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+{"text": "By combining ontologies from different sources the authors developed a novel approach to describing phenotypes of mutant mice in a standard, structured manner. The mouse is an important model of human genetic disease. Describing phenotypes of mutant mice in a standard, structured manner that will facilitate data mining is a major challenge for bioinformatics. Here we describe a novel, compositional approach to this problem which combines core ontologies from a variety of sources. This produces a framework with greater flexibility, power and economy than previous approaches. We discuss some of the issues this approach raises. N-ethyl-N-nitrosourea (ENU) mutagenesis .\" The authors also describe the assays used to record these observations: \"Nesting Patterns: six cages of wild-type and six cages of mutant mice (N = 4 mice per cage) were used to evaluate nesting patterns. A 5 \u00d7 5 cm piece of cotton nesting material  was placed in each cage. After 45 min, photographs were taken of each nest and the nest depth was measured. Nest height data were analyzed using the Student's t test.\"In this section we describe an example of the application of the compositional schema. We chose a phenotype example at random from the MP database: 'nest building' [MP:0001447]. Several descriptions of nest-building patterns can be found in the corresponding reference . For exaFor some users/applications the compound term 'abnormal nest building' might be a sufficient description of this particular phenotypic instance, but this would result in information loss. A human would have to retrieve and read the reference to extract further information. Our schema allows the expression of this information in a machine and human readable manner. In Table Nest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [well-formedPatterns ) {returnNest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [poorly-formedPatterns ) {returnNest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [fluffyPatterns ) {returnWe note here that had the value 'fluffy' not been included in the standard values for a quality assay, it could be captured in the free-text field provided by the schema. To express a nest of 50 mm depth or significantly shallower:Nest building {has_attribute} attribute:absolute_depth {characterized_by} undefined_ absolute_depth_assay {returns_value} 50 mmNest building {has_attribute} attribute: relative_depth {characterized_by} undefined_ relative_depth_assay {returns_value} shallow {has_qualifier} significantWith this information one could go back to a higher level and still be able to express a more general characterization of this phenotype as 'abnormal nest building' but obviously the opposite is not possible.An important unresolved issue concerning the use of ontologies to describe phenotypes arises from the fact that all the ontological structures developed so far are designed to describe individual mice. Mutagenesis experiments usually characterize a number of mutant mice to take into account variable penetrance of the mutation and other stochastic effects. A strategy will therefore need to be developed to describe the generalized phenotypic properties of a cohort of mice. This may involve the use of more sophisticated relations such as {usually characterized by} or even quantitative relations such as {80% characterized by}.The assay plays a central role in our schema Figure . Assays eye {has_attribute} attribute:color {characterized_by} visual inspection {returned_value} pinkOn a practical level, assays can add specificity and functionality to the relationship between entities, their attributes and the corresponding values. Most important, an assay vocabulary allows the entire schema to be dynamic by including new assays and capturing explicit differences between assays in different laboratories. The assay will also allow standardization and definition of values for a given phenotypic character, for example, how abnormal is defined in relation to body position.Our schema can be expressed using a variety of modeling tools and knowledge representation (KR) languages . We chosThe schema was designed to be easily populated using extant core ontologies, such as anatomy, and defining attributes related to each entity. The assay vocabulary can be constructed as required. Permitted values are defined in the range of different assay attributes in part devised in the form of a general scheme and in part built from the output of particular assays. Although we include for demonstration purposes three core ontologies, namely behavior, anatomy, and developmental anatomy Figures  and 4, wFigure et al. [Since most of the ontologies we are planning to use were generated using the DAG-edit  format, et al. , with miDecisions will inevitably have to be made to combine a core ontology with its attributes and then define facets of that relationship, for example, cardinality, attribute value type and attribute range. In our schema, the class hierarchy of all ontologies employed represents an 'is-a' relation. So, mouse social behavior 'is-a' mouse behavior, or mouse social behavior is a 'kind-of' mouse behavior and so forth. All other relationships, including PATO and 'part-of' relationships, are modeled as attributes. However, we note here that efforts are currently being made by the GO consortium to define and formalize the 'part-of' relationship, which is considered vital and special in bio-ontologies, especially anatomy .Because our phenotype ontology and PATO need to be the result of a collaborative effort within the communities, we feel that it is important to set out the basic modeling concepts that need to be applied upon allocating attributes to the core ontologies. Deciding whether to introduce a new attribute or represent this functionality through an entity is often quite difficult. Several things need to be considered in order to make the best decision, although it should be noted that there are no clear distinction as to what is a right or wrong decision.The first thing to take into account is that subclasses of a class inherit all properties of the parent and could have additional properties and different restrictions from the latter. PATO should remain as general as possible, and, when possible, care should be taken to avoid making PATO domain specific. For example, in the behavior ontology there is a class named 'reflexes' that contains children such as 'blinking reflex', 'Preyer reflex' and 'righting reflex'. It might be worth considering having one 'attribute of reflex' available in PATO rather than creating a separate attribute 'of' for each individual reflex, such as 'attribute of blinking reflex', 'attribute of Preyer reflex', and so on. Then again, if one wishes to assign different functionalities to these properties, creating separate attributes might be useful. As a rule though, one should consider that PATO needs to be consistent, usable and interoperable if it is to be applied to the general domain of phenotypes. Repetition between core ontologies and PATO should be avoided where possible.What is also often not clear is whether one should add a new class to represent functionality or assign attributes to already existent classes. For example, think of the entity 'body position'. There are several ways to model this entity in the mouse behavior phenotype ontology. One could declare 'body position' as a child of a class called 'posture'. An 'attribute of body position' could then be assigned to this class with a range of values that might be specific to an assay, for example SHIRPA  allows t'Body position' could also be split into an entity of 'body' and an attribute of 'position'. Again, a new class 'body position' should be assigned, if one considers the objects with different attributes as different kind of object and this distinction important in the domain. As a general rule, before assigning new classes and attributes one should consider the functionality and their role in the domain, creating more distinctions as the depth of knowledge that is required to be expressed in the ontology increases.Classes in the hierarchy should not necessarily have to introduce new properties . AlthougWe have presented here an approach to the use of ontologies in describing mouse phenotypes that could provide a platform for the consistent representation of mouse phenotypic data. We have also described in detail a possible methodology to construct applications of this schema across different domains. We have dealt with modeling issues and provide guidelines to deal with semantic and practical problems.We maintain that such modeling efforts in any domain should be done in a collaborative fashion in the community. Repetition between different parts of the mouse phenotype ontologies is unavoidable. However, the use of consistent IDs, synonyms and records for associated annotations could allow seamless integration of ontology products. The nature of the schema proposed, as well as its components, is extremely dynamic; therefore coordination of efforts is vital.The structure allows extensibility and interoperability. Although an ontology should not cover all possible information about a domain, the main idea behind this concept is to allow the phenotype ontology to cope with novel and unpredictable phenotypes and account for new assays, serving scientific autonomy and information validity and integrity. We have built a software system  which in"}
+{"text": "Salmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest.Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection.Strains were selected from patients with mild infections (n = 9) and patients with severe infections (n = 9) and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3) were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.We investigated clinical significance of known virulence factors of  Salmonella enterica subsp. enterica is one of the leading food-borne pathogens. For example in 2006 in the United States, Salmonella enterica subsp. enterica caused 45.808 registered cases of salmonellosis, corresponding to an incidence of 15 cases/100,000 inhabitants [Salmonella serotype Typhimurium, denoted S. Typhimurium, accounted for 17% of the salmonellosis cases in the USA and 25% of the Danish cases [Globally, abitants . Salmonesh cases . The outS. Typhimurium [Several factors in both the host and the bacteria influence the outcome of an infection. Clearly an important aspect of human infection is the immune state of the patient. It has been shown that immunocompromised patients are more prone to develop a severe infection . Anotherhimurium .Salmonella Pathogenicity Islands (SPIs). A total of 14 SPIs have been described so far [Significant bacterial factors for the outcome of infection are encoded by a wide range of genetic elements, including plasmids, prophages and d so far . SPI-1 ed so far .S. Typhimurium strains share more than 99% genomic content [S. Typhimurium is primarily represented by the prophages in the genome [S. Typhimurium strains, and some of them can even show a high degree of variation in host adaptation [S. Typhimurium, in particular, the Salmonella Virulence Plasmid (pSLT) which was observed more frequently in the strains isolated from blood than the strains isolated from faeces [Different  content . The detaptation . Intra-sm faeces . It has m faeces .S. Typhimurium strains [The recent development of microarray technology has allowed an extensive screening of many  strains -15, but S. Typhimurium strains by DNA microarray analysis. These strains were selected on the basis of a previous epidemiological study where clinical data were obtained by means of patient interviews. The strains were selected from patients with mild infections and from patients with severe infections, and clinical data allowed us to correct for known underlying diseases and patient age. Strains were analyzed for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype, and metabolism. We show that S. Typhimurium strains causing very different symptoms in patients had little genomic variation, and the observed variation does not correlate to the severity of disease.We analyzed a collection of Xba I PFGE profiles of all isolates).All strains were subtyped by Pulsed-field gel electrophoresis (PFGE), Multiple-locus variable-number of tandem-repeat analysis (MLVA) and Multilocus sequence typing (MLST). In general, the PFGE types of the strains correspond to the phagetype. All of the phagetype DT12 strains had the PFGE 22 profile and five out of six DT104 strains had the PFGE 14 profile. The remaining phagetypes showed different PFGE profiles  of the strains were primarily ST19. Only three strains had other STs and these were ST376, ST35 and ST34 .The pathogenicity marker group consisted of 87 markers and the strains had a highly similar pattern of present/absent genes within this marker group. Only the DT104 strains showed variation, as they lacked the The SPI-1 to SPI-5 were present and SPI-7 was absent in all strains  was detected in different phagetypes in this study, but notably this prophage is present in all DT104 strains, and these observations corresponded to previous findings [S. Typhi specific genes STY3672, STY3676 and STY4625. The markers of these genes were detected in three S. Typhimurium strains. The occurrence of these genes could be explained by lacking specificity of the probe, or by point mutations in the S. Typhimurium strains leading to cross-hybridization. Prophages are known to contribute to virulence in mice [Differences between the  strains . The Salfindings . Other g in mice  but presThe mobility marker group also displayed variation between strains, but most variation related to incompatibility groups of plasmids and probes encoding transposons. The variation did not correspond to any phagetypes or disease symptoms.hldD gene and the irsA gene, but these genes have previously been shown to be specific for the DT104 phagetype [S. Typhimurium LT2 strain [S. Typhi [S. Dublin and S. Paratyphi C [S. Typhimurium strains are representative for the Danish S. Typhimurium population regarding the presence of pSLT, as 72% of all Danish S. Typhimurium isolates from 2005 until 2009 carried the plasmid. Out of five strains lacking the pSLT, three had caused severe symptoms. Interestingly, strains can cause infection with severe symptoms even if they lack the plasmid. Furthermore, strains can carry the pSLT and only cause infection with mild symptoms. In this study, the presence or absence of pSLT did not correspond to any phagetypes or disease symptoms.The strains showed highly similar profiles when comparing the virulence associated genes. Some variation was detected between other phagetypes and the DT104 strains which were the only strains containing the hagetype . Also th2 strain . The stuS. Typhi , and simatyphi C . The pSLatyphi C , howeverS. Typhimurium infections.The dendrogram calculated on the basis of the array results showed clustering of the strains into four groups. The clustering confirmed DT104 as being a clonal phagetype, but a number of probes were also designed to target only DT104 strains, and that might emphasize the separate clustering of this phagetype. The clustering of the remaining groups was mainly correlated to the presence or absence of the pSLT and other mobile elements such as transposons. The four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of S. Typhimurium LT2 sequence, but also some additional known genes from other serotypes such as S. Enteritidis and S. Typhi. The presence or absence of additional S. Typhimurium genes, which are not present in the LT2 sequence, could not be assessed in this study. It is possible that the presence or absence of such genes, not present in LT2, are responsible for the observed differences in the patient symptoms. Although this is not likely, as recent publications of sequenced S. Typhimurium strains showed few gene differences to the LT2 sequenced strain [The probes on the array were designed primarily on basis of the d strain ,29.Salmonella strains for the presence of a wide range of known virulence genes, and detected no significant difference in the presence of these genes. The investigated strains were carefully selected, based on epidemiological data, to represent strains causing severe symptoms of disease and strains causing mild symptoms of disease. Although the investigated strains had different genomic contents, this study found no evidence of a correlation between the genomic contents of the S. Typhimurium strains and the symptoms they caused in human cases of salmonellosis. Based on the results of this study, an idea which immediately suggests itself is that the factors and defence mechanisms of the host immune system may play a fundamental role in the different outcomes of infection.We investigated a collection of S. Typhimurium infection, identified by the examination of samples submitted to Statens Serum Institut (SSI) from hospitals and general practitioners. Patients were invited to participate by their own physicians or the relevant hospital department. Individuals who agreed to participate were mailed a questionnaire and asked to complete the questionnaire immediately. Data was collected by a computer-assisted telephone interviewing system (CATI) whilst the subjects were looking at their questionnaire. This method facilitated data collection and allowed standardized probing about relevant exposures and outcomes. Data collected included information on clinical symptoms, treatment, medications  from one month before infection to one month after, underlying illnesses, foreign travel during the two weeks prior to inclusion and basic socioeconomic variables i.e. education, occupation and household income. Cases were excluded for the following reasons: an earlier history of Salmonella infection within six month prior to onset of the current infection, lack