 leaf growth stage with a laboratory chamber sprayer  equipped with 8002 flat fan nozzles delivering 200\u2009L\u2009ha\u22121, at 250 kPa at the height of 50\u2009cm, and at room temperature.Fifty plants from each population were sprayed with 1080\u2009g ae haA visual evaluation of damage ranging from a scale of 0 to 100 was performed to determine the glyphosate resistance at 21 days after application (DAT). A score of 100 meant total glyphosate control, and 0 meant no control. Populations with a score of 85 or over were characterized as being GS, while populations with a lower score were characterized as being GR , and nine GR ones . Twelve plants of each putative L. rigidum were used for molecular analysis. Additionally, twelve reference susceptible plants (L. multiflorum and L. perenne) were included in the study.In this research, AFLP markers were used as a system for EcoRI and MseI restriction enzymes (Invitrogen). The primers for selective amplification are described in Fernandez et al.4.DNA was extracted from the leaf tissue (50\u2009mg), using the Speedtools DNA Extraction Plant kit . The quality and concentration of the DNA was evaluated by spectrophotometer analysis with 260\u2009nM and 280\u2009nM light absorption. AFLP analysis was carried out using the fluorescent AFLP IRDye kit for Large Plant Genome Analysis (LI-COR Biosciences). Template preparation was performed following the protocol included in the kit, including digestions with AFLP products were separated by polyacrylamide electrophoresis by using an automated sequencer (LICOR 4300). Polymorphic AFLP markers and primers were identified and individuals were scored for presence or absence of AFLP fragments, using the computer package SAGAMX 2 GENERATION. UPGMA analysis was performed with AFLP marker data using the computer program NTSYSpc 2.2.L. rigidum populations. Glyphosate applications were applied with a laboratory chamber sprayer under the same conditions described previously. The following glyphosate  rates were used: 0, 62.50, 125, 250, 500, 1000, 2000, 4000, and 8000\u2009g ae ha\u22121. The experiment was arranged using ten replicates per rate and was repeated twice. Plants were cut at the soil surface 21 DAT, and fresh weight reduction (GR50) and survival (LD50) at 50% were measured.Herbicide treatments were applied at the 3\u20134 leaf growth stage of 51. Approximately 50\u2009mg of fresh tissue was transferred to 2\u2009mL eppendorf tubes containing 1\u2009mL of 1\u2009mM NH4H2PO4 (pH 4.4). Glyphosate was added to eppendorfs at following concentrations: 0, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 400, 500, 600, and 1000\u2009\u00b5M. The eppendorfs were incubated in a growth chamber during 24\u2009h under the above conditions. After 24\u2009h, the eppendorfsd were stored at \u221220\u2009\u00b0C until their analysis. They were then removed from the freezer and thawed at 60\u2009\u00b0C for 30\u2009min. 250\u2009\u00b5L of 1.25\u2009N HCL was added to each tube. Again, they were left at 60\u2009\u00b0C for 15\u2009min. A 125\u2009\u00b5L aliquot from each tube was pipetted into a new 2\u2009mL eppendorf, and 500\u2009\u00b5L of periodic acid and sodium metaperiodate (0.25% [wt/v] each) was added. They were incubated at room temperature for 90\u2009min, after which 500\u2009\u00b5L of 0.6\u2009N sodium hydroxide and 0.22\u2009M sodium sulfite was added. All the eppendorf tube contents were then transferred to glass vials. Samples were measured in a spectrophotometer at 380\u2009nm within 30\u2009min. Each glyphosate concentration contained six replicate plants of each population. The assay was repeated twice. The results were expressed as \u00b5g shikimic acid \u00b5g\u22121 fresh weight.Leaf segments (5 mm diameter) were harvested from the youngest fully expanded leaf from a pool of 15 plants per population at the 3\u20134 leaf growth stageet al.51. 14C-glyphosate  was added to commercial glyphosate with a specific activity of 0.834 KBq \u00b5\u22121. The final glyphosate concentration corresponded to 300\u2009g ae ha\u22121 applied in 200\u2009L\u2009ha\u22121. Plants at the 3\u20134 leaf growth stage were treated with a drop of 1\u2009\u00b5L (0.834 KBq plant\u22121) with a micropipette  onto the adaxial surface of the second leaf. The treated leaf was washed with 3\u2009mL of water: acetone (1:1\u2009v/v) solution to remove the non- 14C-glyphosate absorption at 96\u2009h after treatment (HAT). The rinsate was mixed with 2\u2009mL of scintillation cocktail and analyzed by liquid scintillation spectrometry (LSS) on a scintillation counter . The remainder of the plant was carefully removed from the pot, and its roots were gently washed with distilled water. The plant was divided into treated leaf, remaining shoot tissue, and roots. The plant parts thus obtained were dried at 60\u2009\u00b0C for 96\u2009h and combusted in a Packard Tri Carb 307 biological sample oxidizer . Evolved 14CO2 was trapped and counted by LSS in a 18-mL mixture of Carbo-Sorb E and Permafluor E\u2009+\u2009(1:1\u2009v/v) . The amount of radiolabel deposited was checked by washing a treated leaf excised immediately after deposition. There were five replicates, and the experiment was arranged in a completely randomized design. The assays were repeated twice. The proportion of absorbed herbicide was expressed as [kBq in combusted tissue/(kBq in combusted tissue\u2009+\u2009kBq in leaf washes)]\u2009\u00d7\u2009100.The assays were carried out according to methodology described by Fernandez-Moreno 14C-glyphosate translocation was visualized using a phosphor imager . Plants were treated and collected in the same way described in the absorption and translocation assays. The whole plants were gently rinsed, pressed, and then left to dry at room temperature during four days. Next, the dried plants were placed adjacent to a 25\u2009cm\u2009\u00d7\u200912.5\u2009cm phosphor storage film for 13\u2009h and scanned for radiolabel distribution on a phosphor imager. The experiment was carried out with three plants per population.et al.52 was used for EPSPS extraction. The total content of proteins in the extract was measured according to the method of Bradford53 using a Kit for Protein Determination . The specific EPSPS activity in plants from each population was studied in the presence and absence  of glyphosate. The EPSPS activity was determined using a EnzChek Phosphate Assay Kit . The glyphosate concentrations used were: 0, 0.1, 1, 10, 100, and 1000\u2009\u00b5M. Three replicates at each glyphosate concentration were used, and the experiment was repeated three times. The release of phosphate on the bottom level was measured during 10\u2009minutes at 360\u2009nm in a spectrophotometer .Samples of five g of leaf tissue (3\u20134 leaf growth stage) from each population were ground to fine powder using a pestle in a mortar. The methodology by Sammons et al.10. Total RNA was isolated from leaves using TRIzol reagent  following the manufacturer\u2019s instructions. RNA was then treated with TURBO DNase  to eliminate any DNA contamination and stored at \u221280\u2009\u00b0C. cDNA synthesis was carried out from 2\u2009\u03bcg of total RNA using a M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase  in combination with oligo (dT)12-18 and random non namers  according to the manufacturer\u2019s instructions. To amplify the EPSPS gene, primers previously designed by Perez-Jones et al.54  were used. The PCR reactions were carried out using cDNA from 50 ng of total RNA, 1.5\u2009mM MgCl2, 0.2 mMdNTP, 0.2\u2009\u03bcM of each primer, 1\u2009\u00d7\u2009buffer, and 0.625 units of a 100:1 enzyme mixture of nonproofreading (Thermus thermophilus) and proofreading (Pyrococcus furiosus) polymerases  in a final volume of 25\u2009\u03bcL. All PCR reactions were made in duplicate and cycling conditions were: 94\u2009\u00b0C 3\u2009min, 35 cycles of 94\u2009\u00b0C 30\u2009s, 55\u2009\u00b0C 30\u2009s and 72\u2009\u00b0C 1\u2009min; and a final extension cycle of 72\u2009\u00b0C 10\u2009min. An aliquot of the PCR product was loaded in a 1% agarose gel to check the correct band amplification. The rest of the PCR product was then purified using ExoSAP-IT\u00ae for PCR Product Clean-Up  as indicated by the manufacturers. Five purified PCR products per population were sequenced .EPSPS gene sequencing was conducted according to the protocol described by Alcantara-de la Cruz 50), 50% mortality (LD50), or the herbicide rate 50% inhibition of EPSPS activity (I50).y is the above ground fresh weight, the survival, or the enzyme activity expressed as the percentage of the non-treated control, d is the coefficient corresponding to the upper asymptote, b is the slope of the line, g is the GR50, LD50, I50, and x (independent variable) is the herbicide rate.Dose-response and EPSPS enzyme activity data were subjected to non-linear regression analysis using a three-parameter log-logistic equation Equation\u00a0 to deterdrc package55 for the statistical environment R56. Resistance indices (R/S) were computed as R-to-S GR50, LD50 or I50 ratios. To test for a common GR50, LD50, or I50 for R- and S-populations, i.e. Resistance Index equal to 1, a lack-of-fit test was used to compare the model consisting of curves with population-specific g values with a reduced model with common g55.Regression analyses were conducted using the Analysis of variance (ANOVA) was conducted to test for differences between GR and GS populations in the different assays. When needed, differences between means were separated using the Tukey HSD test at P\u2009<\u20090.05. Model assumptions of normal distribution of errors and homogeneous variance were graphically inspected. ANOVAs were conducted using the Statistix (version. 9.0)  software.Supplementary materials"}
+{"text": "Parthenium hysterophorus has developed glyphosate-resistance in both islands, independently. The resistance level and mechanisms of different P. hysterophorus accessions (three collected in Cuba (Cu-R) and four collected in the Dominican Republic (Do-R) have been studied under greenhouse and laboratory conditions. In in vivo assays , the resistance factor levels showed susceptible accessions (Cu-S \u2265 Do-S), low-resistance accessions (Cu-R3 < Do-R4), medium-resistance accessions (Do-R3 < Cu-R2 < Do-R2) and high-resistance accessions (Do-R1 < Cu-R1). In addition, the resistance factor levels were similar to those found in the shikimic acid accumulation at 1000 \u03bcM of glyphosate (Cu-R1 \u2265 Do-R1 > Do-R2 > Cu-R2 > Do-R3 > Do-R4 > Cu-R3 >> Cu-S \u2265 Do-S). Glyphosate was degraded to aminomethylphosphonic acid, glyoxylate and sarcosine by >88% in resistant accessions except in Cu-R3 and Do-R4 resistant accessions , whereas a little glyphosate (<9.32%) was degraded in both susceptible accessions at 96 h after treatment. There were significant differences between P. hysterophorus accessions in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with and without different glyphosate rates. The R accessions showed values of between 0.026 and 0.21 \u03bcmol \u03bcg\u22121 TSP protein min\u22121 basal EPSPS activity values with respect to the S (0.024 and 0.025) accessions. The same trend was found in the EPSPS enzyme activity treated with glyphosate, where a higher enzyme activity inhibition (glyphosate \u03bcM) corresponded to greater resistance levels in P. hysterophorus accessions. One amino acid substitution was found at position 106 in EPSPS, consisting of a proline to serine change in Cu-R1, Do-R1 Do-R2. The above-mentioned results indicate that high resistance values are determined by the number of defense mechanisms (target-site and non-target-site resistance) possessed by the different P. hysterophorus accessions, concurrently.Glyphosate has been the most intensely herbicide used worldwide for decades, and continues to be a single tool for controlling weeds in woody crops. However, the adoption of this herbicide in a wide range of culture systems has led to the emergence of resistant weeds. Glyphosate has been widely used primarily on citrus in the Caribbean area, but a study of resistance in the Caribbean islands of Cuba and the Dominican Republic has never been carried out. Unfortunately,  Herbicide resistance is an evolutionary phenomenon that allows resistant weed biotypes to be exposed to the normal dose of a herbicide undergoing any suffering growth alterations -glycine) is a post-emergent herbicide that is non-selective, highly systemic and widely used for weed control around the world  and target site resistance (TSR)  is a troublesome annual weed of the Asteraceae family that is native to the Gulf of Mexico and other Latin American countries , as well as the seeds's ability to persist in the soil and germinate over a wide range of temperatures, have contributed to the widespread distribution of ragweed parthenium in perennial and annual crops  as well as in surrounding areas  the level of glyphosate resistance of different accessions; (2) the possible NTSR and TSR mechanisms involved; and (3) to find out if the resistance genes may also increase the multiplicative or additive resistance levels in P. hysterophorus.The main objective of this work is a survey of P. hysterophorus seeds were collected from plants not controlled with glyphosate at doses normally used  in areas with perennial crops in two Caribbean Islands. Seeds from Cu-S and Do-S accessions never exposed to glyphosate were collected from adjacent areas and used as a reference control  with a total output volume of 200 L ha\u22121 water at a pressure of 200 kPa. Four weeks after glyphosate treatment plant survival of the resistant accessions was estimated, and seed produced from surviving plants was collected and stored in paper bags for all subsequent trials. In the case of susceptible accessions (Cu-S and Do-S), no plant survival was observed 4 weeks after glyphosate treatment.In 2013, mature P. hysterophorus accessions were germinated in trays (12 \u00d7 12 \u00d7 6 cm) containing the same substrate as described before and placed in a growth chamber of similar environmental conditions controlled as before. One week after germination, individual seedlings were transplanted into pots (6 \u00d7 6 \u00d7 8 cm) and grown under fluctuating 30/20\u00b0C day/night with a 14 h photoperiod and 850 \u03bcmol m\u22122 s\u22121 photon flux density, and 80% relative humidity. As glyphosate (EPSPS inhibitor) is used in early post-emergence, at the four leaf stage, resistant and susceptible P. hysterophorus seedlings were treated with increasing glyphosate doses: 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 g ae ha\u22121 . The experiment were conducted with 10 replications (one plant pot\u22121) of each accession per herbicide dose, and the experiments were repeated twice. Thirty days after herbicide treatment, herbicide effects on plant survival (LD) and above-ground vegetative biomass (GR) were assessed.Seeds of putative resistant  and susceptible (Cu-S and Do-S) of the P. hysterophorus accessions at the 4\u20136 leaf stage . Glyphosate was added to the tubes at the following concentrations: 0, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 400, 500, 600, and 1000 \u03bcM. The Eppendorf tubes were incubated in a growth chamber during 24 h under the previously described conditions. After 24 h, the tubes were stored at \u221220\u00b0C until analysis. Eppendorf tubes were removed from the freezer and thawed at 60\u00b0C for 30 min. Two hundred and fifty micro liters of 1.25 N HCL was added to each tube, and placed at 60\u00b0C for 15 min. A 125 \u03bcL aliquot from each tube was pipetted into a new 2 mL Eppendorf tube, and 500 \u03bcL of periodic acid and sodium metaperiodate (0.25% [wt/v] each) was added. They were incubated at room temperature for 90 min, after which 500 \u03bcL of 0.6 N sodium hydroxide and 0.22 M sodium sulfite was added. The contents of all tubes were transferred to glass vials. Samples were measured in a spectrophotometer at 380 nm within 30 min. For each glyphosate concentration and accession, three replications were stablished and repeated twice.Leaf segments (50 mm diameter) were harvested from the youngest fully expanded leaf from a batch of 15 plants per 14C-glyphosate was mixed with commercially formulated glyphosate to prepare a solution with a specific activity of 0.834 kBq \u03bcL\u22121 and a glyphosate concentration of 1.8 g ae L\u22121 (360 g ae ha\u22121 in 200 L). P. hysterophorus plants at 4-leaf stage were treated with the radiolabeled herbicide by applying one droplet of 1 \u03bcL of glyphosate solution (0.834 kBq \u03bcL\u22121) on the adaxial surface of the second leaf in each plant using a micropipette (LabMate). The 14C-glyphosate unabsorbed in the treated leaf was removed with 3 mL of water: acetone solution  96 h after droplet application. Preliminary assays with two accessions (Cu-R1 and Cu-S) studied had revealed that the glyphosate absorption leveled-off at 96 h after the droplet applications. The rinsate was mixed with 2 mL of scintillation liquid and analyzed by liquid scintillation spectrometry (LSS) . The plants were separated into the treated leaf, rest of the shoot and root after being placed in cellulose cones. The plant tissue was dried at 60\u00b0C over 96 h and combusted in a biological sample oxidizer . The 14CO2 evolved was trapped and counted in 18 mL of a mixture of Carbo-Sarb E and Permafluor  (Perkin-Elmer). Thus, over 95% of the total radioactivity applied was recovered. There were five replications and the experiment was arranged in a completely randomized design, and repeated twice. The proportion of absorbed herbicide was expressed as:Absorption and translocation study was carried out following the methodology proposed by Cruz-Hip\u00f3lito et al.  The 14C-P. hysterophorus plants were treated with a glyphosate rate of 360 g ae ha\u22121 at 4\u20136 leaf stage. At 96 h after treatment (HAT), glyphosate and its metabolites, i.e., AMPA (aminomethylphosphonic acid), glyoxylate and sarcosine, were determined by reversed-polarity capillary electrophoresis following the methodology described by Rojano-Delgado et al. 2SO4 was added to the solution to obtain 45% (w/v) concentration, with stirring during 30 min. After that, the mix was centrifuged at 20,000 g for 30 min at 4\u00b0C. The previous step was repeated to precipitate the protein in the extracts but in that case with a (NH4)2SO4 concentration of 80% (w/v) stirring for 30 min. Finally, they were centrifuged at 20,000 \u00d7 g for 30 min at 4\u00b0C.The enzyme extraction was conducted according to the protocol described by Dayan et al. . Five grns Table  were groAll the pellets were dissolved in 3 mL of extraction buffer and dialyzed in 2 L of dialysis buffer  over 12 h. The protein concentrations were determined by Bradford assay . The assay buffer was composed of 1 mM MgCl2, 10% glycerol, and 100 mM MOPS, 2 mM sodiummolybdate and 200 mM NaF. The experiments were conducted with three replications of each accession per glyphosate concentration and repeated three times. EPSPS enzyme activity was expressed as percentage of enzyme activity in presence of glyphosate respect to the control (without glyphosate).The assay for the determination of EPSPS activity followed the methodology described by Dayan et al.  using thP. hysterophorus accession, and stored at \u221280\u00b0C for the extraction of RNA. Their tissue was ground in liquid nitrogen in a STAR-BEATER 412\u20130167 mill . Total RNA was isolated from leaves as described by Pist\u00f3n (\u22121). An iScriptTM cDNA Synthesis Kit  at 40 \u03bcL reaction volume was used following the manufacturer\u2018s instructions.For RNA extraction 100\u2013200 mg of young leaves were taken from plants of each y Pist\u00f3n , and theBidens-F10 (5\u2032- GGTTGTGGYGGTVTRTTTCC-3\u2032) and Bidens-R11 (5\u2032- GTCCCAASTATCACTRTGTTC-3\u2032) based on EPSPS gene sequences described previously . Transformation was confirmed through PCR using the M13F and M13R primers as described  and Geneious\u00ae versi\u00f3n 8.1.8 . The multiple sequences were aligned by means of the Muscle algorithm incorporated into SeqMan Pro versi\u00f3n 11.The PCR fragments were cloned in the pGEM\u00ae-T Easy Vector System  and transformed into competent cells of 50), 50% mortality (LD50), and inhibition of EPSPS activity by 50% (I50).Dose-Response and EPSPS enzyme activity data were subjected to non-linear regression analysis .Where drc package  to meet model assumptions. Model assumptions of normal distribution of errors and homogeneous variance were graphically inspected. When needed, differences between means were separated using the Tukey HSD test.Analysis of variance (ANOVA) was conducted using Statistix 9.0  to test for differences between R and S accessions in shikimate accumulation at 1000 \u03bcM glyphosate in the leaf segment; and proportion of the different glyphosate metabolites; proportion of applied P. hyterophorus accessions from Cuba island had resistance index (RI) values  that ranged from 2.7 to 24.6, and 6.1 to 27.5 fold resistance, respectively, while on Dominican Republic island values were between 5.4 to 20, and 6.3 to 22.7 fold resistance, respectively (Table Dose-response assays showed the existence of the first case of glyphosate-resistant weeds in the Caribbean (Cuba and Dominican Republic). The two susceptible weeds (Cu-S and Do-S) had similar susceptibility levels Figures ; Table 2in vivo (GR50 and LD50) and the shikimic acid accumulation in leaf disks at 1000 \u03bcM of glyphosate, the resistance order of the P. hystherophorus accessions was Cu-R1 \u2265 Do-R1 > Do-R2 > Cu-R2 > Do-R3 > Do-R4 > Cu-R3 >> Cu-S \u2265 Do-S. There were significant differences at 1000 \u03bcM glyphosate between R and S accessions of Cuba  and Dominican Republic .The fact that plants treated with glyphosate increase shikimic acid accumulation in leaf disks due to the inhibition of EPSPS activity led us to carry out the experiment depicted in Figures P. hysterophorus accessions at 96 h after treatment (HAT)  (Table 14C-glyphosate which was recovered.There were marked differences in glyphosate absorption between the resistant and susceptible glyphosate 5) Table . All accp = 0.0003, DF = 8, n = 45), rest of the shoots , and root  . There were significant differences at 96 HAT in glyphosate metabolism levels between accessions . Glyphosate levels decreased, whereas glyphosate metabolites  increased at 96 HAT in the Cu-R1, Do-R1, Do-R2, Cu-R2, and Do-R3 accessions. Higher glyphosate levels remained in the Cu-R3 and Do-R4 (low resistance), and very high one in the Cu-S and Do-S (susceptible) accessions. In these last accessions, sarcosine was not detected (Table Previous tests demonstrated that the highest glyphosate translocation and metabolism was reached at 96 HAT in the 50 (herbicide dose which reduces the enzyme activity to 50%) values were different in all accessions, ranging between approximately 47.65 in Cu-R1, 25.2 in Do-R1, 22.1 in Do-R2, 1.4 in Cu-R2, 1.2 in Do-R3, 1.2 in the Cu-R3, and 1.1-fold resistance in Do-R4 accessions relative to their susceptible accession, respectively  were lower with 0.024 and 0.025 \u03bcmol \u03bcg\u22121 protein min\u22121, respectively , and Dominican Republic . The Cu-R1, Do-R1, and Do-R2 exhibited 8.8, 7.2, and 4.8-times higher basal enzyme activities than their susceptible accessions, respectively. For Cu-R2, Do-R3, Do-R4, and Cu-R3 accessions the values were similar to those found for their susceptible accessions, respectively.The basal activity of EPSPS enzyme (without glyphosate) in the resistant accessions was between 0.026 and 0.21 \u03bcmol \u03bcgP. hysterophorus plants of resistant and susceptible accessions were sequenced. The fragments were aligned and numbered based on a published EPSPS sequence of Arabidopsis thaliana (L.) Heynh. (GenBank: CAA29828.1). The resistant accessions of P. hysterophorus Cu-R1 from Cuba, and Do-R1 and Do-R2 from Dominican Republic, showed an amino acid substitution at position 106 consisting of a Proline to Serine  led to the emergence of glyphosate-resistant weeds early in the second decade of the 21st century  employed by farmers, and the agro environment conditions , and the glyphosate degradation to sarcosine and inorganic phosphate by a C\u2013P lyase. These steps have been reported by some authors such as Liu et al. ; Komoba L. perenne spp. multiflorum population from Arkansas, no differences were observed in the I50 values, which could be explained as a lack of effective mutations in the binding site of the enzyme  were more highly resistant to glyphosate as a result of showing different concurrent resistance mechanisms, including reduced absorption and translocation, glyphosate metabolism, and EPSPS gene mutation.Results reported here are in agreement with previous works, in which the Proline to Serine substitution was found to confer glyphosate resistance in other weed species such as A. tuberculatus  that are accumulated by these accessions .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": "Chloris genus is a C4 photosynthetic species mainly distributed in tropical and subtropical regions. Populations of three Chloris species occurring in citrus orchards from central Cuba, under long history glyphosate-based weed management, were studied for glyphosate-resistant status by characterizing their herbicide resistance/tolerance mechanisms. Morphological and molecular analyses allowed these species to be identified as C. ciliata Sw., Chloris elata Desv., and Chloris barbata Sw. Based on the glyphosate rate that causes 50% mortality of the treated plants, glyphosate resistance (R) was confirmed only in C. elata, The R population was 6.1-fold more resistant compared to the susceptible (S) population. In addition, R plants of C. elata accumulated 4.6-fold less shikimate after glyphosate application than S plants. Meanwhile, populations of C. barbata and C. ciliata with or without glyphosate application histories showed similar LD50 values and shikimic acid accumulation rates, demonstrating that resistance to glyphosate have not evolved in these species. Plants of R and S populations of C. elata differed in 14C-glyphosate absorption and translocation. The R population exhibited 27.3-fold greater 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) activity than the S population due to a target site mutation corresponding to a Pro-106-Ser substitution found in the EPSPS gene. These reports show the innate tolerance to glyphosate of C. barbata and C. ciliata, and confirm the resistance of C. elata to this herbicide, showing that both non-target site and target-site mechanisms are involved in its resistance to glyphosate. This is the first case of herbicide resistance in Cuba.The  The use of herbicides is the most common weed control method D\u00e9lye, . HoweverThe mechanisms conferring glyphosate resistance are grouped into two major groups  is a C4 photosynthetic species distributed in tropical and subtropical regions . Given that weed control strategies in larger commercial fields are absolutely the focus of glyphosate applications at the post-emergence growth stage, scientific confirmation is necessary. In this way, this study could define the species that are evolving toward to glyphosate resistance or even tolerant species to this herbicide.Studies of herbicide resistant weeds in Cuba are scarce because of lack of knowledge of the issue. This situation is similar to the Dominican Republic, in which studies have already begun to be carried out  establish their taxonomical identity based on morphological and molecular analyses, (2) evaluate their resistance/tolerance levels, and (3) determine the mechanisms involved.This work aimed to characterize suspicious glyphosate-resistant populations of three different Chloris species in citrus orchards in central Cuba. These fields had been repeatedly treated with glyphosate  continuously for over 10 years, and sometimes received more than one application per year .In 2014, our research group (Dr. Rafael De Prado) together with the Weed Science group of the Ministry of Agriculture of Cuba (Dr. Jorge Cueto), prospected for Clhoris species (treated = T) were harvested separately in in citrus orchards from Arimao and Ceiba, in Cienfuegos Province, from at least 20 plants that had been survived to the last glyphosate treatment. Seeds from a population of each species from nearby locations with no known records of exposure to glyphosate were also collected (non-treated = NT).Mature seeds of three suspicious glyphosate-resistant populations of \u22122 s\u22121) and 80% humidity. Subsequently, the seedlings from each population of the different Chloris species were transplanted individually into pots (1 plant per pot) containing the same substrate and placed in a growth chamber under the conditions described above. Furthermore, 20\u201330 plants from each population were placed in a greenhouse until flowering and fruiting.The seeds were germinated in containers using a substrate of sand/peat (1:2 v/v), covered with parafilm, and placed in a growth chamber at temperatures of 28/18\u00b0C (day/night), with a 16 h photoperiod . For the spikelets, we examined the length and width of the lower and upper glumes, the number of sterile florets, the length of hairs surrounding the callus, the length and width of the lemma of the fertile floret (fertile lemma), the presence and length of hairs on or adjacent to the keel and on the margins of fertile lemma, the length of the palea of the fertile floret, the lemma length and width of the basal sterile floret, the lemma length of any additional sterile floret, and the presence and length of awns on lemmas. The characteristics of the caryopses were length, width, thickness, shape, and length of the embryo mark. The shape of caryopses was quantified as the variance in their three dimensions, each relative to length , using the Speedtools Plant DNA Extraction kit (Biotools). The DNA concentration was measured using a NanoDrop ND 1000 spectophotometer. DNA was diluted to a final concentration of 10 ng/\u03bcL.Twenty-four accessions from the 2, and 10 mM Tris-HCl all in a volume of 20 \u03bcl. The optimized thermal cycling conditions were 2 min at 94\u00b0C, followed by 40 cycles of 94\u00b0C for 25 s, 56\u00b0C for 25 s, 72\u00b0C for 25 s and a final extension at 72\u00b0C for 7 min. AFLP fragments were resolved in 25-cm gels (0.25 mm spacer thickness). Electrophoresis and detection were performed on a two-dye, model 4300 LICOR automated DNA Sequencer. Digital AFLP gel images were scored to obtain binary (band presence/absence) data using the SAGA GENERATION 2 software program.Twelve AFLP primer pairs were used . The reaction mix contained 10 ng template DNA, 2.5 U Taq DNA polymerase, 40 pmol primer, 200 \u03bcM dNTPs, 2.5 mM MgClData clustering was conducted for AFLPs with the NTSYS-pc-2.2 software Rohlf,  using JaChloris population were sprayed at the 3\u20134 leaf growth stage. Glyphosate applications were applied with a laboratory chamber  equipped with 8002 flat fan nozzle delivering 200 L ha\u22121 at 250 KPa at the height of 50 cm. The following glyphosate  rates were used: 0, 62.5, 125, 250, 500, 1,000, 2,000, 3,000, and 4,000 g ae ha\u22121. The experiment was design using nine replications per rate and was repeated twice. Plants were cut down at the soil surface 21 days after application (DAT).Plants of each \u22121 fresh tissue.Fifty mg of fresh tissue (4 mm leaf disks) were harvested from the youngest fully expanded leaf at the 3\u20134 leaf growth stage from 15 plants per population. Shikimic acid accumulation was determined according to Hanson et al. . The glyC. elata populations.14C-glyphosate  was added to the commercial herbicide to prepare a solution with a specific activity of 0.834 kBq \u03bcL\u22121. The final glyphosate concentration corresponded to 360 g ae ha\u22121 in 200 L ha\u22121. Plants were harvested at 24, 48, 48, 72, and 96 h after 14C-glyphosate treatment (0.834 kBq/plant). Five plants per populations at each time evaluated in a completely random design were handled according to Fern\u00e1ndez-Moreno et al. (14C-glyphosate absorbed was expressed as [kBq in combusted tissue/(kBq in combusted tissue + kBq in leaf washes)] \u00d7 100.This study was carried out in the two o et al. . Radioac14C-glyphosate in plants of the two C. elata populations was visualized using a phosphor imager .Translocation of C. elata population at 3\u20134 leaf growth stage, were treated with 300 g ae ha\u22121 of glyphosate (as described in the dose-response assays) in a completely randomized design. Untreated plants were used as controls. Leaf tissues were washed with distilled water at 96 HAT, flash-frozen in liquid nitrogen, and stored at \u221240\u00b0C until use. Following the methodology described by Rojano-Delgado et al.  were ground to fine powder in liquid nitrogen a chilled mortar. The enzyme activity was extracted according to the protocol described by Sammons et al. (\u22121 min\u22121 and expressed as a percentage with respect to the control (without glyphosate). The experiment was repeated three times for each samples.Leaf tissue of the s et al. . The basC. elata population and stored at \u221280\u00b0C for RNA extraction. Total RNA was isolated using the TRIzol reagent  according to the manufacturer's instructions. RNA was then treated with TURBO DNase  to eliminate any DNA contamination. cDNA synthesis was carried out using 2 \u03bcg of total RNA and M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase  in combination with oligo (dT)12\u221218 and random nonamers  according to the manufacturer's instructions. To amplify the EPSPS gene, primers previously designed by Perez-Jones et al.  and proofreading (Pyrococcus furiosus) enzymes  in a final volume of 25 \u03bcL. All PCR reactions were completed in duplicate, and the cycling conditions were as follows: 94\u00b0C for 3 min, 35 cycles of 94\u00b0C for 30 s, 55\u00b0C for 30 s and 72\u00b0C for 1 min, with a final extension cycle of 72\u00b0C for 10 min. An aliquot of the PCR product was loaded onto a 1% agarose gel to confirm the correct band amplification. The remainder of the PCR product was then purified using ExoSAP-IT\u00ae for PCR Product Clean-Up  as indicated by the manufacturers. Five purified PCR products per population were sequenced .Young tissue (100\u2013200 mg) was collected from 10 plants of each s et al.  , increase the mortality (LD50), and inhibit the EPSPS activity (I50) by 50% in each Chloris population using the three-parameter log-logistic function: y = ([(d) / 1+(x/g)b]) where y is, depending on the analysis, the above ground fresh weight, survival, or EPSPS-activity expressed as the percentage of the non-treated control, d is the parameter corresponding to the upper asymptote, b is the slope, g is the GR50, LD50, or I50, and x (independent variable) is the glyphosate rate. Regression analyses were conducted using the drc package in the R program version 3.2.5   software.An analysis of variance (ANOVA) was conducted to test for differences between populations in the different assays. When needed, differences between means were separated using the Tukey HSD test at C. ciliata Sw., C. elata Desv., and Chloris barbata Sw. Only the last species listed is an annual species, and it can be easily separated from the other two species by its long-awned lemmas, the glabrous keel of the fertile lemma and the presence of hairs flanking the keel. In addition, caryopses of C. barbata were clearly more elongated in shape than those of the remaining species, as indicated by the higher values of the seed shape index. Distinctive traits of C. ciliata include a low number of racemes in the inflorescences, more than two sterile florets per spikelet, long keel hairs and relatively short awns. Compared to the other two species, racemes of C. elata plants were consistently longer and their fertile lemmas were shorter in length, showing much longer marginal hairs. In addition, the embryo mark in caryopses was shorter in this species , thus providing complementary information to the morphological analysis. Group I contained all samples of C. ciliata whereas Group II consisted of two subgroups, II-1 including C. elata, and II-2 including C. barbata. It is noting that the cluster analysis did not separate T and NT populations of C. ciliata or C. barbata, while the T and NT populations of C. elata were clearly separated  for the NT and T populations of C. elata was achieved at 88.3 and 542.1 g ae ha\u22121, respectively, i.e., T populations were 6.1-fold more resistance to glyphosate than the NT population. The LD50 values of the T populations exhibited 15-fold resistance than the NT population of C. elata. In contrast, GR50 and LD50 values in both C. barbata and C. ciliata species were not different between T and NT populations  and susceptible (S) to glyphosate, respectively. In the following assays, we focused in C. elata.It was determined that 14C-glyphosate recovery was 93.2 and 94.3% for the R and S populations of C. elata, respectively (data not shown). 14C-glyphosate absorption increased slowly in the first 72 HAT. At this time, population S had absorbed 30% of glyphosate, while population R had only absorbed 22%. The maximum glyphosate absorption rate was observed between 72 and 96 HAT, which was the two-fold higher in the S population (64%) than in the R population .For glyphosate absorbed into the R and S \u22121 TPS\u22121 min\u22121 for the R and S populations, respectively   codon instead of a CCG (proline) codon  values can vary between 515 and 703 g ae ha\u22121  compared to the NT population showed the greatest resistance level of this species, similar to other Chloris species, such as C. elata (5.4) from Brazil  and Chloris truncata (2.4\u20138.7) from Australia  of this population. Therefore, the T population of C. elata was characterized as resistant (R) to glyphosate, and the populations T and NT of C. barbata and C. ciliata are tolerant to this herbicide. These results are reflected in those obtained in dose response assays. For this reason, we continued to study the glyphosate resistance mechanisms only in the case of C. elata.Chloris, and the results are contradictory. 14C-glyphosate studies on C. virgata and C. truncata do not show significant differences in the absorption and subsequent translocation of the herbicide, and the resistance was determined by mechanisms within the target site , have been reported in mono- and dicotyledonous weeds, endowing resistance to glyphosate  and target site (Pro-106-Ser mutation) resistance mechanisms to glyphosate.Morphological- and molecular-based analysis allowed the identification of the three Chloris genus can be either resistant or tolerant to glyphosate, supporting the previous reports of both glyphosate statuses in this genus.These results confirm the first case of herbicide resistance in Cuba and strongly suggest that species of the EB and RDP: Idea and designed the experiments; EB, PF-M, MO, FB, HC-H, and RA-dlC: Performed the research. PF-M, FB, MO, RA-dlC, and RDP; Analyzed the results. All authors contributed to write and approve 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": "The intervention of layered CoO2 nanosheets in-between graphene nanosheets is fairly effective in optimizing the pore and composite structures of the Co3O4\u2013graphene nanocomposite and also in enhancing its electrochemical activity via the depression of interaction between graphene nanosheets. The resulting CoO2 nanosheet-incorporated nanocomposites show much greater discharge capacity of ~1750 mAhg\u22121 with better cyclability and rate characteristics than does CoO2-free Co3O4\u2013graphene nanocomposite (~1100\u2009mAhg\u22121). The huge discharge capacity of the present nanocomposite is the largest one among the reported data of cobalt oxide\u2013graphene nanocomposite. Such a remarkable enhancement of electrode performance upon the addition of inorganic nanosheet is also observed for Mn3O4\u2013graphene nanocomposite. The improvement of electrode performance upon the incorporation of inorganic nanosheet is attributable to an improved Li+ ion diffusion, an enhanced mixing between metal oxide and graphene, and the prevention of electrode agglomeration. The present experimental findings underscore an efficient and universal role of the colloidal mixture of graphene and redoxable metal oxide nanosheets as a precursor for improving the electrode functionality of graphene-based nanocomposites.The best electrode performance of metal oxide\u2013graphene nanocomposite material for lithium secondary batteries can be achieved by using the colloidal mixture of layered CoO This prevents the intimate nanoscale mixing between electrode crystals and rG-O nanosheets, and the formation of the open stacking structure of nanocomposite, which diminish the beneficial effect of the hybridization with rG-O nanosheets.Graphene-based nanocomposite is one of the most currently investigated materials in the fields of chemistry, physics, materials science, and nanotechnology because of its intriguing physicochemical properties and promising functionalities12345678910112, [Mn1/3Co1/3Ni1/3]O2, and MnO2 show sufficiently high electrical conductivity and high electrochemical activity2526Currently intense research interest on graphene nanosheet is extended to 2D nanosheets of inorganic solids such as layered metal oxide, layered metal chalcogenide, and layered metal hydroxide1920222 nanosheets on the composite structure, pore structure, and the electrode activity of Co3O4\u2013graphene nanocomposite are systematically investigated. The present strategy is also extended by the incorporation of layered MnO2 nanosheets into the Mn3O4\u2013graphene nanocomposite.Here we report an effective and universal way to improve the electrode functionality of graphene-based nanocomposites using the colloidal mixture of inorganic and graphene nanosheets. The effects of the intervention of layered CoO2 and G-O nanosheets can form stable mixture colloidal suspensions with variable ratios of CoO2/G-O, since they possess very similar surface charge and hydrophilicity each other  analysis  are denoted as CCG0, CCG5, CCG10, and CCG20, respectively. The reduction of precursor G-O to N-doped rG-O during the synthesis is confirmed by C 1s and N 1s X-ray photoelectron spectroscopic (XPS) analysis  oxide phase during the hydrothermal reaction. On the basis of Scherrer equation, the size of Co3O4 particle in the present materials is calculated to be 7.7, 8.2, 8.5, and 9.0\u2009nm for CCG0, CCG5, CCG10, and CCG20, respectively, highlighting a slight increase of particle size upon the incorporation of layered CoO2 nanosheets. The observed minute variation of the particle size of Co3O4 upon the incorporation of CoO2 nanosheets underscores the limited influence of layered CoO2 nanosheets on the crystal growth of Co3O4 nanoparticles.The precursors of exfoliated CoOch other . The effpensions . The sizanalysis . Most ofanalysis . As presCCG nanocomposites commonly exhibit porous morphology formed by the house-of-cards-type stacking of nanosheet crystallites, indicating the formation of many mesopores. Such a mesoporous stacking structure is commonly observed for the self-assembled nanocomposite materials synthesized by the restacking of 2D nanosheets with 0D nanoparticlesAs illustrated in the field emission-scanning electron microscopy (FE-SEM) images of 2 nanosheets with Co3O4 nanocrystals is obviously evidenced by high resolution-transmission electron microscopy (HR-TEM), see 2-free CCG0 material in 2-free CCG0 material in 3O4 phase, indicating the immobilization of the spherical Co3O4 particles on the surface of the nanosheets. For the case of CoO2\u2013incorporated CCG10 nanocomposite, the FFT images of 2 nanosheets with Co3O4 and graphene. This is further confirmed by the observation of clear lattice fringes corresponding to the {110} plane of graphene, the {104} plane of layered CoO2, and the {440} plane of Co3O4, as illustrated in 2, graphene, and Co3O4 in the present CCG10 nanocomposite. As a reference, the rG-O-free Co3O4\u2013layered CoO2 nanocomposite is also synthesized by the same synthetic method as that for the CCG nanocomposites except for G-O and ammonia. Like the CCG nanocomposites, the obtained Co3O4\u2013layered CoO2 nanocomposite displays the formation of spinel-structured Co3O4 nanoparticles anchored on the surface of layered CoO2 nanosheets  spectroscopy, see 2 nanosheets, highlighting an increase of average Co oxidation state caused by the increase of CoO2 content. This result confirms the HR-TEM results showing the presence of CoO2 nanosheets in the present CCG nanocomposites. The successful incorporation of tetravalent CoO2 nanosheets in the present materials is further evidenced by the Co 2p XPS result  type-IV shape and IUPAC H2-type hysteresis loop, suggesting the presence of open slit-shaped capillaries with very wide bodies and narrow short necks. The incorporation of layered CoO2 nanosheets enhances the adsorption of N2 molecule in the low pressure region and also the total amount of N2 molecules adsorbed, underscoring the remarkable increase of micropore volume and surface area. According to the calculation of surface area using the Brunauer\u2013Emmett\u2013Teller (BET) equation, the surface area of the present nanocomposite is estimated to be 32\u2009m2g\u22121 for CCG0, 64\u2009m2g\u22121 for CCG5, 97\u2009m2g\u22121 for CCG10, and 82\u2009m2g\u22121 for CCG20, respectively. This result demonstrates that the surface areas of the present CCG nanocomposites become greater with increasing the content of CoO2 nanosheets upto the composition of CCG10. However, the further addition of CoO2 nanosheets leads to the depression of surface area. The observed lowering of the surface area of the CCG20 nanocomposite is attributable to too high content of CoO2 nanosheet, which is much heavier than the graphene. That is, the increase of the sample mass caused by the addition of heavy CoO2 nanosheets outweighs the accompanying optimization of the pore structure of the nanocomposite. This result clearly demonstrates that, even at a small concentration of CoO2 nanosheets, the incorporation of layered CoO2 nanosheets is fairly useful in expanding the surface area of Co3O4\u2013graphene nanocomposite. As evidenced by the micro-Raman spectroscopy  layerCCG nanocomposites becomes greater with proceeding the cycle. Such an increase of discharge capacity is frequently observed for porous nanostructured materials, which is related to the formation of stable diffusion paths of Li+ ions during the repeated electrochemical cycling123334CCG10 nanocomposite with the largest surface area exhibits the most prominent enhancement of discharge capacity during the cycle. After the 20th cycle, the discharge capacities of CCG nanocomposites are stabilized to ~1230\u2009mAh g\u22121 for CCG0, ~1500\u2009mAh g\u22121 for CCG5, ~1750\u2009mAh g\u22121 for CCG10, and ~1530\u2009mAh g\u22121 for CCG20, highlighting the promising electrode performance of the present nanocomposites with huge discharge capacity and good cyclability. To the best of our knowledge, the observed discharge capacity of the CCG10 nanocomposite is the largest reversible capacity of Co3O4-based materials ever-reported 12CCG10 nanocomposite is estimated to be ~960\u2009mAhg\u22121, which is much smaller that the observed reversible capacity of ~1750\u2009mAhg\u22121. In fact, there are several reports about the larger capacity of graphene-based nanocomposite than the theoretical one3334CCG nanocomposite; (1) the expansion of surface area with the increase of pore volume upon the composite formation results in the additional storage of Li+ ions in the interfacial site of nanocomposite3O4 nanoparticles are changed to amorphous ones, leading to the formation of more accessible active sites for Li-ion insertion2 carbon arrays. The incorporation of CoO2 nanosheets induces the formation of more open stacking porous structure providing more active sites for Li+ ions insertionreported . Since aCCG nanocomposites display high coulombic efficiency of >98%, reflecting the highly stable and reversible insertion/extraction of lithium ions. As shown in the potential profiles of the CCG nanocomposites  nanocomposite.All the present mposites , all the2 nanosheet is more distinct for higher current density condition. The CoO2-incorporated CCG10 nanocomposite exhibits larger reversible capacities for all the current densities applied than does the CoO2-free CCG0 material; the CCG10 nanocomposite shows the discharge capacities of 1684, 1656, 1562, 1288, 1002 and 817\u2009mAh g\u22121 at the current density of 100, 200, 400, 800, 1600 and 3200\u2009mA g\u22121, respectively. However, the CoO2-free CCG0 electrode delivers much smaller discharge capacities of 1232, 1207, 1044, 949, 642 and 428\u2009mAh g\u22121 at the same current densities, respectively. While the discharge capacity of the CCG10 nanocomposite at 100\u2009mA g\u22121 is larger by 136% than that of the CCG0 material, the CCG10 material shows even twice larger discharge capacity compared with the CCG0 one at a higher current density of 3200\u2009mA g\u22121. This finding provides clear evidence for the improvement of rate performance upon the incorporation of CoO2 nanosheets, highlighting the improvement of charge transport property.As presented in 2-incorporated nanocomposite, the extended electrochemical cycling test is carried out for the CCG10 nanocomposite with high current density of 1000\u2009mA g\u22121 and in the voltage window of 0.01\u20133\u2009V vs. Li/Li+. As plotted in \u22121 upto the 500th cycle. For the entire cycle, the coulombic efficiency of the CCG10 nanocomposite is well-maintained to ~99%, verifying the high electrochemical stability of this material. The observed beneficial effect of the incorporation of CoO2 nanosheets on the discharge capacity of the nanocomposite is attributable to the expansion of surface area and the change of pore structure, resulting in the additional storage of Li+ ions in interfacial site of the CoO2 nanosheet-scaffolded nanocomposite. The incorporation of layered CoO2 nanosheets also induces an enhanced nanoscale mixing between Co3O4 particles and rG-O nanosheets, which is responsible for the excellent cyclability and rate characteristics of the present CoO2-incorporated CCG nanocomposites.To examine the long-term stability of the CoO2 addition, the transport property of the present nanocomposites is investigated with electrochemical impedance spectroscopy (EIS). As plotted in ct) at high-to-medium frequencies and a line corresponding to Warburg impedance at low frequencies. The incorporation of layered CoO2 nanosheets gives rise to a significant reduction in the diameter of semicircle, indicating the decrease of Rct. Among the present nanocomposites, the CCG10 material displays the smallest diameter of the semicircle, indicating its most efficient transport property. A further increase of CoO2 content to the CCG20 nanocomposite degrades the electron transport property, which is attributable to the decrease of highly conductive graphene content. The relative order of Rct is in good agreement with the relative electrode performances of the present nanocomposites, underscoring the main role of the improvement of transport property in enhancing the electrode performance upon the incorporation of CoO2 nanosheets. Such a variation of transport properties is further confirmed by the change of the line slope in the low frequency region. The slope of this line for the present nanocomposites becomes steeper in the order of CCG0 < CCG20 < CCG5 < CCG10, reflecting the improvement of transport property caused by the promoted nanoscale mixing of Co3O4 nanoparticles and graphene nanosheets upon the incorporation of CoO2 nanosheets.To better understand the origin of the beneficial effect of CoOCCG10 nanocomposite whereas the CoO2-free CCG0 nanocomposite shows a significant aggregation of electrode particles. Such an aggregation of electrode particles is negligible for the CoO2-incorporated CCG10 nanocomposite, confirming the beneficial role of CoO2 nanosheets in the maintenance of the open structure of nanocomposite. Since Co3O4 experiences severe volume change during lithiation\u2013delithiation process, the depression of particle agglomeration is surely advantageous in enhancing the electrode performance of the nanocomposite. In addition, the electrochemical cycling induces an amorphization of both the CCG0 and CCG10 nanocomposites, see + diffusion via the provision of more diffusion paths2 nanosheets does not induce any significant change in the XRD data of the cycled derivative, indicating negligible effect on the structural stability of the nanocomposite. The present finding strongly suggests that the beneficial role of CoO2 addition mainly originates from the improvement of the morphological stability of composite structure rather than the change of crystal structure. Even though the incorporated CoO2 nanosheets is transformed into cobalt oxide particles during the electrochemical cycling, an intimate mixing between cobalt oxide and rG-O in the CoO2-incorporated CCG10 nanocomposite provides improved diffusion paths for Li+ ions as well as a strong electronic coupling between cobalt oxide and graphene nanosheeets.The effects of electrochemical cycling on the crystal structure and morphology of the present nanocomposites are examined with powder XRD and FE-SEM analyses. As demonstrated in 2 nanosheet-incorporated Mn3O4\u2013N-doped rG-O nanocomposite. The obtained Mn3O4\u2013layered MnO2\u2013N-doped rG-O displays the typical XRD patterns of Mn3O4 phase and porous morphology formed by the house-of-cards-type stacking of sheet-like crystallites, as observed for the present CCG nanocomposites  ions into HCoO24 was reduced to 1/6 of conventional concentration\u22121 by ultrasonication for 0.5\u2009h. An aqueous suspension of exfoliated G-O (48\u2009mL) was reacted with 2.4\u2009mL 0.2\u2009M Co(Ac)2 and 1\u2009mL 30% NH4OH aqueous solution, 1\u2009mL H2O, and layered CoO2 nanosheets (0\u20132\u2009wt% to exfoliated G-O nanosheets). The mixture was stirred at 80\u2009\u00b0C for 10\u2009h. Then the mixture was transferred to an autoclave for hydrothermal reaction. The reaction condition was 3\u2009h at 150\u2009\u00b0C. In this step, G-O was reduced to N-doped rG-O. After the reaction, powdery precipitates were collected by centrifugation, washed with ethanol and distilled water, and then freeze-dried. After the completion of the reaction, only transparent supernatant solution remained. No observation of Tyndall phenomenon for the supernatant solution clearly demonstrated the absence of any precursor colloidal particles in this solution. Additionally, no formation of precipitate upon the addition of hydroxide ions confirmed the complete incorporation of Co2+, CoO2, and graphene reactants into the precipitated nanocomposite materials. On the basis of the present findings, the weight ratios of the components in these materials could be estimated from the starting ratios of the reactants. The weight ratio of Co3O4:CoO2:rG-O components was estimated to 3.7:0:1 for CCG0, 3.7:0.007:1 for CCG5, 3.7:0.015:1 for CCG10, and 3.7:0.03:1 for CCG20, respectively. Since the weight of G-O component was much more convenient and precise to calculate than its molar concentration, the weight ratios of CoO2/G-O were applied for controlling the compositions of the present nanocomposites like many other studies about the graphene-based nanocomposites.The exfoliated layered CoOCCG nanocomposites and their electrochemically cycled derivatives were analyzed by powder XRD analysis  analysis. The crystal morphology of the present samples was examined by FE-SEM (JEOL JSM-6700F) and HR-TEM/SAED . The spatial elemental distribution of the present materials was probed with EDS\u2013elemental mapping analysis. XANES spectroscopic experiment was carried out at Co K-edge at the beam line 10C at the Pohang Accelerator Laboratory (PAL) in Korea. The chemical bonding nature of nitrogen species was investigated with XPS analysis , in which a monochromated X-ray beams was used. All the XPS spectra were calibrated with a reference to the adventitious C 1s peak at 284.8\u2009eV to rule out any possible spectral shift by the charging effect. To avoid the accumulation of charge during the measurement, all the samples were deposited on metallic copper foil. N2 adsorption\u2013desorption isotherms were measured at 77\u2009K using Micromeritics ASAP 2020 analyzer to determine the surface area. Before the measurements, the degassing of the samples was carried out at 150\u2009\u00b0C for 3\u2009h under vacuum. Micro-Raman spectra were obtained with a JY LabRam HR spectrometer using an excitation wavelength of 514.5\u2009nm. The zeta potentials of the pure colloidal suspensions of G-O and layered CoO2 nanosheets, and their colloidal mixtures were measured with Malvern Zetasizer Nano ZS .The crystal structures of the as-prepared \u22121 and a potential range of 0.01\u20133.0\u2009V (vs. Li/Li+). The EIS data were collected in the frequency range of 0.01\u2009Hz\u2013100\u2009KHz. Electrochemical measurements were carried out at room temperature using 2016 coin-type cell of 1\u2009M LiPF6 in an equivolume mixture of ethylene carbonate/diethyl carbonate (EC/DEC\u2009=\u200950:50). The working electrodes were fabricated by mixing 80\u2009wt% active material, 10\u2009wt% Super P, and 10\u2009wt% polyvinylidene fluoride (PVDF) dissolved in N-methyl-2-pyrrolidinone (NMP). The composite electrodes were prepared by coating the anode slurry onto a copper foil as a current collector and drying under vacuum at 110\u2009\u00b0C for 12\u2009h. The test cells were assembled in an argon-filled glove box. All the galvanostatic charge\u2013discharge tests were performed with Maccor (Series 4000) multichannel galvanostat/potentiostat in the voltage range of 0.01\u20133.0\u2009V (vs. Li/Li+) at current density of 100\u20133200\u2009mA.The CV data were collected using an IVIUM analyzer with a scanning rate of 0.5\u2009mV sHow to cite this article: Jin, X. et al. An Effective Way to Optimize the Functionality of Graphene-Based Nanocomposite: Use of the Colloidal Mixture of Graphene and Inorganic Nanosheets. Sci. Rep.5, 11057; doi: 10.1038/srep11057 (2015)."}
+{"text": "Promoting cultural competence of health professionals working with Aboriginal and Torres Strait Islander communities is key to improving health outcomes. Cultural Educators and Cultural Mentors (CE/ CMs) have critical roles in Australian general practice training, yet these are not well understood.Guided by a CE/CM Network, our research team including experienced CE/CMs, used surveys and semi-structured interviews to explore these roles and investigate best practice in employment and support. Participants sampled from stakeholders involved in general practice education across Australia included CE/CMs, Medical Educators, General Practice Supervisors and Registrars, and representatives of Regional Training Organisations, Indigenous Health Training Posts and other key organisations. We undertook thematic analysis using a framework approach, refined further in team discussions that privileged views of CE/ CM members.Participants comprised 95 interviewees and 55 survey respondents. We organised our findings under three overarching themes: understandings about cultural education and mentoring; employment and support of CE/CMs; and delivery and evaluation of cultural education and mentoring. Our findings supported a central role for Aboriginal and Torres Strait Islander CE/CMs in face-to-face Registrar education about culture and history and related impacts on health and healthcare. Cultural education was reported to provide base-line learning as preparation for clinical practice whilst cultural mentoring was seen as longitudinal, relationship-based learning. Mentoring was particularly valued by Registrars working in Aboriginal and Torres Strait Islander communities.Challenges described with employment and support included difficulties in finding people with skills and authority to undertake this demanding work. Remuneration was problematic, particularly for CMs whose work-time is difficult to quantify, and who are often employed in other roles and sometimes not paid. Other improved support recommended included appropriate employment terms and conditions, flexibility in role definitions, and professional development. Recommendations concerning implementation and evaluation included valuing of cultural education, greater provision of mentoring, partnerships with Medical Educators, and engagement of CE/CMs in rigorous evaluation and assessment processes.Our research highlights the importance of the unique CE/CM roles and describes challenges in sustaining them. Professional and organisational support is needed to ensure delivery of respectful and effective cultural education within general practitioner training. Similar to other Indigenous peoples, Aboriginal and Torres Strait Islander people, who constitute 3.1% of the Australian population, suffer worse outcomes in almost all health indicators compared to non-Indigenous Australians . Social In alignment with calls for training in cultural diversity to be introduced in undergraduate medical teaching internationally , AustralIn accordance with these standards, training in this area has been delivered largely in partnership with Aboriginal and Torres Strait Islander people and communities, with individuals selected for educational roles based on their expertise in cultural issues and their leadership roles in their communities. They are described as Cultural Educators (CEs) or Cultural Mentors (CMs) and they often work closely with Medical Educators (GPs working as Registrar trainers) either in regional organisations providing postgraduate GP training (RTOs) or in Indigenous Health Training Posts where general practice registrars (Registrars) undertake clinical placements. Indigenous training posts are health services serving Aboriginal and Torres Strait Islander communities, mostly operating as Aboriginal Community Controlled Health Services . RegistrCurrently there are approximately 70\u00a0CE/CMs employed in general practice training in Australia. Cultural Mentors are usually community appointed  and are not required to have particular qualifications, whilst CEs often will have some education enabling them to provide more formal education sessions. Those recruited to these roles are Aboriginal or Torres Strait Islander people and range from young academics to older community Elders and Traditional Owners. Their remuneration varies from approximately $(AUD) 60/h for CMs to $80/ h for CEs .The contribution of CEs and CMs to general practice training is recognised and their expertise increasingly sought in cultural education as well as in clinical teaching and assessment . HoweverIn this paper we summarise the findings of research commissioned by Australian General Practice Training, Ltd., the national organization that oversaw GP training at the time of this research. Whilst in a comprehensive research report  we addreOur research was strongly supported by Aboriginal organisations with letters of support received from the National Aboriginal Community Controlled Health Organisation and its state and territory based affiliates. It was also approved by five Aboriginal Human Research Ethics Committees as well as the university ethics committee. The research team comprised three experienced CE/CMs , two academic GPs  who also worked as clinicians and educators in Aboriginal Community Controlled Health Services, two consultants  and three university-based academics , one of whom was Aboriginal. The team was supported by a national reference group of senior Aboriginal CE/CMs appointed by the funding organisation. This reference group approved the research plan including selection and recruitment of participant groups, also data collection tools and received regular updates on the progress of the research. Though they did not contribute to analysis and interpretation of the data, this group reviewed and approved the final research report.We used a mixed methods approach collecting qualitative and quantitative data from surveys and semi-structured interviews which were adapted for the different stakeholder groups Table\u00a0. The useThe surveys were piloted over a two month period with stakeholders related to two of the 17 RTOs operating at that time. After minor modifications these were circulated by email to all 17 RTOs with a request for a senior manager to complete the relevant survey on behalf of the organisation and to distribute other surveys to CEs, CMs, Medical Educators and to Indigenous Health Training posts. A link to an on-line survey was circulated by the national General Practice Registrar Association. Surveys were also emailed by the national CE/CM Network to their members. Survey questions were preceded by a statement explaining the purpose of the research and the use of the data and notifying respondents that their completion of the survey would be taken as consent to participate in the research (See Appendix 1). Following the pilot, surveys were in circulation for 3\u00a0months and over that time multiple telephone and email reminders were provided through the networks described, as well as through personal contact by the research team. All survey responses were de-identified, though stakeholder group and RTO affiliations were recorded.Interviews were conducted over a four month period, during visits to six regions purposively selected from the 17 RTOs operating at that time, by two or three members of the research team, usually including one of the CE/ CM team members. The six regions we visited were selected for variation in geography, size of the Aboriginal and Torres Strait Islander population in their region and innovation in this area of training. The initial two site visits were used to pilot the interview guides which were subsequently aggregated into one interview guide (Appendix 2). We also interviewed representatives of seven other selected RTOs, mostly by telephone, to allow sampling from additional regions across Australia. For each RTO region, interviews were conducted with management or their delegate, Medical Educators and other staff employed by the RTO, CE/CMs working with the RTO as well as staff of related Indigenous Health Training Posts, GP Supervisors and Registrars. Identification of potential participants was facilitated by each RTO, who invited relevant staff, Supervisors and Registrars to participate and scheduled interview times or passed potential participant details to the researchers for later contact.In addition to frequent email reminders, recruitment of interviewees was boosted through advertising at the end of surveys and at two national CE/CM meetings, as well as through snowballing, whereby contacts were asked to forward interview requests to others working in the area.Interviews explored understandings of cultural education and mentoring, roles and challenges for CE/CMs, CE/CM training and support, and availability, content and evaluation of cultural training programs. These were generally conducted with a single participant though in some cases, where the preference of the interviewee was for a group interview, this approach was taken. Towards the end of the data collection period we conducted a large group discussion with 59\u00a0CE/CMs and Medical Educators attending a national workshop. The research team developed a discussion guide in order to explore issues where limited data had been collected up to that time and to seek participant views on themes emerging as key research findings (Appendix 3). The lead researcher (JR) presented early findings of the research in a plenary session and then participants gathered in groups including CE/CMs and Medical Educators, in which they were asked to discuss one of the two sets of questions in the discussion guide. A facilitator was appointed for each group, notes of the discussions were recorded and groups presented the results of their discussions in a following plenary session. Written informed consent was received from all interviewees including workshop attendees.In addition, we conducted telephone interviews with representatives of key stakeholder organisations identified as having important roles and perspectives on GP training in Aboriginal and Torres Strait Islander health Table .The qualitative data derived from the surveys was added to the data collected in interviews and both sets of data coded together within the thematic analysis framework described below.All interviews but one were recorded and notes were taken during the interview. Most interviews were transcribed verbatim, with audiotape recordings of a smaller number of interviews directly analysed. Data were provided in an email response from one individual to interview questions. For the larger group discussion notes from the small groups were collected and included with notes taken by research team members , for qualitative analysis.We undertook data analysis of both qualitative survey data and interview data using a framework approach  based onThe views of the CE/CM research team members were privileged at our research meetings and their endorsement of interpretations by non-Indigenous researchers was sought before these were accepted. The final analysis and report of our findings was carefully reviewed and checked not only by CE/ CM members of the research team, but also by the national Reference Group of senior CEs/ CMs in alignment with principles of decolonising research with Indigenous people .Research participants were recruited from all 17 RTOs including nominated representatives of 13. We received 55 valid survey responses, including from seven regional training delegates; 10\u00a0CE/CMs; two training posts; and 36 Registrars Table . InterviOur findings are presented below under the overarching themes of understandings about cultural education and cultural mentoring; the employment and support of CEs and CMs; and delivery and evaluation of cultural education and mentoring.\u2026 the description is basically close to my own but, I guess it is too cold, too clinical \u2026there should be a sacred sense of mystery about a person\u2019s culture, but how in the world would you pass that on? What cultural awareness does and cultural safety does, is give the broader perspective of a completely different outlook that allows us to see that we\u2019re two people in a room, in a consulting room, and your experience may well be hugely different to mine. .A key consideration was respect for the value of Aboriginal and Torres Strait Islander people\u2019s knowledge and intellectual property including that of CEs/CMs and patients.Hopefully [Registrars] will grasp the concept that although they wear a \u2018white coat\u2019 [this] does not necessarily mean they possess more \u2018knowledge\u2019 than that of the Aboriginal patient. .It is important to understand that the intellectual property belongs to the community or the individual and should not be exploited or exported as a right without consent or acknowledgement. .Participants generally supported definitions of cultural education and mentoring developed in consultation with CEs and CMs Fig. . HoweverAllow them more time to spend in community visiting people, visiting significant sites and having hands on approach. Having a real cultural immersion experience instead of a watered down classroom experience. We have this word \u201cAboriginal\u201d that\u2019s actually a coloniser\u2019s word...that puts one thing onto this hugely disparate group of cultures, and then attached to that, we have cultural education and cultural mentoring as if we can teach this one thing, and we can\u2019t. Reflection on one\u2019s own culture was considered a key component of cultural learning.\u2026it helps create awareness of how the culture of our patient and our own cultures  affect our interaction\u2026 .Face to face interaction with Aboriginal and Torres Strait Islander people was seen as a critical element in cultural education, which was recommended to include both cultural and historical impacts on health and health care and to be specific to a local context.[Cultural mentoring] also provides a contact to whom one can direct cultural questions without fear of being culturally insensitive\u2026 .Participants considered cultural education and mentoring delivered different learning experiences. Cultural education was seen to prepare Registrars for work with Aboriginal and Torres Strait Islander individuals and communities, ensuring baseline knowledge. Mentoring involved support of the learner as they worked in a community and was characterised as an ongoing dialogue with both mentor and mentee gaining from the process. From the learner perspective, the mentoring relationship was observed to offer a safe and personalised learning environment.Though the roles of both CEs and CMs were noted to vary according to location, this was particularly characteristic of the CM, described by one CE interviewee as the \u2018extended arm (into the community) of the CE\u2019. Interviewees commented that the CM needed to be the \u2018right\u2019 person, as cultural mentoring requiring particular personal qualities which can\u2019t always be learned and concern the authority given by the community for the CM to take this role.I don't want to be a formal Cultural Mentor or Educator because...I don't feel like it is my right to do that when I'm not from here. Might be different if I'm back home but not here\u2026Maybe it\u2019s about the title. Sometimes it\u2019s just around clarifying the role though, I mean demystifying it a bit because \u2026 it was never meant to be like \u2026 the person who was the owner of all the cultural knowledge stuff. It was just somebody who was Aboriginal and was local, that knew a bit about their local culture that could actually help people out a bit, or even just point them in the right direction. Critically, interviewees highlighted the right of the local community and the training post to define CE/CM roles according to local needs and context and recommended avoiding a constrictive focus on terminology.All participants valued the CE and CM roles and the vast majority also noted that these roles needed to be undertaken by Aboriginal and Torres Strait Islander people.There\u2019s mentoring going on in [the Indigenous Health Training Post], no question. We know that, but we don\u2019t employ them. It just happens. And they\u2019re not getting paid for it. They\u2019re just a health worker and they have to fit that in with all the other thousand things that\u2019s required of a health worker. The challenges in defining and setting limits around the role, including for those who combined cultural mentoring with other work roles, increased the complexity of determining appropriate remuneration and employment parameters.It [cultural mentoring] happens for nothing at the moment\u2026 someone in the community will see that someone is struggling, and \u2026 some bond will form with the health worker or an Aboriginal member of the community \u2026That additional role of CM is too much\u2026[training organisations] have to acknowledge that it\u2019s a special set of skills, and that people should be paid for it. There\u2019s been great difficulty in sort of trying to set up the notion of the more formalised CM program\u2026theoretically you want the person to be available \u201call the time\u201d. You can\u2019t pay someone to be available all the time\u2026so what is it that you need them to be able to do? Interviewees also described challenges related to appointing CMs and the importance of succession planning.How to appoint a Cultural Mentor?\u2026well, I think that it\u2019s a bit tricky\u2026 it does have to be somebody who\u2019s just generally recognised as being someone who\u2019s able to take on that title\u2026 one should be thinking about successors and potential additional people to the pool \u2026 if someone appears to be maybe a good person for the role, they should be \u2026 maybe mentored by the cultural mentor. .However some uncertainty was expressed about who was appropriate to fill the roles and how best to engage and support them. CE and CM roles were reported as filled by a variety of people employed by different organisations, with many providing cultural education or mentoring alongside other substantive roles or, particularly in the case of CMs, in unpaid and sometimes unrecognised positions. The pressure of general practice training on the relatively small Aboriginal and Torres Strait Islander workforce was clearly recognised.\u2026 that relationship can be different and can be pretty dynamic depending on where you are, who you\u2019re working with and who\u2019s giving you the education, or who\u2019s mentoring. \u2026 it must be very hard for an Aboriginal person to do this on an ongoing basis. I have witnessed staff have their personal story questioned and vilified. The ones I see give it their all die just a little every time they run a session, till they say they can do it no more. .The costs for those undertaking these often unremunerated roles were repeatedly emphasised with cultural mentoring seen to be a challenging role. When roles were not clearly agreed by all parties including CMs, Registrar and RTO expectations could be unrealistic. Participants also described the personal cost for CEs and CMs, which put them at risk of burn out and potentially less effective relationships with Registrars.\u2026remuneration is more than just money. For instance, if you work in a community as a doctor you\u2019re supplied with a house to live in, your electricity is paid, your phone is paid, your Internet is paid, your TV is paid for\u2026 Community people\u2026see these people come and go, and have plenty of advantages. It\u2019s not unreasonable, I think, sometimes for them to expect that you might take your car hunting, when it\u2019s provided for you\u2026things like paying the telephone bill for someone who is able to be a CM is a way of saying that you value that they are available on the telephone. The support of local Aboriginal and Torres Strait Islander communities was critical for CEs and CMs.\u2026as an Aboriginal person, I go back to family members. So I speak to my Elders a lot. I suppose I\u2019m pretty lucky, I\u2019m pretty strong in my culture \u2026I\u2019ve been taught a lot. Some recommended recognition of cultural learning and responsibilities in CE/ CM employment terms and conditions.Support for CEs and CMs varied greatly with key support strategies including adequate and appropriate remuneration, recognition in terms and conditions of employment, organisational support, and strong partnerships with Medical Educators.I would like to see all CEs having access to \u2026 a recognised course in Training and Assessment. Many have the cultural knowledge but lack the presentation and assessment skills which could enhance their role. How to recognise cultural stress in a non-Aboriginal person, \u2026[health professionals] tend to get quite directive, because the world is not organised how they are used to it, and then they want to organise it; they want to do that, they start trying to organise all the people, and the Aboriginal mob just turn off. Cultural education skills training also needed to take account of local context and recognise the different learning needs and styles of individuals.I don\u2019t think [formal CE training] will work for everyone because we all talk from the heart\u2026if you have to sit them in a classroom it\u2019d be the worst thing possible and I wouldn\u2019t want to do it. A CE/ CM career pathway was recommended to make these roles more attractive, in the same way that medical career pathways were clearly defined.People should have the opportunity to travel [on a career pathway] as a cultural mentor, to become a cultural educator, to become a senior cultural educator, and then to become a supervisor. Few CE/ CM professional development or career advancement opportunities were described. Although not recommended as employment requirements, professional development such as individualised professional development plans, peer mentoring and national networking were suggested to sustain CE/ CM engagement. Other training suggestions included teaching and mentoring skills training, and an understanding of cross-cultural stress and its manifestations in a non-Aboriginal person.I feel that, in general, cultural education was done well but did not know much about cultural mentoring. .Cultural education was reported to be delivered across all training regions though cultural mentoring appeared less widely available, occurring mostly in training posts.\u2026As a result of some black box of cultural education, cultural mentoring\u2026 we\u2019re producing doctors that patients go, \u201cOh, these guys I can trust.\u201d And then it would lovely to open up the lid of a black box and say, \u201cOkay, what are the components of this that we know produce those outcomes?\u201d At the moment, all we\u2019re asking is, \u201cAre you happy?\u201d \u2026but actually, some of the most effective education won\u2019t make them happy, it will make them uncomfortable and question what they\u2019re doing, because we\u2019ve got it wrong for 200 years. Interviewees described the ideal of cultural competence as a lifelong process supported at organisational level and shaped by continuous cultural education, experience and evaluation and assessment. Assessment, particularly of values and attitudes, was not widely reported and this was observed to make tailoring of cultural education to the individual Registrar difficult. The importance of involving Aboriginal and Torres Strait Islander people in evaluation and assessment was clearly highlighted.Patients need to be asked and communities, \u201cIs this doctor any good? Can you trust them? Can you trust them more after this intervention that we\u2019ve done?\u201d And that\u2019s really hard and quite a long term thing to be able to measure, so it\u2019s not done, but that\u2019s what we need to demonstrate. Integration of cultural and clinical teaching was recommended and noted to be supported by respectful partnerships between CEs/ CMs and Medical Educators as well as supportive structures within RTOs.\u2026integrates the cultural education with clinical education i.e. cultural aspects which can impact on the consultation\u2026 .For some participants however cultural education was also not prioritised sufficiently. Few RTOs extended cultural education to staff and to GP Supervisors. CEs were more often engaged in delivery of programs than in development and evaluation. Evaluative processes could be perfunctory.Aboriginal and Torres Strait Islander people working as CEs and CMs have a central role in face-to-face education of general practice Registrars about local culture and history and related impacts on health. This role was highly valued by all our participants. Whilst cultural education was described as providing base-line learning in preparation for practice with Aboriginal and Torres Strait Islander patients, cultural mentoring was seen by our participants as longitudinal, relationship-based learning and was particularly valued by Registrars working in Aboriginal and Torres Strait Islander communities, though less frequently provided compared to cultural education.Yet there were observed to be challenges in providing this model of training. A key challenge described by our participants is finding people with the skills and authority to undertaken the demanding work inherent to CE and CM roles. Remuneration is also a challenge, particularly for CMs whose work-time is difficult to quantify and who are often employed in other substantive roles and sometimes not paid at all. Appropriate remuneration and employment terms and conditions, provision of relevant professional development, strong partnerships with Medical Educators, as well as organisational support were identified as important for sustaining CE and CM roles.The focus in Australia on health care provider education regarding Indigenous cultures is shared with many other countries , 20 althAddressing this ideal, recent international reviews of cultural diversity training report a range of strategies, including tuition by those whose culture is the subject of the teaching and immersion in communities , 20, 25.Our research, prioritising the voices of those engaged in these roles, focusses on challenges encountered in delivering this model of training and makes a number of recommendations for the Australian general practice training context which are likely to be relevant to those looking for sustainable approaches to delivery of cultural competency training for healthcare providers in other countries and settings.CEs and CMs described a lack of training and confidence and poor health as well as personal costs in re-living the trauma recounted in teaching which have been described previously , 16. OurAlthough a focus on roles over titles could assist in flexibility, lack of a title may hamper promotional opportunities in non-Indigenous organisations and compromise career pathways for these experienced educators. This was a concern for our interviewees as was the lack of professional development for CEs and CMs. Suggestions for professional development included skills and knowledge in medical educational approaches, however context was noted to be important with training such as in use of technologically based educational approaches, unlikely to be culturally appropriate for all CE/CMs, whilst remote and on-line approaches may be useful for others. There is clearly a need to respect and value Indigenous teaching approaches such as story- telling which have also been recommended in both research and clinical environments .This need for respect infuses much of our data. It includes respectful relationships with learners and with Medical Educators and organisational respect for the CEs and CMs. Calls for organisational support for cultural education from our participants echo similar advocacy in the international literature for systems approaches to improving cultural competence in healthcare , 23. TheOrganisational respect was seen to include wider support for Aboriginal and Torres Strait Islander peoples such as Reconciliation Action Plans which aim to make non-Indigenous organisations a safe place for Aboriginal and Torres Strait Islander people to work . RespectIn a cycle perhaps reflective of the cultural context of this research, the CEs and CMs for whom the organisation needs to be culturally safe can assist both in developing this capacity for organisational critical reflection, as well as establishing and maintaining connections to communities that will sustain change over time.A key strength of this research has been the leadership and guidance by CEs and CMs from inception, as well as within the research team and overseeing the research. Whilst this strong engagement with those whose experience was under investigation may be considered by some to be a conflict of interest, it also assisted us in gaining the trust of participants and provided a sound understanding of the research context as well as ensuring the research was culturally appropriate. The wide range of participants both in terms of roles and geography was a unique feature of our research. Though survey respondents were unlikely to constitute a representative sample of the various stakeholder groups, we found substantial concordance between qualitative survey responses and interviews.Our research, undertaken in an Australian general practice training context, reflects on the unique roles of CEs and CMs in this context and provides a valuable opportunity to learn from those engaged in these roles. Their reflections highlight the importance of CE and CM roles as well as challenges in sustaining them. The recommendations of our participants concerning professional and organisational support of those engaged in this work, align with the international literature and provide valuable insights for those seeking to develop respectful and effective cultural education programs."}
+{"text": "Compared to DGEBA (Bisphenol A Diglycidyl Ether)-based material, these biobased thermosets differ by their cross-linking ratio, the nature of the phosphorylated function and the presence of an aliphatic chain. Eugenol has led to thermosets with higher glass transition temperatures due to a higher aromatic density. The flame-retardant properties were tested by thermogravimetric analyses (TGA), a pyrolysis combustion flow calorimeter (PCFC) and a cone calorimeter. These analyses demonstrated the efficiency of phosphorus by reducing significantly the peak heat release rate (pHRR), the total heat release (THR) and the effective heat of combustion (EHC). Moreover, the cone calorimeter test exhibited an intumescent phenomenon with the residues of phosphorylated eugenol thermosets. Lastly, the higher flame inhibition potential was highlighted for the phosphonate thermoset.Epoxy materials have attracted attention for many applications that require fireproof performance; however, the utilization of hazardous reagents brings about potential damage to human health. Eugenol and cardanol are renewable, harmless resources (according to ECHA) that allow the achievement of synthesis of novel phosphorylated epoxy monomers to be used as reactive flame retardants. These epoxy building blocks are characterized by  Epoxy thermosets are among the most used polymers in the world due to their excellent chemical, thermal, mechanical and dielectric properties. They are found in many applications such as coatings, adhesives, composites and high performance materials ,2,3. Des3) and phenols by our team [In order to increase both aromatic density and thermal stability, biobased epoxy trimers have been obtained using a phosphorus linker  leading to materials with a high biobased content. We finally report the characterizations of those biobased high performance thermosets to evaluate the structure-properties relationship. Consequently, the impact of the phosphonate or the phosphate, the functionality as well as the influence of the structure on flame-retardant and thermo-mechanical properties have been studied.The aim of this work is to design biobased epoxy monomers based on eugenol and cardanol to prepare thermosets with a high phosphorus content and two oxidation degrees of phosphorylated functions. Indeed, the presence of the phosphorus atom inside the network allows to obtain excellent intumescent properties without the loss of thermo-mechanical properties, the major drawback of the addition of flame-retardant additives to thermosets. Furthermore, the reactive way prevents migration of flame-retardant compounds out of the network. This original grafting of cardanol and eugenol monomers owing to phosphorylated functions increases the functionality of biophenol monomers. Trimerization or dimerization is followed by the epoxidation of double bonds in mild conditions, before curing with a benzylic diamine  leads to a functionality of 2. The epoxidation reaction was then performed. Meta-chloroperoxybenzoic acid (m-CPBA) was used for the epoxidation reaction of allylic function of eugenol, whereas hydrogen peroxide was used for the epoxidation of internal unsaturations of cardanol. Due of the toxicity of m-CPBA, the use of hydrogen peroxide was favored when relevant. In fact, hydrogen peroxide is used for the epoxidation of internal unsaturations whereas m-CPBA is used for terminal unsaturations [Three monomers from eugenol  and one urations .1H and 31P NMR. The 1H NMR of the TEEP monomer and all the 31P NMR are in the 1H NMR spectra of eugenol, dieugenylphosphate (DEP) and di(epoxized-eugenyl)phosphate (DEEP). In the spectra of eugenol, the signals at 6.7 and 6.9 ppm were assigned to aromatic protons a, b and c. The protons of the double bond can be found at 5.0 (g) and 5.9 ppm (f) as multiplets. The methoxy protons (d) were localized at 3.8 ppm and finally the allylic protons (e) were observed at 3.3 ppm. In the spectrum of DEP, only a few changes are observed. Indeed, the aromatic protons (a) are deshielded when the hydroxyl function is grafted onto the phosphate. Furthermore, the signals of the benzene protons were observed at 7.1\u20137.2 ppm (j1) and at 7.3\u20137.4 ppm (i1 and h1). Finally, in the spectrum of DEEP, the disappearance of both olefinic and allylic protons was observed. They were replaced by the epoxy protons (f2 and g2) and by the glycidyl protons (e2) respectively at 3.0, 2.9 and 2.6 ppm. All of these observations confirmed the chemical structure of DEEP epoxy monomer in a quantitative yield.The structures of the three new monomers, DEEP, DEEP-Ph and TECP  were ch1H NMR spectra of eugenol, dieugenylphosphonate (DEP-Ph) and di(epoxized-eugenyl)phosphonate (DEEP-Ph). In the spectrum of DEP-Ph, the aromatic protons (a) are deshielded when the hydroxyl function is grafted onto the phosphonate. Furthermore, the signals of the benzene protons were observed at 7.4\u20137.7 ppm (i1 and j1) and at 8.0\u20138.1 ppm (h1). Finally, in the spectrum of DEEP-Ph, the disappearance of both olefinic and allylic protons was observed. They were replaced by the epoxy protons (f2 and g2) and by the glycidyl protons (e2) respectively at 3.0, 2.9 and 2.6 ppm.1H NMR spectra of cardanol, TCP and TECP. In the spectrum of cardanol, the signals at 7.1, 6.8 and 6.7 ppm were assigned to aromatic protons 13, 14, 15 and 16. The protons of the terminal unsaturation can be found at 5.8 (2) and 5.0 ppm (1) whereas the internal unsaturations were localized at 5.4 ppm . The protons in \u03b1 position of two double bonds were observed at 2.7 ppm (6 and 3) whereas the protons in \u03b1 position of only one double bond can be found at 2 ppm (9 and 3\u2032). The benzylic protons are localized at 2.5 ppm (12). The protons at 1.6 and 1.3 ppm were assigned to the protons of the aliphatic chain . Finally, the saturated terminal protons (1\u2032) were observed at 0.9 ppm. In the spectrum of TCP, the main change comes from the deshielding of the aromatic protons  when the hydroxyl function is grafted onto the phosphate. Finally, the internal unsaturations were epoxidized using hydrogen peroxide. Indeed, this reagent is well known to hardly epoxidize the terminal unsaturations. This result was confirmed by 1H NMR. Indeed, 29% of terminal and 88% of internal double bonds were respectively epoxidized. Furthermore, the signals of the formed epoxy functions were observed at 3.0 ppm. The COSY 1H-1H NMR was used to assign all the protons  in order to synthesize epoxy thermosets. Both phosphonate and phosphate linkers allow to bind two or three molecules together, and to form epoxy monomers with high aromatic density and functionality. The reaction of these monomers with short diamine leads to rigid thermosets.These four epoxy monomers have been reacted with MXDA has been selected for its compatibility with the original phosphorus-containing epoxy monomers and the stiffness conferred by the aromatic ring. Furthermore, a new biobased synthesis pathway of MXDA has been recently reported by J\u00e9r\u00f4me et al. . In orde1H NMR titration with an internal standard. The results are respectively 195 g/eq for TEEP, 310 g/eq for DEEP, 241 g/eq for DEEP-Ph and 240 g/eq for TECP.A stoichiometric ratio of two epoxy functions for one amine function was used for the synthesis of all materials. The EEW was calculated via The degree of cross-linking of the networks was characterized with gel content measurement . The calg values. We observed some differences for Tg values which are 84, 98 and 109 \u00b0C of TEEP, DEEP and DEEP-Ph materials, respectively. Values of Tg are linked to the mobility of the network and are thus influenced by its rigidity but also linked to the mobility of each segment. With kneecap movements, methoxy groups have shown an important influence on the glass transition temperature of materials [g for TEEP material which possesses three methoxy groups against two for the DEEP material. This also explains the high Tg of the DGEBA material around 115\u2013120 \u00b0C [g of the eugenol-based materials.DSC measurements have been performed on each material. The curves are summarized in aterials ,36. This5\u2013120 \u00b0C ,38. Inde\u03b1 values followed the same trend than Tg values measured by DSC. Narrow peaks of tan \u03b4 for all materials, except the one with DEEP, show a good homogeneity. According to the theory of rubber elasticity [rubbery, \u03bd\u2032 and T\u03b1. According to the values of E\u2032 rubbery and \u03bd\u2032 the stiffness of eugenol-based materials ranged as follows: DEEP > DEEP-Ph > TEEP. The order of T\u03b1 for DEEP and DEEP-Ph is inverted compared to E\u2032 rubbery measurements but the tendency of those results is similar to Tg for which the DSC measurement and explanation has been given earlier. Such a difference in cross-linking density could therefore be explained by the presence of side reactions. Transesterification can occur with phosphate groups and high temperature of curing can promote this side reaction [rubbery could be expected for TEEP but the higher flexibility brought by methoxy groups as well as the space between chains impact the network, and this can explain these lower values. Finally, TECP was the softest material due to its long and dangling aliphatic chains with a E\u2032rubbery of 1.3 MPa at T\u03b1+50 \u00b0C and a crosslinking density of 140 mol/m3.The thermo-mechanical properties of the different materials have been compared by DMA and the results are outlined in asticity ,40, the reaction . Phosphad5%. These degradation temperatures were similar but slightly lower to those obtained from DGEBA. Furthermore, the char yields of DEEP, DEEP-Ph and TEEP-based materials are higher than char yields of TECP-material because they present higher aromatic densities than the cardanol formulation. In all cases, the char yields are higher compared to DGEBA-based thermoset.Firstly, TGA measurements were carried out under nitrogen atmosphere to characterize thermal stability of the materials and the curves are shown in The flame-retardancy of thermosets was then assessed using PCFC B. DGEBA-Interestingly HRR curves show two peaks for TECP material which may be ascribed to the heterogeneity of the network as already discussed. Another hypothesis is that the second peak corresponds to the decomposition of aliphatic chains. Indeed, the temperature of this peak is close to the decomposition temperature of polyolefins as polyethylene  or polypOn the contrary TEEP, DEEP and DEEP-Ph lead to drastic decrease of pHRR and THR and strong enhancement of char. The heat of combustion is also lowered. Such high performances in comparison to TECP thermosets are due to the higher aromatic content of TEEP, DEEP and DEEP-Ph but also to the higher phosphorus content of corresponding thermosets. We observe that there is no significant difference between phosphate and phosphonate-containing materials. The DEEP-Ph thermoset exhibits a complex decomposition pathway and a very broad range of decomposition temperature (270\u2013550 \u00b0C) allowing an important decrease of the heat release rate in comparison to other phosphate-containing thermosets. Char yields measured by PCFC are quite close to those obtained by TGA.In order to get an insight on our results, PCFC data are compared to other comparable data already published. While sample size in cone calorimeter is not standard, it is not possible to compare properly our results.Flammability of epoxy depends greatly on the groups present in the network. According to literature, THR can be expected in a large range from 11 to more than 30 kJ/g for phosphorus-free epoxy thermosets . Note thM\u00e9nard et al. have compared the influence of phosphorus on different epoxy thermosets . In thisEugenol-based thermosets, and especially TEEP and DEEP ones, exhibit flammability data at microscale close to those obtained with a phloroglucinol-containing epoxy thermoset and difurfurylamine : THR andCone calorimeter tests were carried out to give an insight on the flammability at bench scale A. Indeed2) and leaves only a small amount of char. Once again, this is similar to the fire behavior already observed for DGEBA-IPDA.The DGEBA-MXDA thermoset exhibits a very high pHRR  and moderate char content (15.4 wt.%). Interestingly, TECP and DGEBA thermosets have the same char content at microscale (10-12 wt.%) but a large difference is observed in Cone Calorimeter tests . This is due to the presence of phosphorus which improves the thermo-oxidative stability of char avoiding its decomposition at the end of the test when the flame is vanished and pyrolysis becomes aerobic.From cone calorimeter results, the TECP thermoset shows a pHRR close to 700 kW/m2) but a much lower THR (9\u201310 kJ/g) associated to enhanced charring .TEEP, DEEP and DEEP-Ph thermosets show a slightly lower pHRR  is not clear and may be counterbalanced by the higher phosphorus content of DEEP thermoset.An interesting result concerns the influence of phosphorus on gas-phase mechanisms. Indeed, the effect heat of combustion (EHC) parameter highlighted the efficiency of the phosphorus radicals from phosphonate in the gas phase compared to the phosphate ones. Furthermore, while the char contents in the cone calorimeter and at a microscale (PCFC or TGA) are roughly similar, combustion efficiency \u03a7 can be properly calculated as the ratio between EHC and the heat of complete combustion measured in PCFC. The combustion efficiency is around 0.8 for the DGEBA thermoset, which corresponds to quite well-ventilated combustion. Combustion efficiency decreases for phosphorus-containing thermosets, a little  reduction was observed for DEEP and TEEP. The reduction is slightly more significant for DEEP and mostly DEEP-Ph (a 30% decrease was obtained for this last thermoset). This parameter X may be ascribed to the flame inhibition effect, which is quite uncommon for phosphate groups. Indeed, phosphorus with a high oxidation degree is known to act mainly or exclusively as a char promoter . Alterna2 ratio was drawn versus heat release rate during intense burning  to avoid to take into account the oxidative decomposition of char at lower temperature at the end of the test  and the residue content, it is possible to calculate the fraction of phosphorus remaining in the residue after cone calorimeter. meta-chloroperoxybenzoic acid (m-CPBA) (77%), dichlorophenylphosphine oxide, phenyl dichlorophosphate, hydrogen peroxide (30% aqueous), formic acid and p-toluenesulfonic acid (PTSA) were purchased from Sigma Aldrich Merck . Cardanol (Cardolite\u00ae NX-2026) was purchased from Cardolite . Meta-xylylenediamine (MXDA) was obtained from CTP . All materials were used as received. Deuterated solvent CDCl3 was obtained from Eurisotop for NMR study. Ethyl acetate (EtOAc), pentane and toluene were purchased from Sigma-Aldrich.Eugenol, phosphorus oxychloride, triethylamine, 1H and 31P NMR) analyses were performed in deuterated chloroform (CDCl3) using a Bruker Avance 400 MHz NMR spectrometer at a temperature of 25 \u00b0C. NMR samples were prepared as follows: 10 mg of product for 1H experiment in around 0.4 mL of CDCl3. The chemical shifts were reported in part per million relative to tetramethylsilane. Spin multiplicity is shown by s = singlet, d = doublet, t = triplet, m = multiplet.NMR analyses Proton and carbon Nuclear Magnetic Resonance  were performed using a TG 209F1 apparatus (Netzsch) at a heating rate of 20 \u00b0C/min. Approximately 10 mg of sample was placed in a platinum crucible and heated from room temperature to 900 \u00b0C under nitrogen atmosphere (60 mL/min).n-octadecane and n-octane standards. Nitrogen was used as the purge gas. Approximately 10 mg of sample were placed in perforated aluminum pans and the thermal properties were recorded between \u2212100 \u00b0C and 200 \u00b0C for the tri(epoxized-eugenyl)phosphate (TEEP) and tri(epoxized-cardanyl)phosphate (TECP)-based materials and from 25 \u00b0C to 250 \u00b0C for the di(epoxized-eugenyl)phosphonate (DEEP)-based material at 20 \u00b0C/min to observe the glass transition temperature. The Tg values were measured on the second heating ramp to erase the thermal history of the polymer. All the reported temperatures are mean values.Differential Scanning Calorimetry (DSC) analyses were carried out using a NETZSCH DSC200F3 calorimeter. Constant calibration was performed using indium, g \u2212 100 \u00b0C to Tg + 100 \u00b0C, at a frequency of 1 Hz with a fixed strain of 10\u22125 m. These conditions have been chosen to study the elastic behavior of the materials. All the thermosets were analyzed at least two times for repeatability.Dynamic mechanical analyses (DMA) were carried out on Metravib DMA 25 with Dynatest 6.8 software. Samples were tested according to uniaxial tension mode while heating at a rate of 3 \u00b0C/min from \u2248 T1H NMR The Epoxy Equivalent Weight (EEW) is the amount of product needed for one equivalent of reactive epoxy function. It was determined by 1H NMR using an internal standard (benzophenone). A known weight of product and benzophenone was poured into an NMR tube and 500 \u03bcL of CDCl3 were added. It was determined using Equation (1) by comparing the integral of the protons of the benzophenone and the integral of the epoxy moiety.PhCOPh is the integration of the benzophenone protons; \u222bepoxy is the integration of the protons of the epoxy function; Hepoxy refers to the number of protons of the epoxy function; HPhCOPh is the number of protons of the benzophenone; mepoxy is the weight of the product; mPhCOPh refers to the weight of the benzophenone, MPhCOPh is the molecular weight of the benzophenone.Titration of the epoxy equivalent weight by \u03b1 + 50 , and at very small deformations. Under these hypotheses, the cross-linking density (\u03bd\u2032), can be obtained from Equation (2), where E\u2032 is the storage modulus, R is gas constant and T\u03b1 is the temperature, in K, of the transition from vitreous to rubber domain of material determined at the maximum of the tan \u03b4 curve. Calculated values are given for information purposes only, and they can only be compared.Cross-linking density from rubber elasticity theory ,40, the 2 is the mass of the material after swelling in THF and m1 is the initial mass of the material.Swelling index three samples of around 30 mg each were separately put in THF for 24 h. The swelling index (SI) was calculated using the Equation (3) where m3 is the mass of the material after drying and m1 is the initial mass of the material.For the gel content after SI measurements, three samples were dried in a ventilated oven at 70 \u00b0C for 24 h. The gel content (GC) was calculated using the Equation (4), where mPyrolysis combustion flow calorimeter flammability was first investigated using a pyrolysis combustion flow calorimeter which was developed by Lyon and Walters . The sam3 was placed at 2.5 cm below a conic heater and isolated by rock wool. Plates were wrapped in aluminum foil but no retained frame was used. The samples were exposed to a heat flux of 35 kW/m2 in well-ventilated conditions (air rate 24 L/s) in the presence of a spark igniter to force the ignition. HRR was determined according to oxygen depletion (Huggett\u2019s relation) as in the pyrolysis combustion flow calorimeter (PCFC). Note that the dimensions of samples are not standard and their masses are quite low. The standard deviations of cone calorimeter measurements are around 15%.Cone calorimeter fire behavior was also studied using a cone calorimeter (fire testing technology) which is a powerful tool to investigate the fire behavior of polymers at bench scale. Samples were previously conditioned away from air and moisture. A horizontal sample sheet of 70 \u00d7 80 \u00d7 2 mmScanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX). The bulk structure of residues was observed using an SEM microscope (FEI Quanta 200 SEM). Elemental composition was analyzed with energy-dispersive X-ray detector (Oxford) coupled with the SEM microscope. For each residue, three analyses were carried out. Note that in the case of reference epoxy, residue is not expanded and only the surface was analyzed.The synthesis of TEP is described as follows: Eugenol  and triethylamine  was solubilized in ethyl acetate (100 mL). Phosphorus oxychloride  was added dropwise, at 0 \u00b0C, to the solution. The reaction mixture was stirred at room temperature for 24 h. When the reaction was complete, the mixture was diluted with 100 mL of ethyl acetate and washed three times with 100 mL of deionized (DI) water and one time with brine solution (100 mL). The organic layer was dried with anhydrous magnesium sulfate and filtered. The filtrate was purified by silica gel chromatography (8:2 Pentane:EtOAc) to give 10.6 g of TEP as yellow oil (97% yield).1H NMR : \u03b4 (ppm) 7.31 ; 6.67\u20136.76 ; 5.87\u20135.99 ; 5.03\u20135.14 ; 3.75 ; 3.34 .31P NMR : \u221215.78.The synthesis of TCP is described previously with: cardanol , triethylamine , ethyl acetate (410 mL) and phosphorus oxychloride . Purification: silica gel chromatography (9:1 Pentane:EtOAc) to give 40 g of TCP as yellow oil (73% yield).1H NMR : \u03b4 (ppm) 7.21\u20137.25 ; 6.98\u20137.08 ; 5.76\u20135.88 ; 5.27\u20135.49 ; 4.94\u20135.10 ; 2.75\u20132.88 ; 2.54\u20132.63 ; 1.94\u20132.12 ; 1.52\u20131.66 ; 1.16\u20131.45 ; 0.84\u20130.96 .31P NMR : \u221217.66.The synthesis of DEP is described previously with: eugenol , triethylamine , ethyl acetate (750 mL) and phenyl dichlorophosphate . Purification: silica gel chromatography (8:2 Pentane:EtOAc) to give 16 g of DEP as yellow oil (24% yield).1H NMR : \u03b4 (ppm) 7.33\u20137.39 ; 7.17\u20137.25 ; 6.68\u20136.82 ; 5.90\u20136.00 ; 5.04\u20135.16 ; 3.76 ; 3.37 .31P NMR : \u221212.82.The synthesis of DEP-Ph is described previously with: eugenol , triethylamine , ethyl acetate (512 mL) and phenylphosphonic dichloride . Purification: silica gel chromatography (8:2 Pentane:EtOAc) to give 24 g of DEP-Ph as a white solid (52% yield).1H NMR : \u03b4 (ppm) 8.0\u20138.10 ; 7.42\u20137.59 ; 7.17 ; 6.64\u20136.72 ; 5.84\u20135.99 ; 4.98\u20135.10 ; 3.68 ; 3.32 .31P NMR : 12.94.meta-chloroperbenzo\u00efc acid (m-CPBA 77%)  in ethyl acetate (200 mL). The reaction mixture was stirred at room temperature for 24 h. The organic layer was washed several times with saturated sodium bicarbonate, brine and deionized water. The organic layer was dried with anhydrous magnesium sulfate and filtered. The filtrate was distilled under vacuum and purified by column chromatography (Pentane/EtOAc 50:50) to afford 10.3 g of TEEP as brown viscous oil (93% yield).TEEP was synthesized as follows. A solution of TEP  in ethyl acetate (50 mL) was added dropwise to a cooled suspension of 1H NMR : \u03b4 (ppm) 7.30\u20137.37 ; 6.73\u20136.87 ; 3.78 ; 3.10\u20133.16 ; 2.75\u20132.96 ; 2.53 .31P NMR : \u221215.92.p-toluenesulfonic acid  in toluene or EtOAc (4 mL) was heated to 50 \u00b0C. Then, a hydrogen peroxide solution (30% (w/w) in H2O)  was added dropwise. The reaction mixture was stirred at 65 \u00b0C for 24 h. The organic layer was washed several times with saturated sodium bicarbonate, brine and deionized water. The organic layer was dried with anhydrous magnesium sulfate and filtered. The filtrate was distilled under vacuum and purified by column chromatography (Pentane/EtOAc 8:2).TECP was synthesized as follows: A solution of TCP , formic acid , 1H NMR : \u03b4 (ppm) 7.18\u20137.25 ; 6.97\u20137.06 ; 5.74\u20135.94 ; 5.4\u20135.62 ; 4.95\u20135.22 ; 3.03\u20133.21 ; 2.86\u20133.01 ; 2.57 ; 2.14\u20132.45 ; 1.69\u20131.85 ; 1.18\u20131.67 ; 0.82\u20131.03 .31P NMR : \u221217.65.m-CPBA  and ethyl acetate (430 mL). Purification: silica gel chromatography (2:8 Pentane:EtOAc) to give 11 g of DEEP as yellow oil (65% yield).The synthesis of DEEP is described previously with: DEP , 1H NMR : \u03b4 (ppm) 7.35\u20137.39 ; 7.19\u20137.27 ; 6.77\u20136.88 ; 3.79 ; 3.13\u20133.19 ; 2.80\u20132.89 ; 2.56 .31P NMR : \u221216.52.m-CPBA  and ethyl acetate (680 mL). Purification: silica gel chromatography (2:8 Pentane:EtOAc) to give 14 g of DEEP-Ph as a white solid (55% yield).The synthesis of DEEP-Ph is described previously with: DEP-Ph , 1H NMR : \u03b4 (ppm) 8.04\u20138.12 , 7.48\u20137.64 ; 7.21 ; 6.72\u20136.83 ; 3.75 ; 3.12\u20133.18 ; 2.78\u20132.86 ; 2.55 .31P NMR : 13.00.Several formulations were carried out using TEEP, TECP, DEEP, DEEP-Ph, DGEBA and MXDA. In order to prepare the materials, the mass of amine was calculated using the Equation (5). The Amine Hydrogen Equivalent Weight (AHEW) and the Epoxy Equivalent Weight represent the amount of product needed for one equivalent of reactive function.2 have been synthetized to characterize flame retardant properties.The materials were synthesized with a molar ratio epoxy/amine of 2:1. Indeed, two epoxy groups react with each hydrogen of the primary amine. The amine and the epoxy were stirred using a speedmixer under vacuum and then the homogeneous mixture obtained was put in an aluminum mold for three hours at 80 \u00b0C and then cured at 150 \u00b0C for one hour for the eugenol derivatives, while for the cardanol derivative the mixture was heated at 120 \u00b0C overnight and then cured at 150 \u00b0C for three hours. Sheets of 7 \u00d7 8 cmg. These new eugenol-based thermosets exhibit good thermo-mechanical properties with high char yields. The thermal stability of these thermosets was finally characterized by TGA and their flame retardant properties by PCFC and cone calorimeter tests. The incorporation of phosphorus as a linker between phenols confers flame-retardant properties to the networks, without the use of flame-retardant additives. This reactive approach prevents the migration of flame-retardant additives out of the network. Furthermore, phosphonate or phosphate functions do not hang off the main chain of the network and are completely integrated to it. Hence, the incorporation of phosphorus leads to a significant decrease of flammability as evidenced by pHRR and THR values. Furthermore, eugenol lead to monomers with better flame retardant properties than cardanol which contains long aliphatic chain. This chain is unfavorable to the char promotion and contains high carbon amount, contributing to heat release. Furthermore, phosphate and phosphonate groups are both equally efficient to promote char and reduce flammability for these materials. Moreover the residues are expanded, contrarily to residues from DGEBA and TECP thermosets, relating to great intumescence properties. The flame inhibition effect was confirmed as an additional flame retardancy mode-of-action with a better efficiency for the phosphonate function. In conclusion, novel biobased aromatic monomers were synthesized, giving access to high performance thermosets with good thermal, thermo-mechanical and flame retardant properties.In this paper, we synthesized four biobased epoxy monomers from cardanol and eugenol without using BPA or epichlorohydrin. Four different thermosets have been obtained after curing with a benzylic amine (MXDA) and their thermo-mechanical properties have been compared to a petroleum-based epoxy thermoset coming from DGEBA. Due to the high aromatic density of eugenol-based materials, high glass transition temperatures are obtained whereas the long aliphatic chain of cardanol monomers leads to flexible thermosets with low T"}
+{"text": "Mycobacterium abscessus. Multiple resistance to antibiotics and the ability to form biofilm contributes considerably to the treatment failure. The search of novel anti-mycobacterial agents for the control of biofilm growth mode is crucial. The aim of the present study was to evaluate the activity of carvacrol (CAR) against planktonic and biofilm cells of resistant RGM strains. The susceptibility of RGM strains (n = 11) to antibiotics and CAR was assessed by MIC/MBC evaluation. The CAR activity was estimated by also vapour contact assay. The effect on biofilm formation and preformed biofilm was measured by evaluation of bacterial growth, biofilm biomass and biofilm metabolic activity. MIC values were equal to 64 \u03bcg/mL for most of RGM isolates (32\u2013512 \u03bcg/mL), MBCs were 2\u20134 times higher than MICs, and MICs of vapours were lower (16 \u03bcg/mL for most RGM isolates) than MICs in liquid phase. Regarding the biofilm, CAR at concentrations of 1/2 \u00d7 MIC and 1/4 \u00d7 MIC showed a strong inhibition of biofilm formation (61\u201377%) and at concentration above the MIC (2\u20138 \u00d7 MIC) produced significant inhibition of 4- and 8-day preformed biofilms. In conclusion, CAR could have a potential use, also in vapour phase, for the control of RGM.Rapidly growing mycobacteria (RGM) are environmental bacteria found worldwide with a propensity to produce skin and soft-tissue infections. Among them, the most clinically relevant species is  Mycobacterium abscessus is an emerging human pathogen causing lung infection but also responsible for wound, catheter and eye infections and also tattooing , a major constituent of many essential oils of the Labiatae family, is classified among the substances generally recognized as safe (GRAS) and approved for use in food . Severaldermidis \u201321, Salmhymurium , Listeriytogenes  titaniumytogenes  and carbytogenes . Despite0 \u03bcg/mL) .The goal of the present study was to extend the research and to evaluate the antimicrobial activity of CAR, either in liquid and vapour phase, against different species of resistant RGM. Moreover, the antibiofilm activity of CAR, in liquid phase, was also evaluated against RGM species capable to form biofilm.M. abscessus #09716, #29904, #30235, #70513, #73596, #90459, #74600; M. chelonae #74471; M. fortuitum #26647; M. mucogenicum #45646 and M. smegmatis #44041 by line-probe reverse hybridization assay  and conventional biochemical and cultural methods, as suggested by Clinical and Laboratory Standard Institute (CLSI)  wer wer28] wThe final concentration of CAR ranging from 4096 to 8 \u03bcg/mL. Ethanol maximum concentration was 1.25% (v/v). Minimum inhibitory concentration (MIC) was determined by microdilution methods and interpreted following the Clinical and Laboratory Standards Institute guidelines . Growth 4 CFU/mL). Different volumes of CAR were added in a glass slide placed in the upper lid of each Petri dish. The final concentration ranging from 4096 to 8 \u03bcg/mL of the air space. The plates were sealed with adhesive seals and incubated at 30\u00b0C for 4\u20135 days. Each assay was made in triplicate. The lowest concentration of CAR vapours preventing visible growth was recorded as MIC.The effect of CAR vapours was evaluated with an invert Petri dishes method as previously described ,32. BrieMycobacterium strains was tested on 96-well polystyrene flat-bottomed microtitre plates as previously described [The biofilm-forming ability of escribed . Then, t8 CFU/mL by optical density (OD492) measurements and diluted 1:100 was inoculated (100 \u03bcL) into each well of microtiter plates in the presence of sub-MICs of CAR (100 \u03bcL) or medium (control). The correlation between CFU/mL and optical density (OD492) was obtained through the use of a standard curve and the following equation which was developed by plotting the OD values as a function of the log CFU/mL:The effect of sub-inhibitory concentrations (sub-MICs) of CAR (ranging from 1/2 \u00d7 MIC to 1/16 \u00d7 MIC) on biofilm-forming ability was studied . BacteriAfter incubation for 3\u20134 days at 37\u00b0C, the effect on planktonic bacterial growth i) and biofilm biomass ii) was estimated as follows:492 using a spectrophotometer EIA reader ;the medium  was removed from the each well and transferred to wells of a new 96-well polystyrene flat-bottomed microtitre plates in order to evaluate the total mass amount by measuring the OD492 measurements. The biofilm reduction was estimated by the following equation:the biofilm biomass was evaluated as follows: the wells of microtitre plates were washed twice with sterile PBS, dried, stained with 0.1% safranin and washed with water. The biofilm biomass eluted in acetic acid 30% (v/v) was evaluated by ODAll experiments were made in triplicate.6 CFU/mL and were inoculated (200 \u03bcL) in 96-well polystyrene flat-bottomed microtitre plates. After incubation for 4 and 8 days at 37\u00b0C, the planktonic bacterial growth was dislodged and the wells were washed with sterile PBS and filled with twofold dilutions of CAR, ranging from the MIC to a 16-fold the MIC  and the ODBiofilm metabolic activity. The planktonic growth was dislodged and, after washing, each well was treated with the Cell Proliferation Kit II XTT  as previously reported . This asAll experiments were made in triplicate.The reduction of biofilm supernatant growth/biofilm biomass/biofilm metabolic activity was estimated using p value <0.05 was assumed as significant.ANOVA was used to evaluate the significant differences between the samples treated with CAR and the samples without CAR. A M. abscessus strains were resistant to AMK , CIP , CLR after 72 h incubation , CLR after 14 incubation days , LZD , MEM , SX , TGC  and intermediate to AMK , FOX , LZD ; M. chelonae #74471 was resistant to AMK (MIC 64 \u03bcg/mL), FOX (MIC > 128 \u03bcg/mL), CIP (MIC > 4 \u03bcg/mL), MEM (MIC >16 \u03bcg/mL), SX (MIC > 64 \u03bcg/mL) and intermediate to LZD (MIC 16 \u03bcg/mL); M. fortuitum #26647 was resistant to CIP (MIC 4 \u03bcg/mL), CLR after 14 incubation days (MIC > 8 \u03bcg/mL), SX (MIC: > 64 \u03bcg/mL) and intermediate to CLR after 72 h of incubation (MIC 4 \u03bcg/mL), FOX (MIC 32 \u03bcg/mL); M. mucogenicum #45646 was resistant to CIP (MIC > 4 \u03bcg/mL), SX (MIC > 64 \u03bcg/mL); M. smegmatis #44041 was resistant to SX (MIC > 64 \u03bcg/mL).The results of susceptibility tests showed that M. abscessus isolates were 32 \u03bcg/mL (#44041), 128 \u03bcg/mL (#09716), 256 \u03bcg/mL (#29904) and 512 \u03bcg/mL (#30235). The MBC values were 2\u20134 times higher than MICs (from 64 \u03bcg/mL to 2048 \u03bcg/mL) in all RGM strains, except for one isolate (# 45646) for which the MIC was equal to the MBC value. The MIC values obtained using vapour contact assays were lower than those in liquid medium for all the RGM isolates. Specifically, MICs were equal to 16 \u03bcg/mL for all strains except for one of them (#09716), for which the MIC was 64 \u03bcg/mL.The susceptibility patterns of RGM strains to CAR, in liquid and vapour phase are shown in Mycobacterium strains, i.e. M. abscessus  and M. fortuitum  was evaluated in terms of influence on biofilm supernatant growth, biofilm biomass and biofilm metabolic activity . CAR shoM. abscessus #29904, the effective percentage reductions of biofilm supernatant growth , biofilm biomass  and biofilm metabolic activity  were detected with 2 \u00d7 MIC, 4 \u00d7 MIC and 2 \u00d7 MIC, respectively. Moreover, even using increasing CAR concentrations (from 2 x MIC to 16 x MIC), similar OD results for 4 d-biofilm were detected. This evidence is probably related to the higher MIC value of CAR (256 \u03bcg/&mL) respect to most RGM (64 \u03bcg/mL).Specifically for M. abscessus #73596, the effective percentage reductions of biofilm supernatant growth , biofilm biomass  and biofilm metabolic activity  were detected with 8 \u00d7 MIC, 4 \u00d7 MIC and 4 \u00d7 MIC, respectively.For M. abscessus #09716 and M. abscessus #70513. Despite effective percentage reductions  of biofilm supernatant growth of M. abscessus #09716 were achieved with the same concentration of CAR (4 \u00d7 MIC), the significant biofilm biomass and biofilm metabolic activity decrease (> 65%) was detected with 4 \u00d7 MIC for 4 d-biofilm and 2 \u00d7 MIC for 8 d-biofilm. Equally, significant reduction of biofilm supernatant growth (> 60%) of 4 d- and 8 d-biofilm M. abscessus #70513 was achieved with 4\u20138 \u00d7 MIC and 2 \u00d7 MIC, respectively.Differently, a more susceptibility of 8 d- respect to 4 days old biofilm was detected for Curcuma longa, against a lung isolate of M. abscessus [M. avium subsp. paratuberculosis and as a monoterpenic inhibitor of the chorismate mutase enzyme of M. tuberculosis [M. tuberculosis, have efflux pumps activity and induce rough bacillary and agglomerates [Hemophilus infuenzae, Streptococcus pyogenes, Streptococcus pneumoniae, and Staphylococcus aureus pathogens [The search of novel anti-mycobacterial agents for the control of biofilm growth mode is crucial. At this regard, we have recently reported the antimicrobial activity of curcumin, a phenolic compound extracted from the bscessus . CAR hasrculosis , 37. Moromerates . Howeveromerates . The preomerates . In addiomerates ,41. Carvomerates  describeomerates ,44. Specathogens ,45 has bM. fortuitum represent another important finding. Concentrations below the MIC (1/2\u20131/4 \u00d7 MIC) impaired biofilm formation whereas concentration above the MIC (2\u20138 \u00d7 MIC) caused significant disaggregation effect on the biofilm biomass and metabolic viability of cells embedded in a biofilm matrix at two maturation state (4-day and 8-day biofilms). According to Lemos et al. [M. abscessus was more sensitive than the 4-day biofim. The effect could be due to a poorer 8-day biofilm in terms of biomass and metabolic activity. In contrast, the 4- and 8-day biofilm of M. fortuitum showed a similar susceptibility trend. About CAR toxicity, Cacciatore et al. [Rapidly growing mycobacteria have been studied for their ability to form biofilm . CAR emes et al.  classifie et al.  demonstrMycobacterium strains also in a sessile lifestyle and offers interesting applicative prospects related to its volatility such as diffusion and penetration into inaccessible areas.In conclusion, within the limits of the present study CAR could have the potential for implementation of strategies for treating"}