of communication skills in Danish, or if the patient's health care provider deemed the patient unable to complete interview due to an impaired mental state. A parent was interviewed if the patient was under 15 years of age and if the patients were between 15 and 18 years of age they could be interviewed - subject to parental approval. This study was reported to The Danish Data Protection Agency and has been approved by the regional scientific ethical committee of Copenhagen and Frederiksberg Municipality (KEF 01-031/01).Data for the present study was obtained from a prospective cohort study carried out in Denmark from September 2001 to December 2002 . Cases wS. Typhimurium were chosen based on the previously described patient interviews. Strains from patients over the age of 65 years and strains from patients with known underlying diseases were not included in this study. The patients were sorted according to hospitalization data and fever. Two groups were then established: a severe infection group with patients who were hospitalized due to their S. Typhimurium infection and also had a fever; and a mild infection group with patients who were not hospitalized and did not have a fever. From each of these groups nine strains were selected, aiming to represent the same phagetypes in each group . Of the nine hospitalized patients, four had bloody stools. Furthermore, three outbreak strains were included in the study, representing strains with known high virulence potential. All faecal samples received at SSI in Denmark are screened for double infection with frequently occurring intestinal pathogens such as Campylobacter, Shigella, Yersinia and others. All strains used in this study were confirmed as originating from a single-organism infection.Faecal strains of up Table . The phaSalmonella enterica serotype Typhimurium.All strains were previously serotyped at SSI according to the White-Kauffmann-Le Minor scheme  by aggluhttp://www.danmap.org was determined by microbroth dilution and interpreted according to Clinical and Laboratory Standards Institute guidelines [Susceptibility to a standard panel of antimicrobial agents idelines , except Phage typing was performed by the National Food Institute, Technical University of Denmark according to the phage typing scheme developed by Callow  and exteSalmonella. The microarray used gives no information on the location of a gene or target sequence and can only score its presence or absence. Uncertain array results were resolved by PCR using primers described previously [The DNA microarray used in this study was previously described . A set oeviously .Analysis of the DNA microarray data was performed as previously described . A compaMLVA was performed as described previously  to ensurXbaI restriction enzyme according to the Pulse-Net protocol [Xba I PFGE profiles of all isolates).PFGE was carried out with protocol , gels wehttp://mlst.ucc.ie/mlst/mlst/dbs/Senterica/.Multilocus sequence typing (MLST) was carried out as previously described  and the EL carried out the microarray experiments, the MLST analysis, participated in the study design and drafted the manuscript. MT participated in conceiving and designing the study. BM designed the microarray. SH participated in the microarray experiments and participated in drafting the Methods section. MH carried out the patient interviews and the epidemiological analysis and participated in drafting the Methods section. HC participated in conceiving and designing the study. EMN participated in conceiving and designing the study. All authors read and approved the final manuscript.PFGE profiles. Xba I PFGE profiles of all isolatesClick here for fileTyping results of all strainsClick here for fileMicroarray results of all markers. Markers are listed alphabetically within marker groups. A grey box indicates the marker being present and a white box indicates the marker being absent.Click here for file"}
+{"text": "Small intestinal neuroendocrine tumors (SI-NETs) are typically slow-growing tumors that have metastasized already at the time of diagnosis. The purpose of the present study was to further refine and define regions of recurrent copy number (CN) alterations (CNA) in SI-NETs.U test or Fisher\u2019s exact test were used to evaluate associations between tumor groups and recurrent CNAs or clinical parameters.Genome-wide CNAs was determined by applying array CGH (a-CGH) on SI-NETs including 18 primary tumors and 12 metastases. Quantitative PCR analysis (qPCR) was used to confirm CNAs detected by a-CGH as well as to detect CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering was used to detect tumor groups with similar patterns of chromosomal alterations based on recurrent regions of CN loss or gain. The log rank test was used to calculate overall survival. Mann\u2013Whitney The most frequent abnormality was loss of chromosome 18 observed in 70% of the cases. CN losses were also frequently found of chromosomes 11 (23%), 16 (20%), and 9 (20%), with regions of recurrent CN loss identified in 11q23.1-qter, 16q12.2-qter, 9pter-p13.2 and 9p13.1-11.2. Gains were most frequently detected in chromosomes 14 (43%), 20 (37%), 4 (27%), and 5 (23%) with recurrent regions of CN gain located to 14q11.2, 14q32.2-32.31, 20pter-p11.21, 20q11.1-11.21, 20q12-qter, 4 and 5. qPCR analysis confirmed most CNAs detected by a-CGH as well as revealed CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering of recurrent regions of CNAs revealed two separate tumor groups and 5 chromosomal clusters. Loss of chromosomes 18, 16 and 11 and again of chromosome 20 were found in both tumor groups. Tumor group II was enriched for alterations in chromosome cluster-d, including gain of chromosomes 4, 5, 7, 14 and gain of 20 in chromosome cluster-b. Gain in 20pter-p11.21 was associated with short survival. Statistically significant differences were observed between primary tumors and metastases for loss of 16q and gain of 7.Our results revealed recurrent CNAs in several candidate regions with a potential role in SI-NET development. Distinct genetic alterations and pathways are involved in tumorigenesis of SI-NETs. Small intestinal neuroendocrine tumor (SI-NET) arising from enterochromaffin cells is the most common type of gastrointestinal endocrine tumor. SI-NET is also termed midgut carcinoids, well-differentiated neuroendocrine tumor of the midgut, ileal carcinoid or neuroendocrine tumor of the midgut ,2. The tSeveral molecular cytogenetic studies have been performed with the aim of understanding the mechanisms of SI-NET development. Using conventional comparative genomic hybridization (CGH) we and others observed frequent copy number (CN) losses at 18q, 11q, 16q, and gains of 4p ,13. The To further refine and define regions of recurrent CN aberrations (CNAs) we applied a-CGH and genomic quantitative real-time PCR (qPCR) to a panel of SI-NETs from 32 patients and evaluated the findings to the clinical parameters and patient outcome. We also aimed to identify differences in CNA profiles between primary tumors and metastases.Fresh frozen samples from patients with sporadic SI-NETs were included in this study. All tumor samples were obtained from patients operated for SI-NETs at the Karolinska University Hospital-Solna since 1989 and had been collected and stored at the Karolinska tissue biobank. All samples were obtained with informed oral consent and ethical approval from the local ethical committee of Karolinska Institutet. A standardized procedure of tumor collection was employed including macroscopical dissection by a histopathologist, snap freezing and storage at \u221280\u00b0C until use. The histopathological diagnosis was established at routine examination according to published criteria . The immTotally 30 tumor samples (19 primary tumors and 11 metastases) from 29 patients  were collected for the a-CGH screening. For the subsequent qPCR analyses of selected loci the tumor sample panel was extended to a total of 43 tumor samples from 32 patients. The clinical data concerning gender, age at diagnosis of the primary tumor, functioning tumor, previous SI-NET surgery, metastasis and follow-up are detailed in Additional file Genomic DNA was either available from our published series of SI-NETs  or was ihttp://www.lu.se/sciblu) were used. The 33 K and 38 K array slides contained 33,370 and 38,000 BAC clones, respectively (CHORI BACPAC resources) (http://bacpac.chori.org/ genomicRearrays.php) giving a resolution of one clone per 50\u2013100 kb. Information about experimental procedures and data analyses have previously been described in detail . Pooled normal blood DNA  was used as calibrator and a normal mucosal intestine DNA as normal control. CNs were predicted by Copy Caller v1.0 software (Applied Biosystems).qPCR analysis of CNs was applied to all 43 SI-NETs for selected loci with frequent CN losses in 18p  multivariate analysis using Cox proportional hazards modeling was applied to those recurrent regions of CNAs which were significantly associated to survival. Associations between tumor groups and recurrent CNA or clinical parameters were evaluated by Fisher\u2019s exact test and for age at diagnosis by Mann\u2013Whitney We determined genome-wide CNAs in 30 tumors representing 19 primary tumors and 11 metastases from 29 patients with SI-NET using a-CGH. All samples analyzed displayed CNAs, with the largest and smallest extent of total CNAs observed in tumors number 4 (550 Mb) and 29 (2.5 Mb), respectively. CN losses and gains were in many cases extensive and involved entire or almost entire chromosomes. Gains were more common than losses. Recurrent CN losses were found on chromosomes 18, 16, 11, 9 and 13 , 11q (23%), 9 (20%), 16 (17%) and 13 (13%). Fifteen of 30 tumors (50%) had entire or almost entire loss of chromosome 18. In addition, loss of chromosome 18 was the only detectable recurrent CNA in four tumors , three of which were primary tumors. Sub-chromosomal losses of chromosome 18 were observed in six tumors . These tumors shared a recurrent region of 35.5 Mb at 11q22.1-qter (Table\u00a0SDHD and members of the cysteine-aspartic acid protease (caspase) family including CASP4 and CASP12. Whole chromosome 9 loss was found in two tumors and segmental losses in four tumors with recurrent regions at 9pter-p13.2 and 9p13.1-11.2. Loss of entire chromosome 13 was found in one metastasis and two primary tumors showed deletions at 13q11-ter and 13q13-q21.32.Recurrent CN losses were observed on chromosome 16 in 5/30 (17%) tumors. A recurrent region of 34.5 Mb loss which maps to 16q12.2-qter was detected in 5 tumors  , 5 (23%), 7 (13%), 14 (30%) and 20 (37%)  which overlapped with partial or entire gains in five other tumors . This region encompassed several genes among others DHRS4L2, REC8L1, IPO4, PSME1 and PCK2. One tumor (case 25) displayed gain of entire chromosome 14, and four tumors  carried partial or small gain of 14 without harboring copy number losses on chromosome 18. A MOR of 1.4 Mb gain at the telomeric region of chromosome 20 (q13.33) was detected in 9/30 cases (30%) (data not shown). The 20q13.33 region overlapped with several cancer related genes such as CDH4, LAMA5, RPS21, KIAA1510, TNFRSF6B , RTEL, EEF1A2 and PTK6.A MOR of 230 kb gain at 14q11.2 downstream of the EMILIN2 (p11.32-31), DCC (q21.1-2), BCL2 (q21.33) and CDH19 (q22.1). CNs in chromosome 18 were verified in 12/19 (63%) of the tumors for EMILIN2, 11/18 (61%) for DCC, 11/17 (65%) for BCL2 and 11/18 (61%) for CDH19 .All CNAs identified by a-CGH were reviewed to identify recurrent loss or gain regions. The selection of candidate genes for validation were based on the most common CNAs in the present study as well as candidate genes identified from the literature ,14-17,25LIN2 p11.2-31, DCCCDH1 on 16q22.1 and SDHD on 11q23.1 and they were verified respectively in 3/5 (60%) and 4/7 (57%) of tumors with corresponding losses detected by a-CGH , 5 (P = 0.003), 7p22.3 (P = 0.001), 7p22.2-22.1 (P = 0.001), 7q22.1 (P = 0.001), 7q22.3-qter (P = 0.006), 14q11.2 (P < 0.0005), and 14q32.2-32.31 (P < 0.0005). A significant correlation was also observed between group II and gain on 20pter-p11.21 (P = 0.014).The a-CGH data was subjected to hierarchical cluster analysis to detect tumor groups with similar patterns of chromosomal alterations based on recurrent regions of CN loss or gain. Two different tumor groups (I and II) and five chromosomal clusters -(a-e) were identified Figure\u00a0. Tumor gP = 0.016) and harbored gains on 7p22.3 (P = 0.018), 7p22.2-22.1 (P = 0.018), 7q22.1 (P = 0.018), 7q22.3-qter (P = 0.003), 14q11.2 (P = 0.049), and 14q32.2-32.31 (P = 0.016). Loss on 16q12.2-qter was more common in distant metastases compared with primary tumors (P = 0.003), and this loss along with gain on 7q22.3-qter were more common in metastases compared to primary tumors , suggesting their involvement in tumor progression.Cases with extra-hepatic metastases  . CN losses on chromosomes 11 and 16 and gain on chromosome 20 were more frequent in female patients involving 11q23.1-qter (P = 0.039), 16q12.2-qter (P = 0.019), 20pter-p11.21 (P = 0.017) and 20q11.1-11.21 (P = 0.039).Next, we tested possible associations between CNAs in recurrent regions and patient survival. CN gain in the 20pter-p11.21 region was associated with shorter overall survival P = 0.01  had intact chromosome 18 . Thus, CDH1 could represent a potential candidate tumor suppressor gene in this region.Four of 12 (33%) metastases had loss at 16q as compared to 1/18 (6%) primary tumors confirming our previous observations by conventional CGH . This di cancers ,31. InacCN losses within 11q have been reported for many cancer types and have been associated with metastatic disease for example in pheochromocytoma . Loss ofTNFRSF6B, that may inhibit apoptosis and promote cell survival, is over-expressed in gastrointestinal tract tumors [In addition to gain of entire chromosome 20, a 1.4-4.2 Mb region at 20q13.33 was gained in about 33% of tumors. Similar alterations have been reported in e.g. digestive tract tumors and breast cancer ,36, and t tumors , colorect tumors  and gastt tumors . FurtherP = 0.014) and the patients had a shorter overall survival (P = 0.013). Female patients more frequently showed losses on chromosomes 11q and 16q and gains on 20p and 20q. Furthermore, gain on 7q and loss on chromosomes 9p and 18p were associated with younger age at diagnosis.Unsupervised clustering of all CNAs identified two distinct tumor groups (I and II) and 5 chromosomal clusters (a-e). Interestingly, gains of chromosomes 4, 5, 7 and 14 clustered together in tumor group II. This distinct genetic alteration in SI-NETs is in accordance with a previous study . Four meWe identified two recurrent regions on chromosome 18 with a possible role in tumor initiation. Recurrent CN gains of chromosomes 4, 5, 7, 14 are usually concomitant and could together with loss of 16q and gain of 20p be involved in progression of SI-NETs. Distinct genetic alterations and pathways are involved in SI-NET development.SI-NET: Small intestinal neuroendocrine tumor; a-CGH: Array comparative genomic hybridization.The authors declare that they have no competing interest.JH contributed to the study design, performed a-CGH experiments and data analysis, interpreted the results and drafted the manuscript. OF performed qPCR, clustering, statistical analysis and interpreted the results. LS performed a-CGH experiments. MK, AH, CL and JZ provided the clinical information. OF, LS, AH and JZ critically revised the manuscript. CL contributed to conception and study design, interpretation of data and substantially revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/505/prepubClinical data for the 32 patients with SI-NET in the study.Click here for fileExamples of a-CGH profiles showing loss of 16q12.1-ter in case 1, loss of 11q22.1-qter in case 27P, gain of 14q11.1-32.31 in case 32 and gains of 14q11.2 and q32.2-qter in case 28.Click here for fileCopy numbers detected by a-CGH and q-PCR (aCGH/q-PCR).Click here for file"}