+{"text": "W boson pairs is presented. It uses a data sample from proton\u2013proton collisions corresponding to an integrated luminosity of 36.1\u00a0fbA search for doubly charged scalar bosons decaying into  H\u00a0[H and An extension of the scalar sector of the Standard Model (SM) is possible in the context of type II seesaw models\u00a0, originaentclass1pt{minimaoublet H\u00a0. ElectroW bosons, The assumption of a non-zero ument}vt\u00a0, 5. Exteolliders\u00a0, where \\mentclasspt{minima of\u00a0GeV)\u00a0 for a mo of\u00a0GeV)\u00a0. For tha of\u00a0GeV)\u00a0. In contW bosons. The triplet vacuum expectation value is taken to be W bosons. The mixing between the CP-even scalars is taken to be W bosons, and therefore only the region The present paper focuses on the phenomenology of doubly charged scalar bosons The four-boson final states are identified by the presence of light charged leptons (electrons or muons), missing transverse momentum, and jets. The analysis uses three final states defined according to the number of light leptons: same-sign (SS) dilepton channel , additional interactions were generated using Pythia \u00a08.186\u00a0[Monte Carlo (MC) simulation samples were produced for signal and background processes using the full ATLAS detector simulation\u00a0 based onGeant4\u00a0 or, for g system\u00a0. To simua \u00a08.186\u00a0, 26 witha \u00a08.186\u00a0, and thea \u00a08.186\u00a0, and oveThe signal events containing sion 3.4 , which ision 3.4 , 31. Thesion 3.4 , 26 withsion 3.4 . Event srections\u00a0 in QCD wVV, VVqq, and VVV (where V denotes a vector boson W or Z and qq labels the vector-boson fusion production mechanism) was simulated with a NLO QCD matrix element computed by Sherpa and matched to the Sherpa parton shower. The main background contribution in the WZ production, for which the total cross-section prediction is ZZ production with a total cross-section of VV and The SM background processes were simulated using the MC event generator programs and configurations shown in Table\u00a02\u00b11.1\u00a0pb\u00a0. The mai9\u00b10.6\u00a0pb\u00a0, 46, whiin Refs.\u00a0\u201349.W and Z bosons are classified as a prompt-lepton contribution, while processes with at least one of the reconstructed leptons being a misidentified hadron or photon, or a lepton from hadron decays constitute the fake-lepton contribution. The simulated events are not used to evaluate the background originating from charge-misidentified leptons for the The simulated SM contributions in each of the channels considered are separated into prompt-lepton and fake-lepton contributions, depending on the source of the reconstructed leptons at generator level. The processes that contain only reconstructed charged leptons originating from prompt leptonic decays of pp collisions are reconstructed using at least two tracks with transverse momentum Interaction vertices originating from d tracks\u00a0.loose and tight identification working points, described in Ref.\u00a0[Electrons are reconstructed as tracks in the inner detector matched to clusters in the electromagnetic calorimeter, within the region of pseudorapidity |\u03b7|<2.47\u00a0. The can in Ref.\u00a0, are useloose or tight identification criteria which are defined in Ref.\u00a0[Muon candidates are reconstructed by combining tracks formed in the inner detector and in the muon spectrometer, within the region of pseudorapidity |\u03b7|< 2.5\u00a0. Only th in Ref.\u00a0, and whiT (for tight) and L (for loose). The type T leptons are a subset of the type L.Combining the selection criteria mentioned above, two types of lepton requirements are used for both the electrons and muons: type b-hadrons are identified (b-tagged) via a multi-variate discriminant combining information from the impact parameters of displaced tracks with topological properties of secondary and tertiary decay vertices reconstructed within the jet\u00a0[b-tagging algorithm used for this search has an average efficiency of 70% to identify b-jets with Jets are reconstructed from topological clusters\u00a0 of energlgorithm\u00a0, 55 withy vertex\u00a0. Jets co the jet\u00a0. The b-tTo avoid object double counting, an overlap removal procedure is applied to resolve ambiguities among electrons, muons, and jets in the final state. Any electron candidate sharing an inner detector track with a muon candidate is removed. Jets within The missing transverse momentum, with magnitude s object\u00a0.Candidate events are selected using triggers that require at least one electron or one muon to pass various thresholds of ument}pT\u00a0. The higW bosons from one of the doubly charged Higgs boson decays leptonically, while the two W bosons from the other doubly charged Higgs boson decay hadronically. In the W boson decays hadronically and in the W bosons decay leptonically. All channels present significant W boson decays. In the W boson decays originate from the first- and second-generation quarks, and therefore lead to events without b-jets. The event selection is divided into two steps: the preselection and the signal region selection.The signal topologies studied in this search involve the presence of at least two leptons of the same charge and are classified as explained above in three mutually exclusive categories: L requirement. Events are selected only if the absolute value of the sum of charges of the leptons is two, one and zero for the T. Similarly in the T. In the The preselection requirements are summarised in Table\u00a0b-jets Z bosons and neutral mesons decaying into same-flavour opposite-sign leptons (SFOS), the invariant mass of such lepton pairs is required to be greater than 12 (15)\u00a0GeV for the Z boson. For the Z boson invariant mass veto is also applied to Further preselection requirements are based on After this preselection, 562 data events are selected in the The background processes containing only prompt selected leptons are estimated with MC simulations normalised to the most precise cross-section calculation  is misidentified.The composition of the fake-lepton background varies considerably among the analysis channels. Therefore, the methods to estimate the fake-lepton contributions are different for the Fake-lepton contribution estimate for thechannel The fake-factor method assumes that the fake-lepton contribution in a nominal region, which can be the preselection or the signal region, can be computed using an extrapolation factor that is referred to as a fake factor, and is denoted as L but not the type T identification criteria. That lepton is denoted by T identification, divided by the number of fake-lepton events in the same region but where one of the selected leptons is of type The fake factors are calculated in fake-enriched control regions with kinematic selections designed to enhance their content in fake leptons. In the case of the T, and where one is of type T and the other of type C indicates the fake-enriched control region.The muon fake factor is thus computed in the fake-enriched region, where a pair of same-sign muons was selected, as follows:The electron fake factor is computed using the fake-enriched region where a same-sign entclass1pt{minimaR) are obtained by multiplying the fake factors by the number of events in a region with the same selection as the nominal region, but where at least one lepton is of type The muon fake factor is measured to be Fake-lepton contribution estimate for thechannel A method similar to that employed for the mentclass2pt{minimFake-lepton contribution estimate for the channel There are too few data events to apply the fake-factor method in the b-jet. A small component from light quarks is also present. Two data samples designed to contain fake leptons originating from Z boson candidate. For the T region, the fake lepton is assumed to be the lepton with the lower W boson decays are needed to lead to the multi-lepton topologies considered in this analysis, all signal events are expected to feature significant W boson decays for W bosons are emitted close to each other in the laboratory frame. Consequently, the two same-sign leptons from the decays of the two W bosons tend to be close in the The hypothetical signal produces four W boson, or to a lesser extent from a Z boson, and one fake lepton from the hadronic final state produced. The WZ associated with jets, with both bosons subsequently decaying into leptons. This process also produces other features of the signal, such as significant b-jets for most of the production cross-section. For the WZ events, the mass of the same-flavour opposite-sign lepton pair is close to the Z boson mass, while no such resonant distribution is expected for the signal. In the ZZ production. Processes containing top quarks  and VV and 17% for VVV and tZ process predictions are taken from the respective inclusive cross-section measurements\u00a0[The theoretical uncertainties associated with the largest SM backgrounds, VV\u00a0 (includient}tt\u00afV\u00a0, are esturements\u00a0, 64. ForThe uncertainties related to event reconstruction include the lepton\u00a0, 65 and }ETmiss \u00a0. The impb-jet identification algorithms, used in the analysis to veto events containing b-jets, are found to be negligible.The uncertainties related to the efficiencies of electron\u00a0 and muonThe uncertainties originating from data-taking conditions include the luminosity measurement and the pile-up simulation procedure. The uncertainty of the integrated luminosity is  in Ref.\u00a0. The uncee and The uncertainties related to the background contributions from electron charge misidentification are 22\u2013The theoretical and experimental systematic uncertainties described above are assumed to be correlated amongst the various signal regions in the interpretation of the final results. Overall, the sensitivity of the search is dominated by the statistical uncertainty of the event yield in the signal regions.The experimental uncertainties arise from the accuracy of the detector simulation and from the uncertainties associated with the data-driven methods that are used to estimate the instrumental backgrounds. These uncertainties originate from the following sources:A, defined as the number of selected events selected in a given channel divided by the total number of The expected and observed event yields in the signal regions are shown in Fig.\u00a0The statistical analysis of the results is based on a likelihood ratio test\u00a0 using thThe expected and observed upper limits of the W bosons is performed in proton\u2013proton collisions at a centre-of-mass energy of 13\u00a0TeV. The data sample was collected by the ATLAS experiment at the LHC and corresponds to an integrated luminosity of A search for the pair production of doubly charged Higgs scalar bosons with subsequent decays into"}
+{"text": "As interest in Animal-Assisted Interventions (AAI) grows, there is increasing need to differentiate informal activities from formal and professionally directed therapies, including mental health focussed Canine-Assisted Psychotherapy (CAP). There have been no reviews focusing exclusively on CAP and the distinct developmental period of adolescence. The aims of this study were to identify the characteristics of CAP interventions, their impacts and their acceptability, tolerability and feasibility for adolescents with mental health disorders.A systematic review identified studies incorporating canines into mental health treatments for adolescents aged 10\u201319 years. Studies reporting qualitative or quantitative psychological or psychosocial outcomes were included.Seven studies were scrutinised. Intervention characteristics varied, including a range of formats, settings, locations, doses, and facilitators. Information on the role of the canines in sessions was sparse. CAP had a positive impact on primary diagnoses and symptomatology, conferring additional benefits to standard treatments for internalising disorders, post-traumatic stress disorder, and equivalent effects for anxiety, anger and externalising disorders. CAP was associated with positive impacts on secondary factors including increased engagement and socialisation behaviours, and reductions in disruptive behaviours within treatment sessions. Global functioning also improved. There was insufficient evidence that CAP improved factors associated with self-esteem, subjective wellbeing, or coping. Good attendance and retention rates indicated high levels of acceptability. Moderate to high tolerability was also indicated. Feasibility may be limited by additional training and logistical requirements.spontaneous , adjunctive (facilitator-led), and experiential (participant-led).We recommend the development of theoretically informed, standardised  intervention protocols that may subsequently form the basis of efficacy and effectiveness testing. Such protocols should clearly describe canine-participant-facilitator interactions via a formalised nomenclature; There is emerging evidence to suggest that CAP improves the efficacy of mental health treatments in self-selected adolescent populations via reductions in primary symptomatology, and via secondary factors that improve therapeutic processes and quality, such as engagement and retention. The incorporation of animals into human treatments has a long history. As the field has evolved, much work has been done to both define and describe the nature and types of these interventions, and to explore their efficacy (the ability to produce the desired or intended result within a controlled research context) and effectiveness  , 2. SomeGiven the crucial importance of clear definitions to establishing efficacy, considerable work has been done by researchers and international bodies to highlight the need for common terminology \u20136. A numThis review is particularly focused on mental health specific interventions that focus on psychological and psychosocial outcomes. These are sub-sets within AAT, including, for example AAT in counselling (AAT-C)  animal-aA number of literature reviews have been conducted to explore the efficacy of AAI\u2019s on psychological and psychosocial variables. Eight systematic reviews have indicated that multi-species AAIs may be effective as an adjunctive treatment for human health and wellbeing \u201317. AcroAlthough a there are a number of reviews that have focused specifically on equine-assisted interventions \u201323, no sdirected by health and human service providers as part of their profession\u201d .mp. (youth or young person or teen* or adolesc* or child*).mp. 1 and 2 and 3The reference sections of included papers were hand searched by the lead researcher (MJ) for additional studies missed by the database searches. Relevant titles were identified and cross-checked for duplication. Remaining titles were searched, and abstracts reviewed in line with the search strategy. Studies identified for full-text review were downloaded.For each identified study, a range of data were extracted including: study characteristics and design; participant, facilitator and animal characteristics; intervention characteristics; and outcomes and conclusions.Studies were assessed for methodological quality using the National Institute of Health (NIH) guidelines for quantitative studies , 66 and A description of the participants was extracted, including age and sex. Diagnoses or difficulties were reported if identified by the authors . Facilitators were accepted as mental health professionals if they could be identified either by qualification, job title or job role. Descriptions of canines were extracted, including information about their training, certification or evaluation.Details about the intervention, such as program outlines or session activities, were reported as a measure of standardisation or replicability. The nature of engagement between participants and canines was retrieved and coded as structured (facilitator-directed) interactions, semi-structured interactions, and/or spontaneous interactions. The format of interventions was coded as group or individual. The setting of the intervention refers to the location in which the intervention occurred, including information about indoors versus outdoors where available. The dose for each intervention included frequency  and total hours, calculated by adding the duration of each session , and the length of the intervention .Outcomes and conclusions were extracted, including information about the type of assessments used and a summary of the results. Information about the acceptability, tolerability and feasibility were extracted. Limitations, future directions and conclusions drawn by the authors were all documented.A total of 3985 studies were identified for initial screening via database searching. After exclusions see , 88 studAll seven of the studies were published in the last 17 years, with four published in the last 5 years. Three of the studies were conducted in the USA, two in Italy, one in South Africa, and one Israel. All studies had the stated goal of reducing the impact of psychological distress or disorder, and/or to improve the adaptive functioning of the client see .A range of methodological designs were reported, including three randomised controlled trials (RCT), one case-controlled study, one pre-post design and two qualitative studies. Only two studies had \u2018good\u2019 methodological quality, both were RCTs , 72. In Three of the studies , 73, 74 Two of the studies were ranked as \u2018poor\u2019 for methodological quality , 76. LanFour studies reported interventions for participants aged 13\u201316 years, whilst three studies involved participants from the wider developmental period 10\u201318 years or 11\u201317 years see . NarroweParticipants presented with a range of difficulties, including: females with a history of physical or sexual abuse, low achievement in school & interpersonal difficulties ; males aFacilitators were reported to be qualified or student counsellors , 75, 76,Four of the studies included information about the selection, assessment or certification status of the canines employed \u201373, 75. A broad range of interventions were employed in the reviewed studies, including individual counselling, group counselling, and combined interventions in a variety of settings such as a school, hospitals and community settings see .One intervention was reportedly standardised; the HART model developed by Hartwig . This waThe interventions delivered by Stefanini and colleagues , 72 consThe remaining three studies reported group interventions \u201376. BothUnlike the other studies, Hanselman  reportedA summary of outcomes reported by each of the authors is outlined in p = .018) and a significant reduction in the risk for a PTSD diagnosis (p = .046) on the self-report measure PTSD Checklist\u2013Civilian (PCL-C) from pre to post-intervention. At Pre-test the intervention group scored significantly higher on the PCL-C than the comparison group who had no exposure to traumatic events (p = .014); this difference was not present by the end of the intervention (p = .409).Hamama, Hamama-Raz  reportedp = .063). The intervention group had significantly higher rates of depression than the comparison group (without trauma history) pre-intervention (p = .022) which was not evident post-intervention (p = 0.316). Hartwig . It is not clear, however, what type of AAT should be trialled, nor the important elements to include in such an intervention. There are currently no published, clearly defined or recognised CAP protocols which could form the basis for RCTs. We therefore recommend that following the development of a standardised protocol, preliminary trials are conducted to assess acceptability, tolerability, feasibility and qualitative outcomes, prior to moving forward into RCTs.There is evidence to suggest that CAP may improve the efficacy of mental health treatments in self-selected adolescent populations via reductions in primary symptomatology including PTSD and internalising symptoms, and the severity of serious psychiatric disorders. CAP may also confer additional benefits via secondary factors that improve therapeutic processes and quality, such as attendance and retention, positive socialisation, and feelings of connection.Much work still needs to be done to establish the key components of CAP interventions that are most effective. The authors of this review have proposed a clear nomenclature to describe the interactions between canines, facilitators and participants, and provided recommendations for establishing theoretically grounded, standardised , CAP intervention protocols that may subsequently form the basis of efficacy and effectiveness testing via RCTs.We received no funding for this review.S1 Fig(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file."}
+{"text": "The advanced or recurrent endometrial cancer (EC) has a poor prognosis because of chemoresistance. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a glycolytic enzyme, is overexpressed in a variety of human cancers and plays important roles in promoting tumor cell growth. Here, we showed that high expression of PFKFB3 in EC cell lines is associated with chemoresistance. Pharmacological inhibition of PFKFB3 with PFK158 and or genetic downregulation of PFKFB3 dramatically suppressed cell proliferation and enhanced the sensitivity of EC cells to carboplatin (CBPt) and cisplatin (Cis). Moreover, PFKFB3 inhibition resulted in reduced glucose uptake, ATP production, and lactate release. Notably, we found that PFK158 with CBPt or Cis exerted strong synergistic antitumor activity in chemoresistant EC cell lines, HEC-1B and ARK-2 cells. We also found that the combination of PFK158 and CBPt/Cis induced apoptosis- and autophagy-mediated cell death through inhibition of the Akt/mTOR signaling pathway. Mechanistically, we found that PFK158 downregulated the CBPt/Cis-induced upregulation of RAD51 expression and enhanced CBPt/Cis-induced DNA damage as demonstrated by an increase in \u03b3-H2AX levels in HEC-1B and ARK-2 cells, potentially revealing a means to enhance PFK158-induced chemosensitivity. More importantly, PFK158 treatment, either as monotherapy or in combination with CBPt, led to a marked reduction in tumor growth in two chemoresistant EC mouse xenograft models. These data suggest that PFKFB3 inhibition alone or in combination with standard chemotherapy may be used as a novel therapeutic strategy for improved therapeutic efficacy and outcomes of advanced and recurrent EC patients. Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries , with anThe upregulation of glycolysis is one of the major metabolic pathways implicated in cancer progression. One of the rate-limiting steps of glycolysis involves Fructose 2,6-bisphosphate  and is mediated by 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzyme (PFKFB3). PFKFB3 catalyzes the synthesis of F2,6BP, which subsequently activates phosphofructokinase-1 (PFK-1) and upregulates the glycolytic flux , head anIn this study, the antitumor effects of PFKFB3 inhibition in EC were evaluated in type I and type II chemoresistant EC cells in vitro and in vivo using two chemoresistant xenograft mouse models. We inhibited PFKFB3 by genetic silencing as well as chemically with the use of PFK158, a specific inhibitor of PFKFB3, and studied the impact of PFKFB3 inhibition on glycolysis, cell proliferation and chemoresistance in EC cells. Finally, the antitumor effects of PFK158 alone and in combination with chemotherapy on apoptosis, autophagy, DNA repair and the Akt/mTOR signaling pathway were examined.ser461) were determined in both type I and type II EC cell lines. Among the EC cells tested, significant expression of p-PFKFB3 was observed in EN1, HEC-1A, HEC-1B (type I), ARK-2 and SPAC1L (type II) cell lines. Western blot analysis of chemoresistant HEC-1B and ARK-2 cells showed significantly higher levels of both t-PFKFB3 and p-PFKFB3 than the chemosensitive Ishikawa and RL95-2 cells . Compared with the cells treated with the single agents, HEC-1B and ARK-2 cells treated with the combination regimen showed synergistic responses Fig. . FurtherTo determine whether the synergistic growth inhibition induced by the combination of PFK158 and CBPt/Cis was due to apoptosis, apoptotic cells were scored by flow cytometry analysis using Annexin V and PI labeling. The results showed that co-treatment with PFK158 and CBPt /Cis led to a significant increase in the percentage of apoptotic cells in HEC-1B and ARK-2 Fig. . FurtherNext, we sought to investigate whether PFK158 or in combination with CBPt/Cis could induce autophagy. Moreover, we measured the expression of the autophagic protein LC3BII, which is cleaved from the LC3BI protein during autophagosome formation and the levels of p62, which is degraded during autophagosome formation. The results showed autophagy was activated after PFK158 treatment in a dose-dependent manner, as confirmed by LC3BII upregulation and p62 downregulation in PFK158-treated HEC-1B and ARK-2 cells Fig. . Also, wSince the Akt/mTOR signaling pathway has been implicated in the pathogenesis of EC , we wondTo further investigate whether PFKFB3 levels affected the sensitivity of EC cells to chemotherapy, we generated PFKFB3 knocked down (KD) clones in chemoresistant HEC-1B and ARK-2 cell lines using CRISPR/Cas9 targeting PFKFB3 with scrambled CRISPR/cas9 vector as controls and overexpressed (OE) PFKFB3 in chemosensitive cells (Ishikawa) with vector-transfected cells as controls. Efficient KD of PFKFB3 in HEC-1B and ARK-2 cells was verified by western blot analysis Fig. . MTT assResistance to chemotherapy is multifactorial with previous studies that have focused on changes in the glutathione levels, increased activity of drug efflux transporters, altered drug targets playing a role in conferring chemoresistance . In thes6 HEC-1B and ARK-2 cells were subcutaneously injected. Following the detection of palpable tumors, the mice were treated with vehicle, PFK158 alone, CBPt alone, or both for 14 days . For in vitro experiments, PFK158 was dissolved in DMSO, and for in vivo experiments, PFK158 was dissolved in 40% solution of Captisol in ddH2.Cell lines used in this article are presented in Supplementary Table 5 cells/well into 6-well plates, and after 24\u2009h, transfected with 5\u2009\u03bcg PFKFB3 CRISPR KD/OE plasmid  using 4.5\u2009\u03bcl Attractene and up to 250\u2009\u03bcl OptiMEM/well. Two days later, transfection efficiency was confirmed by fluorescence microscopy. Afterward, the transfected cells were selected with G418 for ~2 weeks before individual colonies were isolated. PFKFB3 knockdown and overexpression clones were verified by western blot for cell lysates.To construct PFKFB3 knockdown (KD) and overexpression (OE) in EC cell lines, HEC-1B, ARK-2 and Ishikawa cells were seeded at a density of 1\u2009\u00d7\u200910Western blot analysis was performed as previously described . The blo500 cells were plated onto 6-well plates and allowed to grow overnight. The medium was then supplemented with vehicle alone or the indicated drugs for 72\u2009h. Then the medium was discarded. Following washing, fresh medium was re-added to the plates, and cells were incubated for up to 14 days until colonies became visible. Cells were washed twice with PBS and fixed with 100% methanol, stained with crystal violet, and washed with deionized water. The colonies were imaged and counted using ImageJ software.3 cells per well and incubated for 24\u2009h. Cells were then treated with different doses of the compound for indicated times. The inhibitory concentrations 50% (IC50) values were determined by MTT assays, as previously described  for 30\u2009min in the glucose-free medium. Subsequently, cells were washed, mounted, and analyzed in the Zeiss LSM510 fluorescence microscope. The fluorescent intensities were calculated using ImageJ software.6 cells were seeded in 96-well plates overnight and treated with PFK158 (0\u201320\u2009\u00b5M) for 24\u2009h. According to the manufacturer\u2019s protocol, the LDH released into the medium was transferred to a new 96-well plate and mixed with a reaction mixture . Measurement of mitochondrial ATP production experiment was performed according to the manufacturer\u2019s (BioVision Inc. USA) protocol. LDH activity and ATP production were represented as a percentage of control by normalizing the OD values of untreated control cells.1\u2009\u00d7\u200910To determine synergy, a range of drug concentrations was used, and the CI values were calculated using CompuSyn software using a non-constant ratio approach, according to Chou-Talalay . The CI 6 cells per well and allowed attaching overnight. Then cells were exposed to CBPt (100\u2009\u03bcM), Cis (10\u2009\u03bcM), PFK158 (5\u2009\u03bcM), PFK158\u2009+\u2009CBPt, PFK158\u2009+\u2009Cis for 24\u2009h. Afterward, floating and adherent cells were stained with Annexin V-Pacific blue and propidium iodide (5\u2009\u03bcg/ml). Cells were analyzed by CellQuest Pro software  as previously described [Cells were plated in 6-well plates at a density of 1\u2009\u00d7\u200910escribed .HEC-1B/ARK-2 cells, untreated and treated with PFK158, transiently transfected with Cherry-GFP-LC3B for 48\u2009h or PFKFB3 downregulated cells, were grown on four-well chambered slide for the desired time. After drug treatment, cells were washed with PBS, fixed with 4% formaldehyde, permeabilized using 0.2% Triton X-100 in PBS for 15\u2009min. After blocking with 2% bovine serum albumin (BSA) in PBS for 1\u2009h at room temperature, the cells were incubated overnight with primary antibodies (RAD51 and \u03b3-H2AX) at 4\u2009\u00b0C. The cells were then incubated with fluorescent secondary antibodies for 1\u2009h at room temperature. IFC was performed as previously described [HEC-1B and ARK-2 cells were transfected with pDR-GFP, an HR substrate that generates a functional green fluorescent protein (GFP) upon successful HR by I-SceI cleavage . 48\u2009h la6 HEC-1B or ARK-2 cells subcutaneously into the right dorsal flanks. Treatment was initiated after 5-days post-inoculation when the subcutaneous implants measured 3\u2009mm by 3\u2009mm. Mice implanted with each cell line were randomly assigned to four treatment groups and treated for 2 weeks (n\u2009=\u200910 per group). Groups were treated as follows: (i) HEC-1B v) ARK-2 intraperitoneal injections of 40% Captisol for the control group; (ii) HEC-1B vi) ARK-2 intraperitoneal injections of PFK158 at 35\u2009mg/kg twice weekly  (iii) HEC-1B vii) ARK-2 intraperitoneal injections of CBPt at 50\u2009mg/kg weekly  (iv) HEC-1B viii) ARK-2 combination of CBPt  and PFK158 . Body weight and tumor size were measured every 2 days with a caliper, and tumor volume was calculated using the following formula: V\u2009=\u2009length\u2009\u00d7\u2009width2\u00d7(\u03c0/6) . At the end of the experiments, the mice were euthanized. Harvested tumors were weighed and processed for further analyses .Animal experiments complied with the Institutional Animal Care and Use Committee (IACUC) guidelines at the Mayo Foundation, following approved protocols. Mice were injected with two \u00d710Tumor tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Then the samples were sectioned at 5\u2009\u00b5m thickness and stained with hematoxylin and eosin (H&E). The expression levels of Ki-67 and p-PFKFB3 were evaluated by immunohistochemistry in the paraffin-embedded tumor sections, according to a previously described protocol .Cell apoptosis in tumor tissues was performed by terminal dUTP Nick End-Labeling (TUNEL) staining with an In Situ Apoptosis Detection Kit according to the manufacturer\u2019s instructions.t-test or one-way ANOVA as appropriate. The minimal level of significance was p\u2009=\u20090.05. *p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001; **** or #p\u2009<\u20090.0001. If not specified, the analysis is not significant.Data are represented as mean\u2009\u00b1\u2009SD deviation from at least three independent experiments. All statistical analyses were performed using the GraphPad Prism 7 software. Data were analyzed using either non-linear regression or Supplemental Material"}
+{"text": "RNA\u2013protein interactions are the crucial basis for many steps of bacterial gene expression, including post\u2010transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram\u2010negative bacteria, knowledge about RNA\u2013protein complexes in Gram\u2010positive species remains scarce. Here, we used the Grad\u2010seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of ~\u00a088% of the transcripts and ~\u00a062% of the proteins of this important human pathogen. Analysis of in\u2010gradient distributions and subsequent tag\u2010based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram\u2010positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA\u2010based regulation of virulence\u2010relevant pathways. RNA complexes in Streptococcus pneumoniae via Grad\u2010seq uncovers an unexpected role for the exoribonuclease Cbf1 in stabilizing sRNAs that control bacterial competence for DNA uptake.Comprehensive assessment of protein\u2010 Within the past two decades, our view of bacterial gene regulation has dramatically changed. Once considered as organisms with a protein output that is a straight function of transcription initiation, we know now that bacteria amply use noncoding RNAs (ncRNAs) and RNA\u2010binding proteins (RBPs) in post\u2010transcriptional control networks that impact almost every aspect of physiology  to accelerate the discovery of the major functional RNAs and RBPs in a bacterium of interest  unless in a complex , ribonucleoside\u2010diphosphate reductase (NrdEF), galactose\u20106\u2010phosphate isomerase (LacBA), and an intact 9\u2010subunit cytosolic ATP synthase F1 complex (AtpCDGAH)  Fig\u00a0. SurprisS.\u00a0pneumoniae TIGR4 proteins with the keyword \u201cRNA\u2010binding\u201d, 77 of which were detected in our gradient . Without synthesis of CSP, competence and therefore DNA uptake from the environment cannot be induced  in enteric bacteria  was first described as a host factor for replication of plasmid pT181 in S.\u00a0aureus  when cbf1 is deleted in a different S.\u00a0pneumoniae strain and exogenous CSP is added  acts on a large number of transcripts  and grown overnight at 37\u00b0C and 5% CO2. Strains used are listed in 600\u00a0nm of 0.5 at 37\u00b0C without shaking. Subsequently, the pre\u2010culture was refreshed in THY to a starting OD600\u00a0nm of 0.05 and grown at 37\u00b0C without shaking to a final OD600\u00a0nm of 0.5.S.\u00a0pneumoniae TIGR4 wild type was grown to an OD600\u00a0nm of 0.5, cooled down in an ice\u2010water bath for 15\u00a0min, and then harvested by centrifugation for 20\u00a0min at 4\u00b0C and 4,000\u00a0g. The cells were washed three times in ice\u2010cold 1\u00d7 TBS, resuspended in 500\u00a0\u03bcl ice\u2010cold 1\u00d7 lysis buffer A . Mechanical lysis was performed using the FastPrep\u201024 instrument  in 2\u2010ml tubes with lysing matrix E  at 6\u00a0m/s for 30\u00a0s. To remove insoluble debris and the beads, the lysate was cleared by centrifugation for 30\u00a0min at 4\u00b0C and 16,100\u00a0g. Of the cleared lysate, 10\u00a0\u03bcl was mixed with 1\u00a0ml TRIzol (Thermo Fisher Scientific) for the RNA input control and 20\u00a0\u03bcl was mixed with 20\u00a0\u03bcl 5\u00d7 protein loading buffer for the protein input control.400\u00a0ml of g  using a Beckman Coulter SW40Ti rotor, followed by manual fractionation into 20 590\u00a0\u03bcl fractions and measurement of the A260\u00a0nm of each fraction. 90\u00a0\u03bcl of each fraction and 40\u00a0\u03bcl of the pellet were mixed with 30\u00a0\u03bcl of 5\u00d7 protein loading buffer for protein analysis and stored at \u221220\u00b0C.400\u00a0\u03bcl of the cleared lysate was then layered on top of a linear 10\u201340% (w/v) glycerol gradient (in 1\u00d7 lysis buffer A without DNase I or RNase inhibitor), which was formed in an open\u2010top polyallomer tube (Seton) using the Gradient Station model 153 (Biocomp). The gradient was centrifuged for 17\u00a0h at 4\u00b0C and 100,000\u00a0g. The aqueous phases were collected, and 1\u00a0\u03bcl of GlycoBlue (Thermo Fisher Scientific) and 1.4\u00a0ml of ice\u2010cold ethanol/3\u00a0M NaOAc, pH 6.5 (30:1) were added and precipitated for at least 1\u00a0h at \u221220\u00b0C. The RNA was collected by centrifugation for 30\u00a0min at 4\u00b0C and 16,100\u00a0g and washed with 350\u00a0\u03bcl ice\u2010cold 70% ethanol, followed by centrifugation for 15\u00a0min at 4\u00b0C and 16,100\u00a0g. The lysate RNA sample stored in TRIzol was purified according to the manufacturer's protocol, except that the precipitation was performed using the mentioned ethanol mix. After drying of the RNA pellet, it was dissolved in 40\u00a0\u03bcl DEPC\u2010treated H2O and DNase\u2010digested by addition of 5\u00a0\u03bcl DNase I buffer with MgCl2 (Thermo Fisher Scientific), 0.5\u00a0\u03bcl RNase inhibitor, 4\u00a0\u03bcl DNase I (Thermo Fisher Scientific), and 0.5\u00a0\u03bcl DEPC\u2010treated H2O, followed by incubation for 45\u00a0min at 37\u00b0C. The DNase\u2010treated RNA was purified by the addition of 150\u00a0\u03bcl DEPC\u2010treated H2O and 200\u00a0\u03bcl acidic P/C/I as described above. The purified, DNase\u2010treated RNA was dissolved in 35\u00a0\u03bcl DEPC\u2010treated H2O and stored at \u221280\u00b0C.The remaining 500\u00a0\u03bcl of each fraction was used for RNA isolation by addition of 50\u00a0\u03bcl of 10% SDS (25\u00a0\u03bcl for the pellet) and 600\u00a0\u03bcl of acidic phenol/chloroform/isoamyl alcohol . The fractions were then vortexed for 30\u00a0s and let rest at room temperature for 5\u00a0min before separating the phases by centrifugation for 15\u00a0min at 4\u00b0C and 16,100\u00a0Equal volumes of the gradient RNA samples were separated by denaturing 6% PAGE in 1\u00d7 TBE and 7\u00a0M urea and stained with ethidium bromide. For northern blotting, unstained gels were transferred onto Hybond+ membranes  and probed with RNA\u2010specific radioactively labeled DNA oligonucleotides.S.\u00a0pneumoniae GAPDH (GenBank AAK76079) was raised in rabbit against a synthetic peptide (DPIVSSDIVGMS) corresponding to amino acids 275\u2013286. Synthetic peptide production, immunization, and validation by ELISA were done by Innovagen , following a 41\u2010day immunization protocol including two booster injections. It was further used in previous studies  and probed with an anti\u2010FLAG primary antibody  and an anti\u2010mouse secondary antibody . As loading controls, the membranes were stripped and probed for glyceraldehyde\u20103\u2010phosphate dehydrogenase (GAPDH) using a rabbit antiserum. Immunoreactive serum to 2O. 10\u00a0\u03bcl of the resulting 1:10 dilution was mixed with 10\u00a0\u03bcl of a 1:100 dilution of the ERCC spike\u2010in mix 2 (Thermo Fisher Scientific) and subjected to library preparation for next\u2010generation sequencing (Vertis Biotechnologie). Briefly, the RNA samples were fragmented using ultrasound (four pulses of 30\u00a0s at 4\u00b0C) followed by 3\u2032 adapter ligation. Using the 3\u2032 adapter as primer, first\u2010strand cDNA synthesis was performed using M\u2010MLV reverse transcriptase. After purification, the 5\u2032 Illumina TruSeq sequencing adapter was ligated to the 3\u2032 end of the antisense cDNA. The resulting cDNA was PCR\u2010amplified to about 10\u201320\u00a0ng/\u03bcl using a high\u2010fidelity DNA polymerase followed by purification using the Agencourt AMPure XP Kit (Beckman Coulter Genomics). The cDNA samples were pooled with ratios according to the RNA concentrations of the input samples, and a size range of 200\u2013550\u00a0bp was eluted from a preparative agarose gel. This size\u2010selected cDNA pool was finally subjected to sequencing on an Illumina NextSeq 500 system using 75 nt single\u2010end read length.For RNA\u2010seq, 5\u00a0\u03bcl of the gradient samples was diluted in 45\u00a0\u03bcl DEPC\u2010treated Het\u00a0al, https://doi.org/10.5281/zenodo.250598) and the short read mapper segemehl . For the downstream analysis, only transcripts with a sum of \u2265\u00a0100 reads in all fractions within the gradient were considered. For each fraction, read counts were normalized by calculating size factors following the DESeq2 approach .Based on these normalized values, two analyses were performed: one containing all the detectable transcripts, and one containing only the detectable ncRNAs. For the ncRNAs, t\u2010SNE dimension reduction  diluted in 250\u00a0\u03bcl 1.25\u00d7 protein loading buffer. The samples were subsequently reduced in 50\u00a0mM DTT for 10\u00a0min at 70\u00b0C and alkylated with 120\u00a0mM iodoacetamide for 20\u00a0min at room temperature in the dark. The proteins were precipitated in four volumes of acetone overnight at \u221220\u00b0C. Pellets were washed four times with acetone at \u221220\u00b0C and dissolved in 50\u00a0\u03bcl 8\u00a0M urea, 100\u00a0mM ammonium bicarbonate.For mass spectrometry (MS), the gradient protein samples (diluted in 1.25\u00d7 protein loading buffer) were homogenized using ultrasound . Insoluble material was then removed by centrifugation for 15\u00a0min at 4\u00b0C and 16,100\u00a0et\u00a0al, Digestion of the proteins was performed by the addition of 0.25\u00a0\u03bcg Lys\u2010C (Wako) for 2\u00a0h at 30\u00b0C, followed by dilution to 2\u00a0M urea by the addition of 150\u00a0\u03bcl 100\u00a0mM ammonium bicarbonate, pH 8 and overnight digestion with 0.25\u00a0\u03bcg trypsin at 37\u00b0C. Peptides were desalted using C\u201018 Stage Tips , a database containing the UPS2 spike\u2010in and a database containing common contaminants. The search was performed with tryptic cleavage specificity with three allowed miscleavages. Protein identification was under control of a false discovery rate of 1% on both protein and peptide levels. In addition to the MaxQuant default settings, the search was performed against the following variable modifications: protein N\u2010terminal acetylation, Gln to pyro\u2010Glu formation , and oxidation of Met. For protein quantitation, the LFQ intensities were used  were eliminated. Following this, for each spike\u2010in protein, the median log10 intensity was subtracted from the log10 intensities of each fraction. The fraction\u2010wise median of the resulting values was then subtracted from the log10 intensities for each bacterial protein in the corresponding fractions. Finally, all log10 intensities smaller than the 5% quantile of all intensities in the dataset were replaced by the value of the 5% quantile of all intensities in the dataset.Normalization of the proteins across the fractions was performed using the UPS2 spike\u2010in. For this, only spike\u2010in proteins with detectable intensities in all fractions were used. The spike\u2010in proteins showing the highest variance . Primers used are listed in In vitro transcription was performed using the TranscriptAid kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The resulting RNA was purified using denaturing 6% PAGE in 1\u00d7 TBE and 7\u00a0M urea. An RNA oligonucleotide representing only the 14 nt tag (GGGAGACCUAGCCU) was used as negative control.To pull down proteins using RNA as bait, a modified version of a published protocol . The rest of the protocol was performed at 4\u00b0C. The washed beads were coupled to 4\u00a0\u03bcg of a 3\u2032\u2010biotinylated, 2\u2032\u2010O\u2010methyl\u2010modified RNA adaptor complementary to the 14 nt tag of the bait RNAs (AGGCUAGGUCUCCC\u2010biotin) for 1\u00a0h. The adaptor\u2010coupled beads were washed twice with 1\u00a0ml of lysis buffer B, resuspended in 1\u00a0ml of lysis buffer B, and split into two tubes with 500\u00a0\u03bcl each. One tube was used to couple 10\u00a0\u03bcg per 100 nt of bait RNA overnight, and the other was stored for pre\u2010clearing.For the pull\u2010down, 100\u00a0\u03bcl magnetic streptavidin beads  were washed 3\u00d7 with 1\u00a0ml of lysis buffer B  as described above. To pre\u2010clear the lysate from content that binds the beads unspecifically, the stored beads were incubated with the lysate for 3.5\u00a0h. The beads were subsequently removed by centrifugation for 10\u00a0min at 16,100\u00a0g. The bait RNA\u2010coupled beads were washed twice with lysis buffer B and incubated with the pre\u2010cleared lysate supernatant for 2\u00a0h to capture interacting proteins of the bait RNAs. To get rid of unspecific binders, the beads were washed with 1\u00a0ml each of wash buffer A , wash buffer B (lysis buffer C with 0.1% Triton X\u2010100), and lysis buffer C.To prepare the lysate for the pull\u2010down, 100 ODThe beads were resuspended in 35\u00a0\u03bcl of 1\u00d7 LDS sample buffer (Thermo Fisher Scientific) containing 50\u00a0mM DTT and boiled for 5\u00a0min at 95\u00b0C to elute the bound proteins. Following alkylation as described above, the pull\u2010down samples were run on a 4\u201312% Bolt Bis\u2010Tris plus gel (Thermo Fisher Scientific) using MES buffer (Thermo Fisher Scientific). After staining with SimplyBlue Coomassie (Thermo Fisher Scientific), each lane of the gel was either cut into 11 pieces or specific prominent bands were cut. The gel pieces were prepared for LC/MS\u2010MS by destaining with 30% acetonitrile in 100\u00a0mM ammonium bicarbonate, pH 8 followed by shrinking with 100% acetonitrile and drying in a vacuum concentrator (Eppendorf). 0.1\u00a0\u03bcg trypsin was added per gel piece and digestion performed overnight at 37\u00b0C in 100\u00a0mM ammonium bicarbonate, pH 8. The supernatant was removed, and the peptides were extracted from the gel pieces with 5% formic acid. Finally, the supernatant was pooled with the extracted peptides.et\u00a0al .NanoLC\u2010MS/MS analyses were performed similarly to Braun et\u00a0al  on an LTAnalysis of the pull\u2010down MS data was performed as described for the Grad\u2010seq MS data, except that the search against the UPS2 spike\u2010in database was skipped.et\u00a0al, cbf1\u20103xFLAG strain was grown to an OD600\u00a0nm of 0.5. Half of the culture was irradiated in 50\u00a0ml fractions at 254\u00a0nm in a 22\u00a0\u00d7\u00a022\u00a0cm plastic tray at 0.8\u00a0J. After collection of the cells by centrifugation at 4\u00b0C, the pellets were frozen in liquid nitrogen and stored at \u221280\u00b0C. Cells were thawed on ice, resuspended in 800\u00a0\u03bcl NP\u2010T buffer , and transferred to tubes containing 1\u00a0ml of glass beads. Following lysis for 10\u00a0min at 30\u00a0Hz using a mixer mill (Retsch MM400), the lysates were cleared twice by centrifugation for 15\u00a0min at 16,100\u00a0g and 4\u00b0C and mixed with one volume of NP\u2010T buffer containing 8\u00a0M urea. Finally, the lysates were incubated for 5\u00a0min at 65\u00b0C with shaking at 900\u00a0rpm, put on ice for 2\u00a0min and diluted 10\u00d7 in ice\u2010cold NP\u2010T buffer, and put back on ice.Sample preparation for UV crosslinking and immunoprecipitation followed by RNA\u2010seq (CLIP\u2010seq) followed a previously published protocol  and twice with 2\u00a0ml of NP\u2010T buffer. Subsequently, the beads were resuspended in 100\u00a0\u03bcl NP\u2010T buffer containing 1\u00a0mM MgCl2 and 2.5\u00a0U benzonase (Sigma) followed by incubation for 10\u00a0min at 37\u00b0C with shaking at 800\u00a0rpm. Then, the beads were put on ice for 2\u00a0min and washed once with 500\u00a0\u03bcl high\u2010salt buffer and twice with 500\u00a0\u03bcl CIP buffer  followed by resuspension in 100\u00a0\u03bcl CIP buffer containing 10\u00a0U of calf intestinal alkaline phosphatase (NEB) and incubation for 30\u00a0min at 37\u00b0C with shaking at 800\u00a0rpm. The beads were washed once with 500\u00a0\u03bcl high\u2010salt buffer and twice with 500\u00a0\u03bcl PNK buffer  followed by resuspension in 100\u00a0\u03bcl PNK buffer containing 1\u00a0\u03bcl T4 polynucleotide kinase (Thermo Fisher Scientific) and 1\u00a0\u03bcl \u03b3\u201032P\u2010ATP for 30\u00a0min at 37\u00b0C. Then, 10\u00a0\u03bcl 1\u00a0mM ATP was added and the beads were incubated for 5\u00a0min at 37\u00b0C, followed by two washes with 1\u00a0ml of NP\u2010T buffer and resuspension in 10\u00a0\u03bcl protein loading buffer. The labeled RNA\u2013protein complexes were eluted from the beads by incubation for 5\u00a0min at 95\u00b0C, which was repeated once.To perform the immunoprecipitation, 30\u00a0\u03bcl of anti\u2010FLAG magnetic beads was washed 3\u00d7 with 800\u00a0\u03bcl NP\u2010T buffer, added to the lysate, and rotated for 1\u00a0h at 4\u00b0C. The beads were collected by centrifugation at 1,000\u00a02O.15\u00a0\u03bcl of the eluted samples was separated using 12% SDS\u2013PAGE followed by transfer to a nitrocellulose membrane . Marker sizes of the protein ladder were highlighted using a radioactive marker, and the autoradiogram of the blot was subsequently used as a template to cut out the labeled complexes and their corresponding controls. The membrane pieces were then cut into smaller pieces, transferred to LoBind tubes (Eppendorf), and incubated with 200\u00a0\u03bcl PK buffer  for 1\u00a0h at 37\u00b0C with shaking at 800\u00a0rpm. Then, 100\u00a0\u03bcl PK buffer containing 9\u00a0M urea was added and the incubation continued for 1\u00a0h. The membrane pieces were briefly centrifuged and the supernatant extracted with one volume of P/C/I. The purified RNA fragments were resuspended in 10\u00a0\u03bcl DEPC\u2010treated Het\u00a0al, Library preparation for next\u2010generation sequencing was performed by the CoreUnit SysMed at the University of W\u00fcrzburg, Germany. The protocol used is the same as published before  using READemption  (version 0.1.0) for peak calling in a similar way as described previously . Following elution, the purity was tested on a Superdex 16/600 column . Purified Cbf1 was stored in 100\u00a0\u03bcl aliquots of 20\u00a0mM HEPES, pH 8, 250\u00a0mM NaCl, and 10% glycerol at \u221280\u00b0C (~\u00a01.35\u00a0mg/ml).The purification of recombinant Cbf1 was performed by the Recombinant Protein Expression core unit at the Rudolf Virchow Center in W\u00fcrzburg, Germany. Briefly, to purify recombinantly expressed Cbf1, the CDS was cloned into pet21b+, which adds a C\u2010terminal His\u2010tag to the sequence . After transformation into in\u00a0vitro\u2010transcribed RNAs  were digested with 3.5\u00a0\u03bcg Cbf1 (~\u00a0100\u00a0pmol) in a total volume of 20\u00a0\u03bcl . Primeret\u00a0al, in\u00a0vitro RNase assay with truncated csRNA1 was performed as described above, except that the total reaction volume was scaled up to 200\u00a0\u03bcl containing 4\u00a0\u03bcg of RNA. 50\u00a0\u03bcl of the reaction (1\u00a0\u03bcg of RNA) was taken at the indicated time points and stopped by addition of 150\u00a0\u03bcl acidic P/C/I and 200\u00a0\u03bcl of H2O. As negative control, 1\u00a0\u03bcg of untreated RNA was used. After P/C/I extraction, the RNA was dephosphorylated using 10\u00a0U of calf intestinal alkaline phosphatase (NEB) in 1\u00d7 NEB buffer 3 in 25\u00a0\u03bcl for 1\u00a0h at 37\u00b0C. The RNA was then P/C/I\u2010extracted and precipitated in the presence of 1\u00a0\u03bcl GlycoBlue (Thermo Fisher Scientific) and 250\u00a0pmol of RNA adapter E1. Ligation of the adapter was performed in a 20\u00a0\u03bcl reaction containing 20\u00a0U T4 RNA ligase (NEB), 1\u00d7 T4 RNA ligase buffer, and 10% DMSO overnight at 16\u00b0C. After ligation, the RNA was P/C/I\u2010extracted and reverse transcribed for 5\u00a0min at 50\u00b0C followed by 60\u00a0min at 55\u00b0C using 200\u00a0U SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) and 39\u00a0pmol of adapter E1\u2010specific oligo E3RACE in a 20\u00a0\u03bcl reaction containing 1\u00d7 FS buffer, 2\u00a0mM dNTPs, and 5\u00a0mM DTT. The template RNA was digested by addition of 1\u00a0\u03bcl RNase H (NEB) and incubation at 37\u00b0C for 30\u00a0min. 1\u00a0\u03bcl of the cDNA was then used as template for a Taq PCR using E3RACE and JVO\u201015297 as primers. The PCR products were run on a 3.5% agarose gel, the bands cut from the gel and cloned using the StrataClone PCR cloning kit (Agilent Technologies) following the manufacturer's instructions. For the 5\u2010 and 15\u2010min time points, the inserts of 20 positive clones each were amplified by PCR using the primers M13 and M13rev and analyzed by Sanger sequencing. Primers used are listed in Identification of 3\u2032 ends via 3\u2032 rapid amplification of cDNA ends (RACE) was performed as described before  and snap\u2010frozen in liquid nitrogen. Following RNA isolation using the hot phenol method and DNase treatment, 5\u00a0\u03bcg of RNA was analyzed by northern blotting and quantified using ImageJ  to a final concentration of 100\u00a0ng/ml. For the control, the same volume of sterile H2O was added instead. After 5, 10, 15 and 30\u00a0min of stimulation, samples were collected the same way as for the 0\u00a0min time point. Protein samples were thawed on ice and diluted in 200\u00a0\u03bcl protein loading buffer, and equal volumes (10\u00a0\u03bcl) were subjected to PAGE and Western blotting. RNA isolation was performed using the hot phenol method followed by DNase treatment. 15\u00a0\u03bcg of RNA was loaded on a 1.2% agarose gel, run, and stained with ethidium bromide to visualize 16S and 23S rRNAs as loading control. Finally, capillary blotting on a Hybond+ membrane was performed and the membranes probed with RNA\u2010specific radioactively labeled DNA oligonucleotides.To determine the influence of CSP induction, the gyrA was used as the control gene. Data were analyzed using the comparative \u0394\u0394CT method  and a CFX96 system (Bio\u2010Rad). cbf1::Sp, R6\u0394cbf1::Sp, and TIGR4 cbf1\u20103xFLAG\u2010Sp were constructed by homologous recombination in the genome of a cassette composed of a spectinomycin (Sp) resistance gene flanked with ~\u00a01,000\u00a0bp homologous to the regions bordering the region of interest. Cassettes were assembled by PCR assembly via overlapping regions in the primers used for amplifying individual fragments. Briefly, the upstream (up) and downstream (dwn) of TIGR4/R6 cbf1 were amplified using primers cbf1DF/cbf1DJ1R (up) and cbf1DJ2F/cbf1DR (dwn), and TIGR4 or R6 gDNA as the template. The spectinomycin resistance (Sp) gene, along with its promoter and terminator, was amplified from plasmid pSP72::Sp (courtesy of P. Mellroth) using primers cbf1DJ1F and cbf1DJ2R. The final up\u2010Sp\u2010dwn cassette was amplified using primers cbf1DF/cbf1DR (TIGR4) and cbf1DF2/cbf1DR2 (R6) and transformed into competent TIGR4/R6 cells. The cbf1\u20103xFLAG strain was constructed by assembling cbf1 from TIGR4 gDNA, amplified with primers cbf1VF/cbf13xFLAGR, with the Sp\u2010dwn region of TIGR4\u0394cbf1::Sp strain amplified with primers AbRFLAGF/cbf1DR. The final product was amplified using primers cbf1_nestedF/cbf1_nestedR and transformed into competent TIGR4 cells.Primers used are listed in cbf1 deletion was complemented in TIGR4\u0394cbf1::Sp using plasmid pJWV25 which integrates into the chromosome at the bga locus by homologous recombination between sequences flanking the insert  following manufacturer's instructions and selected on LB plates supplemented with 100\u00a0\u03bcg/ml ampicillin. The resulting vector, pGG8A, was purified using the QIAprep Spin Miniprep Kit (QIAGEN), and 300\u00a0ng of plasmid DNA was transformed into competent TIGR4\u0394cbf1::Sp. All PCR products described above were verified on agarose gel and, when necessary, excised, and purified using the Wizard SV Gel and PCR Clean\u2010Up System (Promega). Transformations were performed as described below for induced competence using 100\u00a0ng/ml CSP\u20101 (R6) or 100\u00a0ng/ml CSP\u20102 (TIGR4). Transformant colonies were isolated on blood agar plates containing the appropriate antibiotics at the following concentration: spectinomycin, 200\u00a0\u03bcg/ml; tetracycline, 2\u00a0\u03bcg/ml (pGG8A). All strains constructed were confirmed by PCR followed by sequencing at Eurofins (Germany).The cbf1::Sp were grown in C+Y  was added, and incubation was continued at 30\u00b0C for 60\u00a0min, followed by 90\u00a0min at 37\u00b0C. Samples were serially diluted and plated on blood agar plates containing 150\u00a0\u03bcg/ml streptomycin to enumerate transformant colony\u2010forming units (CFU) and on blood agar plates without antibiotics to enumerate total viable CFU. Results were expressed as the percentage of total CFU resistant to streptomycin. The rpsL PCR product used in transformation experiments was amplified using primer pair rpsLF/101\u2010rpsLR, gDNA from an S.\u00a0pneumoniae strain carrying a point mutation in the rpsL gene conferring resistance to streptomycin (Muschiol et\u00a0al, For spontaneous competence assays, pre\u2010cultures of R6 (Ottolenghi & Hotchkiss, BH\u2010N, GG, JH, and JV conceived and designed the study. GG, JH, and L\u2010MH performed experiments. KUF, JH, and SDG analyzed RNA\u2010seq and MS data. AS and JTV performed MS and analyzed MS data. BH\u2010N and JV supervised the project. JH and JV wrote the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Table\u00a0EV1Click here for additional data file.Table\u00a0EV2Click here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure\u00a01Click here for additional data file.Source Data for Figure\u00a03Click here for additional data file.Source Data for Figure\u00a04Click here for additional data file.Source Data for Figure\u00a05Click here for additional data file.Source Data for Figure\u00a06Click here for additional data file.Source Data for Figure\u00a07Click here for additional data file."}
+{"text": "Compared with less experienced respondents, the respondents with more than 30 years of work experience applied the caring behaviors on the subscales \u201csensitivity\u201d (p = 0.009), \u201cexpression of emotions\u201d (p = 0.001), \u201cproblem-solving\u201d (p = 0.008), and especially \u201chumanism\u201d and \u201cspirituality\u201d (p < 0.001) more frequently. The results indicate that respondents are more focused on applying skills or carrying out a task than on caring behaviors which is about demonstrating compassion, loving kindness, and relationships.Nurse\u2013patient interactions based on caring behaviors ensure better working conditions and better-quality healthcare. The aim of this quantitative study is to examine how nurses self-assess the frequency of applying caring behaviors in nurse\u2013patient interactions and to identify the differences in the application frequency of caring behaviors in relation to work experience and education level. The respondents were Bachelor of Science (BSc) nurses and nurses with basic training (VET) employed in different clinical departments of the Clinical Hospital Center in Croatia. The survey used the \u201cCaring Nurse\u2013Patient Interactions Scale (Nurse Version)\u201d. The respondents assessed the caring behaviors from the subscale \u201cneeds\u201d as the most frequently applied (median (Me): 4.7; interquartile range (IQR): 4.4\u20134.9), while the least frequently applied were the procedures from the subscale \u201csensitivity\u201d . The VET nurses reported applying caring behaviors to the subscales \u201chope\u201d ( Nursing, like other healthcare professions, is based on the ideals of service to humanity ,2. NurseThe importance of nurse\u2013patient interactions based on caring behaviors has been proven in philosophical discussions, theories, and innovative research by the renowned theoreticians Watson, Leininger, Boykin, and Swanson ,5,6. TheFrom an objective perspective, caring as a concept is hard to define. Caring is what patients expect and should experience to be satisfied with their provided nursing services ,10,11,12Carrying out tasks and reducing expenditure have become a priority in contemporary healthcare. Although nurses may feel as if they are being caring, they often provide healthcare while not actually taking care of what patients expect and want . In addiMoreover, the relevant literature suggests that the perception and implementation of caring behaviors are influenced by factors such as knowledge, formal education , and lenGenerally, there is a difference in nursing between the human caring models, which are primarily aimed at holistic patient care and the reason why many nurses choose this profession, and the biomedical model, which is focused on task completion and maintaining the reality of institutional demands ,38. NursThe theoretical framework for this study is based on Watson\u2019s human caring theory . In WatsThe essence of Watson\u2019s theoretical contribution is captured in 10 carative factors. The first three, in her own words, are the philosophical foundation for the science of care . Table 1Each factor contains a dynamic phenomenological component related to each individual in nursing care. Through these carative factors, nurses perform their basic professional duty. Watson\u2019s carative factors are well-accepted in the profession because they express the humanistic value of care. Watson promoted the concept of 10 carative factors during the clinical caritas processes . These pCaring cannot be measured or quantified objectively . Still, In 2005, Cossete developed a Nurse\u2013Patient Interaction Scale (CNPI-70) as a reliable instrument to assess the quality of nurse\u2013patient interactions . The CNPI-70 consists of 70 items and 10 carative factors/subscales based on Watson\u2019s Theory of Caring . All 70 A Philippine study by Fortuno  used theThe relevant literature identifies nurses\u2019 level of education and exposure to care as key personal factors that affect perception and application of caring behaviors ,31,32,33It can be noticed that there is still an insufficient number of studies that have researched the nursing perception of caring behaviors in nurse\u2013patient interaction using the CNPI-70 scale. This deficit affects the global understanding of the importance of caring behaviors and the importance of the ability to self-assess in order to make personal and professional progress, as well as consequently to improve whole organizations . TherefoThe aim of this study was to research how nurses self-assess the frequency of applying caring behaviors in nurse\u2013patient interactions and to identify the differences in application frequency of caring behaviors in relation to work experience and education level.This cross-sectional study was conducted in the Clinical Hospital Center in Croatia (CHC). The quantitative research approach was used and an anonymous survey was performed using a closed-ended questionnaire.Respondents were selected using the principle of availability according to the defined criteria. Thus, the study included a total of 735 registered nurses who are permanently employed at the clinics and departments, and who are in direct contact with patients and provide 24 h healthcare. According to Watson , the conThe sample size was calculated using the online software Sample Size Calculator from Creative Research Systems . The calData were collected using the translated and standardized 70-item version of the Caring Nurse\u2013Patient Interactions Scale Questionnaire  . This veThe CNPI-70 questionnaire was translated from English to Croatian through the following steps: forward translation by two bilingual experts independently; back translation, without any reference to the original instrument wording; comparison of the original and the translated items by another bilingual expert; and review of the translated questionnaire to comply with the system standards for clinical practice. The reliability of both questionnaires was tested using Cronbach\u2019s coefficient. The reliability of each CNPI-70 subscale ranged from 0.75 (\u201chumanism\u201d) to 0.90 (\u201cproblem-solving\u201d). The overall CNPI-70 questionnaire reliability was 0.97, indicating a high reliability. To examine the frequency of caregiving in clinical practice, the question was highlighted at the beginning of the questionnaire: \u201cHow frequently do you apply the attitudes and behaviors described in each of the following statements?\u201d The self-rating scale used a five-point Likert scoring system. Each item was scored from 1 to 5 points .Data were collected for four months at clinics/departments from the aforementioned institution. The researchers (authors of this manuscript) distributed a questionnaire in the clinical departments to all nurses. Nurses were invited to participate voluntarily in this study by completing and returning the questionnaire in sealed envelopes. Respondents completed the questionnaires using the pencil-paper method. The time for completing the questionnaire was not limited; it lasted on average 30 min. The respondents voluntarily and willingly engaged in the research, as they have witnessed great changes in nursing in the Republic of Croatia. Before each data collection, the researcher thoroughly explained the research\u2019s purpose, the ethical issues, and the questionnaire\u2019s details to the respondents. The respondents had the right to withdraw before and during the questionnaire completion. The anonymity of the participants was guaranteed, and there was no possibility to determine their identity from the responses. Only researchers had access to research data.p < 0.05 was considered statistically significant. Descriptive statistics for nominal variables were expressed as proportions and percentages. The normality of the distribution of numerical variables was tested by the Shapiro\u2013Wilk test. Numerical variables  are not following a normal distribution and are represented by the median (Me) and interquartile ranges (IQR). Therefore, the non-parametric Kruskal\u2013Wallis test was used to compare the median differences among several groups, while the Mann\u2013Whitney U test was used to compare the median differences between the two groups. The statistical analysis of the reliability of each scale/subscale and the overall CNPI-70 questionnaire was conducted using Cronbach\u2019s alpha coefficient.Statistical analysis was conducted using SPSS Statistics for Windows, . The author gave us consent for the translation and use of the CNPI-70 instrument. This study was approved by the Institutional Review Board of the CHC .Of the 735 distributed questionnaires, 697 (94.8%) were valid, thus satisfying the calculated minimal number of respondents. Thus, the study included 697 respondents, of whom 642 (92.1%) were female and 55 (7.9%) were male. The age of the respondents ranged from 19 to 65, with a median (Me) of 37 years (IQR 30\u201348). Regarding education level, 533 (76.5%) respondents completed VET and 164 (23.5%) completed their bachelor\u2019s degree. Years of service in nursing ranged from 1 to 44, with a median (Me) of 17 years (IQR 9\u201327).Of the 10 questionnaire subscales, the highest median score was 4.7 (IQR 4.4\u20134.9) and this was achieved for the subscale \u201cneeds\u201d, while the \u201csensitivity\u201d subscale received the lowest median score (3.8) (IQR 3.2\u20134.3) .n = 307) estimated that they apply significantly less the caring behaviors from the following subscales: \u201chumanism\u201d (p < 0.001), \u201csensitivity\u201d (p = 0.009), \u201cexpression of emotions\u201d (p = 0.001), \u201cproblem-solving\u201d (p = 0.008), and \u201cspirituality\u201d (p < 0.001) compared to respondents with more than 30 years of work experience (Respondents with up to 15 years of work experience (perience .p < 0.001), \u201cproblem-solving\u201d (p = 0.003), and \u201cenvironment\u201d (p = 0.021) more frequently than BSc nurses did  of the CNPI-70 instrument, respondents self-assessed the application of certain caring behaviors as very often or almost always. High subscale scores were described in the study by Delmas et al.  despite Despite the high self-assessment score in this study, data analysis showed a significant difference in the frequency of application in certain caring behaviors. Therefore, respondents gave the highest self-ratings for the subscales \u201cneeds\u201d (4.7) and \u201cenvironment\u201d (4.6), which was only partially supported by the results of other relevant studies ,42,43. AIt is evident that respondents respect the concept of basic human needs. However, the highly assessed subscales (\u201cneeds\u201d and \u201cenvironment\u201d) belong to the clinical aspects of care . By contAccording to Fortuno , key facIn this study, the \u201cproblem-solving\u201d and \u201csensitivity\u201d subscales were rated lowest, which is in full accordance with the results of other studies ,42,43. FIn this study, the respondents with up to 15 years of work experience rated procedures related to \u201chumanism\u201d, \u201csensitivity\u201d, \u201cexpression of emotions\u201d, and \u201cproblem-solving\u201d significantly lower compared to the respondents with over 30 years of work experience. These findings are supported by the results of other studies ,49. VandThe results of our study show that respondents with more years of work experience valued spirituality more highly than the respondents with fewer years of work experience. According to Watson , spirituWith regard to education, BSc nurses in this study assessed the \u201chope\u201d, \u201cproblem-solving\u201d, and \u201cenvironment\u201d subscales lower than nurses with basic training (VET). These results can be explained using specific organizational models in clinical practice in which BSc nurses are oriented toward organizational work, management, administrative work, and communication with other healthcare professionals. Thus, these nurses have less time for direct interaction with their patients. There are no significant differences regarding education in relation to other carative factors. Some studies point out the positive correlation between education level and caring attitudes and behaviors , while oAccording to Watson , the proWe believe that it is important to focus subsequent research on nursing students and their patients to determine which caring behaviors are considered more significant from the perspective of patients, students, and registered nurses. Furthermore, additional longitudinal studies using various research methods  should be carried out in order to identify the impact of carative factors on patient satisfaction. Also, future studies should research the correlation between nursing caring behaviors and the patient outcomes. Moreover, it would be useful to research the effect of time on developing and maintaining the relationship of trust with a patient for the purpose of effective caring behavior.The study had certain limitations. First, research was conducted in a single clinical hospital, and we believe that it should be extended to other clinical centers to gain an overview of application of caring attitudes and behaviors. Second, this study\u2019s results which directly relate to specific items in CNPI-70 scale were compared to only a few available studies which had used CNPI-70 scale for assessing importance and frequency of application of nursing caring attitudes and behavior in nurse\u2013patient interactions. Third, since self-assessment was carried out using the CNPI-70 scale, respondents may have inflated their scores to be viewed as a more caring health professional .This study contributes to the dissemination of knowledge related to caring behavior and a better understanding of nurse\u2013patient interactions on a global level. The results showed that caring behaviors are vital segments in nurse\u2013patient interactions, and their quality needs to be assessed and self-assessed. The CNPI-70 scale can be of immense help to nurses in clinical practice to identify the areas of strength and weakness in nurse\u2013patient interactions, which will lead to self-correction and improve the relationship with patients. Furthermore, a better quality of nurse\u2013patient relationship can improve working conditions, improve patient safety, and ensure a higher level of satisfaction for both nurses and patients, thus providing significantly better healthcare. Moreover, the results of this study can be used to analyze and review the existing nursing curricula and to design curricula that will include the content of caring behaviors so that nursing students will learn their importance through efficient methods.Based on the study results, respondents most frequently apply caring behaviors related to assistance with gratification of human needs (\u201cneeds\u201d), whereas cultivation of sensitivity to one\u2019s self and others (\u201csensitivity\u201d) is the most rarely applied. Moreover, there are differences in the application of attitudes and caring behaviors in clinical practice according to years of experience and education level. BSc nurses in this study assessed the subscales \u201chope\u201d, \u201cproblem-solving\u201d, and \u201cenvironment\u201d lower than nurses with basic training (VET). Respondents with up to 15 years of work experience assessed factors related to \u201chumanism\u201d, \u201csensitivity\u201d, \u201cexpression of emotions\u201d, and \u201cproblem-solving\u201d significantly lower than those with more than 30 years of work experience. The results indicate that respondents are more focused on applying skills or carrying out a task than on caring, which is about demonstrating compassion and lovingkindness in their professional relationships."}
+{"text": "Autism spectrum disorder (ASD) is associated with hyper- and/or hypo-sensitivity to sensory input. Spontaneous alpha power, which plays an important role in shaping responsivity to sensory information, is reduced across the lifespan in individuals with ASD. Furthermore, an excitatory/inhibitory imbalance has also been linked to sensory dysfunction in ASD and has been hypothesized to underlie atypical patterns of spontaneous brain activity. The present study examined whether resting-state alpha power differed in children with ASD as compared to TD children, and investigated the relationships between alpha levels, concentrations of excitatory and inhibitory neurotransmitters, and atypical sensory processing in ASD.Participants included thirty-one children and adolescents with ASD and thirty-one age- and IQ-matched typically developing (TD) participants. Resting-state electroencephalography (EEG) was used to obtain measures of alpha power. A subset of participants  also completed a magnetic resonance spectroscopy (MRS) protocol in order to measure concentrations of excitatory  and inhibitory (GABA) neurotransmitters.Children with ASD evidenced significantly decreased resting alpha power compared to their TD peers. MRS estimates of GABA and Glx did not differ between groups with the exception of Glx in the temporal-parietal junction. Inter-individual differences in alpha power within the ASD group were not associated with region-specific concentrations of GABA or Glx, nor were they associated with sensory processing differences. However, atypically decreased Glx was associated with increased sensory impairment in children with ASD.Although we replicated prior reports of decreased alpha power in ASD, atypically reduced alpha was not related to neurochemical differences or sensory symptoms in ASD. Instead, reduced Glx in the temporal-parietal cortex was associated with greater hyper-sensitivity in ASD. Together, these findings may provide insight into the neural underpinnings of sensory processing differences present in ASD.The online version contains supplementary material available at 10.1186/s11689-020-09351-0. Autism spectrum disorder (ASD) is an etiologically complex, heterogeneous condition affecting 1 in 54 children, making it one of the most prevalent neurodevelopmental disorders . These dBehavioral responses to incoming sensory information are determined, in part, by one\u2019s cortical state . ElectroPrevious studies have focused on neurophysiological differences in ASD, and how they may contribute to the behavioral characteristics associated with the disorder balance and atypical neural oscillations and sensory function in ASD, we also sought to examine the associations between in vivo measures of excitatory and inhibitory neurotransmitters, alpha power, and sensory symptoms. By comparing not only the physiological features of both groups, but mapping those onto measures of ASD symptomatology, findings from the current study may provide a better understanding about the heterogeneous nature of ASD symptoms and their neurophysiological bases.Participants included 31 children with ASD and 31 age- and non-verbal IQ-matched TD children  with a NetAmps 400 high-input amplifier. Data were collected from 124 of 128 possible channel locations. In order to decrease attrition, EOG electrodes (electrodes placed on the face) were not used. Data were sampled at 500\u2009Hz and referenced to the vertex electrode.t(60) = 1.4, p = .156, or components removed , t(60) = 1.4, p = .168. Artifact-corrected data were segmented into 1-s epochs, and epochs containing residual artifacts were rejected. Alpha power (8\u201312\u2009Hz), expressed as decibels (dB), was extracted from three regions of interest (ROI) each of which consisted of 4 locations across midline frontal , central , and posterior electrode locations  completed the imaging protocol (see Supplementary Table https://www.fil.ion.ucl.ac.uk/spm/). All metabolites were CSF-corrected with the exception of GABA, which was tissue-corrected using the method as described in Harris et al. ),One other potential explanation for the absence of any correlations within the ASD may be that the regions examined in the present study do not include the generators of alpha activity that have previously been shown to contribute to differences present in ASD. For example, scalp-recorded alpha power may be associated with GABA-mediated thalamic activity , 55. PriLastly, although we observed associations between alpha levels and Sensitivity and Seeking scores within the TD group, we did not find evidence of a relationship between alpha levels and sensory symptoms within children with ASD. Previous research demonstrating links between alpha and sensory symptoms has focused on frontal alpha asymmetry rather than absolute alpha power , 28. ThuAs previously reported by Edmondson and colleagues , only GlContrary to prior research that has demonstrated associations between GABA levels and sensory processing in ASD \u201335, no aWhy are atypically decreased Glx values within the rTPJ associated with increased hyper-sensitivity in ASD? The rTPJ is a hub for social-cognitive processes and may coordinate communication about the information gathered from one\u2019s external sensory environment with internal model-based predictions . FurtherIn addition, a predictive coding account has also been used to explain differences in responding to sensory input in individuals with ASD e.g., \u201377). In . In 77]).Children and adolescents with ASD in the present study did provide significantly less usable EEG data; however, the amount of usable EEG was not associated with average alpha power across the two groups, nor did results change when percentage of usable data was included as a covariate. Additionally, because only a subset of participants completed the MRS portion of the study, our MRS analyses were limited to restricted number of participants. As such, correlations with MRS measures should be confirmed with a larger cohort of participants. Furthermore, correlational analyses were not corrected for multiple comparisons, and, thus, should be considered exploratory in nature. Finally, our interpretation of Glx values focuses on glutamate; however, Glx includes contributions from both glutamate and glutamine, and so should be interpreted with cautionSensory processing impairments have a significant impact on the quality of life of individuals with ASD and their families. Thus, discovering the neurophysiological underpinnings of ASD symptomatology, including sensory processing differences, has the potential to provide insight into the heterogeneous nature of the ASD phenotype as well as to inform the identification of early diagnostic markers and development of novel intervention approaches. This study presents evidence that resting-state alpha levels are significantly reduced in children and adolescents with ASD compared to their TD peers, supporting a growing body of previous research. However, these differences were not associated inter-individual differences in sensory processing symptoms or region-specific variations in excitatory or inhibitory neurotransmitters. Instead, individuals with ASD exhibited reduced concentrations of Glx in the right temporal-parietal junction, and atypically decreased levels of Glx were associated with greater levels of sensory symptoms. These exploratory findings, which should be confirmed using a larger sample, suggest that a reduction in glutamatergic drive within the temporal-parietal cortex may contribute to hypersensitivity to sensory input in ASD.Additional file 1. Supplementary Tables"}
+{"text": "Surgery holds high risk for iatrogenic patient harm. Correct and sufficient communication and information during the surgical process is a root solution for preventing patient harm. Information technology may substantially contribute to engaging patients in this process.To explore the feasibility of a digital patient-led checklist for cataract surgery, we evaluated the experiences of patients and nurses who have used this novel tool with a focus on use, appreciation, and impact.A multidisciplinary team, including cataract surgeons, nurses, pharmacists and administrative representatives developed a 19-item digital patient-led checklist for cataract patients who underwent surgery in an ambulatory setting. This \u201cEYEpad\u201d checklist was distributed to patients and their companions during their hospital visit via an application on a tablet. It contained necessary information the patient should have received before or during the surgical preparation (8 items), before anesthesia (2 items), and before discharge (9 items). Patients and their companions were invited to actively indicate the information they received, or information discussed with them, by ticking on the EYEpad. Our qualitative research design included semi-structured individual interviews with 17 patients and a focus group involving 6 nurses. The transcripts were analyzed by 2 independent coders using both deductive and inductive coding.All but one of the 17 patients used the EYEpad, occasionally assisted by his or her companion . In several cases, the checklist was completed by the companion. Most patients felt positively about the usability of the EYEpad. Yet, for most of the patients, it was not clear why they received the checklist. Only 4 of them indicated that they understood that the EYEpad was used to determine if there were sufficient and correct information discussed or checked by the nurses. Although most nurses agreed the EYEpad was easy to use and could be a useful tool for improving patient engagement for improving safety, they felt that not all elderly patients were willing or capable of using it and it interfered with the existing surgical process. They also anticipated the need to spend more time explaining the purpose and use of the EYEpad.Our results showed that a digital patient-led checklist is a potentially valid way to increase patient participation in safety improvement efforts, even among elderly patients. It also illustrates the crucial role nurses play in the implementation and diffusion of technological innovations. Increased patient participation will only improve safety when both healthcare workers and patients feel empowered to share responsibility and balance their power. Health care delivery is too often not \u201cfree from accidental injuries,\u201d according to the Institute of Medicine definition of patient safety . In DutcSurgery is a high-risk area for iatrogenic patient harm ,4. IatroThe last decade has seen increasing awareness and focus on patient safety -9. TradiCommunication between patients and professionals is a major issue in safety . The hanAlthough it is known that communication of the \u201cright things\u201d at the \u201cright moment\u201d is important for preventing iatrogenic patient harm, it is difficult to optimize this process because patients are concerned with many things during their care pathways. Information technology may substantially contribute to engaging patients in activities to improve patient safety ,22.To increase patient participation in enhancing safe care, we developed an online checklist called the EYEpad for cataract patients to be used during their admission. Cataract surgery involves removal of opaque lens and replacement with an implanted artificial intraocular lens (IOL) is the most frequently performed surgery in the world . The feaWe used a qualitative approach to explore patients\u2019 and nurses\u2019 experiences with the digital EYEpad checklist. The definition of semistructured interviews by Green and Thorogood is \u201cIn a semistructured interview, the researcher sets the agenda in terms of the topics covered but the interviewee\u2019s responses determine the kinds of information produced about those topics, and the relative importance of them\u201d . At appoParticipants were recruited at the Rotterdam Eye Hospital, the only eye hospital in the Netherlands providing secondary and tertiary eye care. The hospital has a specialized ambulatory cataract pathway where about 6500 cataract surgeries are performed annually.Patients often had questions about how to care for their treated eyes after discharge from the hospital. To prevent this, initially a paper card was designed to relay information to patients before their discharges. The card served as a memory aid for nurses to inform patients about these points, but was rarely used. Subsequently, a checklist for patients was designed. A multidisciplinary team, including cataract surgeons, nurses, pharmacists, and administrative representatives developed a 19-item patient-led checklist for cataract patients who underwent surgery in an ambulatory setting. The items were based on a review of nurses\u2019 current, often inconsistent, and not formally acknowledged, check moments. An initial gross-list of more than 30 items was reduced to 19, all of which were agreed upon by the multidisciplinary team. The checklist was first tested on paper by patients and later modernized into an application for the tablet called EYEpad. This checklist was distributed to patients and their companions via the application EYEpad, on a tablet, during their hospital visits. It contained 3 lists with necessary information the patient should have received during three contact moments with medical professionals on the day of their surgery: before or during surgical preparation (8 items), before anesthesia (2 items), and before discharge . Finally, the patient can add his or her own questions to ensure that these questions are addressed during the dismissal conversation. Patients could also use the tablet for other general, educational, or entertainment functions, such as news services and games.During a period of two weeks, patients who were scheduled first and last in the morning and in the afternoon were approached to participate in the study. A registered nurse recruited participants according to the following inclusion criteria: (1) age older than 18 years, (2) first cataract surgery, (3) ability to understand Dutch, (4) absence of severe comorbidities, and (5) absence of mental or cognitive disorders.The selected patients were approached by phone one day before their hospital visit. Patients were given information about the study and asked whether they wished to participate. It was emphasized that participation was voluntary and their decision about participating in the study would have no effect on their treatment. When patients agreed to participate, the researcher fixed a time for a short interview at the hospital immediately after the patient\u2019s discharge.I Preparation phase (8 items):Patient namePatient date of birthEye to be operated onDiabetes statusIodine allergy statusExplanation on day of surgery proceedingsExplanation on eye balm application on eve of surgeryExplanation of dilatation dropsII Anesthesia phase (2 items):Time-outAnesthetic eye dropsIII Postsurgical phase (9 items):Review of surgical proceedingsPain assessmentPostsurgical patient flyerAvailability and application of eye drops at homePostsurgical telephone review (date and time)Removal of eye bandageChecking of pupil size and formPhoto surgical teamEye drop application information and trainingThe ambulatory surgical center (ASC) manager invited all nurses to a regular department meeting and allowed the researcher to use a part of the meeting for a focus group.Prior to the interview, the participants provided informed consent for participation and for tape recording of the interview. All interviews were conducted by a trained psychologist (JVDS). Interviews took an average of 10 minutes and took place in a separate room, behind closed doors, to preserve the patient\u2019s privacy.During the interviews, an interview guide with 24 open-ended questions, derived from published literature and in consultation with staff members of the hospital and the University of Twente, was used. The interview questions focused on (1) EYEpad utilization: \u201cDid you use the EYEpad?\u201d; (2) appreciation of the EYEpad: \u201cWhat did you like/dislike in the EYEpad?\u201d; and (3) impact of the EYEpad: \u201cWhat does feeling safe in a hospital mean to you?\u201d Patients were asked explicitly to motivate and support their answers. All interviews were audiotaped. Ethical approval was obtained from the ethics committee of the University of Twente (#13196).Prior to the focus group session, the nurses were asked to complete a 10-item questionnaire. This questionnaire was intended to stimulate the participants to think about the topics discussed during the focus group. We chose this approach to prevent group thinking by the participants.All nurses were asked to provide consent for participation and tape recording of the focus group. The focus group lasted 60 minutes. During the focus group session, a script with open-ended questions, derived from published literature and in consultation with staff members of the hospital and the University of Twente, was used. The questions focused, as they did during the patient interviews, on (1) EYEpad utilization: \u201cWhat instructions did you give to patients during handover of the EYEpad?\u201d; (2) appreciation of the EYEpad: \u201cWhat do you consider to be positive and negative aspects of the EYEpad?\u201d; and (3) impact of the EYEpad: \u201cWhat do you consider as benefits of the EYEpad?\u201d. Finally, the focus group addressed (4) the future of the EYEpad: \u201cWhat needs to be changed for sustainable use of the EYEpad?\u201d The minutes of the meeting were included in the analysis.The audiotaped data from the interviews was transcribed verbatim. Transcripts were deductively coded into one of the three main categories: utilization, appreciation, and perceived impact. Next, the fragments in each category were further divided into subcategories, using inductive analysis, meaning the categories were inferred from the data, rather than from the existing literature. Coding was conducted by 2 coders . Differences were discussed until a consensus was achieved .From the 32 selected patients, 19 patients met the inclusion criteria. Seventeen patients accepted the invitation to participate in the study. Two patients refused to participate because they did not feel well enough to be interviewed after their surgeries. Eleven out of 17 (65%) patients were female. The average age was 69 years, ranging from 58 to 88. Almost all patients were accompanied by their partner (n=12) and others by their daughter (n=2), son (n=1), or another relative (n=1). One patient was not accompanied by a companion.Six of the 18 registered nurses participated in the focus group. All nurses were female (n=6). The average age of the nurses was 46 years, ranging from 20 to 57. Most nurses (n=4) worked at the ASC for at least 5 years.Three themes emerged from the data analysis: utilization, appreciation and impact. The subthemes that belong to these themes can be found in All but one patient used the EYEpad. In several cases (n=9), the EYEpad checklist was completed by the companion as the patients could not clearly see because their eyes were dilated or their reading glasses were stored in a locker. The companions only entered the patients\u2019 answers.I have completed it, but yes, actually I have only pressed the buttons. You have completed the answers.Companion 3Three patients completed the EYEpad on their own because they were more familiar with a tablet than their companion was. One patient and his companion did not use the EYEpad because they were not familiar with the use of a tablet device.Most did not completely understand why they received the EYEpad. They took the EYEpad without further enquiries and assumed it was part of the hospital administration or it was for quality improvement. In only four cases, respondents indicated that the EYEpad was meant to validate if all necessary information was given to the patients and if nurses checked important information for patients\u2019 safety.[Silence] No, I assume things automatically; they need to know who you are, and they repeat that often. [Silence] I think that\u2019s part of the administration.Patient 1Besides using the EYEpad application, patients and their companions could use other functionalities on the tablet, like the web browser, playing a game, or watching movies. One patient read the news on the internet, to relax, before her surgery.Yes, I have checked the news that was available, so I had something to read...That killed the waiting time, so I enjoyed it.Patient 17The others did not use the other functionalities on the tablet because they were unfamiliar with tablet functions or did not feel a need for it.\u2026And I was afraid that there was just one application [the EYEpad app], so I thought, yes [laughs], keep it like this, it is functioning well now, and I should not peddle someone else\u2019s tablet.Patient 2During the surgery, I have watched the [intraocular live viewing] monitor, so you don\u2019t need the iPad [tablet] at that time.Companion 3time-out.\u201d Further, it was not clear to all respondents when to use specific checklist tabs, considering there were 3 different tabs. Lastly, one respondent reported there was little time between the nurses\u2019 explanation and the use of the EYEpad; this patient did not have sufficient time to open the EYEpad and get used to it before he had to use it.Fourteen participants, who already knew how to use a tablet, reported that the EYEpad was easy to use. However, some difficulties were experienced. First, some respondents reported that it was hard to fill in their birth date because the scroll menu moved fast. Second, some respondents did not understand the jargon used in the EYEpad, for example, \u201cYes, especially for that age, it was taking an aim.Companion 2Yes, that\u2019s right [laughing], a kind of roulette, as it kept on rotating.Patient 2When asked whether the participants preferred the version of the EYEpad on the tablet or on paper, 14 participants preferred the digital version because of the usability and ability to save data.It is easy to complete, briefly touch and a checkmark appears.Patient 11Two participants preferred a paper questionnaire above a tablet; both reported low eHealth literacy. Both participants were females. One female was aged 82 and was accompanied by her daughter, and the other female was 72 years old and accompanied by her partner.Five of the 6 nurses provided the EYEpad to all patients, except when the patient did not have a companion or when the patient was very old. However, during the focus group, there was a discussion as to whether a patient could be \u201ctoo old\u201d.When someone is very old, I don\u2019t offer him an EYEpad.NurseBut some elderly are very good with tablets, so I think that\u2019s no reason to not give it to an elderly patient.NurseYes, indeed, some elderly are able to handle the EYEpad, and like it very much, so age should not be a discriminator.NurseSome see you approaching them with the piece, and they don\u2019t look very happy.NurseAnother reason for not providing the EYEpad was the workload experienced by the nurses. The nurses were unanimous that the EYEpad was subordinate to their primary work: treating patients.Sometimes you need to assist a colleague, or sometimes you are very busy, or something else needs your attention; the EYEpad is then the first to neglect or skip.NurseFive nurses mentioned that they found it hard to give the correct explanation when they provided the EYEpad to a patient. This was caused by different reasons. They referred to the busy schedule and the number of patients around during the provision of the EYEpad. Also, the perceived patient knowledge of the tablet played a role.If it\u2019s very busy, it\u2019s difficult to give a proper explanation. You have less time.NurseIf the patient is familiar with an iPad, the instructions can be done fast because you don\u2019t have to explain how an iPad works.Nurseanesthetic drops \u201d on the second tab of the checklist.Although nurses were generally positive about the EYEpad usability, they noticed, just like the patients, a few difficulties. First, it was not clear which action was related to the term \u201cI just still do not understand fully what must be ticked at \u201canesthetic drops.\u201d Is that the moment that we tell the patient they receive anesthetic drops and show which ones? Or is it the moment that we give the anesthetic drops?NurseSecond, it was unclear why only \u201ciodine allergy\u201d was in the checklist because other allergies of the patients were also important to know. Finally, they noticed it was not always easy to go back to the top of the checklist when the checklist was finished.\u201cgood\u201d or \u201cfine\u201d (n=12). They especially appreciated the checkpoints. Both patients and companions indicated they felt more involved in the health care process on using the EYEpad.The EYEpad was well appreciated by patients and companions. Most of the respondents reviewed the EYEpad as Well, I thought it is an extra check, for you can\u2019t check these things  often enough.Patient 2We now need to think ourselves, and that was, ehh, you are more involved at least.Companion 13Most nurses did not appreciate the EYEpad for two reasons. The first reason was that the EYEpad caused some agitation for both patients and nurses. According to the nurses, some elderly patients were scared they had to use a tablet. Agitation was also experienced when the patients\u2019 companions moved from the waiting room to the preparation room to complete the second checklist.What I have experienced as troublesome is that the companions of the patients now more often move to the preparation room taking their entire possessions, because they have to complete over there a second list. This is not really the intention and creates a lot of agitation.NurseYes, indeed, previously the companion came just along to the preparation [room] as a patient had some degree of anxiety, but now they all come in to complete the checklist.NurseThe second reason why not all nurses appreciated the EYEpad was because it was time-consuming.The provision and explanation of the EYEpad still just takes a lot of extra time. It is not always the case that you are there with a short explanation, because most of the patients have several questions about it, such as how it exactly works.NurseThey also mentioned positive aspects of the EYEpad. First, the use of the EYEpad improved the reputation of the day center.It seems luxurious and very modern.NurseSecond, they thought it was nice that younger patients were fine with the EYEpad. Third, they were generally positive about the usability. Most of the nurses (n=4) mentioned that the EYEpad was easy to use. Two mentioned that, although they were not completely familiar with a tablet, they always resolved it together with a colleague.I think it is sometimes still quite a bit of a search, even though I know how an iPad works, but fortunately,you will always bring it to an end.NurseMost patients saw no safety benefits associated with using the EYEpad (n=10). They did not know the purpose of the EYEpad. Six patients, however, thought the EYEpad could contribute to safety because of all the extra data checks.Uh, that the EYEpad would help for safer care, here, in the hospital? Well, no, I really don\u2019t see that link directly.Patient 5Yes, that would be possible, I think, or yes, I do not really know. What do you [companion] think?Patient 10Yes, you know, it certainly can, as long as the nurse still take[s] care of those points [unchecked items on the checklists].Patient 4According to the nurses, the contribution of the EYEpad to safety is not yet known. They felt time they spent on the EYEpad was too short to evaluate its contribution to patient safety. They were still uncomfortable with the EYEpad and felt their explanation to the patients was still suboptimal. The nurses named several impact factors associated with the EYEpad. First, they reported that the EYEpad had a positive influence on the empowerment of patients. The patients were more involved in their care process, more alert, and more conscious of their own responsibility.The patient is more involved in his or her surgery process by the EYEpad.NurseBy the EYEpad the patient becomes more alert and sees more things during the care process.NurseWith the EYEpad you make the patient and his or her companion more aware of their own responsibility.NurseSecond, the EYEpad had a positive impact on the patients\u2019 companions because the companions could use the other functionalities of the tablet to relax. Further, the EYEpad influenced the interaction between patients in a positive manner.If a patient does not understand the EYEpad or encounters a problem, patients help each other. This creates more contact between patient and companion. Perhaps this also reduces the patient\u2019s anxiety.NurseNext, the EYEpad could have a negative impact on patients and their companions because they could get distracted by the EYEpad during intake, get surprised following presentation of the EYEpad, and companions could feel obligated to the patient.If you give the tablet, people go straight to work with the tablet, therefore people pay less attention to the nurse.NursePatients do not yet know anything about the EYEpad when they arrive at the day center on the day of their surgery. It can overwhelm them.NurseSome patients often do not dare to say they do not like it [tablet], because we [nurses] offer them from the hospital, and therefore they think that it is obligatory.NurseCompanions are more concerned with the iPad [tablet] than with the patient, making the guidance or support falls away.NurseLastly, the EYEpad may worsen the supportive role of the companion if the companion gives more attention to the tablet than to the patient.This study showed that the use of the EYEpad as a digital patient-led checklist in cataract surgery is feasible. Feasibility has been demonstrated in three ways. First, the EYEpad was well appreciated by patients. Patients were positive about the additional checks and felt more involved in their care processes. Second, we found the EYEpad, beside some practical difficulties, was easy for patients and nurses to use. Third, we found that the EYEpad helped patients feel empowered.However, there remains room for improvement. The EYEpad, with its current instructions, can increase nursing workload. Furthermore, an improved introduction on the rationale and use of the digital checklist is needed because the purpose of the EYEpad was not always clear to the participants. Improved instructions are likely to further enhance patient experience, increasing patients\u2019 abilities to understand and influence their own care. This was suboptimal in this study because some patients participated just because the EYEpad was handed to them and not because they were motivated to use it.This study had several limitations. One limitation was that we held just one focus group with nurses. More focus groups could have yielded more information about the nurses\u2019 viewpoints. Moreover, participation in the focus groups was voluntary and in the end, only 6 of the 18 nurses participated. The nurses who participated likely held viewpoints that differed from nurses who did not participate.Another limitation was that the purpose of the EYEpad was not clear to all patients and nurses at the start of the study. Only 4 patients indicated they understood the purpose of the EYEpad. This may be due to the limited explanation the nurses gave about the checklist. Apparently, a more elaborate explanation is needed to better understand the purpose of the EYEpad, both for patients and nurses. Previous work has suggested the success of checklist implementation largely depends on a clear explanation of the \u201cwhy\u201d and \u201chow\u201d . A betteFurther, although we inquired as to patients\u2019 general experiences with the EYEpad; we did not explicitly address electronic health  or health literacy during this study. Therefore we cannot be sure that needs related to EYEpad use were specifically addressed.In our study, the checklist was patient-led instead of team-led. We found this helped to empower the patients in their own care pathways. We suggested two possible explanations for why patients felt more empowered by using the EYEpad. First, patients may feel more engaged in their own care process. Using health technology makes patients feel more involved in their own care . As indiAujoulat et al described the success factors for patient empowerment. They found that the basics of patient empowerment were to provide reassurance and opportunities for self-exploration on how to manage illness . Second,Besides empowerment, we also found that the EYEpad increased patient participation. Checklists are supplementary tools that encourage critical thinking and conversation . The EYEA surprising finding was that nurses experienced the checklist as \u201cextra work\u201d instead of as a supportive tool for their daily tasks. This may be because the goal of the checklist was not clearly explained. Furthermore, not all nurses were involved in the development of the checklist, which may have made them feel less engaged.Before further development of the EYEpad, some hurdles should be addressed. These include providing clear instruction on the rationale for the professionals involved and an improved introduction and explanation of the purpose of the checklist for patients. Communication about the objective of the new digital technology, both with health care staff and patients, is a vital element for successful implementation. It is important to include nurses and other health care professionals from the early idea generation stage, into development and iteration, to generate support and interest. Communication about the objective of the EYEpad must be clear, both to nurses and to patients. Further, our study showed that the practical implication involved listening closely to the care pathway: Which moments are best for the digital EYEpad checklist to be distributed given the planning of the surgical treatment flow? In the current process the use of the EYEpad sometimes disrupted the existing flow, when it should have contributed to a smoother and high-quality care process.After these hurdles have been considered, the EYEpad can be further developed and implemented. We found that the EYEpad could encourage learning, for example by conscious information acquisition by patients. We did not give specific attention to eHealth and health literacy of participants. More attention to eHealth and health literacy may improve the level of learning.Further, the checklist should relate to the various steps of the current care process. The better the checklist is implemented, the more structural value it will add toward patient participation in enhancing safe care. It would be useful to make a connection between the checklist and the patients\u2019 records to give the professionals insight into the data in a more accessible way. In addition, future studies should make a connection between the checklist and other patient tools to give patients a more complete overview of their care process.In conclusion, we showed that a digital patient-led checklist during surgery was a feasible instrument in cataract care. Our findings suggest that a digital checklist could increase health literacy and provide enhanced guidance on the day of surgery. Our results also demonstrated the crucial role nurses play in the logistics of technological innovations. Increased patient participation will only improve safety as both health professionals and patients feel empowered to share responsibility and balance power."}
+{"text": "N = 15; mean \u00b1 SD: 23 \u00b1 3 years old, 6 \u00b1 5 years training) performed full-body, low-volume, high-load [68\u201390% of one repetition maximum (1RM)] resistance training over 10 weeks. Back squat strength testing, body composition testing, and a vastus lateralis biopsy were performed before (PRE) and 72 h after the 10-week training program (POST). Fiber type-specific cross-sectional area (fCSA), myofibrillar protein concentrations, sarcoplasmic protein concentrations, myosin heavy chain and actin protein abundances, and muscle tissue percent fluid were analyzed. The abundances of individual sarcoplasmic proteins in 10 of the 15 participants were also assessed using proteomics. Significant increases (p < 0.05) in type II fCSA and back squat strength occurred with training, although whole-body fat-free mass paradoxically decreased (p = 0.026). No changes in sarcoplasmic protein concentrations or muscle tissue percent fluid were observed. Myosin heavy chain protein abundance trended downward  and actin protein abundance decreased  with training. Proteomics indicated only 13 sarcoplasmic proteins were altered with training . Bioinformatics indicated no signaling pathways were affected, and proteins involved with metabolism  were not affected. These data comprehensively describe intramuscular protein adaptations that occur following 10 weeks of high-load resistance training. Although previous data from our laboratory suggests high-volume resistance training enhances the ATP-PCr and glycolytic pathways, we observed different changes in metabolism-related proteins in the current study with high-load training.While high-load resistance training increases muscle hypertrophy, the intramuscular protein responses to this form of training remains largely unknown. In the current study, recreationally resistance-trained college-aged males ( High-load resistance exercise involves performing lifts with heavier weights for fewer repetitions [sets consisting of 1\u20136 repetitions at \u226575% one repetition maximum (1RM)]. Moderate-to-higher volume resistance exercise involves performing more repetitions using light to moderate loads (sets consisting of >10 repetitions at \u226465% 1RM). There has been recent enthusiasm surrounding molecular signaling events that occur in skeletal muscle in response to higher load versus moderate-to-higher volume resistance exercise . In geneIndeed, there have been studies that have examined how resistance training affects certain protein adaptations  along with hypertrophy measures  [reviewed in All procedures in this study approved by the Institutional Review Board at Auburn University (Protocol #18-442 MR 1812), and this study conformed to the standards set by the latest revision of the Declaration of Helsinki.Participants were verbally recruited at Auburn University. Eligible participants had to be free of cardiometabolic diseases  or conditions precluding the collection of a skeletal muscle biopsy. Additionally, participants had to self-report resistance training for at least 12 months prior to the study. Interested participants provided verbal and written consent to participate prior to data collection procedures outlined below.The testing session detailed below occurred during the morning hours (0500\u20130900) following an overnight fast, and procedures are described in the order that they occurred. These sessions occurred prior to the 10-week training intervention (PRE), and 72 h following the last training bout (POST). A timeline of the study is provided in At the beginning of each testing session, participants submitted a urine sample (\u223c5 mL) for urine specific gravity (USG) assessment. A handheld refractometer  was used for measurements. USG measurements were performed given that being in a dehydrated state has been shown to affect body composition measures , and beiBody composition was measured by bioelectrical impedance spectroscopy (BIS) using the SOZO device  according to the methods described by Right leg vastus lateralis muscle biopsies were collected using a 5-gauge needle under local anesthesia as previously described . ImmediaThree repetition maximum back squat testing occurred 3\u20137 days prior to PRE testing and during POST testing following the skeletal muscle biopsy. Strict technical parameters were employed during back squat assessments under the direct supervision of the study coordinator who holds a Certified Strength and Conditioning Specialist Certification from the National Strength and Conditioning Association (NSCA). Testing first began with two rounds of calisthenics . Participants then performed one warm-up set of 10 repetitions using the bar only (20 kg) followed by one set of 10 repetitions at \u223c60 kg. Participants then performed three repetitions at a self-estimated 70% 1RM, and began maximal 3RM attempts thereafter. Approximately 3 min of rest was allowed between 3RM attempts, and repetitions were considered successful when a squat depth was accomplished where the hip crease was below the knee joint. 1RM values were estimated from 3RM values using the NSCA\u2019s most recent text .Participants were provided a pre-programed high-load training regimen to adhere to over the 10-week intervention . Partici3 chunks. Chunks (\u223c20 mg) were weighed using a scale with a sensitivity of 0.0001 g  and placed into 1.7 mL polypropylene tubes with five ventilation holes drilled on the caps. Tissue in tubes were then freeze-dried using a laboratory-grade freeze-drying apparatus . Freeze dry settings were \u221250\u00b0C for the condenser and a vacuum setting of 0.1 mBar, and samples were placed in the condenser well for 4 h under constant vacuum pressure. Following freeze drying, muscle was re-weighed on an analytical scale, and muscle fluid content was assessed using the following equation:Skeletal muscle foils were removed from \u221280\u00b0C storage, placed on a liquid nitrogen-cooled ceramic mortar and pestle, and tissue was pulverized into 2\u20134 mmn = 5 independent tissue chucks) in one of our previous publications (n = 5 independent tissue chucks). Both methods yielded similar tissue fluid content values , and the freeze dry method produced a high degree of reliability (CV = 1.7%).This protocol was piloted using muscle tissue obtained from rat triceps  were collected and placed in new 1.7 mL microtubes on ice. As a wash step, the resultant myofibrillar pellet was resuspended in 300 \u03bcL of Buffer 1 and centrifuged at 1,500 \u00d7 g for 10 min at 4\u00b0C. The supernatant was discarded and the myofibrillar pellet was solubilized in 400 \u03bcL of ice-cold resuspension buffer . The solubilized sarcoplasmic and myofibrillar fractions were then stored at \u221280\u00b0C until protein concentration determination and proteomic analyses described below. Notably, these methods differ slightly from what we have previously used, because of the difficulty in resuspending myofibrils. With extensive piloting, we determined the following: (a) using a different buffer 1  kit . Samples were assayed in duplicate (sarcoplasmic protein) or triplicate (myofibrillar protein) using a microplate assay protocol where a small volume of sample was assayed (20 \u03bcL of 5x diluted sample + 200 \u03bcL Reagent A + B). The myofibrillar resuspensions were then assayed for myosin heavy chain and actin protein abundances using SDS-PAGE and Coomassie staining, and the sarcoplasmic fraction underwent proteomics analysis described below. Average replicate coefficients of variation for myofibrillar protein concentrations and sarcoplasmic protein concentrations were 3.3 and 3.9%, respectively.2O), and 25 \u03bcL 4x Laemmli buffer. Samples (5 \u03bcL) were then loaded on pre-casted gradient (4\u201315%) SDS-polyacrylamide gels in duplicate (Bio-Rad Laboratories) and subjected to electrophoresis at 180 V for 40 min using pre-made 1\u00d7 SDS-PAGE running buffer . Following electrophoresis, gels were rinsed in diH2O for 15 min and immersed in Coomassie stain  for 2 h. Gels were then destained in diH2O for 60 min, and bright field imaged using a handheld digital camera . Image files were converted to grayscale using a gel documentation system and associated software , and region of interest bands were drawn at the prominent 220 and 43 kD bands . Given that a standardized volume from all samples were loaded onto gels (5 \u03bcL), myosin heavy chain and actin protein band densities were normalized to input muscle weights to derive arbitrary density units (ADU) per mg dry muscle. All values were then divided by the mean of the PRE time point to depict myosin heavy chain and actin protein abundances. Contrary to conventional Western blotting methods where protein targets are normalized to total protein , this method allows researchers to determine the change in the relative abundances of myosin heavy chain and actin according to muscle weights. In scenarios where myosin heavy chain and actin protein band densities per muscle weight decrease in response to resistance training, this may be suggestive of a disproportional increase in muscle weight relative to myofibrillar protein accretion. Our laboratory has reported that this method yields exceptional sensitivity in detecting 5\u201325% increases in actin and myosin heavy chain protein abundances , and 0.1% formic acid and mobile phase B was composed of 99.9% acetonitrile and 0.1% formic acid. A 60-minute linear gradient from 3 to 45% B was performed. The eluent was directly nanosprayed into the mass-spectrometer. During chromatographic separation, the Q-Exactive HF was operated in a data-dependent mode and under direct control of the Thermo Excalibur 3.1.66 (Thermo Fisher Scientific). The MS data were acquired using the following parameters: 20 data-dependent collisional-induced-dissociation (CID) MS/MS scans per full scan (350 to 1700 m/z) at 60,000 resolution. MS2 were acquired in centroid mode at 15,000 resolution. Ions with a single charge or charges more than seven as well as unassigned charge were excluded. A 15-second dynamic exclusion window was used. All measurements were performed at room temperature, and three technical replicates were run for each sample. Raw files were analyzed using Proteome Discoverer  software package with SequestHT and Mascot search nodes using the 20180308HumanSwissprot.fasta database and the Percolator peptide validator. The resulting.msf files were further analyzed by the proteome validator software Scaffold v4.0 . Final data for each protein were obtained as spectra data normalized to total spectra, and data are presented as such. Proteins at POST which deviated from PRE values  are presented in the results.Proteomics analysis was performed on the sarcoplasmic protein fraction similar to previous work published by our laboratory . Each sa2 was used to ensure artifact was removed .Methods for immunohistochemistry were employed as previously reported by our laboratory and described elsewhere . Brieflyt-tests were performed when comparing dependent variables between the PRE and POST time points, and statistical significance was set at p < 0.05. BIS-derived extracellular water (ECW), and intracellular water (ICW) failed normality testing and were not correctable with log transformation, square root transformation nor cube root transformation. Thus, we performed both parametric and non-parametric comparisons on these variables and proceeded with parametric tests for all other dependent variables. Pearson correlations were also performed on select variables. For proteomics data, proteins that exhibited a significant up- or down-regulation from PRE to POST  were analyzed for functionally annotated pathways using the OFFICIAL_GENE_SYMBOL query in DAVID v6.8  Bonferroni-corrected p-values values would likely yield stringent adjusted p-values, and (b) Benjamini-Hochberg-adjusted p-values would likely yield p-values that were lower than un-adjusted p-values due to there being only 385 proteomic targets. All data herein are presented in figures and tables as means \u00b1 standard deviation values.Statistical analyses were performed in SPSS  and RStudio . Normal distribution of dependent variables was performed using Shapiro-Wilks tests. Non-normally distributed data was log-transformed prior to statistical analysis. Dependent samples VID v6.8 . Notably2, and pre-intervention body fat percentage was 21.2 \u00b1 4.7%. Of the 15 participants, nine individuals logged all of their workouts, four individuals logged >80% of their workouts, and two participants logged fewer than 80% of their workouts. For the two participants that poorly logged workouts, these individuals verbally confirmed that workouts were being completed, but cited time constraints for not completing training logs. Training data for the 13 participants that logged at least 80% of their workouts are presented in The participants\u2019 average age was 23 \u00b1 3 years, self-reported training age was 6 \u00b1 5 years (range: 1.5 to 16 years), pre-intervention fat-free mass index was 21.6 \u00b1 2.7 kg/mp = 0.026), and an increase in squat strength (p < 0.001) were observed. However, parametric statistics indicated no significant changes occurred in ECW  or ICW  (data not shown). Given these variables were the only variables that non-normally distributed, we also performed Wilcoxon signed-rank tests. These tests also indicated no significant changes occurred in ECW (p = 0.131) or ICW (0.410).PRE- and POST-intervention body composition and estimated 1RM squat strength for all participants are presented in p = 0.104) or mean fCSA were observed (p = 0.110) (p = 0.028) (p = 0.085), whereas values for type II fibers were 74.1 \u00b1 10.5 \u03bcm at PRE and 81.9 \u00b1 8.3 \u03bcm at POST (p = 0.020) (data not shown). Fiber counts averaged to be 105 \u00b1 32 at PRE and 108 \u00b1 27 at POST.Pre- and post-intervention type I, type II and mean fCSAs are presented in = 0.028) . Minimump = 0.236) (p = 0.258) (p = 0.654) (p = 0.034) (p = 0.069)   and percent change in actin  (data not shown). Indeed, changes in myofibrillar proteins other than myosin heavy chain and actin may explain these poor associations. However, we believe that these relationships are likely due to the poorer sensitivity of the BCA assay in quantifying myofibrillar proteins and this is discussed in detail below.No significant changes were observed for muscle percent fluid (= 0.236) , myofibr= 0.258) , or sarc= 0.654) . Interes= 0.034) , and a d= 0.069) . Pre- to< 0.001)  suggestir2 = 0.412, p < 0.009) and sarcoplasmic protein concentrations  were both negatively associated with the PRE-to-POST percent change in these metrics . Of this list of 385 targets, the top enriched proteins or protein clusters at PRE that constituted >1.0% of the total spectra are depicted in p < 0.05 from PRE to POST; p = 0.018). All proteomics data can be found in There were 12 sarcoplasmic proteins that were up-regulated from PRE to POST (unadjusted p < 0.05).The purpose of this study was to examine skeletal muscle protein composition adaptations that occur in response to 10 weeks of high-load, moderate-volume resistance training in previously-trained college-aged male participants. Our data suggest 10 weeks of high-load resistance training facilitates the following adaptations in these participants: (a) a robust increase in lower body strength, (b) a modest, but significant decrease in whole body FFM, (c) a significant increase in type II, but not type I, fCSA, (d) a modest, but significant, decrease in skeletal muscle actin protein abundance and a similar statistical trend with myosin heavy chain protein abundance, (e) no changes in muscle fluid content or sarcoplasmic protein concentrations, and (f) marginal alterations in the sarcoplasmic proteome, with only 13 proteins being altered from PRE to POST , in the current study only 12 sarcoplasmic proteins were up-regulated following 10 weeks of high-load training. As stated prior, we queried functional pathways for the 12 up-regulated proteins, but no significant pathways were unveiled. Instead of putting forth speculations of what the change in each of the individual 12 proteins could potentially signify, we elected to discuss the broader theme of our proteomics analysis data relative to the findings from We elected to perform shotgun proteomics analysis on the sarcoplasmic fraction of muscle tissue, rather than the myofibrillar fraction, given that the relative abundances of the two predominant myofibrillar proteins  were assessed using SDS-PAGE and Coomassie staining. Further, while we have previously examined how 6 weeks of high-volume resistance training affected the sarcoplasmic proteome , the altFinally, data in While this study provides novel data regarding skeletal muscle protein composition adaptations to high-load resistance training, there are limitations. First, although training was prescribed and monitored, direct supervision was not implemented. Notwithstanding, we do have good reason to believe participants were compliant due to weekly communication as well as online logging as described above. Additionally, only one training intervention was utilized. In this regard, future research is needed to compare the protein markers assessed herein with high-load versus high-volume training within or between subjects. The training paradigm itself was relatively complex in both exercise selection as well as prescribed load. The intent of this paradigm was to maintain the participants\u2019 interest in adhering to the study while increasing training load. Although both were accomplished, our training paradigm is more difficult to reproduce given the complexity and this is a limitation. There was also a high degree of variation in self-reported training age (range: 1.5\u201316 years), so this too is another limitation. There were slight differences in buffers used to isolate sarcoplasmic proteins between the current study and p = 0.086) and this could have explained why FFM values were lower at POST. Interestingly, the ECW changes observed herein may be related to implementation of low-volume lifting over the 10-week period. In this regard, we recently reported that the high volume lifting implemented in our previous study by One notable finding is the disagreement between our BCA assay and Coomassie data. Notably, the Coomassie data suggests high-load training slightly reduced the relative abundances of actin and myosin (per mg dry tissue), whereas the BCA data suggest that no statstical change in myofibrillar protein concentrations (per mg dry tissue) occurred. While this finding is difficult to reconcile, we feel the BCA assay has limitations. First, the principle of the BCA assay relies upon the presence of only four amino acids in the sample  . ConversThis is one of only a handful of studies to comprehensively examine skeletal muscle protein composition adaptations that occur in response to resistance training in previously-trained individuals, and is the first study to examine how high-load training affects these variables. Given that data in this area are limited, more studies that examine how different loads affect the markers assayed herein are essential.PXD017805).The proteomic data generated in this study have been deposited into the Proteome XChange database (accession: The studies involving human participants were reviewed and approved by Auburn University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.CV and MR designed the study and primarily drafted the manuscript, and CF, PR, VB, and DB provided the critical edits. CV designed the training program for participants, coordinated the study, and performed all analyses except proteomics. VB, BM, and VI prepared the samples for proteomics. RS performed the proteomics analysis. All other authors either assisted with testing, assays, or other aspects of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Arising from the strong interaction between Cl atoms in Ti3C2Clx and the under\u2010coordinated Pb2+ in CsPbBr3 lattice, the expanded perovskite lattice is compressed and confined to act as a lattice \u201ctape\u201d, in which the Pb\u2014Cl bond plays a role of \u201cglue\u201d and the 2D Ti3C2 immobilizes the lattice. Finally, the defective surface is healed and a champion efficiency as high as 11.08% with an ultrahigh open\u2010circuit voltage up to 1.702\u00a0V is achieved on the best all\u2010inorganic CsPbBr3 PSC, which is so far the highest efficiency record for this kind of PSCs. Furthermore, the unencapsulated device demonstrates nearly unchanged performance under 80% relative humidity over 100 days and 85\u00a0\u00b0C over 30 days.The crystal distortion such as lattice strain and defect located at the surfaces and grain boundaries induced by soft perovskite lattice highly determines the charge extraction\u2010transfer dynamics and recombination to cause an inferior efficiency of perovskite solar cells (PSCs). Herein, the authors propose a strategy to significantly reduce the superficial lattice tensile strain by means of incorporating an inorganic 2D Cl\u2010terminated Ti 3C2Clx MXenes are used as lattice \u201ctape\u201d to reduce the defects and release tensile strain located at interfaces and grain boundaries of CsPbBr3 perovskite film, achieving a champion efficiency up to 11.08% with an ultrahigh voltage of 1.702 V for CsPbBr3 perovskite solar cells.Arising from the formation of strong Pb\u2014Cl bonding, chlorine terminated Ti Aiming to address this issue, all\u2010inorganic CsPbX3  perovskites have been gradually regarded as promising alternatives because of their superior environmental tolerances. Among them, tri\u2010brominated CsPbBr3 perovskite presents the best weatherability regardless of their large bandgap around 2.3\u00a0eV comparing to I\u2010containing species, demonstrating a great potential in semitransparent photovoltaics or high\u2010voltage required electronics owing to the ultrahigh theoretical voltage output around 2.0\u00a0V. Up to date, the minimum PCE loss of inorganic CsPbBr3 PSC is still much higher than that of other devices because of the defective interfaces and grain boundaries induced open\u2010circuit voltage (Voc) deficit. As is well known, the soft feature of perovskite film allows for substantial distorted lattices during the phase conversion process under high temperature, which are mainly located at the superficial region and grain boundaries. Apart from the popular positive under\u2010coordinated Pb2+ ions and negative Pb\u2014X antisites (PbX3\u2212), the expanded or compressed