+{"text": "Objectives To assess the associations between both uric acid levels and hyperuricaemia, with ischaemic heart disease and blood pressure, and to explore the potentially confounding role of body mass index.Design Mendelian randomisation analysis, using variation at specific genes (SLC2A9 (rs7442295) as an instrument for uric acid; and FTO (rs9939609), MC4R (rs17782313), and TMEM18 (rs6548238) for body mass index).Setting Two large, prospective cohort studies in Denmark.Participants We measured levels of uric acid and related covariables in 58\u2009072 participants from the Copenhagen General Population Study and 10\u2009602 from the Copenhagen City Heart Study, comprising 4890 and 2282 cases of ischaemic heart disease, respectively.Main outcome Blood pressure and prospectively assessed ischaemic heart disease.Results Estimates confirmed known observational associations between plasma uric acid and hyperuricaemia with risk of ischaemic heart disease and diastolic and systolic blood pressure. However, when using genotypic instruments for uric acid and hyperuricaemia, we saw no evidence for causal associations between uric acid, ischaemic heart disease, and blood pressure. We used genetic instruments to investigate body mass index as a potentially confounding factor in observational associations, and saw a causal effect on uric acid levels. Every four unit increase of body mass index saw a rise in uric acid of 0.03 mmol/L , and an increase in risk of hyperuricaemia of 7.5% (3.9% to 11.1%).Conclusion By contrast with observational findings, there is no strong evidence for causal associations between uric acid and ischaemic heart disease or blood pressure. However, evidence supports a causal effect between body mass index and uric acid level and hyperuricaemia. This finding strongly suggests body mass index as a confounder in observational associations, and suggests a role for elevated body mass index or obesity in the development of uric acid related conditions. Uric acid is a powerful antioxidant and has been proposed to protect against cardiovascular disease and some cancers.5Despite the expectation that the antioxidant properties of uric acid might have a protective effect against cardiovascular disease, studies have reported associations with a greater risk of ischaemic heart disease, higher blood pressure, and an adverse cardiovascular risk profile.A series of hypotheses has suggested why these unexpected positive associations might exist, including the upregulation of renin release and the subsequent cascade related reduction of endothelial function.16SLC2A9 gene  has been found to be robustly associated with increased plasma levels of uric acid, hyperuricaemia, urate excretion, and gout in genome wide association studies.SLC2A9 variation as a proxy measurement in this way allows for differences in disease risk or measured phenotype across the groups to be attributed solely to the differences in average levels of uric acid attributable to genotypic variation.Mendelian randomisation can account for unmeasured confounding and reverse causation by using genotypes robustly associated with the risk factor of interest as instrumental variables. This approach can be used to test and estimate the causal effect between a risk factor and an outcome.We aimed to investigate whether there was a causal effect of uric acid and hyperuricaemia on ischaemic heart disease and diastolic and systolic blood pressure using a mendelian randomisation approach  and codes I21-I22 from ICD-10), based on characteristic chest pain, electrocardiographic changes, or elevated concentrations of cardiac enzymes after changes in diagnostic criteria over time.In both studies, systolic and diastolic blood pressure was measured by clinic assessment at baseline, as described previously.24To exclude the influence of age and sex on our results, we standardised body mass index into age and sex adjusted z scores within each study (web table 1). One z score corresponds to a standard deviation of 4 units of body mass index; thus, for straightforward interpretation of results, estimates in z score units were converted to the scale for body mass index.Plasma uric acid was measured by clinic assessment at baseline, using colorimetry on a Konelab Autoanalyser (Thermo Scientific). In the Copenhagen City Heart Study, participants had up to four examinations, and measurements of plasma uric acid were available from the 1991-94 and 2001-03 examinations. Each participant\u2019s measurement of uric acid\u2014and hence hyperuricaemia status\u2014that was recorded closest before their event of ischaemic heart disease was used. Hyperuricaemia was defined as plasma levels of uric acid greater than 0.4 mmol/L .SLC2A9 (rs7442295) were coded by applying an additive genetic model based on information from a genome wide association study.FTO (rs9939609), MC4R (rs17782313), and TMEM18 (rs6548238)) as instruments for body mass index. We selected these polymorphisms because they have the largest known common effect sizes for association with body mass index in European populations.26Genotyping was conducted blind to phenotypic data using an ABI PRISM 7900HT sequence detection system (Applied Biosystems) with TaqMan. Genotyping was verified by DNA sequencing in at least 30 individuals, and reruns were performed twice\u2014resulting in successful rates of genotype collection of more than 99.96%. Genotypes of In both studies, data were available for age, sex, smoking status, years of education, and income from questionnaires at clinic assessment at baseline. Smoking was categorised as self reported ever smoking. We grouped education according to years spent in school , and annual income by Danish krones . Observational associations were estimated with and without adjustment for potential confounding factors.2 test. Cox proportional hazards regressions was used to estimate observational hazard ratios for ischaemic heart disease per standard deviation change in uric acid concentration, by hyperuricaemia status and by genotype at SLC2A9 (rs7442295). We used linear regression to estimate observational associations between blood pressure and uric acid (and therefore hyperuricaemia status), by genotype at SLC2A9 (rs7442295). Logistic regression estimated associations between genotype and binary covariates. We used ordered logistic regression to estimate associations between genotype and ordered categorical variables.For genetic variants, we investigated deviation from the Hardy-Weinberg equilibrium using a Pearson \u03c7Instrumental variable analysis generated causal effects in a mendelian randomisation framework. Estimates of causal hazard ratios for risk of ischaemic heart disease were calculated by using a ratio estimator. These estimates were derived by first dividing the log of the hazard ratio for genotype-ischaemic heart disease by the genotype-exposure coefficient\u2014the resulting value then underwent exponentiation. Standard errors for the log hazard ratios of instrumental variables were derived using the delta method.28We pooled estimates from the two studies using an inverse variance weighted, meta-analysis model implemented in the user written \u201cmetan\u201d Stata command.The 58\u2009072 eligible participants in the Copenhagen General Population Study included 4890 (8%) with ischaemic heart disease in 141\u2009559 person years at risk, of whom 1682 participants had incident cases. Analyses included 10\u2009602 participants from the Copenhagen City Heart Study from the 1991-94 and 2001-03 examinations, with 2282 (16%) cases of ischaemic heart disease in 79\u2009979 person years at risk, of which 1748 were incident cases.The mean level of plasma uric acid was 0.30 mmol/L (standard deviation 0.09 mmol/L) in the Copenhagen General Population Study, and 0.31 mmol/L (0.09 mmol/L) in the Copenhagen City Heart Study. Following the definition of hyperuricaemia (uric acid >0.4 mmol/L), 6929 (12%) and 1569 (16%) participants were classified as having hyperuricaemia in the Copenhagen General Population Study and Copenhagen City Heart Study, respectively. Mean diastolic blood pressure was slightly lower in the Copenhagen General Population Study than in the Copenhagen City Heart Study, while mean systolic blood pressure was slightly higher in the Copenhagen General Population Study than in the Copenhagen City Heart Study  and 1.34 (1.28 to 1.39) in the Copenhagen General Population Study and Copenhagen City Heart Study, respectively. These ratios gave a pooled estimate of 1.49 . This association was attenuated, after adjustment, to an increase of 1.56 mm Hg (1.45 to 1.67). Participants with hyperuricaemia had an average increase of 4.61 mm Hg (4.31 to 4.90) in diastolic blood pressure . This estimate also attenuated on adjustment (2.66 (2.36 to 2.96)). A similar pattern of observational associations was seen between uric acid and hyperuricaemia and systolic blood pressure (web fig 3).SLC2A9 (rs7442295) that deviated from the Hardy-Weinberg equilibrium (web table 2). The association between variation at SLC2A9 (rs7442295) and uric acid was roughly linear (web fig 4). Mean levels of uric acid showed an increase in standard deviation of 0.26  and 0.25 (0.21 to 0.28) for each additional A allele in the Copenhagen General Population Study and Copenhagen City Heart Study, respectively (table 2SLC2A9 (rs7442295) accounted for about 2% of the variance in uric acid levels. Variation at SLC2A9 (rs7442295) was associated with an increase of about 5% in the risk of hyperuricaemia in both samples.Both the Copenhagen General Population Study and Copenhagen City Heart Study showed no strong evidence for genotypes including SLC2A9 (rs7442295) was not associated with potential confounders: age, sex, body mass index, smoking, education, and income (web table 2). By contrast, ischaemic heart disease, systolic blood pressure, and diastolic blood pressure showed strong associations with these potential confounders (web table 3). Uric acid and hyperuricaemia exposures also showed associations with these potential confounders (web table 4).In the Copenhagen General Population Study and Copenhagen City Heart Study, SLC2A9 (rs7442295) was not associated with ischaemic heart disease in either the Copenhagen General Population Study ) or the Copenhagen City Heart Study (0.96 (0.89 to 1.03)). Similarly, we did not find any associations between SLC2A9 (rs7442295) and either diastolic or systolic blood pressure (table 2).Variation at Instrumental variable analysis, when pooled, gave an estimate of the causal hazard ratio for ischaemic heart disease of 0.93  per standard deviation increase in uric acid (fig 2). The corresponding estimate of the effect of hyperuricaemia status on risk of ischaemic heart disease was 0.62 . In the sensitivity analysis using incident cases of ischaemic heart disease only, we found weaker hazard ratios per standard deviation increase in uric acid and hyperuricaemia (0.87 (0.69 to 1.09) and 0.42 (0.10 to 1.68), respectively; web fig 1).Instrumental variable estimates of the causal effect of uric acid on diastolic blood pressure failed to show convincing evidence of an effect and had wide confidence intervals. A standard deviation increase in uric acid was estimated to increase diastolic blood pressure by 0.63 mm Hg . This point estimate was largely driven by the association in Copenhagen City Heart Study, and was weaker than the adjusted observational estimate (1.56 mm Hg (1.45 to 1.67); web fig 2A). Similarly, owing to a stronger effect in the Copenhagen City Heart Study, the instrumental variable estimate showed an increase of 4.56 mm Hg (0.01 to 9.12) in diastolic blood pressure in patients with hyperuricaemia (web fig 2B).We saw no evidence of a causal effect of uric acid or hyperuricaemia on systolic blood pressure (web fig 3). The instrumental variable estimate of the causal effect of uric acid on systolic blood pressure, when pooled, was 0.65 mm Hg  per standard deviation increase in uric acid. In the Copenhagen General Population Study, the instrumental variable estimate of the causal effect of hyperuricaemia on systolic blood pressure was similar to the observational estimates. However, this instrumental variable estimate was much smaller in the Copenhagen City Heart Study, producing a pooled estimate of 4.31 (\u22123.72 to 12.33).Observational estimates showed an association of body mass index, a potential confounding factor, with both standardised uric acid and hyperuricaemia. This association remained on adjustment for potential confounding factors. For example, a standard deviation elevation in uric acid was associated with a unit increase of 1.06  in body mass index, which increased to 1.43 (1.40 to 1.47) on adjustment for the other potential confounders (web fig 5). Furthermore, when assessing the observational relations between uric acid and hyperuricaemia with risk of ischaemic heart disease, diastolic blood pressure, and systolic blood pressure, inclusion of body mass index as an additional covariable resulted in the attenuation of association. Specifically, associations for a standard deviation elevation in uric acid were reduced to a hazard ratio for ischaemic heart disease of 1.16 (1.12 to 1.19), a change of 0.60 mm Hg (0.49 to 0.71) in diastolic blood pressure, and a change of 1.37 mm Hg (1.18 to 1.56) in systolic blood pressure. Hyperuricaemia was associated with a hazard ratio for ischaemic heart disease of 1.30 (1.21 to 1.39), a change of 1.03 mm Hg (0.74 to 1.33) in diastolic blood pressure, and a change of 2.41 mm Hg (1.91 to 2.91) in systolic blood pressure (pooled estimates) after accounting for body mass index .SLC2A9 (rs7442295) as instruments for these measures, did not suggest a causal effect on body mass index (web fig 5).In a mendelian randomisation analysis, we reciprocallyUsing data from two large prospective cohort studies, we found no evidence of a causal effect of uric acid or hyperuricaemia on the risk of ischaemic heart disease. Unadjusted observational analyses found an increase of 34-55% in risk of ischaemic heart disease per standard deviation increase in uric acid. These estimates attenuated slightly on adjustment for established cardiovascular risk factors. The same trend was present in our estimates of the association of uric acid and hyperuricaemia on systolic blood pressure. However, mendelian randomisation estimates showed no evidence of an effect of elevated uric acid (and hyperuricaemia) on the risk of ischaemic heart disease or elevated blood pressure.In view of the weak evidence supporting causal associations, further explanation for known observational associations was sought through the analysis of body mass index as a confounding factor. Body mass index is an established risk factor for both blood pressure and ischaemic heart disease observationally,37SLC2A9 (rs7442295) was not directly associated with coronary artery disease in a case-control study.SLC2A9 variation as an instrument for plasma uric acid failed to find evidence of a causal effect of uric acid on metabolic syndrome,SLC2A9 variants are unlikely to be associated with blood pressure.SLC2A9 locus (rs16890979) has been associated with blood pressure,The mendelian randomisation estimates presented here broadly agree with several recent studies. Stark and colleagues found that C2A9 rs74295 was nLC2A9 rs72295 was SLC2A9 variation as an instrument for uric acid levels, this approach does encode for a high capacity sugar transporter, and functional analyses have shown it to have a preferential action on urate.Analyses were undertaken within large, ethnically homogeneous, clinically assessed case series with access to control sets of comparable quality. The nature and size of the existing sample place it particularly well for the undertaking of mendelian randomisation experiments. However, the potential for the complicating effects of linkage disequilibrium, pleiotropy, or the nature of genotypic effect are difficult to account for.Another potential limitation was that we did not have data available to identify participants receiving uric acid lowering drugs at the time of measurement. Although this will have been a small fraction of our total sample size and unlikely to have materially altered our main results, it may have led to underestimates of both observational and instrumental variable estimates.Overall, mendelian randomisation estimates found no evidence of causal effects of either uric acid or hyperuricaemia on risk of either ischaemic heart disease or raised blood pressure. Our Mendelian randomisation results alone suggest that uric acid is of limited clinical interest in ischaemic heart disease or blood pressure. However, there is strong evidence for an effect of body mass index on both uric acid and hyperuricaemia\u2014indicating that body mass index is an important confounding factor to observational association analyses of uric acid and hyperuricaemia. This finding contrasts the notion of body mass index operating as a mediator or being on the causal pathway from uric acid to vascular outcomes. In this case, one would expect to find evidence for a causal association both for body mass index and uric acid level, as opposed to just one of these risk factors. This study clearly shows the value of mendelian randomisation to help dissect complex networks of risk factor association by using independent proxies or instrumental variables for risk factors of interest. Furthermore, our findings suggest that interventions to reduce body mass index could help improve management of gout and related conditions such as urolithiasis.Uric acid has been suggested to protect against cardiovascular diseaseObservational studies have suggested that increased levels of uric acid is associated with ischaemic heart disease and blood pressure; but owing to confounding, bias, and reverse causation, such associations can be difficult to interpretSLC2A9 gene has been reliably associated with circulating levels of uric acid, and has been proposed as an instrument to investigate causal associations with blood pressure and ischaemic heart diseaseThe SLC2A9 gene shows little evidence of a causal association between increased levels of uric acid, raised blood pressure, and risk of ischaemic heart diseaseGenetic variation at the However, causal analysis of body mass index shows strong evidence of an effect of body mass index on uric acid levels, suggesting considerable confounding in observational associationsMendelian randomisation analysis suggests that uric acid is of limited clinical interest in ischaemic heart disease or blood pressure. But interventions to reduce body mass index could help improve the management of gout and related conditions such as urolithiasis"}