lattice  in this area also determines the carrier transfer and ion migration. How to heal the defective nanostructure and to realize the surface solidification undoubtedly maximize the device PCE of PSC.Organic\u2013inorganic hybrid perovskite solar cells (PSCs) have made a forward step towards upcoming commercialization due to the rapid increase of power conversion efficiency (PCE) from initial 3.8% to reported 25.6%.5, 6 Inspired by the defect passivation in Si solar cells, many organic chemicals with functional groups such as pyridine, thiophene, and thiourea have been demonstrated to donate peripheral electrons to the under\u2010coordinated Pb2+ defects for an improved efficiency and stability based on Lewis acid\u2010base interaction. Furthermore, the fabrication of \u201cbone\u2010joint\u201d configuration or soft layer by incorporating organic species with flexible chains can also effectively release the detrimental lattice strain. However, the delicate character of organic molecules and their weak secondary bonding with perovskite lattice cause an inactivation under high temperature and longstanding light irradiation, which is incompatible with the all\u2010inorganic concept. Although inorganic passivation materials such as PbSO4, PbS, metal halides, and oxides are employed to increase the performance of PSCs, there is still no effective passivator to simultaneously reduce the above\u2010mentioned defects and control lattice strain. How to make defect\u2010free perovskite film by passivation\u2010engineering and strain\u2010engineering is of great importance to maximize the photovoltaic performances of all\u2010inorganic CsPbBr3 solar cells.Great efforts have been made to heal the perovskite surfaces by regulating the terminations.21 Ins But the prevalent Cl\u2010containing additives are always ionized, and the excess cations will destroy the perovskite lattice and induce unnecessary recombination centers. In this fashion, the cation size should be precisely controlled to avoid the size mismatch induced steric effect. As is well known, the newly emerging MXenes are new family members of the 2D transition\u2010metal carbides/nitrides with metallic conductivity and easily regulated termination with different chemical groups. Similar to graphene, the MXenes are obtained from the corresponding MAX  phases by selective etching A element and replacing the termination with F, Cl, OH, etc., commonly denoted as xT. Previous reports have demonstrated that the Ti3C2xT MXene can effectively accelerate the charge extraction, enlarge grain size and increase the PCEs of PSCs. However, it is still in lack of a better understanding on the lattice strain release and defect passivation for Ti3C2xT MXene tailored perovskite films. Herein, we report an inorganic Cl\u2010terminated Ti3C2 (Ti3C2Clx) MXene to fully heal the defective surfaces and grain boundaries of CsPbBr3 film. The best device achieves an efficiency as high as 11.08% with an ultrahigh Voc of 1.702\u00a0V, to the best of our knowledge, these data are to date the highest PCE and Voc records for the all\u2010inorganic CsPbBr3 PSCs. Apart from the strong passivation interaction between terminated Cl atoms and under\u2010coordinated Pb2+ ions and enlarged perovskite grain size, the bridging connection between Pb2+\u2010Cl\u2010Ti3C2 restricts the expansion of perovskite lattice to act as a lattice \u201ctape\u201d to reduce the tensile strain. As a result, the significantly reduced defect density in CsPbBr3 film allows for the stability advancement under persistent attack by 80% humidity or 85\u00a0\u00b0C in air for the device free of encapsulation.Arising from the strong electronic coupling, Cl atoms display superiority to thermodynamically stabilize the perovskite lattice and regulate the grain growth dynamics owing to the higher formation energy of Pb\u2014Cl antisites at interface.28 But23C2Clx MXene is synthesized using a redox\u2010controlled Al etching of precursor Ti3AlC2MAX\u2010phase in Lewis acidic CdCl2 melts combined with exfoliation and ultrasonication, the details can be found in the Supporting Information. As schematically shown in Figure3AlC2MAX\u2010phase is composed of Ti6C octahedrons interleaved with layers of Al elements. Upon selectively removing Al atoms from interlayers with CdCl2 melts, an accordion\u2010like microstructure , (004), (10l), and (110) lattice planes of the Ti3C2 MXene, respectively. Additionally, the disappearance of Al 2p signal and the appearance of a sharp peak of Cl 2p from X\u2010ray photoelectron spectroscopy (XPS) spectra  based on the XPS results. The element mapping images  image in Figure\u00a02\u2010TiOxCl4\u22122x layer, a 500\u00a0nm monolayer\u2010aligned perovskite overlayer and a 15\u00a0\u00b5m carbon electrode. By carefully optimizing the Ti3C2Clx MXene dosage  of 7.87\u00a0mA cm\u22122, and a fill factor (FF) of 82.7% has been achieved by recording the characteristic photocurrent\u2013voltage (J\u2013V) curves via reverse scan under standard AM 1.5 G illumination, as shown in Figure\u00a0Voc, Jsc, and FF are attributed to the reduction of charge recombination after adding Cl\u2010terminated Ti3C2 MXene. To the best of our knowledge, both the PCE of 11.08% and Voc of 1.702\u00a0V are till now the highest records for state\u2010of\u2010the\u2010art all\u2010inorganic CsPbBr3 PSCs  and Cl atoms is responsible for the reduction of halide ions deficiency or excess induced defects due to the higher formation energy of Pb\u2014Cl antisites at interfaces,. It is worthy to mention that the underlying surface of perovskite film is passivated by the electron transfer layer SnO2/TiOxCl4\u22122x according to our previous report.To reveal the intrinsic mechanism behind the efficiency enhancement, we first explore the effect of Tiy Figure\u00a0,d, whichy Figure\u00a0 and enha3 Figure\u00a0,41]413C2C\u22121) in comparison to 248\u00a0kJ mol\u22121 for Pb\u2014Br into consideration, the Cl terminated Ti3C2 MXene can drag the adjacent perovskite grains closer  planes as a function of incident angle demonstrate the lattices across whole film especially in bottom region are contracted, as shown in Figure\u00a02+ ion in the perovskite, 2D Ti3C2Clx MXene can tightly adhere to the surface of CsPbBr3 grains, like a lattice \u201ctape\u201d to heal the soft perovskite lattice and to relieve the lattice expansion  than that of pristine perovskite film obtained from Kelvin probe force microscopy (KPFM) images  in the perovskite films using a hole\u2010only device according to the space\u2010charge\u2010limited\u2010current (SCLC) model. As shown in Figure\u00a0VTFL) from 0.78 to 0.56\u00a0V for Ti3C2Clx passivated perovskite film means a significantly reduced nt from 5.93 \u00d7 1015 to 1.92 \u00d7 1015 cm\u22123, which can be cross\u2010checked by the increased photoluminescence (PL) peak intensity  for the Ti3C2Clx\u2010tailored PSC implies an enhanced driving force for charge separation  is deterimined to be \u22124.62\u00a0eV, which will upward bend the valence and conduction band of CsPbBr3 at the interface, accelerating the charge extraction.Under light irradiation, the defects and strain in perovskite film will undoubtedly cause a carrier loss during the transfer process, as illustrated in s Figure\u00a0,e, therep Figure\u00a0, a highe3 PSCs is evaluated under persistent moisture or thermal attacks. As shown in Figure3C2Clx\u2010repaired PSC device free of encapsulation delivers a stabilized PCE performance under 25\u00a0\u00b0C and 80% relative humidity (RH) in air over 100 days, which is mainly attributed to the reduced defect and contracted lattice to trigger unwanted phase attenuation and the protection of hydrophobic Ti3C2Clx encapsulant (enlarged contact angle from 50.9\u00b0 to 92.1\u00b0). Owing to the absence of damageable organic species, both two PSCs present excellent thermal stability. As shown in Figure\u00a03C2 as lattice \u201ctape\u201d to enhance the performance of highly\u2010efficient PSCs. It is worthy to note that the performance improvement behavior of PSC device in stability test is related to the film quality improvement after storage for a few days under controlled ambient atmosphere.Finally, the long\u2010term stability of all\u2010inorganic CsPbBr33C2 MXene into the bulk and surface of perovskite film for stable and efficient all\u2010inorganic CsPbBr3 PSCs. Arising from the formation of Pb\u2014Cl bond between the interfacial under\u2010coordinated Pb2+ ions in perovskite lattice and terminated Cl atoms in Ti3C2Clx MXene, the defects and expanded lattices located at the interfaces and grain boundaries in perovskite film are significantly healed, playing a role of surface lattice \u201ctape\u201d, in which the Pb\u2014Cl bond represents \u201cglue\u201d and Ti3C2 immobilizes the lattice. As a result, the best all\u2010inorganic CsPbBr3 PSC achieves an efficiency as high as 11.08% with an ultrahigh Voc of 1.702\u00a0V, which are the highest PCE and voltage records for CsPbBr3 solar cells. Moreover, the unencapsulated solar cell presents an excellent stability under high humidity (80% RH) and high temperature (85\u00a0\u00b0C) conditions over 100 and 30 days, respectively. Our findings open up a new avenue for preparing high\u2010quality defect\u2010free perovskite films, which not only benefits preparing high\u2010performance PSCs but also other optoelectronic devices based on perovskites.In summary, we have demonstrated a novel strategy to simultaneously realize defect passivation and strain release by incorporating Cl\u2010terminated Ti42\u00b72H2O (Alfa Aesar), CH4N2S (Macklin), TiCl4 (Aladdin), Ti3AlC2 , PbBr2 (Aladdin), CsBr (Aladdin), N,N\u2010dimethylformamide , CH3OH (Sinopharm), isopropanol , HCl , FTO glass (12 \u03a9 per square), Zn powder , carbon paste . All materials were used as supplied without further treatment.SnCl3C2 MXene was fabricated through molten salt\u2010based etching of Ti3AlC2MAX phase and surface group substitution/elimination reactions in a N2\u2010filled glovebox. 0.5\u00a0g of Ti3AlC2 was first mixed with 3.768\u00a0g of CdCl2 (molar ratio of 1:8). The resultant mixture was then heated in an alumina crucible at 610\u00a0\u00b0C for 7 h, which involved the following reactions.The Cl\u2010terminated Tim) to remove excess CdCl2 and metallic Cd, followed by washing with deionized water for several times until pH \u2248 6.5. The resultant Cl functionalized MXene powders were dried under vacuum at 45\u00a0\u00b0C. Finally, by liquid phase exfoliation of Cl functionalized MXene powders in IPA, the 2D few\u2010layer Cl\u2010terminated Ti3C2Clx MXene could be obtained. In details, 0.1\u00a0g of Cl functionalized MXene powders was added to 50\u00a0mL of IPA and then ultrasonicated for 25 h at a power of 300 W. The upper dark green suspension was obtained after centrifuging at 10\u00a0000\u00a0rpm for 30\u00a0min to separate the un\u2010exfoliated MXene. Before usage, the obtained Ti3C2Clx MXene suspension was filtered with 0.22\u00a0\u00b5m PTFE filter.After cooling to room temperature, the powders were washed by concentrated aqueous HCl  at 75\u00a0\u00b0C for 30\u00a0min. After washed with deionized water and ethanol, the substrate was then annealed in air at 200\u00a0\u00b0C for 30\u00a0min. The CsPbBr3 film was fabricated by a multi\u2010step spin\u2010coating method. In details, 1 m PbBr2 DMF solution with different amount of Ti3C2Clx was spin\u2010coated on the substrate at 2000\u00a0rpm for 30 s under 100\u00a0\u00b0C and then dried at 100\u00a0\u00b0C for 30\u00a0min. After that, 0.07 m CsBr methanol solution was spin\u2010coated onto PbBr2 film at 2000\u00a0rpm for 30 s, and heated at 250\u00a0\u00b0C for 5\u00a0min. By repeated this process for several times, a high quality CsPbBr3 film was obtained. 1\u00a0mg mL\u22121 Ti3C2Clx suspension in IPA was spin\u2010coated on the surface of CsPbBr3 film at 2000\u00a0rpm for 30 s, and then annealed in air at 100\u00a0\u00b0C for another 5\u00a0min. Finally, a conductive carbon electrode with active area of 0.09 cm2 was coated on the perovskite film by a blade coating method.The FTO\u2010coated glass was etched with Zn powders and HCl aqueous solution to obtain the desired pattern and then ultrasonic cleaned with detergent, deionized water, and ethanol. The cleaned FTO substrates were further treated by plasma for 5\u00a0min. Before deposition, FTO substrates and SnO reports.45 ComJ\u2013V curves of PSCs were recorded on an electrochemical workstation (CHI660E) under irradiation of simulated solar light . The light intensity was controlled at 100\u00a0mW cm\u22122 calibrated by a standard silicon solar cell. The IPCE spectra of various devices were obtained using an IPCE kit from Enli Technology Co., Ltd. The Voc decay curves were measured at open\u2010circuit mode by illuminating the device for several seconds, and then instantaneously switching off the light. The TSPV signals were recorded by a home\u2010made setup using a 355\u00a0nm laser. Capacitance\u2013voltage (C\u2013V) curves were measured at a frequency of 5\u00a0kHz with an amplitude of 5\u00a0mV under dark. Tafel curves of various devices were measured on CHI660E electrochemical workstation.The morphologies were obtained by a field emission SEM (Hitachi S4800) and transmission electron microscope (Tecnai G2 F20). The element analysis was carried out using SEM (FEI QUANTA250 and Zeiss Gemini300). The Raman mapping was recorded using an InVia Raman system  with an excitation laser wavelength of 532\u00a0nm. The film thickness of MXene was obtained by atomic force microscopy . The surface potential was characterized by KPFM . The XRD and GIXRD patterns of perovskite and MXenes were conducted on an X\u2010ray diffractometer (Bruker D8 Advance). PL spectra were recorded via a fluorescence spectrometer excited by a 350\u00a0nm laser and the TRPL spectra were characterized using a Horiba spectrometer with an excitation wavelength of 500\u00a0nm. The characteristic The authors declare no conflict of interest.Supporting InformationClick here for additional data file."}
+{"text": "Surgical sterilisation is a component of free-roaming dog population management programmes worldwide. However, evidence of the population-level impacts of sterilisation are rarely reported in peer-reviewed literature. Using a priority setting partnership process, we identified the most important unanswered questions concerning these impacts from the perspective of those working with free-roaming dogs. We found that there were many uncertainties surrounding the impacts of such programmes, and how they can be achieved. The top 10 priorities were related to changes in dog population dynamics; risks to human health; human perception and behaviour towards dogs; and logistics related to implementation in the field. Addressing these priorities will enable a more comprehensive understanding of if, how, and why canine surgical sterilisation programmes impact on free-roaming dog populations.Surgical sterilisation is a core activity of free-roaming dog population management (DPM) programmes globally. However, there is limited published evidence on its impact at the population level. To support evidence-based decision making in this field, it is important that research conducted is relevant to those involved in working with free-roaming dogs and implementing such programmes. The aim of this study was to adapt the James Lind Alliance (JLA) user involvement approach to systematically identify the top 10 research priorities regarding the impact of canine sterilisation. International stakeholders with experience working in DPM were asked in an online survey what unanswered questions they had regarding the impact of sterilisation programmes. Thematic analysis of survey responses was used to develop a long list of collated indicative research questions (CIRQs). A literature review was performed to identify questions that were \u2018true uncertainties\u2019 (had not been answered by evidence review). These questions were reduced to a shortlist via an online interim prioritisation survey, and a Delphi consensus process determined the top 10 priorities. The top 10 questions related to dog population size and turnover, dog bite incidents, rabies control, implementation in the field and human behaviour change. These priorities were identified and shaped by people with direct experience of canine surgical sterilisation programmes, and as such are an essential resource for directing future funding and research. Addressing these priorities will generate evidence that is directly applicable to policy makers and practitioners who make decisions regarding the management of free-roaming dogs (FRDs) worldwide. Surgical sterilisation (hereafter referred to as sterilisation) is a common component of humane dog population management (DPM) in countries with free-roaming dogs (FRDs). There are often a number of intended impacts associated with sterilisation, such as controlling population size, improving dog welfare, reducing human\u2013dog conflict and reducing public health risks . DespiteThere has been an increased focus across different disciplines on the discrepancy between what users want researchers to study and what researchers actually investigate ,8. In huPrevious research investigating the impacts of sterilisation programmes has demonstrated that many uncertainties remain ,3,4,6. TThe aim of this study was to engage individuals with experience working in DPM to identify their top 10 research priorities related to the impact of sterilisation programmes for free-roaming dog populations. This novel approach will provide insights for future research in this field, framed by those who are in a position to use this evidence.The design, methods, and analysis for this project were adapted from the James Lind Alliance (JLA) priority setting partnership (PSP) process . This isThe PSP was managed by a research team  at the Centre for Evidence-based Veterinary Medicine (CEVM), with guidance from a steering group. The process was supported and guided by RSD who has previous experience of conducting adapted PSPs in the field of veterinary science ,15. The https://www.nottingham.ac.uk/cevm/population-research/companion-animal-health/impact-of-canine-surgical-sterilisation-programs.aspx (accessed on 27 July 2021)) and provide access to the online survey. Participants were recruited via email and social media using convenience, purposive and snowball sampling in line with the JLA\u2019s inclusive approach. Steering group members promoted the survey through personal and professional contacts. Dogs Trust Worldwide and the Global Alliance for Rabies Control (GARC) also facilitated survey dissemination through their networks.The target population was those with current or previous experience of working in the field of DPM. Individuals were eligible for inclusion if they had experience planning, implementing or managing canine sterilisation programmes. A website page was established to advertise the partnership , or temporary mobile field sites.All uncertainties that related to the impact of canine sterilisation programmes on free-roaming dog populations were considered in scope. These could be uncertainties relating to different types of impact, and how to achieve them, or external factors that may affect impacts. Decisions about whether nominated uncertainties were in or out-of-scope were made by the primary researcher (AC) and subsequently verified by the steering group.www.onlinesurveys.ac.uk). The survey was available in English. Participants were asked, \u201cWhat questions do you have about the impact of surgical sterilisation programmes on a population of free-roaming dogs?\u201d If participants were involved in combined sterilisation and rabies vaccination programmes, they were also asked \u201cWhat questions do you have about using surgical sterilisation as part of a canine rabies control programme?\u201d There was no limit to the number of questions that could be submitted. Participants were also asked to provide some basic demographic information .An online survey was designed using JISC online surveys  (www.onlThe survey was pre-tested by members of the Centre for Evidence-based Veterinary Medicine (CEVM) and piloted by the steering group to ensure it was understandable and easy to complete. The text in the survey was refined to provide definitions of key terms used. The final version was circulated via email and social media to organisations and individuals working in DPM by the primary researcher, steering group, Dogs Trust Worldwide and the Global Alliance for Rabies Control (GARC). Participants were also encouraged to share the link with other colleagues or organisations working in this field. The initial survey was open between February and April 2019.The aim of this stage was to categorise and refine the submitted questions, verify them as \u2018true uncertainties\u2019 (defined by the JLA as questions which have not been answered by systematic review) as opposed to \u2018unknown knowns\u2019 , and create a list of collated indicative research questions (CIRQs) that were representative of the original submissions.Responses were downloaded into Microsoft Excel and were anonymised. Each question was screened for relevance by AC based on the inclusion criteria detailed in the protocol . Out of Each CIRQ was then checked against the existing evidence. A literature search was conducted in CAB Abstracts (1910-present), Medline In-Process and Non-Indexed Citations and Ovid Medline (1946-present) using the OVID interface via the library at the University of Nottingham. Details of the search strategy can be found in n = 47) to a shorter and more manageable list. An online survey was created using card-sorting software . The second online survey was distributed using the same methods as the initial survey and was also emailed to all previous participants who had stated that they were willing to be involved in further stages of this study. The refined list of CIRQs was presented in a randomised order to each participant. Participants were asked to read all of the questions and select (by drag and drop) up to 25 questions that they thought were most important for research (they were not asked to rank the questions). The same demographic information as for the previous survey was also collected. The survey was pre-tested by members of the CEVM and piloted by the steering group prior to launch to ensure that the survey was clear and understandable. Adjustments were made to increase the number of questions that participants could choose and a \u2018maybe\u2019 column was added to the \u2018prioritise\u2019 and \u2018exclude\u2019 options to help with the process of working through the questions. All indicative questions that were moved to the \u2018prioritise\u2019 column were ranked based on the frequency with which they were chosen by all participants and by demographic subgroups . The interim prioritisation survey was open from November to December 2019.Over 30 uncertainties were identified in the initial survey, and therefore an interim prioritisation survey was necessary in order to have a manageable number in the final process . The aimwww.optimalworkshop.com) via an email link. The data were managed using Microsoft Excel. The rounds were designed to follow the steps used in a traditional JLA priority setting workshop.In the JLA methodology, the final prioritisation stage is typically conducted using nominal group technique at an in-person one-day workshop. Due to the international diversity, and consequently multiple time zones of participants in this study, it was decided to adapt this stage of the process so that it could be conducted online. The Delphi technique was used involving two rounds of questionnaires to identify the final top 10 priorities. This was conducted electronically; participants were invited to use the online software , participants were asked to rank all of the shortlisted questions on a numerical scale with 1 being considered most important. They also had the opportunity to comment on each question outlining why they considered it to be important, or unimportant. The individual rankings were combined to give an aggregate ranking for each question.In the second Delphi round, each participant was emailed a document with the aggregate ranking and their previous individual ranking for each of the questions. Anonymous comments regarding the reasons why participants considered each question important, or not important were also presented. Participants were asked to re-rank their top 10 questions after consideration of the aggregate ranking and the comments. Suggestions generated in the previous round for rewording of a number of questions were also included and participants were asked to select a preferred version of wording in such cases. Consensus was set at 70% agreement or disagreement for inclusion and exclusion of questions, or for changing original wording to a new version. The individual rankings were combined to form an overall aggregate rank for each question, with the highest scoring questions comprising the top 10. The two rounds were completed between January and April 2020.A flow diagram of the process and results are shown in A total of 152 participants from 62 different countries completed one or more stages of this study see . Full den = 168/644; 26%)  , leavingn = 30) had more than one part or were relevant to more than one of the CIRQs and therefore the total number of contributing questions is more than the overall number of submitted questions (n = 488).Thematic analysis generated nine key themes . These tThe interim prioritisation survey was completed by 107 participants see . Of thesThe first round was sent via email to 71 participants from previous stages who had expressed an interest in joining the Delphi panel. A total of 44 participants completed the first round and 36 completed both rounds see ; Q4 and n = 3), logistics (n = 2), dog behaviour (n = 1), rabies control (n = 2) and human behaviour change (n = 2).There was little change between the two final rounds in the questions making up the top 10, but there were some changes in their order, particularly the middle rankings (Q4\u2013Q7). Only one question that had been in the top 10 after the first round did not make it to the final top 10 . The highest ranking question remained in this position throughout all stages of the process, including after the initial scoping survey as it had the most number of submitted questions contributing to its formation. The final priorities fell within five of the nine identified themes: population dynamics (This is the first time that a stakeholder-led framework (JLA) has been applied to identify research priorities for a veterinary population management intervention. The use of this methodology enabled the effective engagement of DPM practitioners globally, and provided a platform for the involvement of end users in directing the research agenda, through the identification and prioritisation of knowledge gaps from their perspective. The ability to reach a widely geographically dispersed participant population using online survey methods, including the modification of the final prioritisation process, was a strength of this study. Therefore, we are confident that the results represent an international viewpoint. The questions submitted suggest that those implementing sterilisation programmes want practical, actionable evidence that will enable informed decision-making in the field and demonstrate their wider value in a One Health  context.Despite the widespread use of sterilisation for DPM for several decades, many of the questions submitted highlighted that there is still a need to better understand the pathways through which sterilisation is expected to work for DPM, and how these may interact. Many of the assumptions  have not been tested. Guidelines exist for indicators of impact and their measurement  and thisDog population size and turnover, and the most efficient and effective way to achieve changes in these parameters, were considered of highest priority. Questions in this theme accounted for the top 3 questions and were related to 3 of the other top 10 questions  and it is not possible to say how representative the participants are of the entire population of people working with free-roaming dogs. However, this bias towards participants who use the internet, who could understand written English, and those with a particular interest in the topic under investigation is an accepted limitation of the JLA methodology [The survey was only available online and in English, which may have affected participation. The interim survey had a particularly high number of people access the survey and complete the demographic questions but not the ranking questions . This was most evident in the questions concerning the use of sterilisation in rabies control.The use of the Delphi process for final prioritisation was a deviation from the traditional JLA methodology. A disadvantage of using this method in place of the standard nominal group technique was the lack of discussion between participants, which can help generate new ideas and fosters a collaborative approach to reaching a consensus . HoweverThis study was the first step in using formal prioritisation methods to identify what is important to stakeholders, and further understand the issues associated with measuring the effectiveness of DPM programmes. Future work should assess the priorities of other stakeholder groups such as funding partners, communities and dog owners/caretakersThe JLA methodology was successfully adapted to identify the key uncertainties surrounding canine sterilisation interventions from the perspective of those working in the field. Despite their established nature, the implementation of sterilisation programmes and evidence of their impact is hampered by important knowledge gaps. The development of a research agenda to address persisting unknowns is vital to inform and support future interventions to optimise their effectiveness. In addition to impacts of importance, many of the priorities relate to implementation, suggesting that emphasis is needed not only on whether interventions \u2018worked\u2019 in achieving certain impacts, but also on how these impacts can be achieved most effectively, and the mechanisms driving any changes. Addressing these priorities, which are applicable to DPM worldwide, will generate relevant evidence to enable a more comprehensive understanding of if, how and why canine sterilisation programmes impact free-roaming dog populations, in the areas of most importance to the end users. This will minimise research waste, improve the translation of research to DPM practice and ultimately benefit both animal health and welfare, and human health and well-being in countries with FRD populations."}
+{"text": "X-ray analysis unveiled that PCAL and PCAC are nearly totally shielded in the \u03b2-CD wall. Their aromatic rings are vertically aligned in the \u03b2-CD cavity such that the functional groups on the opposite side of the ring  and 1-CHO/1-COOH groups) are placed nearby the O6\u2013H and O2\u2013H/O3\u2013H rims, respectively. The preferred inclusion modes in 1 and 2 help to establish crystal contacts of OH\u22c5\u22c5\u22c5O H-bonds with the adjacent \u03b2-CD OH groups and water molecules. By contrast, the DFT-optimized structures of both complexes in the gas phase are thermodynamically stable via the four newly formed host\u2013guest OH\u22efO H-bonds. The intermolecular OH\u22c5\u22c5\u22c5O H-bonds between PCAL/PCAC 3,4-di(OH) and \u03b2-CD O6\u2013H groups, and the shielding of OH groups in the \u03b2-CD wall help to stabilize these antioxidants in the \u03b2-CD cavity, as observed in our earlier studies. Moreover, PCAL and PCAC in distinct lattice environments are compared for insights into their structural flexibility.Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are catechol derivatives and have broad therapeutic effects associated with their antiradical activity. Their pharmacological and physicochemical properties have been improved via the cyclodextrin (CD) encapsulation. Because the characteristics of \u03b2-CD inclusion complexes with PCAL ( Phellinus linteus : monoclinic, space group P21 (no. 4), a = 15.4213(11) \u00c5, b = 10.0440(7) \u00c5, c = 21.2795(15) \u00c5, \u03b2 = 110.281(2)\u00b0, V = 3091.7(4) \u00c53, Z = 2, T = 296(2) K, \u03bc(MoK\u03b1) = 0.133 mm\u20131, Dcalc = 1.484 g cm\u20133, 60310 reflections measured (1.41\u00b0 \u2264 \u0398 \u2264 30.67\u00b0), 18989 unique  which were used in all calculations. The final R1 was 0.0620 (I > 2\u03c3(I)) and wR2 was 0.1527 .Crystal data for 2 : monoclinic, space group P21 (no. 4), a = 12.5165(7) \u00c5, b = 19.0287(12) \u00c5, c = 14.0626(8) \u00c5, \u03b2 = 109.656(2)\u00b0, V = 3154.2(3) \u00c53, Z = 2, T = 296(2) K, \u03bc(MoK\u03b1) = 0.132 mm\u20131, Dcalc = 1.471 g cm\u20133, 55384 reflections measured (1.54\u00b0 \u2264 \u0398 \u2264 30.61\u00b0), 19,288 unique , which were used in all calculations. The final R1 was 0.0602 (I > 2\u03c3(I)) and wR2 was 0.1134 .Crystal data for Meaningful, reliable structures and thermodynamic data of CD inclusion complexes can be deduced from complete-geometry optimization by the DFT method. This has been successfully demonstrated in our previous works on the \u03b2-CD encapsulation of polyphenolic plants extracts, including tea epicatechins , olive o1 and 2, excluding the hydration water molecules. Before the calculation, all the underestimated X-ray-derived C\u2013H and O\u2013H distances were normalized to neutron hydrogen distances, 1.083 and 0.983 \u00c5 [Starting atomic coordinates of the 1:1 \u03b2-CD\u2013PCAL and \u03b2-CD\u2013PCAC inclusion complexes were taken from the converged crystal structure refinements of  0.983 \u00c5 . The two 0.983 \u00c5  on a DELRegarding the protonated form of PCAC that was used in the DFT full-geometry optimization of the \u03b2-CD\u2013PCAC inclusion complex, PCAC with pKa ~4.5 in a neutral pH solution containing \u03b2-CD was expected to exist in an anionic form. However, it turned out that PCAC and \u03b2-CD co-crystallized to yield a thermodynamically stable adduct in the single-crystalline state. Similarly, benzoic acid with pKa = 4.2 complexed with \u03b2-CD in solution  and the Estb and \u0394Eint) were calculated straightforwardly. For example, \u0394Estb was obtained by subtracting the molecular energies of \u03b2-CD and PCAL/PCAC from the energy of the \u03b2-CD\u2013PCAL/PCAC complex from the full-geometry optimization. Similarly, \u0394Eint considered the corresponding single-point energies in the complexed states. The DFT-derived inclusion structures of the two complexes together with their O\u2212H\u22efO hydrogen bonds, \u0394Estb and \u0394Eint are given in The stabilization energy and interaction energy of the complex (\u0394Estb and \u0394\u0394Eint) are sufficient to interpret the relative thermodynamic stabilities in relation to host\u2013guest interactions and antioxidant properties.Note that no basis set superposition error (BSSE) correction is applied to the DFT-derived energies of the \u03b2-CD\u2013catechol antioxidant complexes listed in 1) and PCAC (2) by a combined study of single-crystal X-ray diffraction and density functional theory (DFT) full-geometry optimization.Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are polyphenol extracts from plants. They contain a catechol moiety and thus having wide therapeutic effects including antioxidant, anti-inflammatory, and anticancer activities. Their physicochemical and pharmacological properties have been improved via cyclodextrin (CD) inclusion complexation, of which their characteristics are still controversial. To address at the atomic-level of the inclusion complexation, we investigate the \u03b2-CD encapsulation of PCAL  and 1-CHO/1-COOH groups) are placed nearby the O6\u2013H and O2\u2013H/O3\u2013H rims, respectively. The preferred inclusion modes in  and OLE . By cont"}
+{"text": "Intestinal obstruction at the level of the colon is rarely caused by a gallstone. Colonic gallstone is more frequently observed in elderly patients and is associated with high mortality due to treatment challenges. Management with less invasive approaches, including mechanical lithotripsy and endoscopy has been evolving. However, the outcomes are variable, and surgery remains the main cornerstone of treatment. We present a case of an 89-year-old male with gallstone ileus at the level of the sigmoid colon in whom treatment with endoscopy was not successful. We performed an extensive review of\u00a0the literature to understand the most common presentation, diagnostic modalities, and treatment approach of the sporadic reported cases of colonic gallstone ileus. Gallstone ileus is a well-characterized pathology in which bowel obstruction occurs secondary to a gallstone impaction. The stone usually travels to the intestine through a cholecystoenteric fistula which typically forms between the gallbladder and the duodenum, but it can form with other sites of the gastrointestinal tract. The most common site of stone impaction is the ileum, however, around 4% can occur in the colon\u00a0. ElderlyAn 89-year-old male with a history of stroke, atrial fibrillation, heart failure, esophageal adenocarcinoma status post radiation, cholecystitis one year prior to presentation (treated with percutaneous cholecystostomy tube that had subsequently been removed), presented to the emergency room with left lower quadrant abdominal pain of two-day duration. He denied nausea, vomiting, diarrhea, constipation, or blood in the stool. He did not have any sick contacts or recent travel. In the emergency room, his temperature was 98.1\u00b0F, pulse 80 beats per minute, respiratory rate 16 breaths per minute, and blood pressure 106/67 mmHg. The laboratory blood tests are described in Table\u00a0A CT of the abdomen and pelvis revealed a fistula between the gallbladder and the large bowel at the hepatic flexure and air in the gallbladder. Marked diverticulosis with wall thickening and fat stranding was seen at the level of the mid to distal descending colon. A large lamellated stone 3 cm x 2.6 cm) was present in the distal descending colon Figure\u00a0. A gastr.6 cm wasGiven the patient\u2019s several comorbidities surgery was deferred, the patient was kept nil per mouth and conservative treatment with an aggressive bowel regimen including mineral oil enemas as well as IV hydration was started. However, after four days he did not have any bowel movement and for this reason, total parenteral nutrition was initiated and an endoscopic approach to remove the stone was planned.\u00a0On the fifth\u00a0day of hospitalization, a colonoscopy was performed which showed a large black pigmented stone that was completely obstructing the lumen of the sigmoid colon, the surrounding mucosa appeared ulcerated Figure\u00a0.Attempts to pass a guidewire proximal to the stone to pursue balloon-assisted dislodgment of the stone or mechanical lithotripsy as well as attempts to capture the stone in a retrieval net were unsuccessful. Consequently, an exploratory laparotomy with partial left colectomy was successfully done, however, the cholecystocolonic fistula was left intact. Later, the pathology results revealed a 3.4 cm x 2.7 cm black stone and a 4.5 cm x 3 cm transmural defect at the colonic wall where the stone was impacted. The patient\u2019s postoperative period was complicated, he did not tolerate oral intake and his nutrition deteriorated substantially despite total parenteral nutrition (TPN). At the request of the patient and the family he opted for inpatient hospice care. Unfortunately, he died 15 days after the surgery.Gallstone ileus is an extremely rare cause of mechanical bowel obstruction and is responsible for less than 5% of cases of bowel obstruction. However, when stratifying according to age, it is the main cause of intestinal obstruction in up to 25% of patients older than 65\u00a0.\u00a0MoreoveThe location of the gallstone entrapment can occur anywhere from the duodenum to the colon. According to the study of Rodr\u00edguez Hermosa et al. the most common location of the gallstone in the gastrointestinal tract was the ileum (62.5%), followed by the jejunum (22.5%), duodenum 7.5%), and colon (2.5%)\u00a0. However.5%, and We performed a review of the literature to further understand the clinical presentation, diagnosis, treatment, and outcomes of patients with gallstone ileus of the colon. EMBASE and PubMed were used to search for articles related to bowel obstruction caused by gallstone impaction in the colon. The following keywords were used: \u201ccolonic gallstone ileus,\u201d \u201cgallstone impaction,\u201d \u201cgallstone bowel obstruction,\u201d \u201csigmoid gallstone ileus,\u201d and \"cholecystocolonic fistula.\" The references of retrieved articles were extensively reviewed for further inclusion of additional cases. A total of 56 cases were found and we also included our case in this review.The mean age was 80 years old and 74% (n=42) were women. The most commonly reported clinical presentation was abdominal pain (84%), vomiting (67%), and constipation (61%). At least 25% of the patients had a reported history of some type of biliary tract disease including, cholecystitis, symptomatic cholelithiasis, and cholangitis. Elevated inflammatory markers are frequent, from the 22 cases that mentioned the initial blood results, the white blood count ranged from 6.7 x\u00a0109/L\u00a0to 27.4 x\u00a0109/L\u00a0and the average was 14.3 x\u00a0109/L. Similarly, from the 16 patients who had a reported C-reactive protein level, 15 had elevated levels which averaged 122 mg/L.\u00a0Given the clinical picture of bowel obstruction, abdominal X-rays are usually performed and the Rigler\u2019s triad  is sometimes present, however, a nonspecific dilation of the small and large bowel is frequent. For this reason, CT of the abdomen is performed most of the times and 81% (n=46) of cases were diagnosed by CT scan. In some cases, due to the hemodynamic instability, emergent laparotomy was required, for this reason in 7% (n=4) of cases the diagnosis was done during surgery. Other diagnostic modalities were plain abdominal X-ray, endoscopy and gastrografin study, each of these were the main diagnostic methods in only 5% (n=3), 5% (n=3), and 2% (n=1) of cases, respectively  of cases, followed by the descending colon (n=4) and transverse colon n=3)  were treated with laparotomy of which four died during the hospitalization, a total of 32 patients had an attempted colonoscopy for treatment but it was only successful in removing the stone in eight patients (14%) and one patient died. Two patients had successful treatment with laparoscopy, one with trephine loop colostomy, five patients had conservative treatment, and one case did not clarify the type of surgery (Table\u00a0Sigmon et al. used an adult diagnostic gastroscope and performed several attempts to capture the stone with a Roth Net which were unsuccessful, they then used an adult therapeutic gastroscope with a Talon grasping device that was again, unsuccessful. With the last gastroscope and a hexagonal AcuSnare they were able to snare the stone and remove it entirely in one piece\u00a0. BalzariThe overall mortality related to gallstone ileus had been decreasing, In the nineteenth century, gallstone ileus had a high mortality rate of around 44% .\u00a0In the Management of colonic gallstone ileus in high-risk patients is challenging, particularly due to the common advanced age and several comorbidities that many of these patients possess which make them poor surgical candidates. Nonetheless, given the low success rate with other therapeutic modalities, laparotomy remains the standard approach which may involve enterolithotomy or even partial colectomy if the intestinal wall is perforated or necrotic. Endoscopic treatment has been reported in the literature with only a few successful cases mainly using mechanical or electrohydraulic lithotripsy. Several factors influence this outcome including stone size, presence of colonic stricture, time for treatment, and lack of expertise or availability. In our patient, the failure of the endoscopic approach was likely a combination of these factors.\u00a0An endoscopic approach using the available tools and expertise should be attempted in the presence of surgical support due to the risk of intestinal perforation and often failed endoscopic attempts. Conservative management is not recommended as the spontaneous evacuation of the stone is rare and treatment delays will most likely lead to severe complications."}
+{"text": "The Organization of Nurse Leaders of New Jersey has had a formal statewide nurse leader mentoring program since 2014 to develop leadership talent. Learn how a mentoring dyad helped them improve competencies in optimizing resource management. The Organization of Nurse Leaders of New Jersey (ONL NJ) has had a formal statewide nurse leader mentoring program since 2014 to develop leadership talent. Mentorship is incorporated into the ONL NJ strategic plan and continues to be active at ONL NJ.4However, not all mentoring dyads work out.The mentee (author Perez) is an administrative director of medical-surgical nursing at a 178-bed general medical and surgical teaching hospital in New Jersey. The mentor (author Caruso) is a healthcare consultant focused on linking financial and clinical data for improved outcomes.-surgical nursing, she wanted to enhance her capabilities in optimizing resources and controlling salary expenses. She sought a mentorship relationship that would help increase her financial understanding and competencies in the fiscal management of human resources, a key managerial competency as described by the American Organization for Nursing Leadership (AONL) in the domain of The Science: Managing the Business.5In the mentee's current position as administrative director of medicalAs members of ONL NJ, both authors as mentee and mentor attended mentoring educational seminars offered through ONL NJ. The mentee reached out to the mentorship committee using a specific form to request a mentor \u201cmatch\u201d based on her goals. The mentor had done mentoring in the past and was looking to mentor another mentee as part of cohort 6 of the ONL NJ Mentoring Program. ONL NJ then sent both authors copies of their respective r\u00e9sum\u00e9s for review and included the mentee's goals. For the past 25 years, the mentor has had an extensive background in nurse staffing and optimizing management of resources, focusing on linking financial and clinical data for improved outcomes. As Goodyear and Goodyear reported, effective mentoring relationships depend on the skills of the mentee and mentor.2In June 2018, the mentee was informed that she'd been selected to participate in cohort 6 of the ONL NJ Mentoring Program and was matched with her mentor. In August 2018, the mentee and mentor scheduled a first meeting to start cultivating the relationship, review goals, and develop a plan of action for future meetings. The first 3 months of meetings in 2018 were focused on the mentee's objective to better understand internal budget and financial reports regarding human resources.The key stated goals were to:manage the operating budget daily, which included all full-time equivalent (FTE) personnel and all expenses for day in/day out operation of her three medical-surgical unitsensure that actual productivity of nursing hours per patient day matched budgeted productivity of nursing hours per patient day and provide further analysis and understanding of variances to budget.Additional objectives added during the first 3 months included to:reduce nonholiday overtime (OT) hours and expenses of RN staffuse newly developed competencies and expertise to teach and mentor her own three directors with their clinical coordinators to better manage their staff resources.Scheduling regular meetings and planned communication is essential to a successful mentoring relationship.3The mentee had a unique opportunity to develop the business knowledge and skills needed to be successful in her career. The mentor helped the mentee identify areas in the nursing budget process that she needed to improve. In working together, the mentor helped the mentee realize that budgets and cost analytics don't have to be as stressful as she thought. The mentor always provided a positive learning atmosphere while providing the mentee with useful tools, valuable knowledge, and new expertise to manage resources.The dyad first reviewed the staffing budgets of the mentee's three medical-surgical units. As the mentor explained, you can't understand a variance report to budget if you don't fully understand the personnel budget.The mentor provided additional tools with an electronic spreadsheet that she developed showing how a master staffing plan can be used for budgeting purposes to fully understand how productivity of nursing care hours is calculated, how staffing patterns dictate the FTEs needed, and how to calculate specific RN positions needed based on weekend needs and total FTEs. This increased understanding gave the mentee the confidence to seek additional information from the finance department on certain data to better understand these data and use them more fully with her manager direct reports. Often, new nurse leaders \u201cdon't know what they don't know\u201d when they try to deal with budgets and new finance and gain understanding as they try to \u201cconnect the dots.\u201d8Next, they reviewed the various reports on staff productivity to increase the mentee's understanding of her own organization's financial reporting tools of variance analysis. She expressed concern with the high nonholiday RN overtime OT usage, not yet understanding what caused the high OT and how to implement procedures to decrease OT expenses.At the November 2018 dyad meeting, the mentee included one of her clinical directors, who was having excessive OT usage, to do a \u201cdeep dive\u201d into the clinical director's unit budget and understand the root cause behind the high OT usage. At the time, this clinical director hadn't fully understood the staffing plans, the budget, or how best to control OT usage. Further analysis revealed that there was a pattern of \u201ccasual\u201d OT (time adding to OT from early punch-ins and late punch-outs). There was also a concern regarding the use of full-time RNs for extra shifts and low use of per diem nurses and part-timers, increasing OT hours.At about 6 months into the mentoring experience, the mentee wanted to put action plans into place with her own clinical directors and their clinical coordinators to begin the process of reducing OT costs for their new fiscal year of 2019. However, before an action plan could be fully designed and implemented, the clinical director, clinical coordinators, and the RN staff formed a task force to work on a performance improvement plan. The leaders have a strong shared governance model in place and realized that the success of this endeavor needed to start with the challenges as viewed by the staff.The task force agreed on the actions needed and began implementation in several areas:/36 (11%) extra paid regular productive hours above what was budgeted. The leaders and RN staff were surprised by how costly these extra hours could be before the hours over 40 were even counted as OT hours. They couldn't believe that this could be an additional 11% above budget when it occurred. They agreed that excessive hours needed to be addressed to control costs, as well as nurse fatigue when working beyond a 12-hour shift.9Reduce \u201ccasual\u201d OT . For example, if a full-time employee punched in early or punched out late for a total of an extra 4 hours a week beyond their scheduled three 12-hour shifts (36 hours) a week, their total paid hours would be 40 hours, 36 of the hours budgeted, but another 4 hours not budgeted. These 4 hours could account for 4Reduce nonholiday OT hours greater than 40 hours a week by scheduling extra shifts in a less costly manner (schedule per diem and part-time staff before use of OT hours from full-time RN staff).Begin end-of-shift huddles about 5 p.m. each day with clinical coordinators on day shifts and RN staff to determine who might be feeling overwhelmed and anticipating not being able to complete all work on time. The clinical coordinators and RN staff then focused proactively on how they could assist their RN colleagues to complete the work as a team. These huddles identified workflow issues, learning needs of staff, and the impact of clinical process issues with other departments that needed to be addressed. Twelve-hour shifts are exhausting and working longer than 12 hours doesn't promote patient and employee safety.9Standards for Establishing and Sustaining Healthy Work Environments, which has six interdependent standards: skilled communication, true collaboration, effective decision-making, meaningful recognition, authentic leadership, and appropriate staffing.The mentee's institution is committed to actively creating and promoting a healthy work environment that fosters and supports excellence in patient care. It's one of 37 New Jersey hospitals participating in an ONL NJ initiative for nursing workplace environment and staffing councils to bring about positive change, with clinical nurses taking the lead. This work is under the conceptual framework of the American Association of Critical-Care Nurses' \u00ae (NDNQI\u00ae) staff satisfaction scores. (See Table During the months of this work together, the leaders and staff continued to monitor their unit quality dashboards and staff engagement. They didn't experience declines in quality measures and improved in some staff satisfaction data as noted from 2 years of National Database of Nursing Quality IndicatorsThe mentee shared personnel budget variance reports with her mentor and had conference calls to determine progress made in implementing plans to control costs from their interventions. The mentee shared how the actions by her clinical directors and their teams decreased \u201ccasual\u201d OT, reduced unnecessary OT, and continued to trend downward. (See Figures As reported in the literature, it's critical for mentees to take responsibility for their own development and be self-reflective of their ability to learn and grow.When it comes to fiscal management, nurses tend to feel intimidated."}
+{"text": "The COVID-19 pandemic has resulted in increased strain on health care systems and negative psychological effects on health care workers (HCWs). This is anticipated to result in long-term negative mental health effects on the population, with HCWs representing a particularly vulnerable group. The scope of the COVID-19 pandemic necessitates the development of a scalable mental health platform to provide services to large numbers of at-risk or affected individuals. The Mount Sinai Health System in New York City was at the epicenter of the pandemic in the United States.The Center for Stress, Resilience, and Personal Growth (CSRPG) was created to address the current and anticipated psychological impact of the pandemic on the HCWs in the health system. The mission of the Center is to support the resilience and mental health of employees through educational offerings, outreach, and clinical care. Our aim was to build a mobile app to support the newly founded Center in its mission.We built the app as a standalone digital platform that hosts a suite of tools that users can interact with on a daily basis. With consideration for the Center\u2019s aims, we determined the overall vision, initiatives, and goals for the Wellness Hub app, followed by specific milestone tasks and deliverables for development. We defined the app\u2019s primary features based on the mental health assessment and needs of HCWs. Feature definition was informed by the results of a resilience survey widely distributed to Mount Sinai HCWs and by the resources offered at CSRPG, including workshop content.We launched our app over the course of two phases, the first phase being a \u201csoft\u201d launch and the second being a broader launch to all of Mount Sinai. Of the 231 HCWs who downloaded the app, 173 (74.9%) completed our baseline assessment of all mental health screeners in the app. Results from the baseline assessment show that more than half of the users demonstrate a need for support in at least one psychological area. As of 3 months after the Phase 2 launch, approximately 55% of users re-entered the app after their first opening to explore additional features, with an average of 4 app openings per person.To address the mental health needs of HCWs during the COVID-19 pandemic, the Wellness Hub app was built and deployed throughout the Mount Sinai Health System. To our knowledge, this is the first resilience app of its kind. The Wellness Hub app is a promising proof of concept, with room to grow, for those who wish to build a secure mobile health app to support their employees, communities, or others in managing and improving mental and physical well-being. It is a novel tool offering mental health support broadly. COVID-19 has resulted in over 41 million infections with a worldwide case fatality ratio of approximately 2.6% as of early November 2020 . With coIn recent years, mobile health  apps on smartphones have become ubiquitous tools for personal health management and behavior tracking ,9. mHealmHealth apps have been rigorously implemented during the COVID-19 pandemic in order to support contact tracing, symptom tracking, and behavioral management for individuals across the globe. These apps have been deployed to support health care systems, governments, and communities to reduce the spread of COVID-19 and its negative long-term effects on the stress and well-being of individuals -18.The growing knowledge and popularity around mHealth apps, in combination with the imperative to support frontline workers ,19, alloThe Wellness Hub app is designed as a tool for general mental health maintenance and resilience and connects users to the mental health services available through the Center for Stress, Resilience, and Personal Growth (CSRPG) and Mount Sinai at large . A team The app is a standalone platform that hosts a suite of tools that users can interact with daily. While initially only available within Mount Sinai, we envisage the app being applicable and deployable in the future to other outside settings where there is a need to offer resilience-building tools to HCWs. Content  could be tailored to specific health care systems.Resilience-focused content and user tasks for the app were adapted from resilience training workshops provided to Mount Sinai Health System staff starting in July 2020, in direct response to the impact of the pandemic on staff well-being. The Center\u2019s resilience workshops draw upon the work of Southwick and Charney, who outlined 10 factors contributing to personal resilience from laboratory research and interviews with trauma survivors, including prisoners of war during the Vietnam War .The Center\u2019s staff developed a peer co-led resilience training curriculum and first piloted a series of eleven 45-minute workshop meetings . VirtualWe defined the app\u2019s primary features based on the mental health assessment and needs of HCWs. Feature definition was informed by the results of a resilience survey widely distributed to Mount Sinai HCWs  and by tMost prominently, app users answer standardized mental health surveys with subsequent score-based feedback that incorporates various exercises for building resilience. Progress is visualized over time based on individual survey scores, showing green (good) or yellow  feedback depending on the score, allowing users to track their mental health in a regular, quantitative, and accessible way. A user will receive randomized feedback text based on their score . If a user scores in a way that suggests a need for mental health care , a feedback screen appears suggesting that they seek additional mental health and wellness resources and lists available resources and tools directly on the screen for easy access. App users can write private digital journal entries and access videos for relaxation, including some created specifically for Mount Sinai employees. Users also have the ability to tailor their resources and videos to suit their mindset and interests by favoriting specific ones for ease of access. The app also offers quick links to Mount Sinai wellness-related resources . The app is intended to be used in conjunction with CSRPG\u2019s resilience workshops and, therefore, includes key information and activities related to resilience development. We selected features based on their usability as it relates to mental health management and their compatibility with behavior change theory ,24. Our baseline fashion, where all questions are offered at one time, in one baseline survey. After completion, users will see each survey individualized on the home screen and can take each survey as many times as they would like. The surveys offered to users are validated measures commonly used in research and clinical practice, though they are available to answer consistently in the app as opposed to on a validated schedule. This is because the surveys are meant to serve as a feedback mechanism for users, where they can regularly assess their well-being and mental health as well as receive consistent feedback based on their results. The surveys were chosen based on prior knowledge about disorders that occur in the context of trauma as well as data derived from a previous survey distributed among Mount Sinai Hospital HCWs [The app contains mental health surveys presented on the main screen. Users are first introduced to the surveys in a tal HCWs . All surEcological mood assessment (\u201cMy Daily Feelings\u201d). We included a daily feelings meter with emoticons indicating sadness to happiness on a scale from 1 to 7 for participants to rate their daily mood. This measure is similar to that utilized in prior research with smartphone apps .PHQ-8  and GAD-2  (\u201cMy Mood\u201d). The PHQ-8 and GAD-2 screen for depression and overall anxiety, respectively, over the past 2 weeks ,27. The PCL-5  (\u201cMy Stress Reactions\u201d). We utilized the PCL-5, a newly published brief 4-item version of the longer 20-item PCL-5, anchored to the COVID-19 experience, to evaluate posttraumatic stress reactions ,30. ItemCD-RISC2  (\u201cMy Coping Resources\u201d). We utilized the CD-RISC2, a previously validated short form of the CD-RISC ,32. The WHO-5  (\u201cMy Well-being\u201d). The WHO-5 is a 5-item globally utilized measure of overall well-being . Items aAUDIT-C (Alcohol Use Disorders Identification Test-Concise) (\u201cMy Alcohol Use\u201d). The AUDIT-C is a widely utilized 3-item screening measure for alcohol misuse . Items aSpiritual struggle items . Based on prior research, two items were included in the app to measure problems with spirituality based on prior research: (1) \u201cDo you struggle with loss of meaning and joy in your life?\u201d and (2) \u201cDo you currently have what you would describe as spiritual or religious struggles?