+{"text": "Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as \u2018emotional fever\u2019. It has been suggested that the capacity for emotional fever evolved only in amniotes , in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2\u20134\u00b0C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes. Those who argue that this is not the case \u20133 do so Oreochromis niloticus) after exposed to a stress situation (confinement) )inement) . Concerninement) ,11; see inement) ,7 for reAstatotilapia burtini can infer the relative social rank of territorial neighbours by observation, using a capacity for transitive inference , togeth than C.  while ca than C. .et al. [et al. proposed that \u2018a common mental pathway\u2019 characterizes consciousness and that uses pleasure (or displeasure) as means to optimize behaviour.Reviews on cognitive abilities and their relation on emotion and consciousness in fishes and across species have been published recently \u201320. Emotet al.  in his ret al. , and it Callopistes maculatus lizards [One trait that has been identified as indicative of sentience and consciousness is the capacity for showing stress-induced hyperthermia (SIH) or emotional fever ,24. Thes lizards . The ext lizards ,39, maki lizards .The importance of emotional fever in the debate about fish sentience and consciousness lies in the fact that earlier studies failed to identify SIH in fishes (goldfish: ) or amphWith this background, the aim of this study was to ask again the question of whether fishes have the capacity to show emotional fever. We used zebrafish held in an experimental tank that allowed fish to move freely through a gradient of temperatures centred on the optimal temperature for this species. This novel experimental set-up provides accurate, dynamic information on temperature preferences in zebrafish and has recently been used to demonstrate that behavioural fever following exposure to a virus protects zebrafish by optimizing the antiviral response at the level of the transcriptome . In the 2.(a)Danio rerio; 0.93 \u00b1 0.22 g mean weight; 43.73 \u00b1 2.44 mm total length; n = 206) were bred in the zebrafish facilities at the Universitat Aut\u00f2noma de Barcelona, Spain. Zebrafish were originally purchased, as juveniles, from a commercial supplier  and held in a recirculating rack system developed by zfbiolabs\u00ae (www.zfbiolabs.com). Fish were kept in standard zebrafish housing conditions: 28 \u00b1 0.74\u00b0C on a 12 L : 12 D photoperiod cycle [\u00ae and SERA tests\u00ae) and always found under recommended levels [Healthy adult zebrafish 3 (210 \u00d7 30 \u00d7 30 cm of size) divided with five transparent Plexiglas screens to create six equal interconnected chambers, as used by Bolta\u00f1a et al. [The experimental thermal gradient tank was 180 ma et al. . Each sc(c)n = 12 for each group) were introduced into chamber 4 (28.75 \u00b1 0.27 \u00b0C) in the evening for acclimation and filming began at 6.00 the next day, providing a minimum of 12 h acclimation in the gradient tank during which the fish could freely choose their preferred temperature.Fish were housed in the laboratory at the standard husbandry temperature of at 28 \u00b1 0.74\u00b0C and so were acclimated to this temperature . During(d)Three of the six groups were placed in the temperature gradient tank the day before to acclimatize  and confinement experiments started always at the same time every morning (10.00), at least 4 h after the lights were turned on. Fish from the confinement group were gently captured from the temperature gradient tank where they had acclimatized overnight and were placed into a small fishing net (20 \u00d7 14 \u00d7 18 cm) hanging inside the water at around 27\u00b0C for 15 min (chamber 3). Netting and restraining are known stressors in zebrafish . Followi(e)Fish were not tagged to avoid extra sources of stress or infection. Scan sampling was used to monitor the distribution of fish within the thermal gradient for a minimum of 8 h after the stressful event in the experimental groups and for an equivalent time in the controls. The distribution of the animals was recorded with three remote control cameras placed each one in front of two chambers, recording for 30 s during each 30 min period for eight diurnal hours . The same observer extracted data from the recordings, after cross validation with an experienced researcher. Every hour recorded was plotted and analysed. The first 4 h were examined for the expression of emotional fever; the remaining 4 h were used to study the dynamics of extinction of the emotional fever response.(f)\u03b1 \u2264 0.05. An offset was also entered into the model (LnExpected) to accommodate differences in the size of the groups of fish, as one fish in each of one treatment and one control group was lost before the commencement of the study. A Mann\u2013Whitney test to compare the different percentages of fish occupation between controls and confined animals for the three-zebrafish groups on the higher temperature chambers (more than 28\u00b0C) were performed. Significance was set up at p < 0.05 for all statistical analysis. This statistical analysis was performed with IBM\u00ae SPSS\u00ae 17 Statistics v19 for MAC\u00ae OS X.As the study had a hierarchical design , and a count as the outcome variable, a Poisson response model was constructed within the multilevel modelling statistical software MLwiN v. 2.35 . The hie3.p < 0.05 and were retained in the model. The parameter estimates and their standard errors for the final model are shown in n = 12 are shown. The groups of 11 show the same patterns of change as the other groups within each treatment, but as their expected counts are adjusted they have been omitted from the figure to retain clarity. It can be seen that the distribution of the confined treatment groups is shifted towards higher temperature chambers. There was a differential effect on the shape of the distributions between the two treatment groups over time\u2014as time increased the confinement groups tended to move from the central chambers to the extremes, while the control groups tended to move in towards the central chambers, from the extremes, over time. This is shown in more detail in the electronic supplementary material, figure S2.The full three-way interaction and all lower interactions terms between treatment, temperature and time were significant at p < 0.05; All groups of zebrafish under confinement also had a greater percentage of occupation of tank areas at more than 28\u00b0C for the first 4 h than control groups  to return to normal thermal preference distribution is indicative of how intense stressful situations affect the behaviour of this species. In other stress experiments applied to this species maybe this effect gets masked by the design of the tanks but in this case where they can have proper time and conditions to express their behaviour we can see how difficult it is for them to recover allostasis and return to normal conditions after the stressful event.2) targets the preoptical area in the anterior hypothalamus and drives internally generated increases of temperature in endotherms [2 levels in the plasma leading to a change in temperature preference and recovery from infection [Drosophila larvae identified thermosensory neurons that drive thermotaxis in a fluctuating thermal environment providing a basis for thermosensory input coupled to environmental navigation [Considerable research effort has been aimed at identifying the physiological and molecular mechanisms that underlie SIH; these have shown that it is a real fever in the sense that it activates the same exact physiological and metabolic pathways \u201355. The dotherms . By contnfection . Howevervigation . This maOur results suggest that, in fishes as in mammals , change5.et al. [Whatever the underlying mechanisms and associated benefits of the phenomenon and notwithstanding the complex relationship between emotion and consciousness, the fact that the fish used in this study are capable of SIH, or emotional fever, means that the absence of this ability cannot be used to argue for a lack of consciousness in this taxonomic group, as proposed by Cabanac et al. . As discet al. \u20137) that"}
+{"text": "Titania membranes were synthesized via a sol-gel method and coated on macroporous alumina tubes followed by exposure to a vacuum between 30 and 1200 s and then calcined at 400 \u00b0C. X-ray diffraction and nitrogen adsorption analyses showed that the crystallite size and particle size of titania increased as a function of vacuum time. All the TM membranes were mesoporous with an average pore diameter of ~3.6 nm with an anatase crystal morphology. Water, glucose, sucrose, and polyvinylpyrrolidone with 40 and 360 kDa (PVP-40 kDa and PVP-360 kDa) were used as feed solutions for MW cut-off and hexadecane solution for oil filtration investigation. The TM membranes were not able to separate glucose and sucrose, thus indicating the membrane pore sizes are larger than the kinetic diameter of sucrose of 0.9 nm, irrespective of vacuum exposure time. They also showed only moderate rejection (20%) of the smaller PVP-40 kDa, however, all the membranes were able to obtain an excellent rejection of near 100% for the larger PVP-360 kDa molecule. Furthermore, the TM membranes were tested for the separation of oil emulsions with a high concentration of oil (3000 ppm), reaching high oil rejections of more than 90% of oil. In general, the water fluxes increased with the vacuum exposure time indicating a pore structural tailoring effect. It is therefore proposed that a mechanism of pore size tailoring was formed by an interconnected network of Ti\u2013O\u2013Ti nanoparticles with inter-particle voids, which increased as TiO Titania membranes are increasingly finding usability in water processing applications. Titania membranes can operate as membrane contactors to separate water from other substances, or as membrane reactors to take advantage of the catalytic properties of titania crystals to destroy organic compounds whilst separating water. The application of titania membranes includes processing textile wastewaters , desalinIn general, titania membranes are prepared from sol-gel methods and there are several strategies pursued by the research community to tailor the pore size of titania membranes. Ayral and co-workers developed mesoporous titania membranes by controlling the sintering temperature of the titania nanocrystal phase  and by uThe majority of the titania membranes are prepared as thin films coated on porous substrates. This creates an asymmetric structure, with the thin film having smaller pore sizes while the substrate with the larger pore sizes provides the desired mechanical strength for the membrane. The sol-gel method is a versatile route for the synthesis of thin films on substrates, generally by dip-coating where the substrate is immersed in a titania solution. The latter adheres to the surface of the substrate upon withdrawal from the solution. Upon calcination, the final structural properties of the membranes will be influenced by the particle size of the deposited titania films  and the Therefore, this work focuses on studying the effect of the vacuum-assisted method in tailoring the pore size of titania membranes. To this end, prepared titania membranes were exposed to varying vacuum times and the resultant materials were fully characterised. Furthermore, the titania membranes were investigated for MW cut-off in various organic substances to determine the correlations between the material structure and membrane performance. The titania membranes were also tested for oil/water separation using an oil emulsion of hexadecane with a high oil concentration (3000 ppm). Finally, a mechanism is proposed to explain the effect of the vacuum-assisted method on pore size variance.2\u00b7g\u22121, of which the latter is for TM1200. The pore volumes also followed a similar trend, consistent with exposure to vacuum at different times. The pore size distribution (PSD) of the TM materials in 2-anatase   \u00d7 100, where R (%) is the rejection, and pC and fC are the concentrations of the samples collected from the permeate and feed streams, respectively.Molecular weight (MW) cut-off studies were carried out in aqueous solutions containing 3000 ppm concentrations of glucose, sucrose, and polyvinyl pyrrolidine (PVP molecular weights of 36 kDa and 400 kDa). The samples collected from the permeate stream were analysed using a Shimadzu UV-2700 UV-Vis spectrometer  to determine their concentration against calibrated curves. The rejection  prepared using different vacuum exposure times (30 to 1200 s) were investigated by molecular weight cut-off and oil emulsion filtration along with their material characterizations. A strong trend was found in that the water flux of the membranes decreased with the increasing molecular weight of the tested substances using water, glucose, sucrose, PVP-40 kDa, and PVP-360 kDa as a direct result of nanofiltration. Furthermore, all the membranes rejected almost 100% of the larger PVP-360 kDa molecules, albeit the membranes were not able to separate the smaller molecular sizes of glucose and sucrose, which indicates that the membrane pore sizes are larger than the kinetic diameter of sucrose of 0.9 nm. The TM membranes were able to separate a highly concentrated oil emulsion (3000 ppm) delivering rejections from 90% to 93%, which increased from a single layer to three layers of membrane coating, respectively, although water fluxes trended oppositely due to increasing mass transfer resistance through the membranes. Characterisation of materials revealed that the structural formation of the TM membranes was affected by the vacuum time which promoted aggregation of the Ti\u2013O\u2013Ti networks, leading to the creation of increased anatase phase TiO"}
+{"text": "Although methylated TWIST1 is a biomarker of colorectal neoplasia, its detection from serum samples is very difficult by conventional bisulfite-based methylation assays. Therefore, we have developed a new methylation assay that enables counting of even one copy of a methylated gene in a small DNA sample amount without DNA bisulfite treatment. We performed this study to evaluate the sensitivity and specificity of serum DNA testing by the new methylation assay in combination with and without the fecal immunochemical test for hemoglobin for the detection of colorectal neoplasia. This study comprised 113 patients with colorectal neoplasia and 25 control individuals. For the new methylation assay, DNA was treated in two stages with methylation-sensitive restriction enzymes, followed by measurement of copy numbers of hTERT and methylated TWIST1 by multiplex droplet digital PCR. The fecal immunochemical test had a sensitivity of 8.0% for non-advanced adenoma, 24.3% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 88.0%. The new assay had a sensitivity of 36.0% for non-advanced adenoma, 30.0% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 92.0%. Combination of the both tests increased the sensitivity to 40.0%, 45.7%, and 72.2% for the detection of non-advanced adenoma, advanced adenoma, and colorectal cancer, respectively, and resulted in a specificity of 84.0%. Combination of both tests may provide an alternative screening strategy for colorectal neoplasia including potentially precancerous lesions and colorectal cancer. Colorectal cancer (CRC) is the second most commonly diagnosed cancer in females and the third most in males in the world . It is eThe main approach to CRC screening throughout the world is the fecal immunochemical test for hemoglobin (FIT), and patients with fecal hemoglobin >20 \u03bcg hemoglobin/g feces  are referred for colonoscopy . AlthougFIT resulted in a sensitivity of 8.0% (2/25) for non-advanced adenoma, 24.3% 17/70) for advanced adenoma, and 44.4% (8/18) for CRC screening, with a specificity of 88.0% (22/25) (Table 7/70 for Increased CEA (\u22656 ng/mL) was found in 1/6 (16.7%) patients in the non-advanced adenoma group, 9/54 (16.7%) in the advanced adenoma group, and 17.6% (3/17) in the CRC group.For the basic performance test of the CORD assay to detect hypermethylated cancer-derived DNA against a background of blood-derived DNA, we spiked DNA from colon cancer cell line HCT116 (control DNA for methylation of TWIST1 and SEPT9) at ratios of 100%, 50%, 10%, 5%, 1.1%, 0.11%, and 0% into DNA extracted from leukocyte DNA (control DNA for unmethylation of TWIST1 and SEPT9) and measured the methylation levels of TWIST1 and SEPT9 for each sample. As shown in Figure 2 = 0.9188, P < 0.0001; Figure There was a linear relationship between DNA concentration and hTERT copy numbers  in the control group, 1.9  in the non-advanced adenoma group, 1.7  in the advanced adenoma group, and 1.8  in the CRC group Figure . We perfThe median copy numbers of methylated SEPT9 were 2.0  in the control group, 2.0  in the non-advanced adenoma group, 3.6  in the advanced adenoma group, and 2.1  in the CRC group Figure . The besThe criterion for a positive result with the combination of FIT and CORD assay is either a positive FIT or CORD assay or both are positive. The combination of FIT and serum CORD assay of methylated TWIST1 resulted in a sensitivity of 40.0% (10/25) for non-advanced adenoma, 45.7% (32/70) for advanced adenoma, and 72.2% (13/18) for CRC, and the specificity was 84.0% (21/25) Table . FocusinFor methylated SEPT9, the combination of FIT and serum CORD assay resulted in a sensitivity of 28.0% (7/25) for non-advanced adenoma, 44.3% (31/70) for advanced adenoma, 42.9% (6/14) for stage I CRC, and 55.6% (10/18) for all-stages CRC, and the specificity was 80% (20/25) Figure .In the present study, serum DNA concentration was significantly higher in the advanced adenoma group and CRC group than in the control group. Although increased amounts of circulating DNA in patients with CRC have been reported by other investigators , 16, an The CORD assay can count copy numbers of a methylated target gene and an internal control gene, hTERT, simultaneously. We found that hTERT copy numbers correlated positively with serum DNA concentration and that they were significantly higher in the advanced adenoma group and the CRC group than in the control group. Thus, the hTERT copy number, as well as serum DNA concentration, may be a biomarker of colorectal advanced adenoma and CRC. As no investigators have reported serum hTERT copy number as a possible biomarker for detecting colorectal neoplasia, this may also be the first report to show that hTERT copy numbers can be a promising biomarker of colorectal neoplasia. However, as hTERT copy numbers are also increased in the plasma of patients with hepatocellular carcinoma , 20 and,Regarding the methylation assay, although bisulfite treatment of DNA is commonly performed in the conventional methylation assays, this reaction introduces various DNA strand breaks and results in highly fragmented single-stranded DNA  and the Release of tumor markers including circulating tumor DNA occurs by vascular invasion into