\u201d . These iWe offer a library of resources to our Wellness Hub users. Whether based on survey feedback, general curiosity, or additional mental health needs, the app provides access to the Mount Sinai mental health support network and offers resources and tools external to Mount Sinai.For individuals looking for direct interaction with a mental health expert or support network, the app provides contact information for CSRPG, chaplaincy, and other behavioral practices and offices at Mount Sinai. We offer a page focused on promoting resilience and building a resilience plan. This page also offers additional CSRPG workshop content and videos.Resilience page in \u201cMy Resources\u201d in order to discern for themselves what experiences are helping them to build their resilience -compliant ecosystem. For quality control, robust data transfer, and data persistence, the Wellness Hub app stores the data in an encrypted environment. The environment is fully encrypted, and the metadata are stored in secure non-Structured Query Language (NoSQL) instances for quality control.The data transferred from the app to the cloud are sent through an https protocol with end-to-end encryption. Authentication protection at the beginning of the data ingestion is performed, and all data requests external to the server are blocked. No personally identifiable information is ever transferred from the app to the back end. The server manager cannot identify the data\u2019s owner, effectively making the ingested data anonymous .The cloud system is only accessible through a secure shell with a public-private key, and IP address restrictions are in place to prevent cyberattacks. The database is restricted only to the app data and does not host any data from outside the app.The app processes the survey scores locally and initializes the data transfer. Scores are given either a valid value or an invalid value, depending on whether the user completes a full survey or not. The app also implements single sign-on authentication, either Apple or Google, depending on which operating system the app is running. Consistent with our approach to privacy, the app has a nonutilization timer that expires if the app is not used for 2 minutes, and the timer forces a log-out when it expires.The \u201cFavorites\u201d tool in Wellness Hub allows users to tailor the content of the app based on what they find most helpful to them. All resources and videos from the \u201cMy Resources\u201d and \u201cMy Calm\u201d pages can be selected as favorites and will subsequently appear as a personalized library in this view. Here, preferred resources can be easily accessible .Beyond tailoring resources, we also allow users to further personalize their experience through design. In addition to the standard Mount Sinai colors, we include a feature allowing users to choose the color scheme they prefer for their app, with numerous options meant to offer a calming effect to the user . This seFor future use, we have included a basic and optional \u201cAbout Me\u201d anonymous demographics survey in the standard profile section of the app in order to improve the personalization of resources based on age, role at Mount Sinai Health System, and other demographic or COVID-19\u2013related features. This section can also help us better understand the breakdown of our user population and develop new or amend existing features to better address and target outreach to an underserved user population.This app is available for both iOS and Android devices and is currently only available to employees of the Mount Sinai Health System. Due to this employment restriction in distribution, we developed this as a custom app that is only available for download using a unique redemption code or link. The code or link can only be used one time, thus limiting the possibility of users distributing the app outside of the health system. In order to verify the current employment status of the user, we created a page on the Mount Sinai intranet in which users must first log in, thus validating their employment, and subsequently select whether they want to download the app for iOS or Android. Next, they enter the email address to which they want to receive installation instructions. The intranet page automatically pulls a unique redemption code from a list on the back end and sends download instructions, including the unique code, to the interested employee. The code is then marked as \u201cused\u201d and unable to be supplied to another person.The Wellness Hub app was launched between June and September 2020, as a Phase 1 pilot in which we manually distributed redemption codes to interested Mount Sinai Health System employees while further developing the app in anticipation of the Phase 2 launch in October 2020, to correspond with Mental Health Awareness Month. At the time of the Phase 2 launch in early October, CSRPG introduced the app to the Mount Sinai Health System community through regular presentations, in-hospital messaging, and email notifications.Prior to app development, we established key performance indicators (KPIs) to measure the success of the product and level of engagement of our app users. Our KPIs included total number of HCWs downloading the app, total number of baseline surveys submitted, number of users submitting additional surveys postbaseline, and general app engagement metrics .This paper outlines the design and launch process for the Wellness Hub app. Development time for the platform was variable between Phase 1 and Phase 2. From initial ideation to the current version, we estimate a total of approximately 2.5 months for development and launch. This time frame is reflective of one software engineer, one product manager, and one designer working on the app. Since the app was developed in-house at Mount Sinai, the platform\u2019s primary cost is for database management, which is a regular ongoing service fee. As of March 2021, there were new downloads each month and no reports of bugs or software crashes, suggesting a successful technical launch of the Wellness Hub app.We analyzed several KPIs to determine the success of the app early after the launch. These early results serve to showcase the initial launch of the app. Between July 27 and October 13, 2020, during our Phase 1 pilot launch, which included manual app distribution and word-of-mouth recruitment, 74 users downloaded the app. We received informal feedback from many of these early users, which allowed us to refine the app and include additional features and adapt existing ones to support our user population. This led to the inclusion of relaxation videos and the improvement of our survey feedback content and progress visualizations.Our broader, Phase 2 launch was on October 14, 2020, and included greater recruitment and easily accessible app distribution methods. Within 3 months of Phase 2, we received 157 additional app downloads, totaling our reach and app use to 231 users over the course of 4.5 months.Of the 231 HCWs who downloaded the app, 173 (74.9%) completed our baseline assessment of all mental health screeners available in the app. As of 3 months after the Phase 2 launch, approximately 55% of users re-entered the app after their first opening to explore additional features, with an average of 4 app openings per person.While the platform is meant primarily to provide individualized feedback to users, and not analyze a cohort of users together, we have compiled a brief overview of baseline results.screen-in cautionary scores on the WHO-5 , AUDIT-C , and spiritual struggle items . This demonstrates a clear need for additional HCW support related to overall well-being and spiritual struggles  surveys (screeners) after completing the baseline assessment. Of all the surveys available on the app, the daily feelings (\u201cMy Daily Feelings\u201d), PHQ-8 (\u201cMy Mood\u201d), and PCL-5 (\u201cMy Stress Reactions\u201d) surveys had been taken the most, with 410, 368, and 318 total completions, respectively. Considering the everyday availability of these surveys for users on the platform, they were not taken on the validated schedule for which the surveys are primarily intended.As our study of app usage proceeds, we will review resilience-related indicators beyond the baseline assessments taken by users. We intend to review additional KPIs that align with longer-term use of the platform.The strength of the app is that the Center was able to quickly deploy a tool based on existing resilience literature in order to address a mental health need within the frontline HCW community amidst the initial wave of the COVID-19 pandemic.The main limitation of the app is that, as it is not a research study in its current version, there is no strict protocol given to users for how they must interact with the app. As a result, there is significant variability in app usage and our ability to track user experiences relative to their demographics. Future versions of the app may include a research intervention that will empirically test the use and effectiveness of the app as a supplement to CSRPG workshops.Additionally, we consider selection bias; users may already have a bias or inclination toward resilience building, or recognition of their need for wellness tools, thus leading them to search for an app-based tool. We may be missing an important HCW population that is both lacking in resources and would not know how to find or use this tool effectively. We have to consider an outreach strategy that will promote greater access or we must consider making these tools available in multiple formats and venues. Lastly, the app is available to a specific cohort with specific access: HCWs in the Mount Sinai Health System. In its current state, it is meant to serve partly as a supplement to CSRPG workshops held at Mount Sinai. This may impact external validation of the app\u2019s efficacy in building resilience outside of this specific cohort.mHealth apps are an increasingly used tool for people to maintain their physical and mental wellness . mHealthThere are many publicly available resilience-building platforms similar to Wellness Hub, such as COVID Coach and Calm. COVID Coach, developed by the US Department of Veterans Affairs, offers education about coping, self-care tools, progress visualization, and national mental health resources . Calm isscreen-in scores, are consistent with previous findings at Mount Sinai in the same cohort via a hospital-wide early pandemic survey [To address the mental health needs of HCWs during the COVID-19 pandemic, the Wellness Hub app was built and deployed throughout the Mount Sinai Health System. To our knowledge, this is the first resilience app targeted to HCWs and applied within the context of a global trauma. It includes validated surveys and questionnaires based on prior knowledge of disorders that occur in the context of trauma, a journal tool to allow users to log their feelings and activities related to resilience, and numerous Mount Sinai\u2013specific resources and videos targeted to those seeking additional support and tools to build resilience. The app\u2019s initial results, showing that more than half of the baseline assessments resulted in c survey .The limited level of app use and engagement thus far may be related to the constrained recruitment strategies to date, considering the lack of in-person advertising, enrollment, and distribution due to the pandemic. Additionally, the slower uptake is consistent with our expectations of an initial app release, especially considering the involved download process and our active and occupied target audience of frontline HCWs and others in the health system.badges that users can obtain in order to encourage them through their resilience journey as well as inclusion of materials from the CSRPG workshop curriculum in specific learning sections and as thought prompts in an expanded version of our journal tool. As the CSRPG continues to grow and COVID-19\u2019s effects continue to be felt across the health care system, we intend to use our KPIs, user feedback, and guidance from the Center workshops to determine the future direction of the app and its content.We intend to continue developing this app by improving features based on feedback from our users, including the translation of the platform into Spanish and the integration of Mount Sinai employee and health care services, such as appointment scheduling. We will move beyond assessment by adding specific resilience-promoting interventions, action items, and tools. Potential future improvements include The Wellness Hub app is a promising proof of concept for those who wish to build a secure mHealth app to support their employees, communities, or others in managing and improving mental and physical well-being. It offers a novel tool to offer mental health support broadly."}
+{"text": "Breastfeeding mothers have had limited access to breastfeeding support throughout the COVID-19 pandemic. This study aims to investigate breastfeeding practices during the COVID-19 period and to determine the factors associated with supporting exclusive breastfeeding. A sequential explanatory mixed methods approach was adopted, including a quantitative method in the first phase and qualitative method in the second phase. Mothers whose babies were aged over 6 months to 24 months old from July to September 2021 in Pekanbaru City were selected as research subjects. Data analysis was performed with multivariate and deductive content analysis. Of 156 participants, 97 mothers (62.2%) exclusively breastfed their babies. Of those, mothers who delivered exclusive breastfeeding worked less than eight hours per day, were aged 17\u201325 and had low education. Though by using exclusive breastfeeding practice as a reference, associated supports, including emotional, instrumental, appraisal and information regarding exclusive breastfeeding practice were insignificant; however, mothers who practice exclusive breastfeeding had higher information support. During the COVID-19 pandemic period, the informational support factor was found to be important to achieve the successful exclusive breastfeeding practice. Supporting mothers to continue breastfeeding during the COVID-19 pandemic is a public health concern. It is globally recommended that breastfeeding should be continued during the pandemic to increase the health and immunity of babies .Exclusive breastfeeding for the first six months of a new-born\u2019s life reduces the risk of infectious diseases in infants, protects them from chronic diseases in adulthood and increases intelligence scores ,4,5,6. TThe WHO has planned to increase the rate of exclusive breastfeeding in the first six months to at least 50% by the year of 2025; however, the percentage of infants under six months who are exclusively breastfed is only 40% globally . IndonesThe progression of COVID-19 cases increased during the early stage of the Covid pandemic, and it appears that COVID-19 will remain endemic . On the The imposition of lockdown and home confinement led to a decrease in exclusive breastfeeding during the COVID-19 pandemic especially among working mothers in urban areas . FactorsStudies have also shown that the support provided to breastfeeding mothers is an essential factor in successful breastfeeding in Indonesia . HoweverThis study obtained using a mixed-method with a sequential explanatory and cross sectional design approaches. Purposive sampling was used to collect quantitative data from 156 breastfeeding mothers with babies older than six months old from two public health facilities between August and September 2021. We used the Slovin formula with 95% confidence interval for sample size study estimation. The Family Support Questionnaire (FSQ) originally was developed by Biswass  to measuThe qualitative study using the semi-structured in-depth interviews enlisted the participation of 12 informants who were breastfeeding mothers with eligible new-borns. Exclusive breastfeeding in this study means that the baby provided only mothers breast milk from birth to 6 months without any additional drinks and food. Research data were collected by fourth year students of the Faculty of Nursing, University of Riau who have been trained as research data collectors. First, the quantitative data was analysed using chi-square test for independence to test the association between sociodemographic variables and breastfeeding practice patterns, and then the multinomial logistic regression analysis was performed to find whether the independent factors included the breastfeeding support were factors associated towards exclusive breastfeeding practice. The outcome in this study has therefore three nominal categories, which were a combination of breastfeeding and formula milk (mixed), breastfeeding and infant formula. Independent variables with the p-value less than 0.05 were assigned as significantly associated with the outcome variable.The qualitative data was analysed using thematic data analysis. Both quantitative and qualitative results were finally integrated. The ethical test was issued by the ethics committee with the number 283/U.19.5.1.8/KEPK.FKp/2021.At the study locations, there were 156 breastfeeding mothers as samples. The study had babies with ages ranging from 8 to 15 months with most of the mothers aged above 25 years old (59.6%). More than a half of the mothers had given only breast milk before the babies were six months old (62.2%). The sociodemographic background had an association with breastfeeding practice among the study participants. These sociodemographic variables were a low education level (junior to middle high school) and working 8 h or less in a day (including housewives) as shown in the There was inadequate evidence that supporting factors, such as emotional, informational, instrumental and appraisal, were associated with the decision to provide exclusive breastfeeding among mixed and infant formula groups. However, mothers in the high informational group were more likely to nurse their baby exclusively with breastmilk compared to those who mixed both breastfeeding and formula milk, respectively .\u201cSaya udah mencoba memberikan ASI, bayi diletakkan di dada\u2026 itupun cuman sebentar sekitar 5 menit lah, bayinya cuman menjilat-jilat aja, memang gak keluar (ASI)\u2026\u201d(P2)\u201cI\u2019ve tried to feed breast milk, the baby is placed on the chest\u2026 it only lasted for about 5 min, the baby just licked it, the breast milk just didn\u2019t come out\u2026\u201dAccording to in-depth interviews, participants had a number of challenges when breastfeeding their babies, such as being late on early initiation of breastfeeding (IMD) and the problem of breast milk that did not come out. Here are the participants\u2019 expressions:\u201c\u2026 Udah dipencet-pencet payudaranya, ASInya gak ada pas pertama lahiran\u201d(P2)\u201c\u2026The breasts have been squeezed but the breast milk is not there at the beginning of giving birth\u201d\u201cgak ada masalah dengan payudara, putingnya ada, bersih, cuman bengkak, apakah karena banyinya sudah kenyang diberi susu (susu formula) sama bidannya, kayak malas gitu menyusu\u201d(P4)\u201cThere is no issue with the breasts, just the nipples are swollen, it might due to the baby is too tired to breastfeed maybe because the baby has already given the milk (formula milk) by the midwife.\u201d\u201cMimiknya (payudara)\u2026 nyeri terus kalau dia nyusu tu, ada luka, dianya (bayi) kuat mimik.. ngisap ujungnya aja, \u2026akhirnya awak jadi takut kalau dia nyusu\u201d(P9)\u201cMy breasts\u2026 when the baby is breastfeeding, there is a lesion, baby sucks aggressively. only sucks the tip, \u2026in the end when he suckles, I feel anxious.\u201d\u201c\u2026 dia gak tidur-tidur, nangis-nangis terus, ha stress lah awak, anak awak ndak tidur-tidur, capek juga, awak ndak tidur kan\u2026 itulah air, air susu tu kurang\u2026\u201d(P12)\u201c\u2026 The infant does not sleep and constantly cries. I\u2019m really stressed. My child isn\u2019t sleeping and I\u2019m exhausted, therefore I don\u2019t sleep. Finally, the milk gets scarce\u2026\u201dBreastfeeding issues were not only during the IMD procedure, mothers were also experiencing breastfeeding challenges dependent on the infant and mother, which included not enough breast milk or low milk supply, soreness experience, pain and swelling. Here are the responses of the participants from mother\u2019s perspectives:\u201cpas bayinya menghisap itu kan kenak, kenak gusinya gitu pentilnya itu\u2026\u201d(P1)\u201cwhen the baby suckes, the baby\u2019s gums force against the nipple\u2026\u201d\u201cpas N cari-cari, gara-gara gak tepat masuk ke mulut katanya, apa yang bulatan tu gak masuk ke bibir\u201d(P3)\u201cwhen breastfeeding, the baby\u2019s mouth does not suck properly on the nipple\u201dBreastfeeding problems arise not only from the mother\u2019s perspective but also from the baby\u2019s standpoint, such as a baby who has difficulty sucking. These following expressions from participants were presented below:\u201ciya, jadi kami juga ada dipijat di punggung gitu lihat di YouTube\u2026 suami awak peragakan langsung gimana gerakannya, memang terasa enak, katanya memperbanyak air susu gitu\u201d(P2)\u201cYes, I had my back massaged. My husband discovered the technique on YouTube\u2026 My spouse performed the steps. I feel better, and it has the potential to enhance milk supply.\u201d\u201ciya mendukung bapaknya, dia belikan makanan yang memperbanyak air susu\u2026 makan tapai (tape singkong) kata orang kan banyak air susu, tempe, tahu, sayur\u2026 sayur katuk, bayam hampir tiap hari\u201d(P10)\u201cMy husband supports me to breastfeed the baby. He buys food to booster breast milk\u2026 People believe that eating tapai (fermented cassava) helps enhance breast milk production, consume tempe, tofu, vegetables\u2026 such as katuk, spinach almost every day\u201d\u201c\u2026 memang iya kalau sama neneknya waktu tu kan neneknya yang ngasih nyediain makan kan selama empat puluh harikan, memang dipantang kan gak dikasih cabe\u201d(P1)\u201c\u2026 it\u2019s true that grandmother served meals for forty days (for mother) during which chilli consumption was prohibited\u201d\u201cmama sangat perhatian, ngasih saran makan aja yang banyak untuk bayinya, sayuran, buah-buahan\u2026 cuman kan kata mama jangan makan cabe dulu\u201d. \u201cdisuruh makan yang pahit-pahit, jangan yang manis-manis\u2026 iya biar cepat kuat aja\u201d(P4)\u201cMy mother is much caring, offering tips on how to feed my baby a lot of veggies and fruits\u2026 Mom urged me not to eat chilies first and to eat the bitter ones instead of the sweet ones so I could grow strong soon\u201d\u201c\u2026 gak boleh pegang sapu, gak boleh kerja berat-berat, perutnya harus diikat biar jangan apa\u2026 kayak apa ya, biar gak turun peranakannya (rahim), ASI nya biar banyak\u201d(P7)\u201c\u2026 not allowed to sweep the floor, not allowed to do heavy work, I have to tighten my stomach to keep uterine prolapse from coming down, then I can have breast milk excessively\u201d\u201c\u2026 mamak bikin kan ikan baung eh ikan apa ikan apa namanya ikan gabus direbus\u2026cepat keringnya \u2026 \u201ciya makan daun katu (katuk), sayur bayam, katanya banyak ASI, disayur bening aja\u201d(P12)\u201c\u2026 my mother cooks boiled cork fish to help the birth canal wound heal rapidly.\u201d \u201cMy mother also suggests eating katu (katuk) leaves and spinach to ensure a plentiful supply of breast milk\u201dParticipants received various forms of support while breastfeeding. All participants briefly stated that their husbands and families provided them with great support. Husbands provide significant support, particularly in early breastfeeding initiation, which includes paying attention to the wife\u2019s food consumption, massaging the wife\u2019s back and offering help. During breastfeeding, families may offer support and assistance. The participants received family assistance, which included guidance to pay attention to the food consumption, assist the mother\u2019s work and dietary items that can aid in the production of a large amount of breast milk. Here are the reactions of the participants with their husband and family supports:\u201c\u2026 gak ada informasi apa-apa bu, dia hanya nolong lahiran, sehabis tu membersihin bayinya\u2026\u201d(P4)\u201cI didn\u2019t get any information from the midwife. she just helped the delivery, after that she cleaned the baby\u2026\u201d\u201ckalau menjelaskan tentang manfaat ASI ndak ada, cuman disuruh nyusuin anaknya aja\u2026 pas sakit pada puting, hanya dikasih salap\u2026 ndak pa pa itu katanya\u2026\u201d(P5)\u201cThere was no explanation regarding the benefits of breastfeeding; I was just instructed to breastfeed the baby. When my nipples hurt, she just advised ointment\u2026\u201dOn the other hand, the healthcare workers were more likely to not provide any support. Some participants also mentioned that the healthcare workers did not assist with further information about breastfeeding after giving birth. Here are the participants\u2019 expressions:This mixed-method study was conducted to assess the relationship between breastfeeding practices and the factors that support exclusive breastfeeding during the COVID-19 pandemic in Indonesia. More than half of mothers with babies aged six months and older delivered breast milk for the first six months of their child\u2019s life.Mothers with children aged 15 to 24 months were shown to be more likely to provide their babies with a combination of breastfeeding and formula milk compared to infant formula and breastfeeding milk. The older age of mothers aged more than 33 years old, a high education level and working more than 8 h per day showed the same evidence with mothers having children aged 15 to 24 months. We found insufficient evidence on any supporting factors regarding exclusive breastfeeding compared to infant formula milk or the combination of both to their babies.The COVID-19 pandemic has caused significant changes in lifestyle and disruption in various fields, including in the health care sector . As showVarious factors may influence the mother\u2019s inability to give exclusive breastfeeding, such as education, work and other support . Other sThis is because respondents with higher education are more frequently employed. Occupation is a predictor of failure in exclusive breastfeeding. Moreover, the higher economic status tends not to give exclusive breastfeeding. This was supported by the affordability to buy extra food, such as milk formula that could be given to infants in addition to breast milk. The study also revealed that the welfare of the family played an important role in the breastfeeding habit . InfantsApart from the education factor, the working hours were identified as affecting exclusive breastfeeding practice. Working mothers could decrease the opportunity for exclusive breastfeeding in which the mother worked all the time were 1.54-times more likely not to give exclusive breastfeeding than mothers who did not work . LikewisWorking long hours weakened mother-to-child intuition, and physical tiredness might disrupt the let-down reflex and reduce milk production in women . This coThe high percentage of exclusive breastfeeding in this research was attributed to the fact that the majority of mothers did not work or worked for less than 8 hour in the COVID-19 period, whereas the last study also showed that mothers who worked more than 7 hour per a day in urban areas during COVID-19 pandemic were likely to stop breastfeeding earlier . FurtherThe success of exclusive breastfeeding during the pandemic cannot be separated from the husband\u2019s support for breastfeeding mothers; this is because the husband\u2019s assessment of support is the most dominant component of exclusive breastfeeding. There are two positive categories of husband\u2019s support: instrumental and assessment support and two other husbands\u2019 supports, namely emotional and informational support . The supMothers will be more capable and confident when given support by those closest to them when breastfeeding their babies  and partDuring the COVID-19 pandemic, breastfeeding mothers faced various challenges and changes in their lives, including breastfeeding practices. Such policies have led to substantial changes for breastfeeding women, including decreased in-person support from extended family and professional services, increased virtual assistance, employment changes and additional duties at home as a result of company and school closures. Participants who believed they received aid from a number of sources, including their spouses and extended relatives, demonstrated this. Although assistance from health personnel was not received, support from family members, particularly informational support, was extremely significant in this study. It seems that online health service consultations during the COVID-19 pandemic were beneficial and effective . Given tThis study provides valuable information on breastfeeding practices in urban areas in Indonesia during the COVID-19 pandemic. However, the study has several limitations. It is difficult to meet breastfeeding mothers because most mothers are afraid to meet in person face-to-face for data collection by questionnaires or interviews. Future researchers need to evaluate the effectiveness of using various information media needed by breastfeeding mothers both during the pandemic and after the pandemic so that breastfeeding information needs can be met.Factors that may influence exclusive breastfeeding were the mother\u2019s age being more than 33 years old, having children aged 15 to 24 months, having a high education level and working more than eight hours. All supporting factors had no evidence toward exclusive breastfeeding practices among mothers. Hence, the informational support factor was found to be beneficial for successfully exclusive breastfeeding during the COVID-19 pandemic period."}
+{"text": "This article presents a regression tool for predicting the compressive strength of fly ash (FA) geopolymer concrete based on a process of optimising the Matlab code of a feedforward layered neural network (FLNN). From the literature, 189 samples of different FA geopolymer concrete mix-designs were collected and analysed according to ten input variables  and the output variable . The developed optimal FLNN model proved to be a powerful tool for predicting the compressive strength of FA geopolymer concrete with a small range of mean squared error (MSE = 10.4 and 15.0), a high correlation coefficient with the actual values (R = 96.0 and 97.5) and a relatively small root mean squared error (RMSE = 3.22 and 3.87 MPa) for the training and testing data, respectively. Based on the optimised model, a powerful design chart for determining the mix-design parameters of FA geopolymer concretes was generated. It is applicable for both one- and two-part geopolymer concretes, as it takes a wide range of mix-design parameters into account. The design chart  will ensure cost- and time-efficient geopolymer production in future applications. Manufacturing approximately 1 ton of cement produces 1 ton of CO2 gas due to the energy consumption and decarbonisation process of limestone [The high COPromising alternatives for replacement cement are novel sustainable binders, namely the so-called geopolymer  binders consisting of amorphous silico-aluminate phases such as metakaolin, ground granulated blast furnace slag (GGBFS), silica fume or fly ash (FA) ,3,4. GeoGenerally, in the mix-design of geopolymer concrete, the same method is utilised as in traditional Portland cement concretes, taking the compressive strength of the materials as the cardinal design characteristic. The compressive strength (f\u2019c) of concrete is a function of its mix-design proportion and of the water\u2013cement (w/c) ratio of the matrix, and thus it can be predicted according to the proportion of the particular ingredients . Most deLloyd and Rangan  proposedStatistical analysis is another approach to geopolymer concrete mix-design that is based on determining the relationship between the composite\u2019s mix ingredients and its compressive strength. These methods are the Taguchi method ,13, lineMachine learning techniques are powerful tools for solving statistical problems in engineering challenges. The most prominent techniques are artificial neural networks (ANNs) ), multivariate regression (MR) and gene expression programming (GEP). So far, they have been successfully utilised in different areas of civil engineering and building materials science ,16,17,18The single-layer perceptron type of NN is only suitable for linear relationships, while a multilayer perceptron allows more complex computations such as nonlinearity. The choice of NN depends on the learning task ,20. SincIn regression problems, a feedforward neural network (FNN) is more suitable than other types of machine learning (MR and GEP) because it is powerful in computation. It is a very strong and robust tool in the analysis of error signals ,18,20. O2 and 35.5 MPa2 and 89.0% and 82.6% for the training and testing subsets, respectively. The statistical parameters of RMSE and R did not achieve good values and could be improved, and the model is only applicable for two-part geopolymers. Furthermore, their model was built by considering a linear relationship, which is not the best representation, as has been proven by Ahmed et al. [2 and Al2O3, and it is only applicable for two-part geopolymers.Hardjito and Rangan  and Lloyd et al. . Ahmed ed et al.  undertooSo far, no comprehensive NN model has been published in the literature for predicting the compressive strength of FA-based geopolymer concretes that is applicable to both one- and two-part geopolymer concretes and that simultaneously considers the wide range of the composite\u2019s possible design parameters.This research aimed to build a comprehensive design model by optimising a code based on the FLNN as a regression tool to predict the compressive strength of FA-based geopolymer concrete using Matlab R2020b software provided by The MathWorks, Inc.; 1 Apple Hill Drive Natick, MA, USA under the license\u2019s number \u201840552969\u2019 . The sigBased on the optimal FLNN model, a powerful practical design chart for determining the mix-design parameters of both one- and two-part geopolymer concretes has been generated by taking the wide range of possible mix-design parameters into account. The design chart will enable cost- and time-efficient production of geopolymers, thus supporting engineers in broader applications of this novel material in the future practice.A graphical outline of the study design is depicted in In x; w) is the model response, x is the input vector, w is the vector of the free parameters (weights and biases), H is the number of neurons in the hidden layer, g is the activation function in the hidden layer, D is the input number, 1 and 2 are the numbers of layers, h = 1 \u2212 H, j = 1 \u2212 D and w0j and w0 are the bias parameters for the first and second layers, respectively. The architecture of this kind of neural network is depicted in According to the universal approximation theorem , one hidx is the input vector, d is the output vector and f(x) is an unknown vector-valued function. However, the training set (T) is responsible for providing the required knowledge about the f(x) function and is described as follows:The compressive strength of geopolymer concrete (output variable) is a nonlinear function of the proportion of the ingredients (input variables). The following equation describes the functional relationship between the input and output variables :(2)d=f(xF(x)) which is close enough to the unknown function (f(x)) by utilising the input\u2013output mapping as follows:w) of the structure of the FLNN. Therefore, the function approximation is expressed as follows:The goal of the neural network is to construct an approximation function (iy) and the experimental output vector (id) provides the error signal vector (ie) [By training the FLNN to produce the system model, the difference between the FLNN output vector (tor (ei) . When thtor (ei) . The opttor (ei) ,26:(6)E(w (the vector of the free parameters). Thus, the objective function is a function of w. Since the goal is to minimise the objective function, the optimisation problem will be unconstrained. The mean squared function represents the error surface, and the free parameters are the coordinates in the learning process. The most important criteria for all learning algorithms are the objective function gradient and the Hessian matrix (H). The gradient is the first partial derivative of the objective function. Still, the Hessian matrix is the second partial derivative of the objective function with respect to the free parameters w, as stated in Equation (7) [The only unknown is tion (7) :(7)H=\u22072Ew(k)) can be calculated as follows [H\u22121 is the inverse of the Hessian matrix of E(w), and G is the gradient of E(w).The updated values of the free parameters (\u0394 follows :(8)\u0394\u00a0w\u00a0(xi) and output variable (di), is used to calculate the MSE function described by Equation (6). The optimisation process involves a continuous updating of the free parameters (weights and biases) from the initial values until reaching convergence between the approximated response (yi) and the true output (di). The initial values of all biases are set to zero. The initial values of weights should be in the range of the activation function\u2019s boundaries. The desirable values of the weights are calculated on the basis of the uniform distribution to have a mean of zero, and the variance is equal to the reciprocal of the number of connections at the hidden neuron. When H is a positive definite matrix, the convergence is simply performed by Newton\u2019s method. However, this is not always the case, so a scalar parameter (\u03bc) can be added to the diagonal elements of H to modify it. The value 1/(2\u03bc) is called the learning rate (\u03b7), which has to be a very small value but must never reach zero. It is set to an initial value less than one. When the MSE is reduced, \u03bc(k+1) = \u03bck/2; otherwise, \u03bc(k+1) = 2\u03bck if the MSE is increased. The convergence criterion is formulated on the basis of the local or global minimum of the error surface. If the updated values of wi achieve a local or global minimum, then the gradient of the updated free parameters (G) is zero [H matrix and to find an optimisation solution even when the H matrix is not a positive definite matrix. Another important advantage of the FLNN is that it has the ability to deal with the nonlinearity of a function by processing computations in the hidden layer and various activation functions [The training subset, which has corresponding pairs of input variables  and the corresponding response   was collected. The input variables for geopolymer concrete were, however, not unified, and the authors did not follow a unified method. Through simple calculations, the datasets were converted into equivalent variables, which enabled a design method for FA geopolymer concrete that was similar to that of Portland cement concrete [Based on extensive research in the peer-reviewed literature ,41,42,43concrete .The chemical composition of the FA:The following key points were applied to select the input variables on the basis of the chemical composition of the FA, the proportions of the ingredients and the curing conditions:2, Al2O3 and Fe2O3, as well as the alkaline oxides Na2O and CaO [2 and Na2O) from the activator [It has a significant effect on compressive strength. Five oxides can affect the chemical composition. The backbone oxides of the three-dimensional geopolymerisation network are SiO and CaO . The relctivator . Accordictivator , around 2.\u25cf3 in the design-mix as the most solid component of the geopolymer solids (GS) [FA ash content: The weight of the FA was taken in kg/mids (GS) ,44.\u25cfagg/Tagg (kg/m3)), which reflects the effect of aggregate gradation. The second parameter was the geopolymer solids\u2013total aggregate ratio (GS/Tagg (kg/m3)), which reflects the effect of the interaction between the total aggregate  and the geopolymer solids (matrix) in the composite.The effect and interaction of the aggregate are similar to those of Portland cement concrete ,44. Ther\u25cfThe superplasticiser was taken as a weight percentage of the raw material (FA) ,44.\u25cf2SiO3) composition [2SiO3) [2O% and SiO2/Na2O were directly calculated from the Ms. From a practical point of view, it is more feasible to convert the alkali activator solution\u2019s variables  to the equivalent of Na2O%, SiO2/Na2O ratio and water in the alkali solution rather than converting the solid alkali activator\u2019s variables . Calculations of the solid parts of the alkali activation solution were based on the chemical composition, molecular weight and weight of each component in kg/m3, as described by [SH is the weight percentage of the sodium hydroxide solids in the sodium hydroxide solution, Na2O is the sodium oxide, Alkali activator and water: In the case of a two-part geopolymer, different terms have been used to define the variability of the alkali activator, such as the molarity of sodium hydroxide (M), the content of sodium hydroxide (SH), the sodium silicate module (Ms) and the sodium silicate\u2013sodium hydroxide ratio (SS/SH). Both the sodium hydroxide solution and the sodium silicate solution were usually mixed 1 day prior to mixing the geopolymer concrete, producing the final sodium silicate  . Thereforibed by ,44:(10)Ingredient proportions:2O% and the SiO2\u2013Na2O ratio to make the model applicable for both one-part and two-part FA geopolymer concrete. Another advantage of separating the alkali activator\u2019s oxides from water is that water has a negative effect on compressive strength, while Na2O and SiO2 have positive effects on compressive strength [Therefore, the alkali activator was represented in this research as the Nastrength ,21. Henc3.Curing conditions:FA geopolymer concrete is usually associated with heat curing at an early stage . The curIn the experimental determination of the ultimate compressive strength of the geopolymer concrete (as is the case for ordinary Portland cement concrete), the shape and size of the test specimens (cubes or cylinders), as well as the boundary conditions and speed of the test, influenced the results obtained . Therefotrainrp, trainlm, traincgp, traincgb, trainbfg, trainos, traincgf and traingda) and eight activation functions  already built into the Matlab software [Random seeds (S): In general, the error surface has many local minima; as a result, FLNN models would have a set of S that could yield better solutions than others . Therefoxi) and output vector (di) [Defining the input and output variables: After the dataset had been sorted into the required input and output variables, the dataset was stored in the target directory of the potential Matlab code in the form of a Microsoft Excel file. The relevant operations of matrices and arrays were used to define the input vector (tor (di) ,49.Optimising the number of neurons: The number of hidden neurons was changed from 1 to 80. For each FLNN with a specific number of hidden neurons, the MSE was calculated for the training, validation and testing subsets. This was carried out by generating a for-loop considering the number of hidden neurons, which varied from 1 to 80. The solution was evaluated through the cross-validation technique by plotting the MSE versus the number of hidden neurons ,50. The Determining the best performing learning algorithm: After choosing the number of hidden neurons corresponding to a specific local or global minimum, the performance of the FLNN was examined for all 12 learning algorithms through a cross-validation of the MSE. Only the training and validation data subsets were applied at this stage. Nevertheless, an interesting learning algorithm, the Bayesian Regularisation, is self-optimised and does not require a validation subset, but only needs to be assigned the training and testing subsets ,24. TherOptimisation for the choice of activation function: After selecting the best performing learning algorithm, the Matlab code ran this learning algorithm through all eight activation functions to identify the optimal choice of the activation function type.Selecting the optimal model: By determining the number of the hidden neurons, the learning algorithm type and the activation function, the optimal model criteria such as MSE and R were recorded for each local or global minimum.Through the definition of the FLNN, the optimisation process is governed by the number of hidden units (neurons), the choice of the learning algorithms and the choice of the activation function. In the optimisation process, 12 learning algorithms  were evaluated for only the training and testing subsets. The MSE values were 10.4 and 15.0 for the training and testing subsets, respectively. The root mean squared error (RMSE) values were calculated by taking the root of the MSE values, thus giving \u00b13.22 MPa and \u00b13.87 MPa, respectively. As shown in In the first step of the optimisation, the Matlab code was run 201 times by changing S (0\u2013200). The local and global minima were investigated each time by selecting the corresponding hidden neurons. After that, the learning algorithm and activation function choices were examined to determine the optimal model. The best performance was achieved when the default seed number (S = 0) was applied. The results of S = 0 are presented in function ,24. As mSince the RMSE value was \u00b13.87 MPa for the testing data (data that were unseen in the training process), the optimal model could be generalised for future data with an average error of \u00b13.87 MPa. 2 and 46.0 MPa2, and 90.2% and 86.7% for the training and testing subsets, respectively, indicating high error signals. Therefore, MSE increased by 206% for the testing subset . This finding agrees with Hardjito and Rangan [It has been repeatedly reported that the w/GS ratio is the most significant factor controlling the compressive strength ,21. Howed Rangan , who stad Rangan .2, 4.56 and 4.44 MPa and 92.3% and 94.1% for the training and testing subsets, respectively, indicating a considerable increase in the error signals. Thus, MSE increased by 31.3% for the testing subset . Toufigh and Jafari [Researchers have not reached common ground about the chemical composition of the raw material used in geopolymer concrete, although it indisputably has a considerable effect on compressive strength ,23. Thisd Jafari  that a nPracticing civil engineers do not have the required computers, software and knowledge in programming. Thus, a reliable and feasible design chart with a wide range of input variables could strongly support the design procedure of geopolymers\u2019 structural elements under a compression load. Aiming this, a design chart was generated by the optimal model developed by the Matlab code of the FLNN, which presents the relationship between the compressive strength (output variable) and the most significant input influencing variable w/GS ratio for a specific level of all other input variables.2O and Na2O are directly connected in the geopolymerisation process [+) required to balance the negative sign of tetracoordinated Al(4) atom [Starting from the minimum values of each input variable and ending with the maximum values, there were nine levels (L1\u2013L9) of each input variable in equal steps. In addition, the 10th level (L10) was established for the mean values of each input variable.  process . By incr process ,44. By a(4) atom  also pla(4) atom .y-axis and horizontally projected onto the individual curves of the input variable levels (L1\u2013L10) depicted in x-axis).The specified target compressive strength of the FA-based geopolymer concrete  is selected on the The designers have the flexibility to choose one of the mix-design combinations (based either on the preferred w/GS ratio or any other requirements/restrictions of the mix-design parameters). The particular values of each mix-design parameter (input variables) are then read out from The design procedure is as follows:The design chart developed here is a powerful tool for structural engineers, supporting them in the selection and calculation of the mix-design parameters of FA-based geopolymer concretes with the composites\u2019 targeted compressive strength.Lloyd and Rangan  generateGeopolymer production can be profitable only when the dosage of the alkali activator is controlled . An incrTwo-part geopolymer production is considered very challenging in many practical applications due to the difficulties of preparing the alkali activator solution and the process of mixing this with the solid precursor, which requires stringent preventative safety requirements . On the In this research, the compressive strength of FA-based geopolymer concrete was predicted by an optimisation process via Matlab code using the FLNN technique. The training and testing datasets were separated into ten input variables  and into the output variable (compressive strength). The Matlab code was run 201 times. Each time, the hidden neuron number at the local and global minima were checked to determine the learning algorithm type and to choose the activation function, respectively.The FLNN network proved its superior behaviour over other machine learning techniques by obtaining better performance criteria for predicting the compressive strength of FA-based geopolymer concrete. The proposed optimal model achieved reductions in the RMSE values of 33.3% and 35.1%, while the R values increased by 7.9% and 18.1% for the training and testing subsets, respectively, compared with the best performing model in the literature .The proposed model is capable of predicting the compressive strength of FA geopolymer concrete at 28 days of age within a small range of deviation from the actual values (\u00b13.87). It has a high correlation with the actual values and a relatively small error on average, with a very high R value (97.5%) for the unseen data.2O percentage, the SiO2\u2013Na2O ratio in the activator, the geopolymer solids\u2013total aggregate ratio, the water\u2013geopolymer solid ratio, the percentage of superplasticiser and the curing time and temperature.The optimal model is applicable to both one- and two-part geopolymer concretes. Moreover, it considers a wide range of mix-design parameters, such as the chemical composition index, the FA content, the coarse aggregate\u2013total aggregate ratio, the NaAs a result, the following main conclusions can be stated:Additionally, based on the proposed optimal model, a powerful design chart, which is applicable to both one- and two-part geopolymer concretes, has been generated that considers a wide range of mix-design combinations for the targeted compressive strength of the FA geopolymer concrete. The design chart provides a strong support for other experts in the design of the mixtures, simplifying and facilitating the design process itself in a cost- and time-efficient way, while leaving the designer high flexibility."}
+{"text": "The literature demonstrates an association between aspects of caregiving and support with caregiver burden and differences by race. Our objective was to examine correlates of caregiver wellbeing, and if the effect is moderated by race.The National Study of Caregiving (NSOC) is a survey of unpaid and familial caregivers affiliated with participants in the National Health and Aging Trends Study, a nationally representative survey of Medicare beneficiaries. A total of 899 participants were examined cross-sectionally with logistic and multinomial logistic regression models to obtain adjusted odds ratios (aOR) and 95% confidence intervals (CI) for NSOC Round 3 (2017), stratified by race, to determine the association between aspects of caregiving and support variables with the two outcomes, three-level caregiving gains, and response to the statement \u201clife has meaning and purpose.\u201dAmong black caregivers with no family or friends to help, there were lower gains compared to very high gains . Black and white caregivers who endorsed lower ratings regarding being appreciated by the care recipient had lower gains for \u201clife has meaning and purpose\u201d . Black caregivers with lower ratings regarding being appreciated had lower gains compared to very high gains . White caregivers endorsing lower ratings to the same question had lower gains compared to very high gains , and those with more help had lower gains .The relationship between various correlates and positive aspects of caregiving is moderated by black and white races. Further study on the impact of aspects of caregiving and support networks for caregivers may shed light on factors contributing to racial differences and areas for intervention. With the increasing aging population of the United States, the population of unpaid and familial caregivers has also increased \u20133. CaregThis study identifies key correlates of wellbeing and potential moderation regarding domains of caregiving and wellbeing. Most literature on unpaid caregiving for older adults focuses on the burden of care, and positive experiences and impacts on caregivers warrant attention as well , 13\u201316. This study aims (1) to determine the correlates of caregiver wellbeing through analysis of the impact of the domains duration of care, aspects of caregiving, and support environment on caregiver wellbeing and (2) to study the wellbeing of caregivers based on both personal-level factors such as race and gender as well as domains of caregiving duration of care, aspects of caregiving, and support environment.Data were obtained from the National Study of Caregiving (NSOC) for the calendar year 2017 for this cross-sectional analysis. The NSOC is a nationally representative survey of family and other unpaid caregivers for older persons in the USA, and it has been conducted three times in conjunction with the National Health and Aging Trends Study (NHATS), which samples Medicare enrollees aged 65 and older  or are unrelated and unpaid  of the caregiver; this will be derived from dichotomous questions regarding if the caregiver has ever had a heart attack, heart disease, high blood pressure, arthritis, osteoporosis, diabetes, lung disease, cancer, difficulty seeing, difficulty hearing, chronic pain, breathing problems, limited strength in limbs, or fatigue. The number of comorbidities will be divided into the following categories: none, one, two, or three, and four or more , 18.Associations between measures of caregiving gains and purpose in life with each potential exposure will be examined using the chi-square test. Linearity was assessed with age and ordinal variables as well as collinearity to identify any variables that were correlated. Due to collinearity with the hours help daily variable, how often help with chores, shopping, getting around the home, and help with personal care were omitted from the aspects of caregiving models. Whether the caregiver had attended a support group was omitted from the support model due to collinearity. Due to small cell sizes, certain categories were collapsed. For caregiving gains, the three lower strata were collapsed into one, including moderate, low, and very low gains. For \u201cLife has meaning and purpose,\u201d the responses \u201cAgree somewhat\u201d and \u201cDisagree\u201d were collapsed into one category. Other relative and non-relative relationships to the care recipient were collapsed into one category, with immediate family or spouse being the other category. Responses \u201cA little\u201d and \u201cNot at all\u201d to questions regarding the caregiving enjoying being with the care recipient, feeling that the care recipient appreciates them, and the frequency the caregiver speaks to the care recipient's medical provider were collapsed into one category for the analysis due to small cell sizes.Nominal logistic regression and logistic regression analyses accounting for sampling weights will be performed to obtain adjusted odds ratios with 95% confidence intervals. We will also perform a Hosmer\u2013Lemeshow test for goodness of fit of the logistic regression models to determine whether the model is adequate or whether there are correlates with a significant lack of fit. Variables included in this investigation will reflect hypotheses regarding the correlates of caregiver wellbeing, and analyses will be stratified by both race and gender, which were both seen to affect the modifiers of caregiving outcomes in prior studies \u20138. All aMultivariate regression results of the support models, which included correlates with family or friends to talk to, family or friends help caregiving, received financial help, and received caregiver training, found significant correlates among black caregivers. The \u201cLife has meaning and purpose\u201d and support model did not converge after accounting for sampling weights, and the caregiver training was completely determined for the unweighted model. Among black caregivers who had no friends or family to talk to, the unweighted analysis indicated there was 3.43 times the odds of endorsing \u201cSomewhat Agree\u201d or \u201cDisagree\u201d rather than \u201cStrongly Agree\u201d to the statement \u201cLife has meaning and purpose\u201d compared to those who did have family or friends to talk to, adjusting for education, number of comorbidities, caregiver age, and caregiver gender . The covariate received caregiver training predicted black caregivers' responses to \u201cLife has meaning and purpose\u201d perfectly. For the \u201cLife has meaning and purpose\u201d and support covariates model, family or friends to help caregiving, having received financial help, and having received caregiver training were not significant for black caregivers in the unweighted model, and no correlates were significant for white caregivers in the weighted model .In the weighted analyses, among black caregivers with no family or friends to help with caregiving, there was 2.82 times the odds of having moderate to very low gains rather than very high gains compared to those with family or friends to help caregiving, adjusting for education, number of comorbidities, caregiver age, and caregiver gender . Among black caregivers who had not received caregiving training, there was 4.52 times the odds of having high rather than very high gains compared to those with family or friends to help with caregiving, adjusting for education, number of comorbidities, caregiver age, and caregiver gender . For the caregiving gains and support model, having family or friends to talk to and having received financial help were not significant correlates of caregiving gains for black caregivers, and no correlates were significant for white caregivers .The aspect of caregiving models includes variables relating to whether the caregiver has helped with medical tasks, talks to medical providers, their relationship with the care recipient (immediate relative or spouse vs. other), if they believe the care recipient appreciates them, and the hours they help the care recipient daily. For the model aspects of caregiving and \u201cLife has meaning and purpose,\u201d among black caregivers, those who endorsed responses \u201cSome,\u201d \u201cLittle,\u201d or \u201cNot at all\u201d to the statement that the care recipient appreciates them had 2.45 times the odds of endorsing \u201cAgree somewhat\u201d or \u201cDisagree\u201d rather than \u201cAgree strongly\u201d to the statement \u201cLife has meaning and purpose\u201d compared to those who said the care recipient appreciates them \u201cA lot,\u201d adjusting for education, number of comorbidities, caregiver age, and caregiver gender .Among white caregivers, those who endorsed responses \u201cSome,\u201d \u201cLittle,\u201d or \u201cNot at all\u201d to the statement that the care recipient appreciates them had 1.65 times the odds of endorsing \u201cAgree somewhat\u201d or \u201cDisagree\u201d rather than \u201cAgree strongly\u201d to the statement \u201cLife has meaning and purpose\u201d compared to those who said the care recipient appreciated them \u201cA lot,\u201d adjusting for the same demographic covariates . Across the aspects of caregiving and \u201cLife has meaning and purpose\u201d models, the caregiver helping with medical tasks, talking to medical providers, relationship with the care recipient, and hours spent helping daily were not significant correlates of response to the statement \u201cLife has meaning and purpose.\u201d Both models were weighted .For the models relating to aspects of caregiving and caregiving gains, among black caregivers who only \u201cSomewhat,\u201d \u201cA little,\u201d or \u201cNot at all\u201d spoke to medical providers, there was 2.15 times the odds of very low to moderate gains rather than very high gains compared to those who talked to medical providers \u201cA lot\u201d . Among white caregivers who only \u201cSomewhat,\u201d \u201cA little,\u201d or \u201cNot at all\u201d spoke to medical providers, there was 1.65 times the odds of very low to moderate gains rather than very high gains compared to those who talked to medical providers \u201cA lot\u201d .Among black caregivers who endorsed \u201cSome,\u201d \u201cLittle,\u201d or \u201cNot at all\u201d to the statement that the care recipient appreciates them, there was 5.04 times the odds of very low to moderate gains rather than very high gains compared to those who endorsed the care recipient appreciates them \u201cA lot\u201d . Among white caregivers who endorsed \u201cSome,\u201d \u201cLittle,\u201d or \u201cNot at all\u201d to the statement that the care recipient appreciates them, there was 3.27 times the odds of very low to moderate gains compared to very high gains compared to those who endorsed the care recipient appreciates them \u201cA lot\u201d .Among black caregivers helping the care recipient at a one-category increase of hours  per day, there was 0.72 times the odds of very low to moderate gains rather than very high gains compared to those who helped the care recipient for a lower time category . Among white caregivers helping the care recipient at a one-category increase of hours per day, there was 0.81 times the odds of very low to moderate gains rather than very high gains compared to those who helped the care recipient for a lower time category . Helping with medical tasks and relationship with care recipients were not significant correlates of caregiving gains for black or white caregivers. Both models were weighted .Many of the observed associations aligned with our expectations. In the caregiving gains and aspects of caregiving models, for both races, those who endorsed lower frequencies of helping with medical tasks were more likely to have moderate, low, and very low compared to very high caregiving gains. This relationship warrants further study.The models showed differences by black and white races, which was expected. Previous research using NSOC data found differences in caregiver outcomes by race . For theHigher caregiving intensity was represented by the hours help daily variable, which was found to be significantly associated with lower categories of caregiving gains for white caregivers. The relationship between caregiving intensity and caregiver burden has been previously established , 8. SociThis study has a few limitations. The constructs of caregiver wellbeing have not been widely studied, and other positive outcomes may be more appropriate. Causal relationships cannot be determined from this cross-sectional analysis. For both predictor and outcome variables, categories had to be collapsed due to small strata, but the resulting categories were still meaningful. There was a high proportion of missing data, which may cause bias and a lack of precision, and there may be a lack of generalizability because only black and white races were included in the analysis, while the non-Hispanic Other and Hispanic categories were excluded due to small cell sizes.This is a preliminary analysis, and a future study will utilize multiple time points of the NSOC for longitudinal analysis to see whether the relationship persists across three time points when the third wave of longitudinal data is available. Studies on the impact of aspects of caregiving and support networks for caregivers may shed light on factors contributing to racial differences. In addition, further study may identify any areas for intervention and causal explanation for some of the associations identified in this study.www.nhats.org with funding from the National Institute on Aging [Grant numbers R01AG054004 (NSOC III) and R01AG062477 (NSOC IV)].Publicly available datasets were analyzed in this study. This data can be found here: National Study of Caregiving. Produced and distributed by The studies involving human participants were reviewed and approved by Columbia University Research Compliance and Administration System IRB. The patients/participants provided their written informed consent to participate in this study.TM and LP were involved in conceptualizing data analysis, manuscript preparation, interpretation of data, and critical revisions of the manuscript. LP performed data analysis. TM contributed to and supervised data analysis. All authors contributed to the article and approved the submitted version."}