blood during the progression from pre-invasive polyps through the advancing stages of CRC , and theIn the current study we also established serum CORD assay of SEPT9 because Epi proColon is not available in Japan. Compared to SEPT9, the serum CORD assay of TWIST1 showed better performance for colorectal neoplasia screening, especially for non-advanced adenoma. In addition, the serum CORD assay of methylated TWIST1 showed moderate or better sensitivity as compared with those in previous reports of blood-based testing of methylated SEPT9 including the Epi proColon, in which the sensitivity is 7% for non-advanced adenoma, 11% for advanced adenoma, 48%-68% for CRC  , 28. TheThe sensitivity of FIT for the detection of non-advanced adenoma and advanced adenoma is, in general, quite low  because It is reported that resection of adenomatous polyps of the colon and rectum by colonoscopic polypectomy reduces the incidence of CRC by 76\u201390%  and prevIn this study, detection of methylated TWIST1 from serum samples appeared to be useful for colorectal neoplasia screening. However, we admit that hypermethylation of TWIST1 is associated with different types of cancer: breast, uterine cervix, ovary, bladder, gastric, lung, bone, pancreas, and brain \u201344. ThusIn conclusion, the combination of TWIST1 methylation analysis by serum CORD assay and FIT showed higher sensitivities for the detection of non-advanced adenoma, advanced adenoma, and early-stage CRC without great difference in the specificity as compared to those by FIT alone. Because this study suggests that the combination of serum DNA testing of methylated TWIST1 and FIT may be useful to detect individuals with colorectal neoplasia, confirmatory studies using independent data sets are needed to support our findings.We enrolled 148 participants of whom 138 had results that could be fully evaluated Figure . Serum wParticipants received illustrated Japanese-language instructions on sampling feces from one bowel movement by briefly sweeping the tip of a probe several times though the feces . Fresh fSerum carcinoembryonic antigen (CEA) was measured in 76 patients with colorectal neoplasms using the \u201cTOSOH\u201d II CEA commercial immunoassay kit  and an AIA-2000 automatic immunoassay analyzer (Tosoh Corporation) in the laboratory division of Yamaguchi University Hospital. The cutoff value of the serum CEA levels was set at 6 ng/mL following the manufacturer's instruction.DNA from peripheral blood leukocytes was used as a control for unmethylated TWIST1 and unmethylated SEPT9, and DNA from CRC cell line HCT116 was used as a control for hypermethylated TWIST1  and hype2, 10 units of Hha I, 10 units of Hpa II, and 20 units of exonuclease I (Exo I) . Exo I was added to eliminate single-stranded DNA that would escape digestion by the restriction enzymes and to avoid PCR amplification of the undigested fraction [We performed CORD assay consisting of two-step treatments of DNA with multiple methylation-sensitive restriction enzymes followed by multiplex digital PCR . In the fraction . In the We performed multiplex droplet digital PCR to count the absolute copy numbers of hTERT, methylated TWIST1, and methylated SEPT9. The PCR reaction solution consisted of 8 \u03bcL of enzyme-treated DNA , 1 \u00d7 ddPCR Supermix for Probes , 0.25 \u03bcmol/L of each primer of a target gene and an internal control, and 0.125 \u03bcmol/L of each probe of a target gene and an internal control in a total volume of 20 \u03bcL. The sequences of the primer and probe set of TWIST1 were as follows: forward primer, 5\u2032-TCCAAAGGCCAAACCGC-3\u2032; reverse primer, 5\u2032-CCGGGACGCAAATCCTC-3\u2032; probe, 5\u2032-FAM-CTGAAGACGTGGCCGCGCC-TMARA-3\u2032. The PCR amplicon length is 92 bp from 19,157,854 to 19,157,945 of chromosome 7 (human assembly GRCh37/hg19). Those for hTERT were forward primer, 5\u2032-GGGTCCTCGCCTGTGTACAG-3\u2032; reverse primer, 5\u2032-CCTGGGAGCTCTGGGAATTT-3\u2032; probe, 5\u2032-VIC- CACACCTTTGGTCACTC-MGB-3\u2032 . The PCRU test, chi-square test, Fisher's test, and linear regression analyses were used. A P value of less than 0.05 was considered statistically significant. Statistical analyses were performed with GraphPad InStat Ver. 3, and GraphPad Prism Ver. 6 statistical software .To compare variables, the Mann\u2013Whitney"}
+{"text": "Glioblastoma (GBM) is maintained by a small subpopulation of tumor-initiating cells (TICs). The arduous assessment of TIC frequencies challenges the prognostic role of TICs in predicting the clinical outcome in GBM patients. We estimated the TIC frequency in human GBM injecting intracerebrally in mice dissociated cells without any passage in culture.in vivo but only 54% of them grew in vitro as neurospheres. We demonstrated that neurosphere formation in vitro did not foretell tumorigenic ability in vivo and frequencies calculated in vitro overestimated the TIC content.All GBMs contained rare TICsand were tumorigenic Our findings assert the pathological significance of GBM TICs. TIC number correlated positively with tumor incidence and inversely with survival of tumor-bearing mice. Stratification of GBM patients according to TIC content revealed that patients with low TIC frequency experienced a trend towards a longer progression free survival. The expression of either putative stem-cell markers or markers associated with different GBM molecular subtypes did not associate with either TIC content or neurosphere formation underlying the limitations of TIC identification based on the expression of some putative stem cell-markers. Although the past years have witnessed an improvement in the understanding of early molecular events in malignant primary CNS tumors, and a plethora of new therapies targeting these events are now tested in clinical trials, effective treatments for most primary malignant CNS tumors are still lacking . Among hTumor heterogeneity depicts the leading feature supporting tumor robustness and represents the main obstacle to overcome to develop therapeutic strategies. It is then essential to establish successful assays enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations driving the tumorigenesis and delineating clinically relevant targets.Many cancers  depend for their continued growth and propagation on a population of cells called cancer stem-cells (CSCs) or tumor-initiating cells (TICs): these cells are slow-dividing, endowed with unlimited proliferation capacity, functionally defined by their tumorigenic capability when engrafted in mice, unresponsive to standard treatments, thus highly competent in repopulating the tumor \u201311.TICs appear to be relatively rare in most human cancers, ranging from 0,0001% to 0,1% of the bulk tumor cell population , 6], , \u201315. Howe, \u201315. Hoin vitro cell manipulation before transplantation, the use of agents (i.e. matrigel) sustaining tumor cell transplantation, the extent of the immunodeficiency of the recipient host, the duration of the experimental period for tumor formation following tumor cell injection and the experimental procedure implemented for TIC isolation. With this regard, although the exact contribution of each cell-surface marker in identifying the TICs is puzzling and unclear, current protocols are still based on the expression of putative stem-cell markers that could distinguish a small subpopulation of cells with tumorigenic potential from the majority of non-tumorigenic cells. In addition, the gold standard to determine TIC frequency within a tumor is the limiting dilution cell transplantation assay (LDA) [Of note, different aspects may influence TIC estimate: ay (LDA) . NeverthThe variability in TIC frequencies assessment challenges the prognostic role of TICs in predicting the clinical outcome in cancer patients. Thus far, the prognostic value of TICs has been linked in several types of tumors to the presence of stem-related features, such as the expression of stem-cell markers, genetic features, and tumorsphere formation \u201322. Simiin vitro neurosphere assay we look for the existence of any correlation between the sphere-forming capability and the in vivo tumorigenic potential of cells from the same human sample. Moreover, we investigate the effect of in vitro culturing primary GBM cells as neurospheres on their TIC content.Here, we specifically estimate GBM TIC frequency employing limiting dilution transplantation of cells isolated from freshly-dissociated human GBMs. We believe that, owing the lack of definitive markers, only functional criteria applied on freshly-dissociated human GBMs will allow an unbiased assessment of the TIC content within parental GBMs. Furthermore, through in vitro manipulation to evaluate the ability to generate tumors. In parallel, cells were also plated in non-adherent serum free stem-cell medium to allow neurosphere formation. The 86% of the specimens analysed (n=24) was tumorigenic in vivo (Table in vitro while the remaining 46% (n=11) did not (Table in vitro (data not shown). Thus, the inability to grow in culture cannot predict in vivo tumorigenicity.Immediately after surgical removal, GBM specimens (n=28) were enzymatically and mechanically dissociated and viable cells immediately injected in the mouse brain without any vo Table , giving vo Table . Of thesng 46% n=1 did notot Table . Interes5 to 10 cells [in vitro (n=9) comprised a rare, highly variable number of TICs  was 10-fold higher than in vivo frequency  (Paired samples t-test: P=0.0014**)   Figure . Further) Figure .t-test: P=0.0067**)  Figure , fluctua) Figure .in vivo experiments performed with either freshly-dissociated hGBMs and hGBM-NS, we analysed the relationship between the total number of cells intracerebrally injected in immunocompromised mice and tumor incidence. In both conditions, we observed an increment of tumor incidence increasing the number of injected cells: at the maximum cell concentration (105 cells), 100% of incidence was measured only with hGBM-NS . The difference may be due to the smaller proportion of TICs within the freshly-dissociated hGBMs compared to hGBM-NS . Thus, TICs contained in freshly-dissociated hGBMs and hGBM-NS and extrapolated through in vivo LDA have the same tumorigenic potential.By pooling together the S Figure , in compS Figure   derived from hGBM-NS and freshly-dissociated hGBMs. The survival curve of hGBM-NS was significantly shorter . Notably, when either freshly-dissociated hGBMs  under \u201cC. Besta\u201d research ethics committee approval. The specimens were analysed by pathologists and classified as primary GBM (WHO IV). Tumors displayed characteristics consistent with those reported in the literature concerning age, sex distribution, dimensional range, Karnofsky performance status scale (KPS) and invasiveness .Overall survival and progression-free survival have been calculated as elapsed time from surgery to death or from surgery to the diagnosis of recurrence/progression. The patients enrolled in the study were 60% men and 40% women, with a median age of 58 years  and a mean Karnofsky performance score of 80 (range 50-90). The mean follow up was 17 months (range 10-30); 7 patients were lost to follow up, whereas for the others mean progression free survival (PFS) was 8+/\u22125.8 months, while overall survival (OS) was 11.8+/\u22125.1 months, in accordance to RANO criteria . Tumor iwww.invitrogen.com), 20 ng/ml epidermal growth factor, 10 ng/ml basic fibroblast growth factor . Human GBM neurospheres were grown as spheroid aggregates as previously described [TICs were isolated from GBM surgical specimens as previously described . BrieflyTo evaluate the capacity to form neurospheres, cells were resuspended in Dulbecco's modified Eagle medium/F12 medium containing methylcellulose  and seeded on a minimum of three 35 mm culture plates (3000 cells/dish). Two weeks after plating, the number of clones was counted. The ratio between neurospheres formed and number of single cells plated corresponds to the percentage of TICs in the plate.5-10 cells) derived from both freshly-dissociated GBM cells and hGBM-NS were resuspended in 2 \u03bcl of phosphate-buffered saline (PBS) and stereotaxically injected into the nucleus caudatus  of 5 weeks old female nu/nu CD1 mice . Mice were intraperitoneally anesthetized with tribromoethanol (0.1 ml/10 g of body weight). The experiments were performed in accordance with the Italian laws (D.L.vo 116/92 and following additions), which enforce EU 86/609 Directive . The mice were maintained until development of neurologic signs, and the brains of killed mice were collected.Decreasing cell concentrations . The incidences of tumors per number of injected cells and injected TICs were compared by means of Log-linear analysis . In Kaplan\u2013Meier curves, survival differences were compared by Log-rank test. P-values less than 0.05 were considered statistically significant (**) unless otherwise indicated. All statistical tests were two-sided.Paired samples"}
+{"text": "Correction to: Cell Death and Disease10.1038/s41419-019-1837-1, Published online 08 July 2019.Following publication of this article, it was realized that the ordering of the last two authors was reversed and required correction. The correct ordering of the authors for the paper is as follows:Xiaoqing Han, Huifang Shi, Yingying Sun, Chao Shang, Tao Luan, Dake Wang, Xueqing Ba (*) & Xianlu Zeng (*)[(*) indicates corresponding author].This has been corrected in both the PDF and HTML versions of the Article.We apologize for any inconvenience this may have caused the readers."}
+{"text": "We show that in addition to the motor properties the natural asymmetry between microtubule plus- and minus-end growth critically contributes to the organizational potential of the motors. We identify two control parameters that capture system composition and kinetic properties and predict the outcome of microtubule network organization. These results elucidate a fundamental design principle of spindle bipolarity and establish general rules for active filament network organization.During cell division, mitotic motors organize microtubules in the bipolar spindle into either polar arrays at the spindle poles or a \u201cnematic\u201d network of aligned microtubules at the spindle center. The reasons for the distinct self-organizing capacities of dynamic microtubules and different motors are not understood. Using   \u2022Kinesin-5 and kinesin-14 can produce nematic and polar microtubule networks\u2022Self-organizing network architecture depends on both motor and microtubule properties\u2022Two system-level control parameters determine which cytoskeletal network forms\u2022General rules explain the organizational capacities of mitotic motors in the spindle In vitro reconstitutions and computer simulations identify rules that govern microtubule network organization and reveal a design principle underlying bipolar spindle formation. The internal organization of eukaryotic cells depends on cytoskeletal networks. Dynamic microtubules and actin filaments, motile crosslinkers, and other associated proteins drive active networks into a variety of organizational states required for distinct cell functions . PolarizDuring cell division, microtubule crosslinking motors organize microtubules into bipolar spindles, an architecture that is crucial for correct chromosome segregation. The role of motors is particularly evident in female meiosis, when the bipolar spindle self-organizes from randomly oriented microtubules nucleated locally in the vicinity of chromosomes . Minus-ein\u00a0vitro  . Experimin\u00a0vitro .in\u00a0vitro when short, static microtubules were combined with purified artificial kinesin-1 clusters in the presence of crowding agents that promoted microtubule bundling  (To establish asymmetric microtubule growth dynamics MSAP3-C) A and S1 MSAP3-C) . AdditioMSAP3-C) B and 1C MSAP3-C) . CAMSAP3MSAP3-C) C and 1D.We next investigated how purified human plus-end-directed microtubule crosslinking kinesin-5 KIF11  organizeProtein concentrations were: tubulin - 30\u00a0\u03bcM, mCherry-CAMSAP3-C - 1000\u00a0nM, and KIF11-mGFP \u2013 27\u00a0nM. Time is in min:s. Imaging was carried out at 33\u00b0C.Reducing the CAMSAP3-C concentration lowered the efficiency of nematic network formation by dynamic microtubules and KIF11 A. To undTo understand KIF11-dependent network assembly, we explored the experimental phase space of microtubule organization. Keeping the CAMSAP3-C concentration fixed (500\u00a0nM), we lowered the tubulin concentration to reduce the microtubule plus-end growth speed A and theProtein concentrations were: tubulin - 10\u00a0\u03bcM, mCherry-CAMSAP3-C - 500\u00a0nM, and KIF11-mGFP \u2013 27\u00a0nM. Time is in min:s. Imaging was carried out at 33\u00b0C.Protein concentrations were: tubulin \u2013 7.5\u00a0\u03bcM, mCherry-CAMSAP3-C - 500\u00a0nM, and KIF11-mGFP \u2013 82\u00a0nM. Time is in min:s. Imaging was carried out at 33\u00b0C.These results demonstrate that the same motor can form either locally extensile nematic or locally contractile polar networks simply depending on the protein composition of the system. High tubulin and low KIF11 concentrations promote nematic network organization . In contTo understand mechanistically how dynamic microtubules and crosslinking motors drive network self-organization, we investigated the organizational phase space with three-dimensional numerical simulations using Cytosim  Figure\u00a0. Global Simulations allowed us to separate the effects of microtubule growth speed and microtubule number that vary concurrently in experiments when the tubulin or CAMSAP3-C concentration is changed. Systematically varying key parameters revealed a rich phase space with two distinct stable network organizations; a nematic network of aligned, mixed-polarity microtubule domains A and a pp links) and anti-parallel microtubules (Hap links). In this regime, microtubule growth speed was comparable to the motor speed; motors did not efficiently