+{"text": "To analyze the prognostic impact of staged nursing interventions on the treatment of patients with chronic obstructive pulmonary diseases (COPD) combined with type II respiratory failure.  120 patients with COPD combined with type II respiratory failure admitted to our hospital between January 2021 and January 2022 were divided into a control group and a study group, with 60 patients in each group. The control group received conventional strategy interventions, and the study group received staged nursing interventions. Pulmonary function, blood gases, health impairment, knowledge, mood, hope level, and quality of survival were evaluated before and after patient care, and satisfaction and the impact on patient prognosis were assessed. P < 0.05). Health impairment and mood scores were lower in the study group compared to the control group aftercare, and the difference was statistically significant (P < 0.05). Knowledge awareness, hope, and quality of survival scores were higher in the study group compared to the control group aftercare, with statistically significant differences (P < 0.05). The rate of excellent prognosis and satisfaction was higher in the study group compared with the control group, and the difference was statistically significant (P < 0.05).  The improvement of pulmonary function and blood gas in the study group was better than that in the control group aftercare, and the difference was statistically significant ( The implementation of staged nursing interventions during the treatment of patients with COPD combined with type II respiratory failure can significantly improve patient prognosis and has a high application value. Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease with high mortality and recurrence rates, which is characterized by decreased lung function and incomplete reversible airflow obstruction. Studies have concluded  that theP < 0.05), and the hospital anxiety and depression scale (HADS) was lower than that of the control group after psychological intervention. The general self-efficacy scale scores of experimental group were obviously improved after receiving the intervention, and the scores in the experimental group were much higher than the control group after receiving the intervention, namely P < 0.05 [The main contents of staged nursing intervention include admission education and management, operation period nursing, early postoperative rehabilitation nursing, and continuous rehabilitation nursing guidance. According to different surgical approaches and repair methods, targeted care, especially postoperative rehabilitation care and functional exercise, can greatly promote the recovery of patients' functions and shorten the patient's hospital stay. In contrast, routine care mainly reffered to admission guidance, simple health education, regular physical examination, prevention of complications, and guidance of patients on the use of COPD medication, which was demonstrated to be less efficient than staged nursing intervention . As showP < 0.05 . InspireP > 0.05). The inclusion criteria are as follows: meeting the clinical diagnostic criteria for COPD and type II respiratory failure, complete information, voluntary participation, and all treated with the same treatment protocol. The exclusion criteria are as follows: having other pulmonary pathologies such as pulmonary embolism; lung cancer; aberrant cardiogenic pulmonary edema; liver, kidney, and another important organ insufficiency; blurred consciousness; and no communication ability.120 patients with COPD combined with type II respiratory failure who met the inclusion and exclusion criteria and were admitted to our hospital between January 2021 and January 2022 were selected and divided into a control group and a study group of 60 cases each according to the random number table method. The general information of the two groups is shown in 2O to 8\u2009cm H2O as the initial inspiratory pressure, and 2\u2009cm H2O to 3\u2009cm H2O as the initial inspiratory pressure, and the patients were closely monitored for changes in their condition during treatment, and the parameters were adjusted in time. The maximum inspiratory air pressure was 5\u2009cm H2O~6\u2009cm H2O, and when the patient's condition was in a stable state, the inspiratory air pressure and suction pressure were adjusted down, and the inspiratory oxygen level was 40% during this process, and the flow rate was 4\u2009L/min~6\u2009L/min, and ventilation was performed for 4\u2009h\u2009~6\u2009h each time.Both groups were treated with conventional interventions, including nutritional support and water-electrolyte balance, and were treated with a dual-level noninvasive ventilator, with 12 breaths/min to 20 breaths/min, 6\u2009cm HIn the control group, the general routine strategy interventions such as disease monitoring and complication prevention were performed, disease monitoring was done according to medical prescriptions, and patients were closely monitored for the presence of sputum in the respiratory tract and cleared in time to reduce the occurrence of adverse events.The study group applied the stage nursing strategy intervention and formed a stage nursing team, which consisted of a department physician, a department nurse manager, and 5 nurses with more than 5 years of experience in respiratory medicine and excellent service attitude. The nurse manager, as the team leader, discussed the nursing interventions with the team members and formulated the stage nursing measures, which were specifically implemented by the nurses. The specific measures are shown in 2, SaO2, and PaCO2 levels.Pulmonary function and arterial blood gas stability were measured using a pulmonary function and fully automated blood gas analyzer (GEM Premier 4000) before the patients were admitted to the group (before care) and before they were discharged from the hospital (aftercare). Pulmonary function was evaluated based on FEV1, FVC, and FEV1/FVC, and arterial blood gas stability was evaluated based on PaOThe health impairment of patients before and after patient care was evaluated based on the St. George's Respiratory Questionnaire (SGRQ) , which iBefore and after the patients' care, the patients' knowledge was evaluated based on the scores of our self-made pulmonary rehabilitation knowledge questionnaire, which consisted of 5 items: diet, medication, disease, exercise, and oxygen therapy. The entire scale consisted of 24 questions, where the lowest and highest scores of the scale were 0 and 24, respectively, with lower scores indicating more desirable patient perceptions of the disease.The patient's mood and hope level were evaluated before and after patient care using the Self-Rating Scale for Depression (SDS), Self-Assessment Scale for Anxiety (SAS), and Herth Hope Scale developed by Zung, respectively; SDS and SAS both contain 20 questions and are rated on a 4-point scale, with lower scores indicating weaker depression and anxiety. The Herth Hope Scale consists of 12 questions, with scores of 12-23 being low, 24-35 being moderate, and 36-48 being high, with higher scores indicating a higher level of hope.The quality of survival was evaluated on the Quality of Survival Scale before and after patient care, with a total of 26 questions on a 5-point scale, with each question scoring 0-5, with higher scores indicating better quality of survival.The occurrence of adverse events such as multiple organ failure, cardiovascular disease, and death that occurred before the patient was admitted to the hospital for treatment until discharge was used as a prognostic evaluation criterion, and the occurrence was considered a poor prognosis, and the opposite was considered an excellent prognosis.Patients in both groups were assessed for nursing satisfaction by our questionnaire, which included 4 items, namely, very satisfied, satisfied, dissatisfied, and very dissatisfied, and (very satisfied + satisfied)/the total number of cases \u00d7100%\u2009=\u2009total satisfaction.t-test was used for comparison between groups, and the paired t-test was used for comparison before and after patient care; median and quartile spacing M  were applied for those not conforming to the normal distribution, and Wilcoxon's comparison was used between groups. Count data were expressed as n (%), \u03c72 test. P < 0.05 was considered statistically significant.SPSS22.0 statistical software was used for analysis and processing. The Kolmogorov-Smirnov test was applied to test whether the data conformed to a normal distribution, and the measurement data conforming to normal distribution were described by the mean\u2009\u00b1\u2009standard deviation (P > 0.05); after care, the pulmonary function of the two groups was higher than before care, and the increase was more significant in the study group compared with the control group (P < 0.05). See The comparison of pulmonary function between the two groups before care was not significant (P > 0.05); after care, the blood gas indexes of the two groups were higher than before care, and the increase was more significant in the study group compared with the control group (P < 0.05). See The comparison of blood gases between the two groups before care was not significant (P > 0.05); after care, the SGRQ scores of health impairment in both groups were lower than before care, and the reduction was more significant in the study group compared with the control group (P < 0.05). See The comparison of SGRQ scores of health impairment between the two groups before care was not significant (P > 0.05); after care, the knowledge scores of the two groups were higher than before care, and the increase was more significant in the study group compared with the control group (P < 0.05). See The comparison of the knowledge scores of the two groups before care was not significant (P > 0.05); after care, the hope scores of the two groups were higher than before care, and the increase was more significant in the study group compared with the control group (P < 0.05); after care, the mood scores of the two groups were lower than before care, and the decrease was more significant in the study group compared with the control group (P < 0.05). See The comparison of mood and hope scores between the two groups before care was not significant (P > 0.05); after care, the quality of survival in both groups was higher than before care, and the increase was more significant in the study group compared with the control group (P < 0.05). The comparison of the quality of survival scores between the two groups before care was not significant (P < 0.05). See The study group had a higher rate of excellent prognosis than the control group (P < 0.05). See Nursing satisfaction was higher in the study group than in the control group (Currently, the treatment of patients with COPD combined with type II respiratory failure is based on noninvasive ventilation, which is the most commonly used treatment for this disease, and this method has the advantages of easy operation, ready access to the machine, and low trauma to the patient's organism, which can continuously improve the patient's hypoxia and respiratory function after application , 4, 5. HMost of the conventional nursing tools currently used in the clinic are based on the patient's condition in order to maintain treatment, ignoring to some extent the differences in patients' stage needs \u20139. StageIt has been reported  that somIn conclusion, staged nursing interventions can significantly improve pulmonary function and blood gases and improve treatment prognosis in patients with COPD combined with type II respiratory failure with ideal results."}
+{"text": "The potential role of cocoa supplementation in an exercise context remains unclear. We describe the effects of flavanol-rich cocoa supplementation during training on exercise performance and mitochondrial biogenesis. Forty-two male endurance athletes at the beginning of the training season received either 5 g of cocoa (425 mg of flavanols) or maltodextrin (control) daily for 10 weeks. Two different doses of cocoa  were tested in a mouse exercise training study. In the athletes, while both groups had improved exercise performance, the maximal aerobic speed increased only in the control group. A mitochondrial DNA analysis revealed that the control group responded to training by increasing the mitochondrial load whereas the cocoa group showed no increase. Oxidative stress was lower in the cocoa group than in the control group, together with lower interleukin-6 levels. In the muscle of mice receiving cocoa, we corroborated an inhibition of mitochondrial biogenesis, which might be mediated by the decrease in the expression of nuclear factor erythroid-2-related factor 2. Our study shows that supplementation with flavanol-rich cocoa during the training period inhibits mitochondrial biogenesis adaptation through the inhibition of reactive oxygen species generation without impacting exercise performance. Physical activity generates various biochemical signals that trigger exercise adaptations in almost every organ of the body . One of 2max) and power output during an incremental cycle test in untrained participants [2max in active males [Studies in humans have indicated that sub-chronic and chronic cocoa intake reduces the oxygen cost of cycling, thereby improving exercise performance , and incicipants . Other sve males , or in yve males . Moreoveve males .To date, there is no clear evidence for the usefulness of cocoa on the performance of endurance athletes. Furthermore, few studies have characterized the compounds contained in cocoa supplements, so it is difficult to establish which components are responsible for the observed outcomes. Our study aimed to determine whether the intake of flavanol-rich cocoa increases mitochondrial biogenesis and sports performance in athletes and in a murine exercise model.Maltodextrin was purchased from Prozis . High-flavanol cocoa (83 mg/g) was purchased from Chococru . The polyphenolic characterization of high-flavanol cocoa has been described previously ; the maiClinicalTrials.gov (accessed on 22 June 2022) (ID: NCT04301583). Participants were recruited from recreational sports clubs across Madrid through visits, mail and telephone calls. A total of 56 athletes were screened for the study, of whom 54 met the following the inclusion criteria: (1) male endurance cross-country athletes, (2) 18\u201350 years of age, (3) high physical condition (VO2 \u2265 55 mL/kg min\u22121), and (4) body mass index 18\u201325 kg/m2. The exclusion criteria were as follows: (1) consumption of any kind of nutritional or ergogenic supplement, (2) smoking, (3) chronic medication, and (4) gastrointestinal surgery, or any diagnosed disease. Written informed consent was obtained from all participants.We designed a randomized, parallel-group, placebo-controlled trial. The research ethics committee on drugs of the Comunidad de Madrid, Spain, approved the study (reference: 07/694487.9/17). All procedures were in accordance with the 1964 Helsinki Declaration and its later amendments, and the study was registered on Subjects were randomized into two equal-size treatment groups using the RAND function of Excel . The study lasted 10 weeks, which was enough time for the exercise to produce physiological adaptations . AthleteParticipants were individually scheduled to attend the test laboratory between 8:00 am and 12:00 am. They refrained from any physical activity 24 h before the day of the physical test. The protocol followed has been described in a previous study , which oTM Series, MGC Diagnostic Corp., St. Paul, MN, USA) that was calibrated according to the manufacturer\u2019s instructions. The volume calibration was performed at different flow rates with a 3 L calibration syringe that allowed an error of <3%. The calibration of the gas analyzer was automatically performed by the system, using the reference values of ambient gases (16% O2 and 4% CO2). During the progressive test, HR was measured with a Polar Sport-tester , and oxygen consumption was constantly monitored and associated with the speed at which the first ventilatory threshold, second ventilatory threshold, and maximal aerobic speed  were identified, as previously described [2maxABS), MAS, VT1, and VT2.During the tests, gas exchange data were continuously collected using an automatic breath-by-breath measurement system  with thiobarbituric acid (TBA). The TBA reagent was prepared just before the assay and consisted of an equal part mixture of 0.375% TBA in water and 1 M phosphoric acid. A total of 50 \u00b5L of plasma were mixed with 450 \u00b5L of TBA reagent, and the mixture was heated at 85 \u00b0C for 60 min in a dry bath, cooled on ice for 5 min, and then centrifuged at 13,000 rpm for 2 min. Finally, 200 \u00b5L of the mixture was transferred to a black 96-well plate and the fluorescence intensity  was measured on a Jasco FP-8300 spectrofluorometer . Data are expressed in terms of MDA concentration, using as a standard the MDA obtained from the hydrolysis of tetraethoxypropane .Protein carbonylation levels were measured in plasma as described . BrieflySuperoxide dismutase (SOD) activity was measured in plasma using a published protocol  involvinPlasma interleukin-6 (IL-6) levels were measured with a commercial assay .A total of 60 male C57BL/6J mice , 8\u201310 weeks old, were randomly divided into the following 6 groups (n = 10 per group): (1) untrained control group receiving standard rodent chow (CONT), (2) untrained group supplemented with cocoa at 8.2 mg/kg body weight (b.w.) (CO), (3) untrained group receiving high-dose cocoa at 24.6 mg/kg b.w (HCO), (4) control exercise-trained group (CONTEx), (5) exercise-trained group supplemented with cocoa at 8.2 mg/kg b.w. (COEx), and (6) exercise-trained group supplemented with high-dose cocoa at 24.6 mg/kg b.w (HCOEx). Cocoa doses were equivalent to the daily intake of 5 g or 15 g of cocoa for a 70 kg person according to an allometric scale . The cocThe exercise protocol was established according to the previous protocol , in whic\u00ae EDTA tubes and stored at \u221280 \u00b0C until use. For DNA extraction, 200 \u00b5L of whole blood were treated with proteinase K , and DNA was then extracted using phenol/chloroform/isoamyl alcohol (24:24:1) and precipitated with 3 M sodium acetate (pH 5.2) and isopropanol. Finally, the pellets were dissolved in sterile, nuclease-free water. Quadriceps tissue samples were extracted with TrizolTM Reagent . DNA concentration and purity were evaluated by using the absorbance ratios 260/280 nm and 260/230 nm on a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific).Drawn blood samples were collected in VacutainerPPARGC1A Gly482Ser (rs8192678) polymorphism using a pre-designed TaqMan\u00ae SNP genotyping test (ID: C_1643192_20). Amplification was carried out using the StepOne\u2122 real-time PCR system  with a denaturation stage at 95 \u00b0C for 10 min, 50 denaturation cycles at 92 \u00b0C for 15 s, anneal/extension at 60 \u00b0C for 1 min and a final 30 s extension stage at 60 \u00b0C.We performed allelic discrimination analysis for the COX1 and the \u03b2-globin HBB gene  content in human samples was assessed using quantitative real-time PCR by measuring the threshold cycle ratio of the mitochondria-encoded gene cytochrome c oxidase I Nd1 and CytB mitochondrial genes and the constitutive ribosomal gene 18S  were determined by real-time RT-PCR. Total RNA was extracted from quadriceps muscle using the E.Z.N.A.\u00ae Total RNA Kit  and its concentration was quantified in a NanoDrop spectrophotometer (ThermoFisher Scientific). RNA was reverse transcribed to cDNA using the PrimeScriptTM RT Master Mix (TAKARA Bio. Inc.). Quantitative real-time PCR was performed using a SYBER\u00ae Green PCR Master Mix  on the CFX96 Touch Real-Time PCR Detection System . Thermal cycling parameters were as follows: 95 \u00b0C for 30 s, then 40 cycles of 95 \u00b0C for 5 s and the primer-specific annealing temperature .Statistical analysis was conducted using SPSS 27.0 software . The distribution was checked for normality (Kolmogorov\u2013Smirnov test) before analysis. Student\u2019s t-test was applied when the data distribution was assumed to be normal. When more than two groups were compared differences were assessed by ANOVA with the Tukey-Kramer correction. The Wilcoxon range test, the Kruskal\u2013Wallis test, or the Mann\u2013Whitney test was used when the distribution could not be assumed to be normal. The \u03c7A total of 56 participants were enrolled in the present study, of which 42 completed the study: 20 in the CT group and 22 in the CO group. Seven subjects in the CT group and 5 in the CO group did not complete the study because of muscle injury or because they did not follow the training program .No significant differences were found in the age, body weight, height, or body mass index between groups .2 = 2.54; p = 0.281)  .The analysis of the energy, macronutrient and fiber data obtained from the food frequency questionnaire and the three 24 h recalls revealed no significant intergroup differences in T1 and T2 , and bot2max, VT1 and VT2 after the 10 weeks of training (p < 0.001), whereas the CO group did not (p = 0.572). No significant differences were found between the CT and CO groups at either T1 or T3 in the intergroup analysis. These results indicate that cocoa treatment had no overall effect on performance parameters, but it seemed to affect the aerobic capacity of the CO group.Both groups showed improvements in VOtraining ; howeverp = 0.001). By contrast, no increase in the mtDNA copy number was found in the CO group   A, sugges= 0.981) B.Cytb , which serve as signals to trigger exercise adaptations  and promp = 0.048, CO group p = 0.042). The same analysis after 10 weeks of supplementation and training revealed that the exercise bout did not significantly increase TBARS levels (T3 vs. T4) , likely due to the period of adaptation to the exercise provided by the training over the 10 weeks. Moreover, when we assessed the increase in the basal levels of TBARS initially and after the 10-week exercise and supplementation period, we found that the increase was significantly less pronounced in the CO group than in the CT group  than in the CT group (p = 0.478) (p = 0.055)  B, sugges= 0.055) B.p = 0.042) but not in the CO group (p = 0.501)  .Nfr2, a redox-sensitive transcriptional regulator that is required for mitochondrial biogenesis and for the antioxidant transcriptional response to acute exercise and exercise training [Nfr2 in the quadriceps femoral muscle of mice, finding that exercise induced an increase in its expression in the CONTEx group in comparison with the CONT group  correlation between the increase in the mtDNA copy number and the chronic increase in plasma MDA levels. The linear regression analysis confirmed that the increase in the mtDNA copy number was dependent on the increase in plasma MDA levels (R = 0.353 (p = 0.027)). The regression indicated that the predictors of performance (t1km) are relative to the VO2 (\u03b2 = \u22120.401: p = 0.07) and the IL-6 levels at T3 , but not to the mtDNA copy number.We performed correlation and linear regression analyses to better understand the relationship between mitochondrial biogenesis and the other variables. The correlation results showed a statistically positive rho = 0.477 and a significant  such as fruits and vegetables (data not shown), so supplementation with cocoa may have provided an extra amount of antioxidants that was not necessary. Cell signaling depends on the levels of ROS in the organism, so a balance between the levels of oxidative and antioxidant species must be maintained. However, finding this balance is very complex, as the biological dose\u2013response curve in oxidative stress is unpredictable and depends on many factors that must be considered, including age, genetics, health status, and level of physical training, among others , so it bWe also observed that the levels of IL-6 at rest increased after 10 weeks of training in the CT group but not in the cocoa-supplemented group supplementation. Exercise causes an immediate release of IL-6 from the muscle that increases lipolysis and energy uptake in the muscle , and augThe current study had some limitations that warrant discussion. A chronic inhibition of mitochondrial biogenesis was observed in athletes receiving cocoa during the training period. Our study design did not allow us to establish whether supplementation for shorter periods or at different times  would influence these findings. The cocoa used in the present study had a high content of flavonoids that are not found in standard cocoa supplements, so the values cannot be extrapolated to other commercial cocoa powders. The study of the athletes used a single dose of cocoa, and it might have been interesting to test multiple doses to investigate dose\u2013response effects. In addition, we did not study the bioavailability of cocoa or the plasma metabolite profile, which might be an important factor given the different bioavailability of the bioactive compounds present in cocoa. Indeed, an analysis of the plasma metabolite profile would have allowed us to test for potential links between cocoa metabolites and the observed effects. The measurement of lipid oxidation was carried out by using the TBARS method since previous studies showed the inhibition of this parameter by cocoa compounds. However, the TBARS assay is too unspecific and can involve several artifacts; therefore, other markers of lipid oxidation should be used to confirm our lipid-peroxidation-related results. Finally, the inhibition of mitochondrial biogenesis was established in blood cells of athletes and not in the muscle , and the mechanism of inhibition should be investigated in future studies.Chronic cocoa supplementation during the training period of endurance athletes inhibits mitochondrial biogenesis by decreasing oxidative stress levels, without impacting aerobic capacity or exercise performance. At the same time, cocoa prevents the long-term increase in IL-6 that results from continued exercise, which might be protective against muscle damage. The period in which the supplementation occurred, during which time great adaptations to exercise were taking place, the framework of the athletes\u2019 diets, which were rich in fruits and vegetables, and the type of cocoa used in the study, could be determining factors in the observed effects of cocoa on athletes. For this reason, future studies are needed that consider all of these factors and others such as the type of exercise. Nutritional supplement recommendations should follow personalized strategies and be carefully tailored to well-characterized population groups."}
+{"text": "Robotic systems have emerged in dental implant surgery due to their accuracy. Autonomous robotic surgery may offer unprecedented advantages over conventional alternatives. This clinical protocol was used to show the feasibility of autonomous robotic surgery for immediately loaded implant-supported full-arch prostheses in the maxilla. This case report demonstrated the surgical protocol and outcomes in detail, highlighting the pros and cons of the autonomous robotic system. Within the limitations of this study, autonomous robotic surgery could be a feasible alternative to computer-assisted guided implant surgery. Successful outcomes of dental implant restorations are mainly determined by precise prosthetically driven implant placement, especially in full-arch implant rehabilitation . ImmediaA precise implant placement can be achieved either by static computer-assisted implant surgery (s-CAIS) or dynamic computer-assisted implant surgery (d-CAIS). The s-CAIS technology uses a surgical implant guide supported by teeth, bone, or mucosa for the drilling process and insertion of the implant ,9. AdditRobotic technology has been reported to be a significant innovation in dentistry ,16. In tThis clinical case report aimed to demonstrate the feasibility of immediately loaded full-arch implant restorations through a semi-active and task-autonomy robotic system and to determine whether robot-guided precise implant placement results in an appropriate primary implant stability through bicortical fixation.A 58-year-old female was referred to the Center of Oral Implantology, Stomatological Hospital, Southern Medical University  with a poor masticatory function due to a removable partial denture. The patient had no systemic diseases and was a non-smoker. No apparent abnormalities were detected in extraoral examinations. As shown in After the patient signed an informed consent form, a commercial image-guided robotic oral surgery system  was used to perform immediately loaded full-arch fixed implant rehabilitation. The autonomous robotic surgery system mainly included a robot arm, an optical tracker, a positioning marker, and an operating software system, as shown in For the preoperative planning, first, the patient underwent a cone-beam computed tomography  examination with a voxel size of 0.2 mm. The image file was exported to the standard digital imaging and communications in medicine (DICOM) format. Next, the file was input into a virtual implant planning software . A preoperative full-arch rehabilitation was virtually created. In addition, considering the availability of supporting bone, the implant position was confirmed, and the sites of teeth #16, #14, #24, and #26 were set for bicortical stabilization . Finally\u00ae . Then, the patient underwent a CBCT examination , as shown in mination e, and thBefore the surgery, the first DICOM (implant planning) and second DICOM (patient with the positioning marker) were transferred to the robotic surgery system and merged. This protocol could help design prosthetically driven implant placement during the preoperative plan, minimizing the intraoperative time. The 3D areas of interest, such as the alveolar ridge and maxillary sinus, were segmented. The osteotomy plan and target implant position were prepared and visualized. Furthermore, the optical tracker was placed and fixed above the patient\u2019s head. The registration between the positioning marker and the robotic arm was performed. Additionally, the calibration was carried out automatically. The surgeon moved the robotic arm near the oral cavity during the operative phase. Next, the three-dimensional position of the robotic arm was automatically adjusted according to the planned implants. With a flapless procedure, the robotic arm automatically performed the implant osteotomy according to the surgical and osteotomy planning a,b. BaseA standardized 3D voxel-based registration for superimposed images of the virtual implant plan and the postoperative CBCT image was used to assess the implant accuracy. Both DICOM files were imported to the robotic surgery verification system, and this registration process was further performed. In addition, the errors between the planned and placed implant positions were measured, as previously described in detail ,21. BaseSix implants were placed in the maxilla. The bone quality ranged from type III to type IV, according to the Lekholm and Zarb classification . The flaA precise implant placement is a prerequisite for dental implant restorations to restore esthetics and function and to maintain a healthy performance. Computer-assisted implant surgery has been developed and used to meet clinical requirements ,23. HoweTo the best of our knowledge, this clinical case is the first to report the feasibility and accuracy of the immediately loaded implant-supported maxillary full-arch prosthesis through an autonomous robotic surgery system. The clinical results showed relatively lower deviations using the robotic surgery system, achieving bicortical stabilization in the posterior area. Then, the patient received a successful full-arch immediately loaded prosthesis. Accordingly, the overall accuracy of the robotic surgery system met the clinical requirements.The robotic surgery system\u2019s high accuracy for implant placement can be mainly attributed to the following three factors. First, it is critical to automatically register the surgical and image space in this robotic surgery system before the surgery. Compared to conventional registration, automatic registration and calibration with the optical-system method have higher accuracy, meeting the clinical requirements . Second,Admittedly, the robotic surgery system still has deviations, which can be caused by the following factors. First, the CBCT scan errors were determined by voxel size, exposure time, field of view, and metal artifacts, which influenced the initial data acquisition . SimilarThe autonomous robotic surgery system is regarded as a novel technology, providing the following main advantages for implant surgery: (1) precise implant osteotomy; (2) where applicable, minimal invasiveness (flapless implant surgery); (3) the efficient avoidance of anatomical risks, such as the alveolar nerve; and (4) real-time feedback on the drilling force to predict the bone quality. However, the main disadvantages of this robotic surgery system should still be considered: (1) an unsatisfactory cost-effectiveness and time-consuming nature; (2) an additional learning curve for the system; (3) an inability to perform implant osteotomy in patients with a limited mouth opening; (4) the optical tracker cannot be moved during surgery; otherwise, the registration should be repeated; and (5) extra radiation: the preoperative CT scans were performed twice in this treatment protocol.Undoubtedly, a limitation of this case report is that no comparative cohort was enrolled. Additionally, the current treatment protocol can be optimized. For instance, the first CT scan for the template design may be replaced by digital dental impressions to minimize radiation exposure. Meanwhile, implant planning may be performed before surgery. To date, the use of commercially available robotics has not been widely validated in clinical trials, especially in implant dentistry ,16. RegaThis case demonstrated the feasibility of autonomous robotic surgery for immediately loaded implants in the maxilla. Robot-assisted implant surgical systems may open up new horizons for dental implant surgery, enabling accurate and minimally invasive patient-specific procedures. However, further clinical trials are required to provide hard clinical evidence."}
+{"text": "On August 4, 2022, the U.S. Department of Health and Human Services declared the U.S. monkeypox outbreak, which began on May 17, to be a public health emergency  with reported classification by health departments as being travel-associated or locally acquired, 178 (74%) were classified as locally acquired. The percentage of locally acquired cases increased from 51% (33 of 65) during May 17\u2013July 2 to 82% (145 of 175) during July 3\u201322.Among 358 (30%) men (cisgender and transgender) with information on recent sexual behaviors and gender of sex partners available, 337 (94%) reported sex or close intimate contact with a man during the 3 weeks before symptom onset; 16 (4%) reported no such contact. Among 291 men who reported information about their male sexual partners during the 3 weeks preceding symptom onset, 80 (27%) reported one partner, 113 (40%) reported two to four partners, 42 (14%) reported five to nine partners, and 56 (19%) reported 10 or more partners. Among 86 men with information reported, 33 (38%) reported group sex, defined as sex with more than two persons, at a festival, group sex event, or sex party.The most frequently reported signs and symptoms included rash (100%), fever (63%), chills (59%), and lymphadenopathy (59%) . ReporteRash was most frequently reported on the genitals (46%), arms (40%), face (38%), and legs (37%); among 718 persons with monkeypox who reported body regions with rash, 238 (33%) reported rash in one region, 126 (18%) in two regions, 98 (14%) in three regions, and 256 (36%) in four or more regions. Among 104 persons with information on the number of lesions, 88% of cases involved fewer than 50 lesions.Among 334 persons with data available on HIV status, 136 (41%) had HIV infection. Among 954 persons with hospitalization data available, 77 (8%) patients were hospitalized because of their illness. No deaths were reported. Among 339 persons with vaccination status available, 48 (14%) reported previous receipt of smallpox vaccine, including 11 (23%) who received 1 of 2 JYNNEOS doses during the current outbreak, 11 (23%) who received pre-exposure prophylaxis at an unknown time before the current outbreak, and 26 (54%) who did not provide information about when vaccine was administered. Among the recently vaccinated persons with monkeypox, at least one experienced symptoms >3 weeks after their first JYNNEOS dose.Current findings indicate that community transmission of monkeypox is widespread and is disproportionately affecting gay, bisexual, and other men who have sex with men; this is consistent with data reported from other countries  had been allocated to jurisdictions, and approximately 14,700 courses of oral tecovirimat (TPOXX) had been distributed to jurisdictions and providers. Because of long-standing investments in medical countermeasures for potential smallpox events, licensed vaccines and therapeutics for monkeypox are held in the U.S. Department of Health and Human Services Strategic National Stockpile. A national vaccine strategy was developed to equitably expand vaccination in areas experiencing high numbers of monkeypox cases and contacts. Two vaccines are available in the United States.The findings in this report are subject to at least three limitations. First, this analysis includes only 41% of U.S. monkeypox cases reported through July 22 and might not be representative of all cases. Jurisdictions with high numbers of cases without submitted case report forms were more racially and ethnically diverse according to U.S. Census Bureau data; therefore, persons from racial and ethnic minority groups might be more disproportionately affected than indicated by these data. Second, even on submitted case report forms, data for variables such as timing of vaccination, sexual behaviors, HIV status, reason for hospitalization, and whether cases were travel-associated were frequently missing; data might also not reflect symptoms or outcomes occurring after the interview. Finally, persons with monkeypox who have mild symptoms might be less likely to seek care or initiate testing and could be underrepresented in this analysis.CDC is continually evaluating new evidence and tailoring response strategies as information on changing case demographics, clinical characteristics, transmission, and vaccine effectiveness become available. Public health efforts should prioritize gay, bisexual, and other men who have sex with men, who are currently disproportionately affected for prevention and testing, address equity, and minimize stigma, while maintaining vigilance for transmission in other populations. Clinicians should test persons with rash consistent with monkeypox, regardless of whether the rash is disseminated or was preceded by prodrome.A global monkeypox outbreak began in 2022.Among U.S. monkeypox cases with available data, 99% occurred in men, 94% of whom reported recent male-to-male sexual or close intimate contact; racial and ethnic minority groups appear to be disproportionately affected. Clinical presentations differed from typical monkeypox, with fewer persons experiencing prodrome and more experiencing genital rashes.Public health efforts should prioritize gay, bisexual, and other men who have sex with men, who are currently disproportionately affected, for prevention and testing, address equity, and minimize stigma, while maintaining vigilance for transmission in other populations. Clinicians should test persons with rash consistent with monkeypox, regardless of whether the rash is disseminated or was preceded by prodrome"}
+{"text": "Based on the limited resource model of self-control, we construct a chain mediation model to examine the relationship between parental phubbing and adolescents\u2019 academic burnout, and whether social anxiety and self-control play a mediating role in it. We used 4 questionnaires to investigate parental phubbing, social anxiety, self-control, and adolescents\u2019 academic burnout among 828 high school students in Wuhu and Huangshan City, Anhui Province, China. The findings indicated that: (1) parental phubbing, social anxiety, and self-control all significantly predict adolescents\u2019 academic burnout directly and (2) parental phubbing could indirectly influence adolescents\u2019 academic burnout through three pathways: the separate mediating effect of social anxiety and self-control, and the chain mediating effect on both. The results of this study help parents understand how their phubbing actions affect adolescents\u2019 academic burnout and the mechanism of action. The emergence of functions such as online shopping, takeout, and even online grocery shopping has made people today increasingly inseparable from their devices. And the emergence of the WeChat work group has also allowed people to respond to work messages timely even from home. However, researchers have also found that the overuse of mobile communication devices has a negative impact on users\u2019 daily lives . NowadayIn recent years, excessive cell phone use has become a widespread societal issue, giving rise to the term \u201cphubbing,\u201d which refers to the phenomenon of individuals who are too preoccupied with their mobile phone or iPad to ignore others . PhubbinIt is undeniable that parents are the main factor influencing adolescent development. Based on the Ecological System Theory, the most significant microsystem influencing adolescent development is the family, and as parents are the primary members of the family, their behavior has a significant influence on adolescent development . StudiesParental phubbing is perceived by children as a type of carelessness and refusal , while P1.1.Academic burnout is a chronic psychological disorder in which students experience physical and mental exhaustion and poor academic achievement . AcademiFamily environment affects adolescents\u2019 levels of academic burnout . Parents1.2.Based on Ecological System Theory , in addiSocial anxiety is one of the most prevalent anxiety symptoms in adolescents and is characterized by a strong and irrational feeling of embarrassment in social situations . IndividAdolescents with social anxiety shy away from speaking up in class discussions and talking with classmates outside of class, and are afraid to ask teachers and peers for help when they are struggling academically, consequently leading to learning problems. 1.3.The preceding discussion explained the relationship between parental phubbing, social anxiety, and adolescent academic burnout, but there exists another key factor that could affect adolescents\u2019 learning. Researchers have found that self-control is a protective factor for academic burnout . ActuallSelf-control is the capacity that individuals to suppress or curb their desires and impulses and regulate their inherent ways of thinking or behavioral habits to conform to social norms, such as resisting temptation, delaying gratification, and resisting impulses . In adolThe act of learning requires a large number of control resources, and people must exercise more self-control during learning in order to suppress the interference of irrelevant information and maintain their attention on the learning task . That isSelf-control can be influenced by the environment , for ext1.4.Except for the external environment , the individual\u2019s negative emotional state  can also deplete the individual\u2019s self-control resources . Social In conclusion, previous studies have found that parental phubbing will lead to academic burnout in adolescents , but the2.2.1.We used random cluster sampling to recruit adolescents from three full-time secondary schools in Anhui province, China. With the consent of teachers and guardians, students filled out the questionnaire during their weekly class meetings. The participants were informed of the anonymity, voluntary, and confidential nature of the questionnaire.\u03c72(1)\u2009=\u20093.89, p\u2009=\u20090.143.1,032 adolescents were recruited to participate in this study. Invalid questionnaires that failed polygraph questions and consistent responses were excluded. Finally, 828 valid questionnaires met the last criteria, with an effective rate of 80.1%. Among them, 376 (45.4%) were male, and 452 (54.6%) were female; 333 (40.2%) were grade 10, 295 (35.6%) were grade 11, and 200 (24.2%) were grade 12. Their mean age is 16.04 (SD\u2009=\u20090.95), ranging from 15 to 18. There were 315 (38.0%) participants who were only children and 523 (62.0%) participants who were not only children. 429 (51.8%) participants reported being urban residents and 399 (48.2%) reported being rural residents. The gender distribution of participants and their grades did not significantly correlate, 2.2.2.2.1.\u03b1 0.81).Using the Chines version of Parental phubbing Scale from 2.2.2.\u03b1) in this research was 0.85 for the total scale, and for each of the three subscales, it was 0.78, 0.85, and 0.84.Using the Adolescents\u2019 Academic Burnout Scale developed by 2.2.3.\u03b1) of the total scale was 0.93 and for each of the three subscales, it was 0.92, 0.84, and 0.83.To measure adolescents\u2019 social anxiety, we utilized the Chinese translation of the Adolescent Social Anxiety Scale, which was amended by 2.2.4.\u03b1) for the overall scale was 0.89, and was 0.85, 0.66, 0.72, 0.61, and 0.72 for the 5 subscales in the current study.We used the Chinese version Self-control Scale amended by 2.3.Data entry, correlation analyses, and descriptive statistics were carried out using SPSS 26.0 and mediated effects analysis was performed using Mplus 8.1. We first screened the collected data and performed descriptive statistics and correlation analysis between variables after eliminating invalid data. Based on the correlation analysis, we controlled for the effects of gender and grade level and established a direct pathway of parental phubbing to adolescents\u2019 academic burnout. To test the mediating effect, we then incorporated social anxiety and self-control in the mediation model and constructed a pathway of influence from social anxiety to self-control to generate a chain mediation model. Bootstrap testing was applied to estimate confidence intervals and test for indirect effects. And the chi-square value, RMSEA, CFI, TLI, and SRMR was used to evaluate the model fit index.2.4.\u03c72/df\u2009=\u20099.77, RMSEA\u2009=\u20090.103, CFI\u2009=\u20090.39, TLI\u2009=\u20090.37, and SRMR\u2009=\u20090.117, suggesting that the CMB was not serious in this study.This study used only questionnaires for administration, which may have created common method bias (CMB) due to the same data collection method. We emphasized the anonymity and confidentiality of the questionnaires to the participants before they filled out the questionnaires, and used methods such as positive and negative scoring in the questionnaires to preliminary control this bias. Further, two statistical methods were used to test for CMB. (1) Harman\u2019s single-factor test. 20 factors were extracted with eigenvalues bigger than 1, and the first one was able to account for 16.46% of the total variation, indicating that the CMB was not serious in this study for the normal criterion of 40% was not exceeded. (2) Using confirmatory factor analysis  to test 3.3.1.3.2.Since social anxiety, self-control, and academic burnout all contain multiple dimensions, it is necessary to pack these three scales for controlling the impacts of random errors . The spe\u03c72/df\u2009=\u20093.70, RMSEA\u2009=\u20090.057, CFI\u2009=\u20090.93, TLI\u2009=\u20090.91, SRMR\u2009=\u20090.039, and a significant positive impact of parental phubbing on academic burnout .The impact of gender and grade was controlled for getting a more accurate effect. We first tested the direct path of parental phubbing on adolescents\u2019 academic burnout. The results revealed a good model fit: \u03c72/df\u2009=\u20094.29, RMSEA\u2009=\u20090.063, CFI\u2009=\u20090.89, TLI\u2009=\u20090.87, and SRMR\u2009=\u20090.053. The path analysis indicated that the direct effect remained significant , and parental phubbing significantly predicted social anxiety positively  and self-control negatively ; social anxiety predicted academic burnout positively  and self-control negatively ; self-control predicted academic burnout negatively  . Parental phubbing and adolescents\u2019 academic burnout were separately mediated by self-control, and this impact was significant . In the meantime, the serial mediation effect of social anxiety and self-control was also significant . Specific indirect effects and 95% CI are shown in Furthermore, a bias-corrected Bootstrap test for mediating effects and confidence interval estimation was used with 1,000 times repeated samples. Finally, the findings demonstrated that the separate mediation effect of social anxiety between parental phubbing and adolescents\u2019 academic burnout was significant  parental phubbing and adolescents\u2019 academic burnout were significantly and positively associated. (2) Parental phubbing can influence adolescents\u2019 academic burnout by the separate mediation effects of social anxiety and self-control. Social anxiety was positively associated with adolescents\u2019 academic burnout, whereas self-control can negatively predict adolescents\u2019 academic burnout. (3) Parental phubbing predicts adolescents\u2019 academic burnout through the chain mediation effect of social anxiety and self-control. Social anxiety affects self-control negatively. These results provide some practical inspiration for parents and teachers to understand the reasons for adolescents\u2019 declining interest in learning and lack of motivation, and how to intervene in their academic burnout. Firstly, parents need to take the problem of declining academic performance and academic burnout among adolescents seriously enough, but instead of just blaming the students, they should understand that adolescents\u2019 development is influenced by their parents and look more for the causes of the problem from themselves. Secondly, parents should pay attention to reducing the use of internet devices to prevent addiction and consciously avoid using cell phones in front of their children. Even if they cannot avoid using cell phones in the family due to work, they should let their children know the real reason and set a good example for them to work actively and study hard. Thirdly, parents should pay attention to more in-depth communication with their children, encourage them to communicate with others, and develop good social skills. At the same time, schools should also pay attention to shaping a learning atmosphere conducive to interpersonal communication and promoting good peer relationships among students. Fourthly, parents must emphasize the critical role that self-control plays in their adolescents\u2019 learning difficulties. They need to concentrate on improving their kids\u2019 capacity for self-control to strengthen their risk tolerance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Ethics Committee of Anhui Normal University. Written informed consent to participate in this study was provided by the participants\u2019 legal guardian/next of kin.YJ and RH contribute to the title selection and design of this research. RH undertook the guidance and revision of the article. YJ wrote the first draft of the article. LL and YJ constructed the database, performed statistical analysis, and translated the article. 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": "This study aims to investigate the relationship and mechanisms of action among academic stress, academic anxiety, academic self-efficacy, and academic burnout among adolescents.A study of 929 Chinese adolescents  was conducted using the Study Stress Questionnaire, Academic Anxiety Subscale, Junior Middle School Students\u2019 Learning Weariness Scale, and the Academic Self-efficacy Questionnaire.\u2460Academic stress was significantly and positively correlated with academic anxiety and academic burnout, and significantly and negatively correlated with academic self-efficacy. \u2461Academic anxiety partially mediated the relationship between academic stress and academic burnout. \u2462Academic self-efficacy significantly moderated the direct effect of academic stress on academic burnout, and higher academic self-efficacy could buffer the negative effect of academic stress. \u2463Academic self-efficacy significantly moderated the second half of the mediated model\u2019s path (academic anxiety \u2192 academic burnout), that is, low academic self-efficacy amplified the risk effect of academic anxiety on academic burnout.Academic anxiety partially mediates the relationship between academic stress and academic burnout, and this mediating role is moderated by academic self-efficacy. Results of a survey conducted by the China Care for the Next Generation Working Committee in 2020 showed that more than 30% of elementary school students were bored with school and more than 70% of adolescent students have academic burnout, and many students experienced severe burnout or even refused to go to school due to the home quarantine during the epidemic that interrupted the normal rhythm of school attendance for adolescents . RegardiPrevious studies have shown that students\u2019 own poor learning foundation, low self-evaluation, and lack of interest and initiative in learning can lead to academic tiresome . AcademiAcademic anxiety is a negative emotional state that students experience more frequently in academic situations , encompaAcademic self-efficacy (ASE) is the judgment and confidence in an individual\u2019s ability to believe that he or she can successfully complete a specific academic task at a specific stage of learning  and is tFurthermore, academic self-efficacy is considered to be a predictor and protective factor for adolescents\u2019 internalizing and externalizing problems . TherefoWe applied a survey design to achieve our research objectives. We used a whole-group sampling method and approached a junior high school in Shandong Province to collect data from their students, a sample of adolescents from this junior high school that was well represented. Participants were invited to provide their voluntary consent and then complete these measures. Participants did not provide any personal information that would allow researchers or organizations to identify them. Participants were led into the school\u2019s microcomputer room by a schoolteacher and completed an electronic questionnaire on a computer after a trained master examiner read the instructions. All participation was voluntary, and each participant received a ballpoint pen as payment at the end of the test.Using the whole-group sampling method, all students in grades 6 and 7 of a junior high school in Shandong Province, a total of 982 adolescents, were selected to complete the questionnaire survey, and a total of 929 valid questionnaires were returned, with a valid return rate of 94.60%. Among them, 499 (53.71%) were male and 430 (46.29%) were female; 488 were in the first grade and 441 were in the second grade, aged 11 to 15 (11.94 \u00b1 0.77). These adolescents came from urban, rural and rural areas, and it can be confirmed that the sample drawn is highly representative of the adolescent population.p < 0.001, RMSEA = 0.075, SRMR = 0.042, GFI = 0.882, AGFI = 0.851, CFI = 0.922, TLI = 0.911, indicating that the results of CFA had good fitting indicators.This study used the study stress questionnaire for middle school students  to measuIn this study, the academic anxiety subscale of the Mental Health Diagnostic Test (MHT), adapted by Zhou Bucheng , was usep < 0.001, RMSEA = 0.081, SRMR = 0.046, GFI = 0.906, AGFI = 0.874, CFI = 0.938, TLI = 0.927, indicating that the results of CFA had good fitting indicators.This study used Junior Middle School Students\u2019 Learning Weariness Scale  to measup < 0.001, RMSEA = 0.082, SRMR = 0.064, GFI = 0.865, AGFI = 0.834, CFI = 0.922, TLI = 0.912, indicating that the results of CFA had good fitting indicators.The academic self-efficacy questionnaire, developed by First, the common method deviation test was performed using SPSS 22.0, and descriptive statistics and correlation analysis were performed for the main variables. The common method bias test calculated according to Harman\u2019s one-way test showed that there were 10 factors with eigenvalues greater than 1. The first principal component explained 29.89% of the total variance, which is below the critical value of 40%, so it can be concluded that there is no significant common method bias problem in this study. After that, Model 15 in SPSS macro program process v3.3 prepared by As shown in \u03b2 = 0.38, t = 13.40, p < 0.001), and the predictive effect of academic stress on academic burnout remained significant when the mediating variable academic anxiety was introduced , academic stress had a significant predictive effect on academic anxiety , and academic anxiety had a significant positive predictive effect on academic burnout . The upper and lower limits of Bootstrap 95% confidence intervals for the direct effect of academic stress on academic burnout and the mediating effect of academic anxiety did not contain 0  = 323.24, p < 0.001) and learning stress was a significant positive predictor of learning anxiety . Equation 2 was significant overall  = 169.84, p < 0.001), with learning stress significantly and positively predicting academic burnout  and learning anxiety significantly and positively predicting academic burnout , thus learning anxiety mediated the relationship between learning stress and academic burnout. The interaction term between academic stress and academic self-efficacy was a significant negative predictor of academic burnout , and the interaction term between academic anxiety and academic self-efficacy was a significant positive predictor of academic burnout , indicating a significant moderating effect of academic self-efficacy. The results are shown in Model 15 in the process macro program prepared by Hayes was used to test for a moderating mediating effect. The results showed that equation 1 was significant overall (Z \u2266 -1SD) and high (Z \u2267 1SD) subgroups according to the standardized scores controlling for academic self-efficacy for simple slope analysis. First, we explored how academic self-efficacy moderated the direct effect of academic stress on academic burnout. The results found  and insignificant for adolescents when the level of academic self-efficacy was high . This indicates that the lower the academic self-efficacy of middle school students, the greater the effect of academic stress on academic burnout, and conversely, at high levels of academic self-efficacy, the effect of academic stress on academic burnout was not significant.After standardizing the study variables, the study divided the subjects into low ; for adolescents with high academic self-efficacy, academic anxiety was a significant positive predictor of academic burnout . This suggests that the higher the academic self-efficacy of middle school students, the greater the effect of learning anxiety on academic burnout may be, and on the contrary, at low levels of academic self-efficacy, the effect of learning anxiety on academic burnout may not have a significant effect.The study further went on to analyze the moderating effect of academic self-efficacy on the relationship between academic anxiety and academic burnout in the second half of the mediated model path see . It was \u03b2 = 0.011, SE = 0.027, 95% CI = \u22120.040 to 0.065; medium level of academic self-efficacy: \u03b2 = 0.038, SE = 0.015, 95% CI = 0.010 to 0.067; high level of academic self-efficacy: \u03b2 = 0.064, SE = 0.017, 95% CI = 0.032 to 0.100). The results support the hypotheses developed in this research.The bias-corrected bootstrap confirmed that the indirect relationship between academic stress and academic burnout through anxiety was moderated by academic self-efficacy. To be specific, with the improvement of self-efficacy, the indirect effect was stronger  suggests that the protective effect of protective factors is undermined when risk factors are too high , and leaIn this study, a moderated mediation model was constructed to examine the mediating process of \u201cacademic stress \u2192 academic anxiety \u2192 academic burnout\u201d and the moderating role of academic self-efficacy. The results showed that the mediating effect of academic anxiety between academic stress and academic burnout was significant, and the moderating effect of academic self-efficacy in the direct path and the second half of the mediating path was significant, which helps to understand the relationship between learning stress and academic burnout and its internal mechanisms. There are several limitations of this study that could be improved in further research. First, this study used a cross-sectional design and was unable to test the stability of this mechanism of action across time; future studies could be administered to these participants again after a certain period of time to explore the stability of this mechanism of action across time; second, all of the variables explored in this study were related to academics, and according to ecosystem theory , home enNevertheless, this study has strong practical implications. Because of the current high levels of academic burnout among some adolescents, this study can provide targeted suggestions and insights for preventing and intervening in adolescents\u2019 academic burnout. The results of the study show that, first, we can reduce the level of academic burnout by reducing the academic stress of adolescents, such as reducing extracurricular assignments. Second, we can equip adolescents with ways to regulate their emotions and reduce academic boredom by reducing academic anxiety. In addition, according to the results of the study, academic self-efficacy is effective in boosting confidence and coping with external stress effectively, but if students have high levels of academic anxiety, instantly students with high academic self-efficacy will increase the expression of academic burnout as their anxiety level increases. Therefore, for adolescents with high academic self-efficacy, although they can cope with external pressure effectively, they need to pay attention to the regulation of their academic anxiety, so it is more important to provide them with emotional psychological guidance to alleviate their academic anxiety levels; for adolescents with low academic self-efficacy, they need to stimulate their academic self-efficacy as well as pay attention to their own internal emotional regulation. In conclusion, the intervention and prevention of academic burnout in junior high school students should not focus on one aspect only, but should be carried out in an integrated and systematic way from three aspects: academic stress, academic anxiety, and academic self-efficacy.The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the ethics committee at Shandong Normal University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.XG: conceptualization, methodology, supervision, validation, resources, data curation, formal analysis, investigation, validation, and writing\u2014original draft.This study was financially supported by National College Students\u2019 Innovation and Entrepreneurship Training Program of China [grant number: 202210445011]. Shandong Normal University Undergraduate Research Fund Project in 2023 [grant number: BKJJ2022083].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": "Type 2 scleredema on the background of monoclonal gammopathy of undetermined significance (MGUS) is a rare and progressive connective tissue disorder with very few cases reported to date. It is characterized by chronic and diffuse induration of the skin that begins in the upper back and neck and progresses proximally to distally, involving the shoulders, trunk, and arms; the hands are usually spared. Here, we present an unusual case of long-standing scleredema that progressed to involve the hands and fingers. This case was further complicated by new-onset Raynaud\u2019s phenomenon, splenomegaly, lymphadenopathy, the development of a plasmacytoma, and eventual progression to multiple myeloma. We highlight the differential diagnoses for his complex presentation, the workup that was completed, and current treatment options. Scleredema is a rare and progressive connective tissue disorder characterized by chronic and diffuse induration of the skin . A physiScleredema has been classified into three types based on etiology . Type 1 There is no specific treatment for scleredema, although steroids, immunosuppression, phototherapy, and intravenous immunoglobulin have been used with varying success -4. For sThe prognosis of scleredema varies based on subtype . Type 1 This case report was previously presented at the University of North Dakota's Frank Low Research Day on April 12, 2023.A middle-aged man with a three-year history of MGUS presented to the clinic with a history of slowly progressive skin tightening and thickening, the recent onset of Raynaud\u2019s phenomenon without digital ulcers, dysphagia with progressive choking of liquids and solids, gastroesophageal reflux disease (GERD), dyspnea, and numbness and tingling in the feet. The cutaneous symptoms began 10 years prior to his initial presentation with skin thickness over his neck and chest, which progressed slowly to involve his shoulders, upper back, torso, and upper arms. His skin progressively hardened over time; he described early and significant limitations in the range of motion at his shoulders. He had previously been diagnosed with scleroderma, per his recollection at the onset of symptoms, and had taken methotrexate, prednisone, and pentoxifylline. He did not note any benefit from these medications and was eventually lost to follow-up. His condition continued to progress, with thickening over his forearms and fingers over the last one to two years.On exam, he was found to have thick skin over the thorax, face, upper back, upper arms, forearms, and fingers, with proximal sites thicker than distal sites Figure . The lowEvaluation of his other presenting symptoms included a pulmonary function test to work up his dyspnea, which was consistent with a restrictive physiology. A chest CT showed clear lungs without evidence of edema, ground-glass opacities, infiltrative processes, or interstitial lung disease. An echocardiogram showed a left ventricular ejection fraction of 55% and increased left ventricle wall thickness. An amyloid scan ruled out transthyretin amyloidosis. A right and left cardiac catheterization revealed normal cardiac hemodynamics at rest and exercise, without evidence of cardiac output limitation or evidence of right or left heart failure. There was no atherosclerotic disease of the coronary arteries. Dyspnea was ultimately attributed to restrictive lung physiology, with contributions thought to be multifactorial and secondary to cutaneous disease of the thorax and the patient\u2019s weight (BMI 37 kg/m2). A barium esophagram to work up his progressive choking of liquids and solids noted normal pharyngeal function, normal peristaltic waves, and no strictures. Esophagogastroduodenoscopy was recommended but not performed at that time. Electromyography (EMG) was ordered to investigate the sensory changes in the feet but was ultimately not performed.The patient had a previous diagnosis of MGUS with 0.4 g/dL immunoglobulin G (IgG) kappa monoclonal proteins and a kappa light chain-restricted plasma cell dyscrasia occupying 5% of his bone marrow. Repeat hematologic testing revealed an M-spike increase to 0.8 g/dL IgG kappa with 5% to 9% plasma cells in the bone marrow. Renal function tests were normal, and no light chains were detected in his urine. Hemoglobin and serum calcium were also normal. A bone survey showed no osseous abnormality with the exception of a small, non-specific oval lucency at the vertex; a follow-up CT of the head did not identify a lesion. He was offered treatment designed to eliminate the plasma cell clone, although the impact on scleredema could not be guaranteed. He opted not to pursue treatment.Two years following his initial presentation, the patient presented to internal medicine with a weight loss of 60 pounds and associated abdominal pain, nausea, vomiting, and right-sided lumbar spinal pain. Imaging revealed a 16.8 cm enlarged spleen Figure , mild gaThe patient underwent a biopsy of the right inguinal mass, which was negative for infectious or malignant causes and was deemed a reactive process. The patient did not note further progression or worsening of his skin thickening during this time and did not elect to follow up with rheumatology or dermatology for a repeat assessment. Lab results at the time of the plasmacytoma diagnosis included a serum calcium level of 9.3 mg/dL, hemoglobin of 16.3 g/dL, and an estimated creatinine clearance of 195.4 mL/min. Thus, the patient did not have evidence of end-organ damage attributable to the underlying plasma cell disorder and did not meet the criteria for multiple myeloma at this time according to the revised International Myeloma Working Group Criteria for the diagnosis of multiple myeloma .Radiation to the L5 plasmacytoma was pursued. In total, the patient received 3750 cGy in 23 fractions, with 250 cGy per fraction, using image-guided radiation and 3D conformal radiation therapy. Following radiation treatments, the patient noted a marked improvement in spinal pain. A timeline depicting the patient\u2019s clinical course is described in Figure Clostridium difficile\u00a0colitis.A repeat PET-CT was obtained in the context of recurrent back pain seven months after his initial PET-CT. This showed an osteolytic lesion  and two indeterminate lesions . With the history of the plasmacytoma and the single lytic rib lesion, along with two further indeterminate lesions, it was felt he met the classification criteria for multiple myeloma according to the revised International Myeloma Working Group Criteria [The patient is set to receive four to six 28-day cycles of chemotherapy consisting of daratumumab and hyaluronidase-fihj and lenalidomide. Dexamethasone will also be given prior to daratumumab and hyaluronidase-fihj for hypersensitivity reaction prophylaxis. Cycle one will consist of daratumumab and hyaluronidase-fihj 1800 mg subcutaneous injections with 20 mg of dexamethasone on days 1, 8, 15, and 22. He will then begin taking lenalidomide 25 mg capsules daily starting on the second cycle. The main side effects anticipated with these chemotherapeutic agents include myelosuppression, increased risk of DVT, hypersensitivity reactions, and injection site reactions.This patient presented with an unusual course of scleredema in that there was additional cutaneous thickening and edema of his hands and fingers, along with Raynaud\u2019s phenomenon. The involvement of his hands in combination with Raynaud\u2019s phenomenon raised the question of concurrent systemic sclerosis in the background of scleredema. However, cutaneous changes progressed from proximal to distal; cutaneous changes secondary to systemic sclerosis progressed from distal to proximal. In Raynaud's disease associated with systemic sclerosis, nailfold capillary abnormalities are seen with a prevalence near 80% . ExtracuOther differentials for late-onset (age over 30) Raynaud\u2019s phenomenon can include systemic lupus erythematosus (SLE), Sj\u00f6gren\u2019s disease, idiopathic inflammatory myopathies, cryoglobulinemia, paraproteinemia, pheochromocytomas, carcinoid syndrome, thyroid disease, or vasospasm secondary to pharmaceuticals \u00a0. While cThe pathogenesis of scleredema is not well understood but is likely due to increased fibroblast production of type 1 collagen and glycosaminoglycans in the reticular dermis, as well as mucopolysaccharide production in the interfibrillar dermal space ,17,2. KeSeveral systemic treatments have been trialed for scleredema, including corticosteroids, pentoxifylline, cyclosporine, methotrexate, hydroxychloroquine, and intravenous immunoglobulin; however, none are universally accepted and are often not successful . In scleScleredema is a rare and progressive connective tissue disorder that has a poorly understood pathogenesis. It can occur following a febrile illness or in association with type II diabetes, MGUS, or multiple myeloma. The disease's insidious onset can hinder a timely diagnosis, and its symptoms can readily be mistaken for other sclerosing skin conditions. Differential diagnoses for the disease include systemic sclerosis, scleromyxedema, and POEMS. Features that help distinguish scleredema from other sclerosing skin conditions include the location of skin involvement and the pattern of progression. While an overlap of limited cutaneous systemic sclerosis cannot be entirely excluded in this patient with the involvement of his hands and fingers, the negative serological testing, normal nailfold capillaries, absence of other cutaneous stigmata, including telangiectasias and calcinosis, absence of interstitial lung disease, and absence of objective signs of esophageal dysmotility by barium esophagram make this less likely. Therefore, an atypical presentation of scleredema with involvement of the hands, rather than the manifestation of two rare diseases, is favored. It is possible that the long duration (over 10 years) resulted in more extensive cutaneous involvement than previously reported in type 2 scleredema. Scleredema with MGUS or multiple myeloma has been shown in case studies to improve with systemic chemotherapy, notably with cyclophosphamide, vincristine, melphalan, thalidomide, or bortezomib."}