reach microtubule plus-ends but dwelled on the microtubules\u2019 sides. Equal numbers of Hp and Hap crosslinks therefore dominate in the nematic network state  Microtubules only are displayed, color-coded according to their orientation . (Right) Only motor crosslinks are shown color-coded according to their type . Parameter values, as in Figure\u00a04A: 17920 microtubules, 40960 motors, microtubule growth speed\u00a0= 30\u00a0nm/s, motor speed\u00a0= 30\u00a0nm/s. For all other parameter values see Table S1. Simulated time is in min:s.p links move inward on the asters\u2019 spokes  Microtubules only are displayed, color-coded according to their orientation . (Right) Only motor crosslinks are shown color-coded according to their type . Parameter values, as in Figure\u00a04B: 2560 microtubules, 40960 motors, microtubule growth speed\u00a0= 5\u00a0nm/s, motor speed\u00a0= 30\u00a0nm/s. For all other parameter values see Table S1. Simulated time is in min:s.max, and a polarity-sorting parameter P, based on the composition of motor crosslinks in the network and its connectivity. Color-coding the network types allowed us to visualize the different organizational states in the phase space , producing three planar sections through the multi-dimensional parameter space A. NematiMot/NMT)  B. This sg/vm) as a second combined parameter  that was previously shown to selectively inhibit microtubule plus-end growth d growth C We investigated the determinants of polar versus nematic cytoskeletal network organization using mitotic motor proteins and dynamic microtubules. We focused on these two prototypical active filament network states because of their importance for bipolar spindle organization required for chromosome segregation during cell division. Compared to previous self-organization assays , severalNematic microtubule networks were previously observed only for rather artificial conditions compared to the situation in the central spindle . Here, wWe identified two control parameters that determine microtubule/motor network organization and reflect underlying mechanistic driving forces; (1) the ratio of motor number per microtubule number that captures the organizational capacity of the system, and (2) the ratio of microtubule growth speed per motor speed that captures the competition between end-bound and side-bound motor crosslinks. Both control parameters combine a microtubule and a motor characteristic, emphasizing the importance of system-level properties for determining the outcome of active network self-organization. Together, these two control parameters define a phase space of reduced dimensionality that can be used to predict the organizational outcome of a microtubule/motor system A. PrevioThe rules for microtubule/motor network organization derived here are independent of the directionality and detailed molecular domain structure of the motors, which differ between plus-directed kinesin-5 KIF11 and minus-directed kinesin-14 HSET . Hence, Our work reveals that the asymmetry of microtubule growth properties is an important morphogenetic determinant in the spindle and responsible for motors of opposite directionality having different preferences for network organization. Fast and dynamically growing microtubule plus-ends and static minus-ends favor nematic versus polar network organization by plus- and minus-motors, respectively. This basic design principle puts a strong constraint on the structure of the bipolar spindle that can be conceptualized as a central nematic network coexisting stably with two polar networks B. BalancWe observed the gradual transition between nematic and polar organization in our experimental and simulated phase spaces. This may imply that, in the spindle, the control parameters can change gradually as the degree of microtubule polarity-sorting changes from spindle center to pole . Our resOur rules for motor-mediated cytoskeletal network organization also provide explanations for several spindle phenotypes observed in mitotic cells or meiotic cell extract resulting from a variety of perturbations B. When pThe physiological importance of relative microtubule growth and motor speeds is also supported by the observation that an\u00a0artificial kinesin-5 that is fast enough to accumulate at microtubule plus-ends has been shown to prevent normal spindle formation in meiotic cell extract by separating half-spindles, forming a central inverted pole instead of a nematic network .Finally, the importance of the number of motors per microtubule for network organization may explain why a variety of perturbations that lead to a reduction of microtubule numbers (by either reducing microtubule nucleation efficiency or microtubule stability) without affecting motor abundance, disfavor the formation of the central nematic zone and hence induce monopolar (or multipolar) spindle phenotypes .Hence, the concepts developed here not only explain the contributions of asymmetrically growing microtubules and different motors for normal spindle shape, but also for commonly observed phenotypes when motor activities or the numbers of the major molecular constituents of the spindle network are unbalanced. The next challenge will be to reconstitute and model\u00a0more complex active networks, extending the concepts developed here. A major aim will be to gain a quantitative understanding of the conditions allowing the unique coexistence of nematic and polar networks in multi-motor systems such as the bipolar spindle. Furthermore, it will be interesting to see to what extent these principles can also be extended to other cytoskeletal systems in cells such as dynamic actin networks .thomas.surrey@crick.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Thomas Surrey  medium in the presence of appropriate antibiotics.Spodoptera frugiperda strain Sf21 grown in suspension at 27\u00b0C in Sf-900TM III SFM (1x) Serum Free Medium (GIBCO). Absence of mycoplasma contamination was verified regularly.For expression of recombinant proteins in insect cells we used BC136474.1 (ImaGene); for HSET NCBI reference Sequence: NM_002263.3 (Origene), for CAMSAP3-C NCBI Refernece Sequence: NM_001080429.2 (Origene)). The C-terminal fragment of CAMSAP3 was chosen based on its ability to preferentially bind to and stabilize the microtubule minus end and inhibit its growth (3G5-mGFP (pJR303) having KIF11 C-terminally fused to an alanine (A) \u2013 glycine (G) linker followed by monomeric GFP  and StrepTagII-mGFP-G5A-CAMSAP3-C (pCT011) having the C-terminal fragment of CAMSAP3 N-terminally fused to either mCherry or mGFP separated by a GA-linker. The StrepTagII in these constructs could later be removed by Tobacco Etch Virus (TEV) protease cleavage. The final protein products are referred to as KIF11-mGFP, untagged HSET, mCherry-HSET, mCherry-CAMSAP3-C, and mGFP-CAMSAP3-C throughout the manuscript. All constructs were verified by sequencing. Baculovirus preparation and protein expression in Sf21 insect cells (Spodoptera frugiperda) were carried out according to manufacturer\u2019s protocols .The full-length protein coding sequences of human kinesin-5 KIF11 (aa 1 \u2013 1056) and human kinesin-14 HSET (aa 1 - 673), and the C-terminal fragment of human CAMSAP3 (CAMSAP3-C: aa 757 - 1276) were amplified by PCR using the respective cDNAs as templates , pH 8.0) supplemented with protease inhibitors (Roche), DNase I , and avidin (10\u00a0mg per liter of culture to capture biotin from insect cell media). Resuspended cells were lysed by douncing (40 strokes) and the lysate was clarified by ultracentrifugation . Clarified lysate was then passed through a StrepTrap HP column . The column was washed with CAMSAP3 lysis buffer containing 0.5\u00a0mM ATP and then with CAMSAP3 lysis buffer. The protein was eluted in CAMSAP3 elution buffer  supplemented with protease inhibitors. The N-terminal StrepTagII was removed by overnight TEV protease cleavage on ice. The protein was then passed through HiPrep Desalting columns  to exchange the buffer to CAMSAP3 storage buffer . The protein was then run once more over a StrepTrapHP column to remove unspecifically binding contaminants. The flow-through was then subjected to size-exclusion chromatography using a Superose 6 Increase column  equilibrated in CAMSAP3 storage buffer. The mGFP-CAMSAP3_C or mCherry-CAMSAP3-C containing fractions were pooled, concentrated , ultracentrifuged , aliquoted, snap frozen, and stored in liquid nitrogen until use.Sf21 cells expressing recombinant KIF11-mGFP were resuspended in ice-cold KIF11 lysis buffer  supplemented with protease inhibitors, DNase I (10\u00a0\u03bcg ml/ml), and avidin (10\u00a0mg per liter of culture). Resuspended cells were lysed by douncing (40 strokes) and the lysate clarified by ultracentrifugation . Clarified lysate was then passed over a StrepTrap HP column. The column was washed with KIF11 lysis buffer and protein was eluted in KIF11 elution buffer  supplemented with protease inhibitors. The N-terminal StrepTagII was removed by overnight TEV protease cleavage on ice. The protein was then purified further by size-exclusion chromatography using a Superose 6 XK 16/70 column  equilibrated in KIF11 storage buffer . The KIF11-mGFP containing fractions were pooled, concentrated , ultracentrifuged , aliquoted, snap frozen, and stored in liquid nitrogen until use.HSET and mCherry-HSET were purified like CAMSAP3 proteins, however HSET lysis buffer , HSET elution buffer  and HSET storage buffer  were used instead of the corresponding CAMSAP3 buffers.Escherichia coli BL21 pRIL, purified, and labeled with SNAP-Surface AlexaFluor546 (NEB)  and stored in EB3 storage buffer  as described recently , CF640R-NHS (Sigma-Aldrich), or biotin-NHS (Thermo Scientific), as described previously (Porcine brain tubulin was purified as described . Purifieeviously . Labelin2 was a kind gift from Marcel Knossow and Andreas Pl\u00fcckthun (Purified recombinant DARPin (D1)l\u00fcckthun .2) or dimers (tubulin).Protein concentrations were determined by Bradford assay or by spectroscopy measurements (absorption at 280\u00a0nm) for tubulin. Concentrations refer to protein monomers 2 (3000 Da) (Rapp Polymere) was used. This modification together with biochemical improvements allowed exploring a considerably wider range of protein activities than in previous self-organization experiments , 1.5\u00a0mM ATP, 1\u00a0mM GTP, 5\u00a0mM 2-ME, pH 6.8) and warmed up to 33\u00b0C on a metal block. Meanwhile the final assay mix was prepared on ice and ultracentrifuged at 278,088 x g for 10\u00a0min at 4\u00b0C. The supernatant was transferred to a fresh Eppendorf tube, allowed to come to room temperature, and then flowed into the pre-warmed flow chamber on a 33\u00b0C metal block. The sample was then transferred to the spinning disc confocal microscope and imaging was started 2-4\u00a0min after flowing the sample into the warm chamber.Flow chambers were assembled from a glass slide and a cover glass separated by a double sticky tape. Unlike in previous self-organization\u00a0studies with microtubules and motors, for improved surface passivation here both glasses were silanized and reacted with polyethylene glycol as described  except teriments . A flow For KIF11-mediated microtubule organization in the presence of CAMSAP3-C, the final assay mix consisted of 64% SAB, 27.2% BRB80 containing oxygen scavengers, BSA and recycled and fluorescently labeled tubulin, 3.3% mCherry-CAMSAP3-C solution in CAMSAP3-C storage buffer and 5.5% KIF11-mGFP solution in KIF11-mGFP storage buffer. The final protein concentrations in the assay were 164\u00a0\u03bcg/ml catalase, 684\u00a0\u03bcg/ml glucose oxidase, 1\u00a0mg/ml BSA, 7.5 \u2013 30\u00a0\u03bcM recycled and fluorescently labeled tubulin (containing 3.5 \u2013 7% Atto647- or CF640R-labeled tubulin), 250 \u2013 1000\u00a0nM mCherry-CAMSAP3-C, and 9 \u2013 273\u00a0nM KIF11-mGFP, as indicated in the text.For HSET-mediated microtubule organization in the presence of CAMSAP3-C, the final assay mix consisted of 63.5% SAB, 27.2% BRB80 containing oxygen scavengers, BSA and recycled and fluorescently labeled tubulin, 4.8% mGFP-CAMSAP3-C or mCherry-CAMSAP3-C solution in CAMSAP3-C storage buffer, and 4.5% mCherry-HSET solution in HSET storage buffer. The final protein concentrations in the assay were 164\u00a0\u03bcg/ml catalase, 684\u00a0\u03bcg/ml glucose oxidase, 1\u00a0mg/ml BSA, 10 \u2013 30\u00a0\u03bcM recycled and fluorescently labeled tubulin (containing 3.5 \u2013 7% Atto647- or CF640R-labeled tubulin), 250 \u2013 1000\u00a0nM mGFP-CAMSAP3-C or mCherry-CAMSAP3-C, and 3.1 \u2013 400\u00a0nM mCherry-HSET or untagged HSET.2 (leading to inverted microtubule dynamics), the final assay mix consisted of 50.5% SAB, 44% BRB80 containing oxygen scavengers, BSA and recycled and fluorescently labeled tubulin, 4.5% mCherry-HSET solution in HSET storage buffer, and 1% DARPin (D1)2 solution in DARPin (D1)2 storage buffer. The final protein concentrations in the assay were 164\u00a0\u03bcg/ml catalase, 684\u00a0\u03bcg/ml glucose oxidase, 1\u00a0mg/ml BSA, 60\u00a0\u03bcM recycled and fluorescently labeled tubulin (containing 3.5% CF640R-labeled tubulin), 2.9\u00a0\u03bcM DARPin (D1)2, and 100\u00a0nM mCherry-HSET.For HSET-mediated microtubule organization in the presence of DARPin (D1)p 4,000, Sigma-Aldrich) to facilitate microtubule positioning near the coverslip for imaging.The flow chamber assembly and all sample preparation steps were performed similarly to the self-organization assay except that imaging was performed now by total internal reflection (TIRF) microscopy and SAB contained 0.15% (w/vol) methylcellulose , and 500\u00a0nM mGFP-CAMSAP3-C.2 (3000 Da) and biotin-CONH-PEG-NH2 (3000 Da), both Rapp Polymere) (2 (3000 Da)) counter glass prepared as described above for improved passivation, separated by double sticky tapes. The assay itself is a modification of the protocol developed earlier   and a si earlier . In shor earlier . The chaFor evaluating microtubule growth speeds in the presence of CAMSAP3-C, the final assay mix was composed of 68%SAB containing Alexa546-EB3, 27.2% BRB80 containing oxygen scavengers, BSA and recycled tubulin, and 4.8% mGFP-CAMSAP3-C solution in\u00a0CAMSAP3-C storage buffer. The final protein concentrations in the assay were 164\u00a0\u03bcg/ml catalase, 684\u00a0\u03bcg/ml glucose oxidase, 1\u00a0mg/ml BSA, 25\u00a0nM Alexa546-EB3, 7.5 \u2013 30\u00a0\u03bcM recycled tubulin, and 250 \u2013 1000\u00a0nM mGFP-CAMSAP3-C, as indicated in the text.2, the final assay mix was composed of 50.5% SAB containing Alexa546-EB3, 44% BRB80 containing oxygen scavengers, BSA and recycled tubulin, 4.5% HSET storage buffer, and 1% DARPin (D1)2 solution in DARPin (D1)2 storage buffer. The final protein concentrations in the assay were 164\u00a0\u03bcg/ml catalase, 684\u00a0\u03bcg/ml glucose oxidase, 1\u00a0mg/ml BSA, 25\u00a0nM Alexa546-EB3, 60\u00a0\u03bcM recycled tubulin, and 2.9\u00a0\u03bcM DARPin (D1)2.For evaluating microtubule growth speeds in the presence of DARPin (D1)2, pH 6.8) containing oxygen scavengers, bovine serum albumin (BSA), and GMPCPP-stabilized fluorescently labeled microtubules (prepared as described previously (Flow chambers were assembled from a poly-(L-lysine)-PEG (SuSoS)-passivated counter glass and an untreated cover glass separated by double sticky tape as described previously . The floeviously , 3.5% CFMicrotubule self-organization assays were imaged either on a 3i Marianas spinning disc confocal fluorescence microscope described earlier , or on aMicrotubule gliding assays, microtubule dynamics assays, and microtubule nucleation assays were imaged by total internal reflection fluorescence (TIRF) microscopy either on an iMIC TIRF microsocope (FEI Munich) described elsewhere  or on a Imaging conditions  were always kept constant within a set of experiments to allow for direct comparisons between samples. All imaging was performed in a heated chamber at 33\u00b0C \u00b1 1\u00b0C.http://www.cs.cmu.edu/\u223ckangli/code/Image_Stabilizer.html). Microtubule growth speeds and microtubule gliding velocities were determined by kymograph analysis. A mean microtubule growth speed was the average of the speeds of individual growth episodes of Alexa546-EB3-marked microtubules (or mGFP-CAMSAP3-C marked microtubules at lower tubulin concentrations where high concentrations of mGFP-CAMSAP3-C decorated the microtubule lattice and prevented EB3 accumulation at the ends) observed in the evanescent field of the TIRF microscope . This analysis provides only a rough estimate of microtubule growth speeds as microtubules frequently appear and then grow out of the evanescent field, and as opposed to surface-bound microtubules, can move around considerably even while growing in the evanescent field. The total length of microtubule growth trajectories for the nematic network regime was estimated from the same dataset by measuring the total EB3 comet displacement per microtubule by kymograph analysis . This analysis provides a lower limit of the actual microtubule lengths, because these measurements were conducted between 2-12\u00a0min after initiation of nucleation by temperature shift whereas the self-organization experiments were considerably longer in duration (up to 90\u00a0min) and likely contained longer microtubules. In addition, the EB3-marked growing microtubule ends frequently appear and then grow out of the evanescent field instead of staying near the glass surface throughout the whole microtubule lifetime. Mean microtubule gliding speeds were calculated as the average gliding speed of individual microtubules on the motor-coated glass surface. The total number of growth episodes, growth trajectories and microtubule gliding speeds measured for each condition are stated in the respective figure legends.Fluorescence microscopy images were processed and analyzed in Fiji and in MATLAB. Raw TIRF microscopy images were aligned as described earlier , and alsThe model used is as previously described . In brienuc with initial length L0. Dynamic\u00a0instability of microtubule plus-ends is implemented with a two-state model without rescue defined by a constant catastrophe frequency kcat, a constant shrinkage speed vs and a force-dependent growth speed vg  and otherwise used measured values from literature where possible see . The dimIn scanning the parameter space to identify control parameters we varied key parameters over reasonable ranges. The number of motors was explored up to a maximum of 16 motors per microtubule. The microtubules in the three phase spaces in p links connecting parallel microtubules sides at an internal angle of 0\u00a0\u2264 \u03b8\u00a0< \u03c0/3, Hap links connecting anti-parallel microtubules sides at an internal angle 2\u03c0/3\u00a0< \u03b8 \u2264 \u03c0 and X links connecting microtubule sides at an internal angle of \u03c0/3\u00a0\u2264 \u03b8 \u2264 2\u03c0/3 .g/vm can be explained through mathematical analysis of the motor distribution profile along a single growing microtubule (g. (2) Motor movement along MTs with a constant velocity vm. (3) Binding and unbinding of motors to the side of the microtubule with rate off respectively. (4) Motors that reach the plus-end of the microtubule do not detach immediately but remain bound and unbind at a rate kend. Analytical results for a more general version of this\u00a0model based on a continuous approximation of the classic TASEP model of a driven lattice gas  relating to https://doi.org/10.17632/s8wz47nc9p.1.The computational model was implemented in Cytosim, publicly available at"}