+{"text": "Associations between PET positivity and related clinical information were assessed using t-tests and \u03c72 tests. The significance of variables differentiating benign lesions from malignant FDG-avid lesions was evaluated by logistic regression analysis. Survival probabilities were derived from Kaplan-Meier curves and compared using the log-rank test. Results: A total of 185 patients (235 post-transplantation PET/CT scans) were enrolled in our present study. Compared with those with true-positive PET results, patients with false-positive PET results exhibited a better prognosis. For the autologous SCT group, false-positive cases were more commonly seen when FDG-avid foci appeared outside the sites of the original disease (p = 0.004), and the integrated CT imaging showed negative results (p = 0.000). In multivariate logistic regression analysis, integrated CT results were the only significant factor. For the allogeneic SCT group, false-positive cases were significantly more commonly seen when DS = 4 (p = 0.046), FDG-avid foci appeared outside the sites of the original disease (p = 0.022), and the integrated CT imaging showed negative results (p = 0.001). In a multivariate logistic regression analysis, whether FDG-avid foci were in the sites of the original disease and integrated CT results were both significant factors. Conclusion: False-positive FDG uptake in post-transplantation PET was not uncommon. Several variables could provide an important reference to differentiate false-positive from true-positive post-SCT PET results for lymphoma involvement. Trial registration number: ChiCTR2300067355.Purpose: The purpose of this study was to differentiate between false-positive and true-positive positron emission tomography (PET) results after hematopoietic stem cell transplantation (SCT) for lymphoma involvement by analyzing several clinical variables and specific imaging features. Patients and Methods: Patients with lymphoma who received SCT and underwent post-transplantation  The cur in 2007  have sho18F-FDG PET in diagnosing lymphoma is that it is sometimes difficult to distinguish whether the FDG-avid foci are lymphoma involvement or inflammatory changes [18F-FDG reflects glucose metabolism of both cancer cells and immunologically competent cells in the tumor [18F-FDG PET [18F-FDG PET/CT imaging results after transplantation. Previous studies have shown a high rate of false-positive PET after transplantation [However, one disadvantage of  changes ,11,12 behe tumor . Another-FDG PET . Conside-FDG PET ,16,17, iantation ,20. Whet18F-FDG PET/CT images.In the present study, we aimed to differentiate between false-positive and true-positive post-SCT PET results for lymphoma involvement by analyzing several clinical variables and specific imaging features and optimizing the interpretation of post-SCT 18F-FDG PET/CT scans within 6 months after auto-SCT or within 12 months after allo-SCT. The exclusion criteria were: (1) patients who received systemic therapy for lymphoma other than maintenance therapy within the time window between day 0 of transplantation and post-transplantation PET. (2) Patients who had an incomplete follow-up. Status before and after transplantation was determined according to the International Working Group criteria (IWGc) [The inclusion criteria were as follows: (1) pathologically confirmed lymphoma (including NHL and HL); (2) received SCT between January 2013 and April 2021 at the First Affiliated Hospital of Soochow University; (3) underwent post-transplantation a (IWGc) ,21.Indications for HSCT  vary by disease type and remission status according to disease-specific NCCN Guidelines ,23,24. I18F-FDG PET and integrated CT. 18F-FDG PET/CT results were compared with the findings of the pathological examination and follow-up practice (more than 6 months). Overall survival (OS) was defined as the interval from day 0 of SCT until the time of death from any cause or last follow-up.We calculated the diagnostic efficiency of Approval was obtained from the institutional review board of the First Affiliated Hospital of Soochow University. The trial registration number was ChiCTR2300067355. The requirement for written informed consent from patients was waived.18F-FDG PET/CT on a GE Discovery STE16 PET/CT . All patients had fasted for at least 6 h, and their blood glucose levels were less than 11 mmol/L before injection. Whole-body PET images (from skull base to mid-thigh) were acquired approximately 60 min after injection of 18F-FDG (0.11\u20130.14 mCi/kg). Emission data were acquired for approximately 2 min in each bed position, with an average of 7\u201310 bed positions per scan. CT examinations were obtained with the following scan parameters: 3.5 mm/slice, 140 kV, 120 mA. PET data were reconstructed with CT-based attenuation correction by using an iterative algorithm. All patients underwent whole-body Patients were assigned into 2 groups based on the results of post-SCT PET using the Deauville 5-point scale ,25: (1) According to the CT-Based Response on the Lugano Classification, patients were divided into two 2 groups: (1) the negative CT group, target nodes/nodal masses that regressed to \u22641.5 cm in the longest transverse diameter of a lesion (LDi); (2) the positive CT group, target nodes/nodal masses still >1.5 cm in LDi or/and a new node > 1.5 cm in any axis or a minimum of 1 cm in LDi of new extra-nodal lesions. All the PET/CT images were specifically reviewed by two nuclear medicine physicians (with at least 5 years of experience in PET/CT). In case of conflicting findings between the two observers, an independent panel of PET physicians with 10 years of experience in PET/CT would review the data and make the final decision.18F-FDG PET/CT and integrated CT results using the kappa consistency test. A t-test was used for continuous variables, and the \u03c72 test was used for categorical variables when assessing the associations between PET positivity and related clinical information. The significance of variables differentiating benign lesions from malignant FDG-avid lesions was evaluated by the logistic regression analysis. Survival probabilities were derived from Kaplan-Meier curves and compared using the log-rank test.The current study assessed the consistency of p less than 0.05 was considered statistically significant.Statistical analyses were carried out using IBM SPSS Statistics (version 26.0). All tests were two-sided, and the value of n = 40), mantle cell lymphoma (n = 8), follicular cell lymphoma (n = 4), B lymphoblastic lymphoma (n = 10), Burkitt lymphoma (n = 4), small B-cell lymphoma (n = 1), anaplastic large cell lymphoma (n = 17), and peripheral T-cell lymphoma (n = 21), NK/T-cell lymphoma (n = 9), and T lymphoblastic lymphoma (n = 53).The enrolled 185 patients (235 post-transplantation PET/CT scans) included 101 patients (105 post-transplantation PET/CT scans) with autologous SCT and 84 patients (130 post-transplantation PET/CT scans) with allogeneic SCT. The multivariate logistic regression analysis on 185 patients (235 post-transplantation PET/CT scans) was carried out. The results showed the integrated CT results, whether FDG-avid foci were in the sites of the original disease and Status at SCT were the significant factors for differentiating false-positive PET results from true-positive PET results . However18F-FDG PET-CT were 100% (19/19), 75.6% (65/86), 47.5% (19/40), 100% (65/65), and 80.0% (84/105), respectively, and those values of integrated CT were 84.2% (16/19), 84.9% (73/86), 55.2% (16/29), 96.1% (73/76), and 84.8% (89/105), respectively. The consistency of 18F-FDG PET-CT and integrated CT was moderate . Among eight scans of positive CT results but negative PET results, all FDG-avid foci were demonstrated to be a benign process rather than lymphoma by pathological examination and follow-up practice.Out of 105 post-transplantation PET/CT scans (101 patients), the presence of lymphoma foci was confirmed in 19 scans by pathological examination and follow-up practice. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of post-SCT p = 0.015, On Kaplan-Meier analysis in patients with false-positive PET results, the 2-year OS rate was 95.2%, and the 5-year OS rate was 95.2%, in contrast to 77.1% and 51.4%, respectively, in those with true-positive PET results. (p = 0.004), and the integrated CT imaging showed negative results (p = 0.000). Multifocal foci were less common in patients with benign FDG-avid lesions than in those with lymphoma involvement, while the difference was not significant (p = 0.055).p < 0.1 were taken into the regression analysis. In the multivariate logistic regression analysis, integrated CT results were the only significant factor for differentiating false-positive PET results from true-positive PET results (Variables with  results .18F-FDG PET-CT were 100% (21/21), 90.8% (99/109), 67.8% (21/31), 100% (99/99), and 92.3% (120/130), respectively, and those values of integrated CT were 90.5% (19/21), 89.9% (98/109), 63.3% (19/30), 98.0% (98/100), and 90.0% (117/130), respectively. The consistency of 18F-FDG PET-CT and integrated CT was moderate . Moreover, among seven scans of positive CT results but negative PET results, all FDG-avid foci were demonstrated to be a benign process rather than lymphoma by pathological examination or follow-up imaging.Out of 130 post-transplantation PET/CT scans (84 patients), the presence of lymphoma foci was confirmed in 16.2% (21/130) scans by biopsy or follow-up imaging. The sensitivity, specificity, PPV, NPV, and accuracy of post-SCT p = 0.033, A total of eleven patients died during the follow-up, including nine because of relapse or progression, one because of severe graft-versus-host disease (GVHD), and one due to serious infection. On Kaplan-Meier analysis in patients with false-positive PET results, the 2-year OS rate was 85.7%, and the 5-year OS rate was 85.7%, in contrast to 53.2% and 39.9%, respectively, in those with true-positive PET results. (p = 0.046), FDG-avid foci appeared outside the sites of the original disease (p = 0.022), and the integrated CT imaging showed negative results (p = 0.001) (p = 0.074) (= 0.001) . FDG-avi= 0.074) .p < 0.1 were taken into the regression analysis. In the multivariate logistic regression analysis, whether FDG-avid foci were in the sites of the original disease and integrated CT results were the significant factors for differentiating false-positive PET results from true-positive PET results  and 67.8% (21/31), respectively, which means the false-positive FDG PET/CT was seen more frequently in patients with autologous SCT than patients with allogeneic SCT. This inconsistency requires further research to confirm.Over the past several decades, PET has emerged as a critical approach for the accurate staging and restaging of lymphoma ,26,27. Hpatients . Normallpatients . Anotherpatients . Our stu18F-FDG-PET/CT in patients with diffuse large B cell lymphoma (DLBCL). A study on lung cancer comes to similar conclusions that false-positive lymph nodes are associated with non-swollen nodes on 18F-FDG PET scans in lung cancer [18F-FDG PET results [18F-FDG uptake of one or more lesion(s) is increased without a concomitant increase in lesion size or number, it is mandatory to obtain a repeat imaging but not considered as true PD. However, it is mainly used for immunotherapy rather than SCT. Our results revealed that false-positive cases were significantly more commonly seen when the integrated CT imaging showed negative results .In our study, Kaplan-Meier analysis showed that patients with false-positive PET results, as compared with those with true-positive PET results, were associated with a decreased rate of death. False-positive PET cases were significantly more commonly seen when the integrated CT imaging showed negative results. In addition, for the allogeneic SCT group, the possibility of false positives should also be considered when FDG-avid foci appeared outside the sites of the original disease. Noa Lavi  has propg cancer . In rece results  and mand results ,35. It iMost previous studies have used DS4 and DS5 as the PET-positive group when assessing treatment efficacy for lymphoma ,37,38,3918F-FDG avidity of the original lesion markedly increased in the liver, it is defined as score 5a, and any new FDG-avid lesion score 5b. The result showed that at the end of treatment, the DS-5a score was highly suggestive of residual disease. However, participants with DS4 and DS-5b require histopathological confirmation before any change in treatment strategy to rule out false-positive [A recent study has divided DS5 into two situations: if positive . Their tpositive  has showp = 1.00), respectively. The false positive rate of PET within 3 months and within 3\u201312 months after allogeneic SCT was 5/12 and 5/19 (p = 0.447), respectively. Undoubtedly, further research is needed to establish the optimal time window for post-SCT PET.In this study, some patients underwent post-transplantation PET/CT more than once. This study included all PET examinations that met the inclusion criteria, not only the first PET examination after transplantation. The original intention was to observe if the false-positive rate of PET was related to the different time windows of post-SCT PET. However, the results showed that the false positive rate of PET within 3 months and within 3\u20136 months after autologous SCT was 10/20 and 11/20 (18F-FDG PET/CT showed positive results. Second, other methods of PET/CT response assessment, such as \u0394SUVmax or metabolic tumor volume, might provide different information from our current study. Furthermore, for the autologous SCT group, it was interesting to find that only CT results were predictable of PET/CT findings. Previous studies showed that multiple factors were associated with false or true positive PET results, such as status at SCT and DS score. This discrepancy might be caused by the lack of uniformity in the patient\u2019s pathological type, treatment protocol, and the number of previous treatments. It is also another limitation of this study. However, it seemed to be certain that CT results played a significant role in determining whether the FDG-avid foci were disease recurrence.This study has certain limitations. First, the presence of lymphoma foci was confirmed in most cases by follow-up imaging but not biopsy when the post-SCT 18F-FDG PET/CT had a high sensitivity to detect lymphoma-involved lesions. However, false-positive 18F-FDG uptake in post-transplantation PET was not uncommon. When post-transplantation PET showed suspicious FDG-avid foci, integrated CT results were an important factor in identifying true or false positives in both autologous and allogeneic SCT cases. In addition, FDG-avid foci that occurred outside the sites of the original disease would likely be diagnosed as non-lymphoma involvement after allogeneic SCT.Taken together, post-transplantation"}
+{"text": "Musa spp.) is a major global economic fruit crop. However, cross-pollination from other Musa cultivars grown in nearby plantations results in seeded fruit that exceeds market demand. This study investigated pollen viability and germination and examined the expression profiles of pollen development-related genes across seven Musa genomes . Twenty-three Musa cultivars were assessed for pollen viability using lacto-aceto-orcein and triphenyltetrazolium chloride staining methods. Results revealed that pollen viability obtained from both methods was significantly different among all the studied cultivars. Cultivars carrying BB (diploid) genomes had higher viability percentages than AA (diploid), AAA, BBB, AAB and ABB (triploid) and ABBB (tetraploid) genomes. Germination of the studied cultivars was also investigated on pollen culture medium, with results showing significant differences between the pollen of each cultivar. The best germinating cultivar was TKM (11.0 %), carrying BB genome. Expression profiles of pollen development-related genes by RT\u2013qPCR indicated that both TPD1A and MYB80 genes were highly expressed in triploid Musa genomes but the PTC1 gene showed down-regulated expression, resulting in non-viable pollen. Pollen viability, pollen germination and pollen development-related genes differed across Musa cultivars. This knowledge will be useful for the selection of male parents for Musa cross-breeding programs. Pollen viability should also be considered when planning Musa production to avoid seeded fruit.Banana ( Musa genus) possess high pollen viability and germination rates, resulting in the production of seeded fruit, which is undesirable for the market. Our study found that bananas with a BB genome revealing exceptionally elevated pollen viability and germination rates compared to other variants. Moreover, we investigated the expression of three pollen development-related genes. Notably, the TPD1A and MYB80 genes showed significantly high expression in bananas with a triploid genome. However, the PTC1 gene exhibited down-regulated expression, suggesting its role in promoting non-viable pollen. These findings hold significant promise for selecting superior male parents in cross-breeding programmes. By considering pollen viability, banana production can be optimized to meet market demands for seedless fruit and avoid the presence of unwanted seeded varieties.Bananas ( Musa spp.), belonging to the monocotyledon Musaceae family, originated in Southeast Asia  and Musa balbisiana . These hybrids have resulted in high genetic diversity among Musa cultivars. All Musa cultivars can be divided into three genomic groups based on polyploid level as diploid , triploid  and tetraploid  , \u2018Kluai Hom\u2019 (AAA) and \u2018Kluai Namwa\u2019 (ABB) are popularly grown for commercial production with high market demand because seedless fruit has a good taste and sweet smell and is viable for industrial processing. However, Musa plantations often face problems with seed formation caused by cross-pollination from other Musa cultivars grown in nearby areas. The seeded Musa fruit has low market demand and is difficult to process. To solve this limitation, Musa pollen viability and development require study as a useful guideline to prevent seed formation and cross-breeding in mass Musa production to create the desired cultivars.Banana  polyploid levels that impact pollen viability and germination percentages. The diploid Musa genome has the highest pollen viability at 11 and 3 folds higher than triploid and tetraploid genomes, respectively (Musa (AA and BB genomes) has higher viability and pollen germination than triploid genomes  and male (pollen) parents, with success depending on two main mechanisms involved in pollen development (pollen viability) and pollen tube germination. Musa genomes.The staining of pollen using triphenyltetrazolium chloride (TTC) and lacto-aceto-orcein (LAO) has been widely employed to assess pollen viability due to its simplicity and effectiveness . TriphenMusa. Musa itinerans (Ma) was significantly regulated by the functions of three key genes: tapetum determinant 1 (MaTPD1A), persistent tapetal cell 1 (MaPTC1) and myeloblastosis 80 (MaMYB80). The expression of the MaTPD1A gene plays an important role in pollen development by controlling the expression of MaMYB80 and MaPTC1 genes in male flowers. The up-regulated MaTPD1A gene promotes high expression of the MaMYB80 gene but reduces the expression of the MaPTC1 gene, resulting in pollen sterility in male flowers, producing small seedless fruit. However, the expression profile of the MaTPD1A gene in mediating pollen development has not been studied in Musa cultivars of Thai germplasm.Few reports have assessed the molecular mechanisms of pollen development and formation in in vitro germination, and expression profiles of pollen development-related genes across Musa genome groups . Increased knowledge of pollen viability can benefit the selection of male parents for Musa cross-breeding programs to produce seedless fruit.This research determined pollen viability by TTC and LAO staining and Musa cultivars consisting of seven genomes  (Twenty-three Thai  1 ABBB)  were useMusa pollen viability was assessed by staining with two assays. The 1 % LAO assay acted as positively stained chromosomes, modified from Musa pollen grains, modified from Musa  from individual assays was dropped on a glass slide and covered with a glass slip. Pollen viability was observed under a polarized light microscope  with \u00d74 magnification and photographed at 25 fields per sample.Images were recorded to count the total number of pollen grains and viable pollen grains. Pollen grains stained with either LAO or TTC that expressed as a round shape and were dark/light red were considered as viable, while pollen grains that expressed as an irregular or wrinkled shape that were pale red or transparent were considered non-viable.Musa cultivar, while individual cultivars were performed for at least six biological replicates.Numbers of pollen grains from individual samples were collected from five anthers per Musa pollen germination was accomplished on pollen culture medium following the modified assay of Musa samples were placed into 1.5 mL Eppendorf tubes containing 1 mL of sterilized pollen culture medium, and the pollen was isolated from its anther by cutting with a needle. The mixture was incubated at room temperature in the dark for 2 h. At the end of the culture period, the pollen was well mixed and 100 \u00b5L was dropped onto a glass slide and covered with a glass slip. Germination was observed under a polarized microscope (Olympus BX53M) with \u00d74 magnification and photographed at 25 fields per sample.The assessment of Musa cultivar, and individual cultivars were performed for at least six biological replicates.Images were taken to count the total number of pollen grains and number of germinated pollen grains . Pollen grains from the individual samples were collected from five anthers per Musa cultivars, 6 with high (T167 and TA15), medium (KKP and KPB) and low (NMO and NNJ) percentages of pollen viability were selected to determine the gene expression profiles involved in Musa pollen development.Of the 23 studied Musa cultivars, following the modified CTAB method of Musa anthers (100 mg) per sample were ground to fine powder with liquid nitrogen in a mortar and pestle. The powder was transferred into a 1.5 mL Eppendorf tube containing 800 \u00b5L of cetyltrimethylammonium bromide (CTAB) buffer . The mixture was gently vortexed, incubated at 65 \u00b0C for 45 min, allowed to cool at room temperature for 5 min and then centrifuged at 7000\u00d7g at 4 \u00b0C for 5 min. The supernatant was transferred into a new Eppendorf tube and an equal volume of phenol:chloroform:isoamyl alcohol  was added. The mixture was vortexed for 5 min, then centrifuged at 17 500\u00d7g at 4 \u00b0C for 15 min and the supernatant (top layer) was transferred to a new Eppendorf tube. An equal volume of C:I at a ratio of 24:1 was added, mixed gently by turning the tube back and forth several times, and then centrifuged at 17 500\u00d7g at 4 \u00b0C for 15 min. This step was repeated once by adding an equal volume of C:I ratio (24:1). The supernatant (top layer) was then transferred to a new Eppendorf tube and 10 M LiCl at 1/3 volume was added to the supernatant. The mixture was mixed well by inverting the tube and incubated at 4 \u00b0C overnight. It was then centrifuged at 17 500\u00d7g at 4 \u00b0C for 30 min. The suspension was discarded and the total RNA pellet was washed and collected two times with 500 \u00b5L of 70 % ethanol by centrifuging at 17 500\u2005\u00d7\u2005g at 4 \u00b0C for 15 min. The total RNA pellet was air-dried for 10 min and dissolved in 30 \u03bcL of RNase-free water. Subsequently, total RNA was assessed for quality and integrity by gel electrophoresis, with concentration quantified by absorbance measurements at A260 and A280 nm using a NanoDrop Lite Spectrophotometer . Purity, assessed by the ratio of A260 nm and A280 nm, ranged from 1.8 to 2.0. The total RNA was treated with DNase l  to remove genomic DNA contamination according to the manufacturer\u2019s instructions.Total RNA was extracted from the anther samples collected from six 18 primer, 4 \u00b5L 5X Reaction buffer, 1 \u00b5L RiboLock RNase Inhibitor (20 U \u00b5L\u22121), 2 \u00b5L 10 mM dNTP Mix and 1 \u00b5L RevertAid M-MuLV RT (200 U \u00b5L\u22121), with the final volume adjusted to 20 \u00b5L using nuclease-free water. The reaction mixture was incubated as three steps of 25 \u00b0C for 5 min, 42 \u00b0C for 60 min and 70 \u00b0C for 5 min. The first strand cDNA was immediately subjected to RT\u2013qPCR amplification and stored at \u221280 \u00b0C until required for further use.The total RNA template (500 ng) was reversed to the first strand cDNA in a 20 \u00b5L reaction mixture using a RevertAid First Strand cDNA Synthesis Kit  containing 1 \u00b5L Oligo (dT)TPD1A (tapetum determinant 1), PTC1  and MYB80 (myeloblastosis 80), while the CAC (Clathrin adaptor complexes medium) gene was used as the internal control reference for normalization.Four primer pairs designed by the Oligo Analyzer program   were useThe RT\u2013qPCR reaction (12.5 \u00b5L) was performed in Thermo Scientific Maxima SYBR Green qPCR Master Mix (2X)  (6.5 \u00b5L), 10 \u00b5M of each forward (0.5 \u00b5L) and reverse (0.5 \u00b5L) primer, first strand cDNA (1 \u00b5L) and nuclease-free water (4 \u00b5L), using the Eco48 Real-Time PCR system .The RT\u2013qPCR amplification was carried out under the following conditions: 1 cycle of 50 \u00b0C for 2 min and 1 cycle of 95 \u00b0C for 10 min, followed by 40 cycles of 95 \u00b0C for 15 s, 60 \u00b0C for 30 s and 72 \u00b0C for 30 s. Finally, a melting curve was realized by progressively heating the reaction mixture from 55 to 95 \u00b0C using 0.3 \u00b0C increments every 0.75 s to check the purity of the RT\u2013qPCR product. All reactions were technically repeated five times with three biological replicates.CAC as the endogenous control (set to 1). Relative expression of the three target genes was analysed using the comparative Ct method , where \u0394Ct1 represented different expression between target and reference gene in samples of five Musa cultivars , \u0394Ct2 represented different expression between target and reference genes in NNJ samples and \u0394\u0394Ct showed different expression levels between \u0394Ct1 and \u0394Ct2. Normalized target gene expression level was calculated by the comparative Ct method (2\u2212\u0394\u0394Ct) using Microsoft Excel 2010 program.The baseline correction was automatically calculated to determine the cycle threshold (Ct) value in each reaction using Eco 48 Study Software installed in the instrument. Data were normalized with Pollen viability percentage was calculated using the formula of Pollen germination percentage was calculated by the formula of The diameters in micrometres of viable and non-viable pollen grains were measured using the ImageJ program .Pollen tube length in micrometres was measured using the ImageJ program .Musa genotypes) were assessed by Duncan\u2019s Multiple Range Test (DMRT) at P-value\u2005\u2264\u20050.1 statistical significance using the Statistical Product and Service Solution version 17.0 software . All values were expressed as mean\u2005\u00b1\u2005standard error (SE) of three to six biological replicates.All data  were statistically assessed using ANOVA. Mean comparisons between multiple treatments  showed complete male sterility with non-pollen viability detected by TTC assay  with pollen grains smaller than 100 \u00b5m (When comparing the size of the pollen grains (\u00b5m) under a polarized light microscope, viable pollen grain diameter (\u00b5m) in all studied n 100 \u00b5m  and B.Musa cultivars carrying the BB genome  showed highest percentages of pollen viability (98.3 % in LAO and 73.1 % in TTC assay) and number of pollen grains per flower (11 682). By contrast, the Musa cultivar TPR, belonging to ABBB genome had the lowest percentage of pollen viability (8.9 % in LAO and 0 % in TTC assay), with pollen grains per flower (16.1) (Pollen size was related to the pollen viability percentage. The r (16.1) .Musa pollen germination was investigated on pollen culture medium following the modified assay of Musa cultivar had significantly different pollen germination percentages. Musa cultivar TKM gave the highest germination percentage at 11.0 %  showed low pollen germination percentage , medium  and low  were selected to analyse the expression profiles of TPD1A, MYB80 and PTC1 genes by RT\u2013qPCR assay. All data obtained from these target genes were normalized by the CAC reference gene. Overall, the TPD1A gene  in two Musa cultivars  showed higher expression, with statistically significant differences from the other four Musa cultivars   .MYB80 gene showed the highest up-regulation in Musa cultivar NMO (ABB genome), followed by Musa cultivar NNJ (AA genome), which was significantly different at P-value\u2005\u2264\u20050.01  (For genes associated with the middle pathway of pollen development (microspore stage), the e\u2005\u2264\u20050.01 . The MYB genome) .PTC1 gene expression, required in the late stage of pollen development (mature pollen stage), was highly up-regulated in all cultivars with statistical differences. Among the six Musa cultivars, KPB with AAA genome had the highest PTC1 gene expression followed by Musa cultivar T167, which was significantly different from the TA15 cultivar from the BB genome and KKP from the AA genome  gave the lowest PTC1 gene expression (The A genome . Two Muspression .TPD1A and MYB80 gene expressions in Musa triploid genome (ABB and AAA) strongly increased compared to the diploid genome (AA and BB) . Conversely, low TPD1A or MYB80 expression might not decrease expression of the PTC1 gene, suggesting pollen grain development as male fertility.In summary, the  and BB)  and B. Bd genome . The relMusa inflorescences contain two types of female and male flowers. Of these, the male-fertile Musa with viable pollen enabled cross-pollination with the female ovary, resulting in fully seeded fruit that was surplus to commercial market demands but a valuable source for further Musa cultivar improvement in cross-breeding programs  stained specific DNA molecules that were present throughout whole cells, such as mitochondrial and chloroplast DNAs in cytoplasm and chromosomes in the nucleus , medium (16\u201350 %) and low (0\u201315 %)  belonging to the BB genome gave the highest levels of pollen viability at 77.0, 65.5 and 76.7 %, respectively, while two cultivars (TPN and KKP) belonging to ABB and AA genomes had moderate percentages of pollen viability at 18.4 and 16.0 %, respectively. The remaining 18 cultivars exhibited low pollen viability. Results indicated that Musa cultivars carrying diploid genomes (AA and BB) had higher pollen viability than Musa cultivars containing triploid genomes. Similarly, the pollen viability of diploid M. acuminata \u2018Pisang Rejang\u2019 (AA) was higher than triploid, tetraploid and mixoploids  . From TTMusa acuminate parents with different diploid genomic groups (such as A1 and A2 chromosomes)  of Musa impacted production of either partially sterile or fertile pollen. This insight into male sterility located on Musa A and/or B genome requires further investigation.Interestingly, our results showed that LMN, NT and KKP cultivars with AA diploid genomes had different levels of pollen viability. These cultivars were produced by interbreeding of Musa cultivars was not related to pollen viability, concurring with previous findings that pollen size (most non-viable pollen) in triploid and tetraploid Musa was larger than diploid Musa (most viable pollen)  and M. acuminata cultivars KKP and NT (AA genome composition) had high pollen viability and pollen germination percentage, with the longest pollen tube compared to the other genotypes. This finding was supported by observations that Musa AA and BB genomes had high pollen viability and pollen tube germination, with a few differences among them (Musa cultivars (carrying AA or BB genome) might be useful as male parents in Musa breeding programs because both diploids had high percentage of pollen viability and pollen tube germination. These diploid cultivars should also avoid plantations nearby the commercial field of edible Musa production (mostly triploid AAA and AAB genomes) to reduce cross-pollination and set seed in their fruit. The role of pollen development-related genes requires further study to better understand the mechanisms of male fertility and sterility in Musa.Similar results for viable pollen were also observed for higher pollen tube germination under culture medium condition. Among the studied Musa cultivars could be classified into three groups of high , medium  and low . The expression profiles of pollen development-related genes were further evaluated using the RT\u2013qPCR assay. TPD1A, MYB80 and PTC1 were characterized as pollen-specific genes involved in early (microspore mother cell), middle (microspore) and late (mature pollen) pollen development stage, respectively (TPD1A and MYB80 genes were highly expressed in the Musa triploid genome (ABB and AAA) compared to the diploid genome (AA and BB). By contrast, the PTC1 gene in the Musa triploid genome was less expressed than in the diploid genome. One possible explanation of these gene associations is that up-regulated TPD1A and MYB80 genes in the Musa triploid genome might reduce PTC1 gene expression, resulting in non-viable pollen or abnormal pollen development.Molecular aspects of pollen development in flowering plants involve several transcription factors that are tightly regulated by dynamic changes in gene expression (TPD1A overexpression in M. itinerans (AA genome) promoted MYB80 gene expression but suppressed PTC1 gene expression, resulting in absence of pollen in male flowers with the fruit small and seedless  gave the highest germination percentage assessed by a pollen culture method. For the expression of pollen development-related genes, the TPD1A and MYB80 genes were up-regulated but the PTC1 gene was down-regulated in the Musa triploid genome, resulting in non-viable pollen. Knowledge of pollen viability is important when selecting male breeders for Musa cross-breeding programs to prevent seed formation in the fruit.This study assessed 23 plad052_suppl_Supplementary_DataClick here for additional data file."}
+{"text": "This study explores the relationship between perceived school climate and exercise behavior among obese adolescents, as well as the multiple mediating effects of perseverance qualities and exercise benefits.t-test, bivariate correlation analysis, descriptive statistical analysis, and structural equation model (SEM).A survey was conducted on 586 obese adolescents in Beijing, with an age range of 13\u201318\u2009years old and an average age of 15.40\u2009\u00b1\u20091.824, among who 337 were male, 249 were female, 303 were high school students and 238 were middle school students. A standard scale was used to evaluate perceived school climate, exercise benefits, perseverance qualities, and exercise behaviors. The data was analyzed by independent samples Z\u2009=\u20092.870, p\u2009<\u20090.01), perseverance qualities  and exercise benefits ; perseverance qualities positively predicted exercise behavior in obese adolescents ; exercise benefits positively predicted the obese adolescents\u2019 exercise behavior . (2) Perseverance qualities  and exercise benefits  play a mediating role in the obese adolescents\u2019 perceived school climate and exercise behavior, respectively. These two factors have parallel multiple mediating effects between obese adolescents\u2019 perceived school climate and exercise behavior, with mediating effects accounting for 16 and 25%, respectively. The mediating effect of exercise benefits is greater than that of perseverance qualities. (3) There is no difference in the specific indirect effects of perseverance qualities and exercise benefits .(1) Perceived school climate among obese adolescents positively predicted exercise behavior (Obese adolescents\u2019 perception of school climate can effectively enhance their motivation to participate in exercise behavior and indirectly influence exercise behavior through exercise benefits and perseverance qualities, cultivate good physical exercise behavior among obese adolescents, and effectively prevent and intervene in the occurrence of obesity. The increasing prevalence of obesity among adolescents is a serious public health issue. Obesity is a chronic disease, mainly caused by an energy imbalance between excessive calorie intake from food, calories consumption or insufficient physical activity, resulting in the enlargement or increase of fat cells .The Report on the Nutrition and Chronic Disease Status of Chinese Residents (2020) shows that the overweight and obesity rate of children and adolescents aged 6\u201317 has risen to 19%. The China Children\u2019s Obesity Report points out that the overweight and obesity rate of children and adolescents in China is also constantly rising, and if without intervention, the number of overweight and obese children aged 7 and above will rise to nearly 50 million by 2030 , but also influenced by environmental stimuli . For teePerseverance qualities refer to an individual\u2019s tendency to strive to achieve long-term goals and maintain passion and effort, especially in situations of challenges and setbacks, which can help individuals better adapt to the environment . CurrentExercise benefits perception refers to the obese adolescents\u2019 perception of various benefits of physical activity, and physical exercise benefits refer to an individual\u2019s intuitive evaluation and perception of the results of accompanying physical exercise behavior . A reasoIn summary, there is currently limited empirical research in the field of physical education on the impact of perceived school climate on students\u2019 psychological aspects of exercise behavior among obese adolescents. From the perspective of the psychology of exercise behavior, this study constructs a path model for the influence of exercise behavior intention among obese adolescents with dual mediating effects of exercise benefits and perseverance qualities, and analyses the mechanism of action to enrich the theory of exercise behavior among obese adolescents, and ultimately, it encourages them to consciously form the practical significance of adherence to physical exercise. Based on the above theoretical assumptions, a theoretical model of the relationship between perceived school climate and physical exercise behavior was constructed see .2, and BMI\u2009\u2265\u200928 can be determined as obesity. Sixty eight teaching classes from 10 ordinary high schools in Beijing were selected, and 600 obese adolescents were screened through the results of the physical fitness test and physical education teacher sampling to conduct a questionnaire survey on the relationship between perceived school climate and exercise behavior. After removing invalid questionnaires, 586 valid questionnaires were obtained, with a valid questionnaire rate of 98%. The age range of the subjects was 13\u201318\u2009years old, with an average age of 15.40\u2009\u00b1\u20091.824, including 93 senior 1 students, 119 senior 2 students, 91 senior 3 students, 142 junior 1 students, 85 junior 2 students, and 56 junior 3 students; 337 male students (57.5%) and 249 female students (42.5%), 303 were senior high school students, and 238 were junior high school students.The Body Mass Index (BMI) method was adopted as the standard for selecting obese adolescents, and the calculation formula is: BMI = weight[kg] \u00f7 (height[m])Declaration of Helsinki. Students were informed prior to the survey that the questionnaires were answered anonymously, emphasizing voluntary completion. The content is strictly confidential and the results are only used for scientific research. All questionnaires were collected on the spot. All subjects were informed of the purpose and characteristics of the study and signed an informed consent form.This study was approved by the Ethics Committee of Xiangya School of Public Health, Central South University . and conducted in accordance with the guidelines of The Physical Activity Rating Scale (PARS-3) revised by The Perceived School Climate Scale developed by Adopting the top 5 items in the study by t-test was used to analyze the differences in direct quantitative data between two different clusters. The obtained data was tested using the Harman\u2019s single-factor method. Then, bivariate correlation analysis was used to analyze the correlation between various dimensions and measure the degree of correlation between the two variable factors. The statistical description is expressed in terms of mean and standard deviation (M\u2009\u00b1\u2009SD).Firstly, SPSS 26.0  was used to input and organize the collected data. Independent samples p\u2009<\u20090.05 and Z\u2009>\u20091.96.Structural Equation Model (SEM) was then used to estimate the correlations between physical exercise, potential influencing factors and pathways. The AMOS 24.0  maximum likelihood estimation method was used for SEM, and the applicability of the model was determined through a variety of indices, including the ratio of the minimum difference to degree of freedom (CMIN/DF), the Goodness of Fit Index (GFI), Adjusted Goodness of Fit Index (AGFI), Comparative Fit Index (CFI) and Standardized Root Mean Square Residual (SRMR) . Direct Finally, a two factor mediating effect analysis was conducted to determine whether the relationship between perceived school climate and exercise behavior was indirectly influenced by perseverance qualities and exercise benefits. 5,000 Bootstrap multiple mediating effects tests were conducted using the syntax in AMOS 24.0, including direct effects, indirect effects, and specific indirect effects to compare differences. The test results are mainly determined through the bias-corrected 95% and Percentile 95% estimation methods to determine whether the confidence interval obtains contain 0.t-tests to determine whether there were differences between different genders in 4 dimensions of perceived school climate, perseverance qualities, exercise benefits and exercise behavior. The results showed that there were no significant differences between different genders and these 4 dimensions at the 0.05 level, as detailed in The study used independent sample t-tests were used to determine whether there are differences between different grades in 4 dimensions of perceived school climate, perseverance qualities, exercise benefits, and exercise behavior. The results show that there is no significant difference between different grades and these 4 dimensions at the 0.05 level, as shown in Independent sample Common method biases refer to the artificial covariation between predictor and criterion variables due to the same data source or rater, measurement environment, project context, and project characteristics. In order to prevent the possibility of common method bias in the collected data, corresponding control measures have been taken during the testing process. The Harman single factor method was used to test for covariance. The test criteria were: there were more than one factor with a characteristic root greater than 1 and the explanatory degree of the maximum factor\u2019s variance was less than 40%. The results of this study show that there are a total of 7 factors with eigenvalues greater than 1 and the variance explained by the first factor was 24.17%, which is less than the critical criterion of 40%. The extraction amount of common variance is higher than 70% (74.02), which indicates that there is no common method bias .In this study, the AVE method was used to test the discriminant validity of each dimension. The AVE method was proposed by Fornell and Larcher, which means that the average variance extraction amount of each dimension must be greater than the square value of the correlation coefficients between each dimension. However, since the AVE is a square value, it must first be converted to the same square unit if it is to be compared with Pearson correlation between dimensions. Therefore, the AVE value was opened and rooted before comparison could be made. Therefore, the AVE value was marked with a root before it could be compared. If it is higher than the Pearson correlation value between dimensions, it can be declared that the dimension has differential validity. In this study, except for the exercise behavior dimension of 0.458, all other dimensions were greater than 0.5, which is in line with the criteria proposed by r\u2009=\u20090.098\u20130.438, p\u2009<\u20090.01 or p\u2009<\u20090.05). Peer support is significantly and positively correlated with autonomy opportunity, exercise behavior and exercise benefits . Autonomous opportunity is significantly positively correlated with unremitting effort, persistent enthusiasm, exercise behavior and exercise benefits . Unremitting effort is significantly positively correlated with persistent enthusiasm  and exercise behavior . Persistent enthusiasm is significantly positively correlated with exercise behavior  and exercise benefits . Exercise behavior is significantly positively correlated with exercise benefits .The correlational analysis of obese adolescents\u2019 perceived school climate , perseverance qualities , exercise benefits, and exercise behavior  shows thThe model fitting test result of the relationship model between perceived school climate and exercise behavior among obese adolescents constructed in this study are good. CMIN/DF\u2009=\u20091.78\u2009<\u20093; GFI\u2009=\u20090.887\u2009>\u20090.8 acceptable, AGFI\u2009=\u20090.875\u2009>\u20090.8 acceptable, IFI\u2009=\u20090.970, CFI\u2009=\u20090.970, TLI\u2009=\u20090.968, RMSEA\u2009=\u20090.036\u2009<\u20090.08, SRMR\u2009=\u20090.036\u2009<\u20090.05, and above empirical path analysis goodness-of-fit indicators all meet the empirical rule of thumb criteria suggested by general scholars . It can p\u2009<\u20090.05 and Z\u2009>\u20091.96.The direct path results of the relationship between perceived school climate and exercise behavior among obese adolescents see  show thaThe standardized path coefficient mainly evaluate the significance of each path coefficient. The results showed that among the path coefficients of exercise behavior as the dependent variable, the absolute value path coefficient of exercise benefits was the highest, with a standardized absolute value path coefficient of 0.274, accounting for 23.9% of the total effect. The path coefficient of perseverance qualities was 0.261, accounting for 22.8% of the total effect. The minimum path coefficient of perceived school climate was the smallest, 0.169, accounting for 14.8% of the total effect. In the direct influence path coefficient of perceived school climate as an independent variable, the maximum path of exercise benefits in the standardized coefficient is the highest, 0.265, accounting for 23.1% of the total effect. The path coefficient of perseverance qualities is 0.176, accounting for 15.4% of the total effect. The above data shows that the proportion of coefficient effects and total effects of each path is different, with H5\u2009>\u2009H4\u2009>\u2009H3\u2009>\u2009H2\u2009>\u2009H1.Z-test are also statistical methods for testing the existence of mediating effects. In the causal path test , with skewness and kurtosis not zero , with a Z-value greater than 1.96 and a 95% CI confidence interval that does not include 0, suggesting that perceived school climate is a significant factor in predicting exercise behavior. The mediating effect of perseverance qualities on perceived school climate and exercise behavior , and the indirect effect of exercise benefits on perceived school climate and exercise behavior also exists . Among them, the perceived school climate has the greatest direct effect on exercise behavior, with an effect value of 0.330, accounting for 59%. In the indirect effect, the perceived school climate -\u2009>\u2009exercise benefits -\u2009>\u2009exercise behavior path effect value is 0.141, accounting for 25%. The perceived school climate -\u2009>\u2009perseverance qualities -\u2009>\u2009exercise behavior path effect value is 0.089, accounting for 16%. There was no significant difference in the mediating effect of exercise benefits compared to perseverance qualities . The Z-value was less than 1.96 and the 95% CI confidence interval include 0. However, the mediating factor of exercise benefits was still more important than the mediating factor of perseverance qualities (0.141\u2009>\u20090.089).This study used Bootstrap 5,000 to test the mediating effect and whether there were differences in specific indirect effects. 5,000 Bootstrap samples were extracted and put back in the original data to form an approximate sampling distribution, and indirect effects, direct effects and specific indirect effects were tested for differences. The test results are as follows see : Perceiv\u03b2\u2009=\u20090.97, p\u2009<\u20090.001), this study perceived that the school climate environment positively predicted exercise behavior . The lack of school climate is not effective in intervening in physical exercise among adolescents . Obese adolescents perceive that school climate has a positive contributor to exercise behavior, and intervention in the environment can effectively increase their participation in physical exercise. The research has pointed out that school based physical exercise interventions mainly focus on students\u2019 motivation, basic psychological needs, goal orientation, enjoyment and stimulating teaching atmosphere in physical education  are associated with physical exercise among adolescents. In one study, students, teachers, principals and parents recognized that the overall school climate indeed promotes children\u2019s participation in physical activity . The clilescents . Perceivlescents , and phylescents . There ilescents , persevelescents  (\u03b2\u2009=\u20090.2ducation . Exercist\u2009=\u20095.092). Teachers are able to help students establish correct beliefs and behaviors by constructing supportive relationships  , and some studies have shown a certain relationship between family environment and perceived exercise benefits  , consistbenefits . This stbenefits , with anThis is a cross-sectional study that is consistent with the research methods of Insufficient physical activity is not only common in high-income countries, but also in low-income and middle-income countries (LMICs). According to reports, the physical activity levels of Asian school-age children and adolescents are also low . DiversiSchool policies can also reduce unhealthy dietary behaviors and obesity risk among children in mega cities in China . SchoolsThis study focuses on the impact of perceived school climate on exercise behavior, advancing relevant research in the fields of perceived school climate and exercise behavior. By constructing a dual mediation model and starting from the perspective of exercise psychology, it explores the relationship between obese adolescents\u2019 perceived school climate and exercise behavior, which has practical significance in promoting their participation in physical exercise. Firstly, the impact of school climate on exercise behavior among obese adolescents was confirmed, and the relationship between perceived school climate and exercise behavior among obese adolescents was interpreted from the perspectives of perseverance qualities and exercise benefits. Secondly, the differences in mediating effects and specific indirect effects were examined.There are also limitations in this study. Firstly, self-reporting may raise the correlation between variables and lead to common method biases. Therefore, the study tests for common method bias to avoid this limitation as much as possible, but there are still some uncontrollable factors that affect the research results, such as: self-expectation and participants\u2019 response emotions. In the future, longitudinal tracking or experimental intervention designs can be applied to more effectively explain the impact of perceived school climate on exercise behavior among obese adolescents. Secondly, this study only examined the mediating effect of perseverance qualities and exercise benefits. Future studies will continue to examine variables closely related to exercise behavior, such as self-efficacy, sensory seeking and psychological resilience, in order to comprehensively reveal the relationship model between perceived school climate and physical activity behavior among obese adolescents.Obese adolescents\u2019 perceived school climate positively predicts exercise behavior, perseverance qualities and exercise benefits. Perseverance qualities and exercise benefits positively predict exercise behavior among obese adolescents. Perseverance qualities and exercise benefits play a mediating role between perceived school climate and exercise behavior among obese adolescents, respectively. These two factors have parallel multiple mediating effects between obese adolescents\u2019 perceived school climate and exercise behavior, with mediating effects accounting for 16 and 25%, respectively. The mediating effect of exercise benefits is greater than that of perseverance qualities. There is no difference in the test results of specific indirect effect between perseverance qualities and exercise benefits. Therefore, in the process of improving the exercise behavior among obese adolescents, it is recommended to start by strengthening their perception of the school climate, focusing on enhancing their perception of the exercise benefits as well as their willpower to persist in exercise. By improving their cognitive level of willpower and exercise benefits, it is recommended to promote the improvement of their exercise behavior, cultivate lifelong exercise awareness, and develop a healthy lifestyle.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by the Ethics Committee of Xiangya School of Public Health, Central South University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants\u2019 legal guardians/next of kin.YY contributed to the evaluation and interpretation of data, writing the first drafts, final version of the manuscript, and scrub data and maintain research data for initial use and later re-use. YQ contributed to the statistical analysis of data and development of the review concept, critically reviewed the manuscript, and participated in the interpretation and synthesis of data. CZ contributed to application of statistical to analyze and synthesize study data. CQ and ZC contributed to verification of the overall replication of results and other research outputs. All authors have read and agreed to the published version of the manuscript."
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