+{"text": "Orthopoxvirus (OPXV) genus but was highly diverged from currently known species, including Akhmeta virus. Here, we present the complete 210,797 bp genome sequence of the Alaska poxvirus isolate, containing 206 predicted open reading frames. Phylogenetic analysis of the conserved central region of the genome suggested the Alaska isolate shares a common ancestor with Old World OPXVs and is diverged from New World OPXVs. We propose this isolate as a member of a new OPXV species, Alaskapox virus (AKPV). The AKPV genome contained host range and virulence genes typical of OPXVs but lacked homologs of C4L and B7R, and the hemagglutinin gene contained a unique 120 amino acid insertion. Seven predicted AKPV proteins were most similar to proteins in non-OPXV Murmansk or NY_014 poxviruses. Genomic analysis revealed evidence suggestive of recombination with Ectromelia virus in two putative regions that contain seven predicted coding sequences, including the A-type inclusion protein.Since the eradication of smallpox, there have been increases in poxvirus infections and the emergence of several novel poxviruses that can infect humans and domestic animals. In 2015, a novel poxvirus was isolated from a resident of Alaska. Diagnostic testing and limited sequence analysis suggested this isolate was a member of the  Poxviridae is a family of large, double-stranded DNA viruses that infect a broad range of animal hosts, from insects to vertebrates. The Orthopoxvirus genus contains some of the best-characterized poxviruses, including Variola virus  and Vaccinia virus . Orthopoxviruses (OPXVs) differ greatly in their host range and virulence [irulence ,2,3. SomOPXV species [Cases of human infection by OPXVs have been increasing in recent years, with increases in cases of cowpox in Europe ,5, outbr species ,20, and  species . OPXV species: two isolated in Europe and one isolated in North America. Orthopoxvirus Abatino (OPVA) was isolated in Italy during an outbreak in captive macaques in 2015 and from a fatal infection in a cat in 2017 [OPXV genus but was highly diverged from known OPXV species [Recently, several novel poxviruses have been discovered after infecting humans and/or domestic animals ,29,30,31 in 2017 ,29,31. A in 2017 ,27. In 2OPXV genus contains two distinct clades of viruses in which genomic sequence similarity correlates to their historic geographic distribution in either the Old World or New World. Old World OPXVs contain seven currently recognized species, including VARV, VACV, MPXV, CPXV, Camelpox virus, Ectromelia virus (ECTV), and Taterapox virus [CPXV may encompass several species [Raccoonpox virus (RCNV), Volepox virus (VPXV) and Skunkpox virus (SKPV) [The ox virus , althoug species ,34. Thers (SKPV) ,35. Newls (SKPV)  and AKMVs (SKPV)  cluster s (SKPV) . The novs (SKPV) . The nins (SKPV) , which wOPXV species, Alaskapox virus (AKPV).The divergence of the Alaska isolate and the seeming disagreement between its isolation in the New World and sequence similarity to Old World OPXVs highlighted a need for further investigation. Here, we present the whole genome sequence of the poxvirus isolated from an Alaska resident  and propAccession numbers for reference OPXV genomes used in this study are listed in \u00ae Terminator v3.1 Cycle Sequencing Kit  on an ABI 3130XL Genetic Analyzer. Called sequences were analyzed using Seqman in the Lasergene Package . Consensus sequences were used to close gaps in the draft genome. Raw reads were mapped back to the gap-filled genome to inspect assembly accuracy.Sample details and collection information was described previously . The AKPab initio approach [Gene prediction was conducted using both an approach  and simiapproach  and GeneAll alignments presented were generated using mafft v7.308 ,39 in Gehttps://github.com/rambaut/figtree/) and edited in Inkscape (https://inkscape.org/). Percent nucleotide identities were reported from Geneious version 9.1.4  based on alignment where all columns containing gaps were stripped.The region from VACV-COP-E9L (DNA polymerase) to A24R (DNA-dependent RNA polymerase subunit rpo132) was extracted from AKPV, reference OPXV genomes, and NY_014, Murmansk, and Yoka poxvirus genomes  and aligA reciprocal blast approach was used to compare the AKPV genome with CPXV-BR and AKMV-2013. All predicted CDS were extracted from the AKPV, CPXV-BR, and AKMV-2013 genomes. CPXV-BR or AKMV-2013 CDS were queried against AKPV CDS, and AKPV CDS were queried against the CPXV-BR or AKMV-2013 CDS using blastn (ncbi-blast+ v2.4.0). Hits were determined using a cutoff Evalue of 0.01. To identify the closest annotated proteins for each predicted AKPV protein, all 206 predicted ORFs were extracted from the AKPV genome and translated into predicted amino acid sequences. The 206 predicted proteins were queried against the \u2018nr\u2019 database using blastp (ncbi-blast+ v2.4.0). Data reflects BLAST search performed on 12 February 2019. Top hit for each query sequence was determined based on bit score using default blastp parameters.To investigate if poxvirus homologs of T4 and B5R were under positive selection in AKPV, dN/dS was examined using PAML . T4 sequB5R and T4 alignments were analyzed for evidence of selection in AKPV using branch-site models in the CODEML package in PAML 4.5 . Log likA region from AKPV143 (DNA helicase) to AKPV175   was aligned to corresponding regions from AKMV-2010, VARV-BRA, HSPV, CPXV-A, CPXV-C, CPXV-E3, TATV, CMLV, ECTV-Moscow, ECTV-Hamptead, ECTV-Naval, OPVA, VPXV, SKPV, and RCNV-85A reference genomes using MAFFT v7.308   of 2.4 kb. The genomic terminal loop was not sequenced, so the leftmost nucleotide was arbitrarily assigned to be the first nucleotide. The A+T content was 67.2%, lower than the A+T content of New World OPXVs such as VPXV (68.7%), SKPV (68.5%) and RCNV (67.7\u201367.9%), but higher than that of most Old World OPXVs (66.3\u201367.3%) . In the The AKPV ITR was 2.4 kb, which is shorter than most OPXVs except VARV, where ITRs range from 0.1 to 1.2 kb  to 19 kbThe central region of OPXV genomes is highly conserved and contains genes involved in essential processes such as transcription, DNA repair and replication ,51,52. Tab inito gene prediction and similarity search revealed 206 predicted genes  were most closely related to an Old World OPXV sequence, including AKMV and OPVA . Eight predicted AKPV proteins returned top hits to New World OPXV proteins, including AKPV011 , AKPV013 (ankyrin repeat-containing protein), AKPV100 (Poly(A) polymerase small subunit), AKPV102 (late 16 kDa membrane protein), AKPV112 (virion core protein), AKPV123 (trimeric virion coat protein), AKPV126 (S-S bond formation pathway protein) and AKPV191 (kelch-like protein) . AKPV011The majority of the 206 predicted proteins (97%) were most similar to annotated OPXV proteins; however, seven predicted AKPV proteins were most similar to proposed proteins in the newly described Murmansk or NY_014 poxvirus genomes: AKPV009, 010, 024, 025, 203, 204, and 205 . MurmansAKPV024 was most similar to CKM51_gp196 (Ankyrin) in the NY_014 genome, with 92.5% nucleotide identity. AKPV025 was most similar to NY_014 CKM51_gp195 , but AKPV025 is much shorter, at 132 nt compared to 552 nt for NY_014 CKM51_gp195. The 38 nt at the AKPV025 N-terminus exhibited 92.1% nucleotide identity with position 30\u201367 in NY_014 CKM51_gp195, and the AKPV025 C-terminus was 91.5% identical to 393\u2013486, producing an alignment with a 325 bp gap in AKPV025. AKPV203, 204, and 205 were most similar to annotated genes in the Murmansk genome. AKPV203 was most similar to poxvirus protein B22R (surface glycoprotein). AKPV204 and 205 were most similar to Murmansk-186 and ankyrin-like protein Murmansk-194 (90.8% and 71.9% amino acid identity), respectively. Murmansk-194 has no known or predicted function, and there is no known homolog to Murmansk-194 or 186 in any poxvirus or other genome. The next best BLAST hit for AKPV205 had <50% amino acid identity and was a truncated ankyrin-like protein in the Yoka poxvirus genome. The next best BLAST hit for AKPV204 also had <50% amino acid identity and was IL-1 beta receptor from AKMV-2013. Interestingly, while AKPV203, 204, and 205 are consecutive in the AKPV genome, the homologs Murmansk-186, 194, and 007 (B22R) are not syntenic and are dispersed in the Murmansk genome. Only three of the predicted proteins in the AKPV genome exhibited less than 60% amino acid identity with known poxvirus proteins: AKPV182, 187, and 190. All three genes shared similarity and synteny with poxvirus genes encoding proteins with predicted functions in immune modulation, virulence, and/or host range, including hemagglutinin (AKPV182), EEV type-I membrane glycoprotein VACV-COP-B5R (AKPV187), and poxvirus T4 protein (AKPV190). Alignment of AKPV182 (hemagglutinin) with OPXV hemagglutinin homologs revealed an insertion in AKPV192 . FurtherAlignments of AKPV187 (B5R) and AKPV190 (T4) with OPXV homologs did not reveal any large insertions or deletions. These genes were analyzed for evidence of selection using PAML. Analysis using the two-ratio branch model revealed that AKPV187 (B5R) was significantly more likely to contain a different dN/dS than reference OPXV B5R homologs. Further investigation using branch-site models (model A and A1) revealed that AKPV187 was more likely to contain sites exhibiting positive selection than the reference B5R homologs . Bayes eThe AKPV genome contained host range/virulence genes typical of the OPXV genus, including homologs of VACV-COP-E3L, K3L, K1L, P28/N1R, B5R, C7L, T4, C3L, CrmB, and serpins SPI-1, 2 and 3 . The AKPThe AKPV genome contained an intact predicted A-type inclusion protein gene (AKPV150) of 3324 nucleotides (1107 amino acids). The A-type inclusion protein was shorter than that from CPXV-B (1279 amino acids), AKMV-2013 (1213 amino acids), and RCNV-Herman (1221 amino acids) due to deletions in the middle of the predicted protein . AKPV150Comparison of proposed AKPV coding regions with annotated genes from other OPXVs revealed AKPV168, 169, and 170 had high similarity to ECTV 140, 141, and 142, with AKPV 169 and ECTV 141 sharing >99% amino acid identity. Due to the high similarity across several sequential genes, a region from AKPV143 (DNA helicase) to AKPV175  of the AKPV genome  was examined for recombination. Potential recombination events were investigated using an alignment of this region of the AKPV genome with corresponding regions from AKMV-2010, VARV-BRA, HSPV-MNG, CPXV-A, CPXV-C, CPXV-E3, TATV, CMLV, ECTV-Moscow, ECTV-Hamptead, ECTV-Naval, OPVA, VPXV, SKPV, and RCNV-85A reference genomes. Recombination analysis using distance plot and bootscan in RDP4 software identified two potential recombinant regions between AKPV and ECTV . The firBased on the results of the recombination analysis using RDP4, AKPV143 to AKPV175 was split into four regions: two predicted recombinant regions, an intermediate region, and a flanking region . Each rePhylogenetic analysis of the potential recombinant regions revealed significant rearrangement compared to phylogenies produced from other areas of the genome. When comparing either the core region of the genome  or a regCowpox virus has led to the hypothesis that extant OPXVs evolved from a CPXV-like ancestor through gene loss and modification [The isolation and original characterization of the poxvirus isolated from a patient in Alaska suggested that this isolate represents a novel, divergent OPXV capable of infecting humans. The complete genome sequence of the AKPV isolate provided unique observations when compared with other OPXVs. The large genome and broad host range of fication ,59. CompPhylogenetic analyses and sequence similarity of conserved core genes indicated that AKPV is more closely related to Old World OPXVs than to North American OPXVs, in agreement with previous findings using nine conserved genes . AdditioThe success of a poxvirus infection depends on the virus\u2019s ability to evade the host immune response. Several genes in the poxvirus genome are known to play a role in modulating the host antiviral response. It has been hypothesized that the presence or absence of such genes as well as sequence differences may underlie the host range and virulence of a given poxvirus. In general, these genes are located at the terminal regions of the genome and often exhibit lower sequence identity and lineage-specific distribution ,58. The Careful analysis of the AKPV isolate genome revealed unexpected recombination with ECTV. ECTV was first identified in 1930 in a laboratory mouse and is the causative agent of mousepox, a disease found in mouse colonies in Europe, Asia, and the Americas ,62. AlthOrthopoxvirus genus. The discovery of new diverse species can strengthen existing understanding, and provide further insight or improve the resolution of previous analyses. It is very likely that the wealth of sequencing information reflecting OPXV diversity will continue to increase. This information can be used to inform diagnostics and may provide increasingly more accurate information about OPXV evolution and origins.Taken together, this study highlights the need for future studies of natural poxvirus circulation in wildlife to generate a better understanding of OPXV ecology and better preparedness for zoonotic infections in humans. The discovery of new, divergent OPXVs introduces the opportunity to re-evaluate the"}
+{"text": "Nodular lymphoid hyperplasia (NLH) is one of the most common non-neoplastic splenic lesions in dogs, especially in old ones, showing a splenic enlargement. More recent studies have been focused on Contrast Enhanced Ultrasonography (CEUS) analysis of the spleen for establishing normal perfusion patterns and blood pool phase peculiarities of focal lesions.The aim of the study was to evaluate the qualitative and quantitative CEUS analysis of the canine splenic NLH, characterizing the CEUS pattern of this pathology on 20 clinical cases.A prospective, observational study was performed using a system equipped with contrast-tuned imaging technology. Mechanical Index was set from 0.08 to 0.11; the contrast medium was a second generation contrast medium composed of sulphur hexafluoride encapsulated of a shell of phospholipids (SonoVue\u00ae). Qualitative and quantitative assessment of the enhancement pattern of splenic NLH were performed.Cytology and histology identified 20 splenic NLH. All of the benign hyperplastic lesions assessed were isoechoic with a homogeneous pattern than the surrounding normal spleen, during the wash-in phase (10\u201320\u2009s) of the CEUS exam. Before finishing the wash-in phase, 20\u201345\u2009s from the contrast medium inoculation, 19/20 benign nodules became markedly hypoechoic to the adjacent spleen. Sensitivity of hypoechoic pattern for NLH was 95%.These findings should prove useful in the evaluation of focal splenic masses in dogs. Since enhancement and perfusion patterns of NLH seem to coincide with some neoplastic lesions of the spleen previously reported, in clinical practice attention must be paid to the final diagnosis of canine splenic lesions using only the CEUS exam. Morphological changes of the splenic tissue related to the presence of focal lesions are common in aging dog .Nodular lymphoid hyperplasia (NLH) is one of the most common non-neoplastic focal splenic masses, especially in old animals showing enlarged spleen \u20134.Ultrasonography is the imaging technique of choice for the detection of splenic disease, even though it has a low specificity.Nodular hyperplasia has a variable appearance and cannot be differentiated on the basis of ultrasonography alone. Echogenicity of the nodules may be similar to the remainder of the spleen; irregular with a hyperechoic central region surrounded by a thin hypoechoic rim; well-demarcated with centralized hypoechoic areas; hyperechoic . SometimApplication of Doppler ultrasound allowed to evaluate the characteristics of large vasculature network within and adjacent to the focal splenic lesions  .At Computed Tomography, malignant splenic masses had significantly lower attenuation values than nonmalignant splenic masses, on both pre- and postcontrast images , althougThe development of microbubbles contrast media and the use of harmonic technique allowed to assess microcirculation. Moreover, by enhancement kinetics, contrast ultrasound provided a mean of quantification of tissues perfusion .More recent studies have been focused on Contrast Enhanced Ultrasonography (CEUS) analysis of the spleen for establishing normal perfusion patterns and blood pool phase peculiarities of focal lesions, with the purpose of overcoming the limitations of the standard ultrasonography altogether and replace invasive or very expensive procedures in discriminating between malignant versus benign splenic lesions \u201315.Several enhancement patterns have been observed in the benign focal lesions: isoechogenicity with the surrounding normal spleen in most of them; hypoechogenicity  throughout all blood pool phases and hyperechogenicity in the early arterial phase have also been detected in a few of them , 14.Morphological or functional modifications of the vascular network have been supposed to be responsible of the enhancement pattern variations in splenic lesions . AlteratBased on the aforementioned concerns, NLH enhancement and perfusion patterns during CEUS analysis thus far appear to be neither specific nor characterized in depth and deserve more attention in order to avoid misdiagnosis \u201314.To testing a previously developed diagnostic criteria, we performed a CEUS examination in 20 dogs affected by NLH, we also reported the quantitative CEUS in order to provide the quantification of blood flow parameters of this lesion.All dogs included in the study were considered healthy and their haematological and haematochemical parameters were within reference range.On B-mode examination, dogs enrolled had a single focal lesion with a mean diameter of 1,86\u2009cm (range: 0.45\u20133.04\u2009cm). 5/20 nodules were located near the splenic capsule, giving an alterated splenic border. 10/20 nodules were homogeneous and hypoechoic than the splenic parenchyma, 8/20 nodules had mixed echogenicity, only 2 lesions were isoechoic with small hypoechoic areas.Color and Power Doppler showed detectable vessels only in 10/20 lesions, with small vessels entering the lesion and in 3/10 with vessels followed the contour of the lesion.Contrast-enhanced ultrasound of the normal splenic tissue showed rapid enhancement of the small splenic arteries (10\u201313\u2009s), a heterogeneous phase of enhancement of the spleen that became homogeneous at peak enhancement and a slow decrease of enhancement.During the wash-in phase (10\u201320\u2009s) of CEUS exam the benign hyperplastic lesions (20/20) were isoechoic with a homogeneous pattern than the surrounding normal spleen.Starting 20\u201345\u2009s from the contrast medium inoculation, when enhancement of the normal spleen was becoming homogeneous until peak enhancement (about 40\u2009s after injection), 19/20 (95%) benign nodules became markedly hypoechoic to the adjacent spleen. Inner thin vessels enhancement were visible at this time, even though they gradually disappeared during the wash-out phase of the normal spleen, so that lesions acquired anechoic feature .Post-processing quantitative analysis showed, a TIC characterized by a faster wash-in and an early washout than normal parenchyma Fig.\u00a0.Fig. 2QuThe ascending segment of the curve was steep and then the curve rapidly descended to baseline.The three-dimensional images of Time to Peak provided a more intuitive visual representation of the contrast medium flow patterns, along the time, both in the nodular tissue and the surrounding normal spleen Fig.\u00a0.Fig. 3ThDescriptive statistical analysis of the quantitative CEUS-derived parameters revealed a normal distribution for each value. The perfusion values were significantly lower in the nodule tissue compared with the values of the normal spleen. The values of median, range and P statistical significance of perfusion parameters for both lesion and normal splenic tissues are provided in Table\u00a0All the examined smears showed cytological features common in all the investigated cases.They showed a variably degree of cellularity but even optimal quality and moderate hemodilution.All smears were characterized by a mixed lymphoid population mainly represented by a predominance of medium and small lymphocytes, a certain presence of lymphoblasts, rare plasma cells or other kind of cells. No signs of malignancy were observed.These findings recalled the features of a typical hyperplastic lymph node response; thus, on this basis and on mere cytological observation, they strongly suggested a diagnosis of hyperplastic nodular reaction of the spleen.None of dog had adverse reaction during the procedure. B-mode follow up examination after one month showed unremarkable changes of nodules.In human medicine, contrast-enhanced ultrasound (CEUS) has been applied in different clinical settings and guidelines for CEUS study of the spleen has been established in a specific study . ContrasAlso in veterinary medicine, studies about the CEUS perfusion pattern of the normal spleen and of focal lesions were performed and most benign splenic lesions showed a perfusion pattern similar to the adjacent parenchyma, so that the lesions were isoechoic to the surrounding normal spleen after the first few seconds and remained homogeneously isoechoic in the wash out phase \u201314. The In the present study we applied CEUS by using the contrast agent sulfur hexafluoride microbubbles in order to determine contrast medium behavior in cytological and histological diagnosed focal splenic NLH in 20 dogs.The appearance of a markedly hypoechoic enhancement to the adjacent spleen 20 to 45\u2009s following the contrast medium inoculation in 95% of examined lesions was the main finding.In addition, by quantitative analysis, faster TTP and MTT associated to a lesser RBF, and lower PI and RBV in the lesions compared with the surrounding normal parenchyma have been detected, suggesting increased blood velocity and a lower blood volume.The occurrence of an early wash-in and early wash-out in the NLH observed in our study, does not agree with results of the previous surveys \u201314.The histologic knowledge about rearrangements of the microvascular environment which can occur in course of nodular lymphoid hyperplasia give us an explanation for the differences on tissue perfusion assessed by Contrast-enhanced ultrasound in NLH lesions and surrounding normal spleen .To justify high perfusion values detected by CEUS analysis in the hyperplastic nodules, Ohlerth et al. (2008) have proposed that distortions of the marginal zone, occurring in the NLH , may leaHeterogeneous echogenic patterns, characterized by isoechoic regions to the normal spleen, interspersed among anechoic areas, were observed in case of NLH associated with splenic hematomas, and even hematomas without hyperplastic foci were found hypoperfused during CEUS evaluation , 29. HowModifications of the leukocytes environment of the spleen have been seen to affect vascular organization and blood dynamics, nevertheless, hyperplastic lymphoid tissue is known to own a dense vascular bed and to be highly vascularized , 20, 26.This morphological feature do not agree with the isoechoic appearance of NLH to the surrounding parenchyma during all blood pool phases.In our study, as many as 19 out of a total of 20 focal splenic nodules had early wash-in and wash-out phases than the normal parenchyma. These findings suggest a variation of the blood dynamics in parallel with the reorganization of the parenchyma vascular architecture. The isoechoic feature of the lesions to the normal spleen in the arterial phase prompts us to suppose the lack of alterations of the vascular-type structures responsible for blood supply. By contrast, hypoenhancement of the focal masses at the peak-enhancement and during the wash-out phase of the spleen, and the lower values of RBV and RBF in the lesions compared with the normal surrounding, probably reflect considerably decrease of the vascular spaces, specifically of the venous sinusoids. Other authors have proposed similar vascular arrangements to be responsible of the faster wash-in and wash-out phases in the malignant tumors to the surrounding normal spleen parenchyma and have speculate that the lack of normal sinusoids combined with neoplastic angiogenesis might be one of the causes of a malignant hypoechoic pattern during the late vascular phase , 13. In About the differences during parenchymal phase about hepatic nodular hyperplasia and splenic nodular hyperplasia, Nakamura et al. (2010), although the precise mechanism of splenic parenchymal phase imaging is not fully determined, speculated that the contrast defect during the parenchymal phase created by splenic nodular hyperplasia might be because of a decrease of splenic macrophages .Other factors such as transducer frequency, scanning parameters, contrast agent features, multiple injections of the contrast medium, patient-related factors and operator skills might influence both the subjective assessment and the measurements of perfusion , 27\u201329.About the contrast agent dose, we used 0.04\u2009ml/kg of body weight, this dosage was lightly different from 0.03\u2009ml/kg previously used in spleen studies with sulphur hexafluoride contrast medium , 30\u201332. Another limitation of this study is that the diagnosis of splenic Nodular Lymphoid Hyperplasia was performed in most cases by ultrasound-guided cytologic aspirate and not from histologic samples of the specific lesion. However, also in 48 out of 60 subjects studied by Ohlerth et al. and in benign lesions reported by Rossi et al., the diagnosis derives from ultrasound-guided cytology , 14.Although cytologic diagnoses often reflect histologic results, if missampling or incomplete sampling occurs or tissue architecture is required to distinguish between reactive and neoplastic conditions, accurate diagnosis with fine-needle aspiration may not be possible .However, in a clinical environment and on incidentally detected splenic nodules, although percutaneous fine-needle aspiration and needle core biopsy can be performed safely in dogs with sonographic splenic changes , ultrasoTo date, there are not specific cytologic pattern to determine with high specificity the nature of splenic focal lesion and most of them have been investigated only on histopathology. In fact, cytology, especially if performed as a \u201cquick\u201d and routine diagnostic method, lacks of important features, like, for example, the architecture and distribution of cells as they really appear in the organ or in the lesion. Furthermore, in a routine work, as cytology is often involved, it is quite impossible to discern, with the usual staining methods, the T or B nature of cells, thus, in case of tumor suspect, other techniques must be applied and, therefore, the patient must be otherwise considered. In this order, immunocytochemistry, cytofluorimetry or Polimerase Chain Reaction for antigen receptor rearrangement (PARR) could represent further methods to consider beside the cytology to differentiate a reactive lymphocyte population from a neoplastic one especially when a malignancy is suspected .In this study a scheduled follow up ultrasound of one month after the diagnosis was performed for all dogs; 12/20 subjects have been checked a second time, during a routine clinical examination over the next 6\u2009months. Certainly, a regular control of the lesion for a longer time could have provided greater strength to the citological diagnosis of NLH.Finally, the CEUS pattern observed in this canine benign splenic lesion was similar to that reported by some authors for splenic malignant neoplastic lesions \u20139. This The CEUS pattern observed in this canine benign splenic lesion study was similar to that previously reported for splenic malignant neoplastic lesions. Consequently, in clinical practice, attention must be paid to the final diagnosis of canine splenic lesions using only the CEUS exam and only accurate anatomo-vascular studies can clarify the coincidence of similar patterns of the contrast-enhanced ultrasound study in different splenic lesions.Informed owners\u2019 consents were obtained. All treatments, housing, and animal care were in compliance with EU Directive 2010/63/EU on the protection of animals used for scientific purposes and with Department\u2019s Animal Ethics Council approval (protocol number: 13/2017).n\u2009=\u200916) or histopathologic (n\u2009=\u20094) samples taken during splenectomy.A CEUS prospective, observational study was performed (October 2015\u2013December 2017) in 20 consecutive dogs with an incidentally detection of a splenic nodular lesion during routine abdominal ultrasonography. Conclusive diagnosis of NLH, after CEUS study, was performed by ultrasound-guided fine needle aspirates ; the bodyweight ranged from 5 to 28\u2009kg (mean 14\u2009kg).Examinations with B-mode, Doppler ultrasonography and CEUS were performed on all dogs.B-mode and Doppler ultrasonography of the abdomen were performed by the same operator (C.M.), holding a GPCert (Diagnostic Imaging), during her PhD course and under supervision of a professor of veterinary medical imaging (MDM), using microconvex (5.0 to 8.0-MHz) and linear (10 to 12-MHz) transducers. Spleen tissue was considered normal if the contours were regular and smooth; if the parenchyma was finely textured, homogeneous and more echogenic than liver and the cortex of left kidney. Lesions of the spleen were assessed for their size, number, echo pattern , and for the presence of cavitary areas within them .Color Doppler evaluation allowed to evaluate the splenic vascularization, assessing the path of blood vessels in and around the lesions.CEUS examinations was performed by the same investigator (MDM), using a system equipped with contrast-tuned imaging technology  in the not sedated dogs.After detecting the lesions with standard ultrasound, CEUS was performed with a linear (5.0\u2009MHz) transducer with contrast agent capability; a Mechanical Index (MI) from 0.08 to 0.11, persistency off, a wide dynamic range, and a single focal zone was set deeper to the lesion, including in the image part of normal splenic parenchyma surrounding the lesion.Eleven exams were done with a machine that allowed to have a double image (B-mode and CnTI images simultaneously); nine exams with a machine with a single image . The contrast agent used was a sulphur hexafluoride signal enhancer , and it was prepared following the manufacturer\u2019s recommendations.An aliquot  of the contrast medium was injected by a second operator (MP), in accordance with a methodology previously reported , 38. EacThe splenic focal lesions were classified according to their echogenicity compared to normal tissue in hyperenhancing, isoenhancing, hypoenhancing or not enhancing (anechoic). The presence of homogeneous or heterogeneous lesion patterns, rim enhancement or inner vessels were also evaluated.0 (injection time) until the maximum SI of the contrast agent. And again, the Regional Blood Flow (RBF) is defined as the ratio between Regional Blood Volume (RBV) and Mean Transit Time (MTT). RBV is proportional to the area under the curve, defined as the area under the time intensity curve during the wash-in and wash-out phase; Mean Transit Time (seconds) was defined as the time interval between half of the maximum SI of the contrast agent in the ascending phase of the curve (wash in) and the same value of SI in the descending phase (wash out).Post processing quantitative analysis of video-clips was performed using a dedicated image-analysis software  during wash in, peak and wash out phases. For each dog, one Region of Interest (ROI), as large as possible, was manually drawn around the entire lesion. A second one ROI, of a similar size to the previous one, was drawn on the normal parenchyma for comparison. For each ROI, a Time Intensity Curve (TIC) was generated, which is a parametric curve of time  versus SI, Signal Intensity, . During CEUS the tissue perfusion was evaluated based on video SI variations over time. The maximal SI was defined as a white band in the grey scale bar (8 bit). Within the selected ROI, the software generated the following parameters: Peak Intensity (PI) defined as the percentage increase in SI reached during bolus transit at time T, from baseline intensity to maximal SI; Time to Peak (TTP), defined as the time, in seconds, from TFor each of the 16 cases where FNA was performed, at least three smears were prepared and stained with MGG. In order to assess a cytological judgement and considering the cytology of such lesion overlapping a lymph node cytology, the following criteria were taken into account: cellularity, homogeneous or mixed lymphoid population, more represented cytotype, cytological elimination criteria for neoplasia versus other differential diagnoses , 40.t-test was used. When data were non-parametric the Mann-Whitney\u2019s u-test was used. To assess a significant association between the type of enhancement of a lesion with the initial diagnosis of benign lymphoid hyperplasia during the blood pool phases, sensitivity with 95% confidence intervals (CI) was calculated. Statistical significance was assessed at the P\u2009<\u20090.05 level.Statistical analysis was performed with a standard computer software program . All data were expressed as median and range. To compare perfusion values of normal splenic tissue to the values of the lesion tissue, the Student\u2019s"}
+{"text": "To investigate the effect of therapeutic listening on state anxiety andsurgical fears in preoperative colorectal cancer patients. A randomized controlled trial with 50 patients randomly allocated in theintervention group (therapeutic listening) (n = 25) or in the control group(n = 25). The study evaluated the changes in the variables state anxiety,surgical fears and physiological variables . In the comparison of the variables in the control and intervention groups inpre- and post-intervention, differences between the two periods for thevariables cortisol (p=0.043), heart rate (p=0.034) and surgical fears(p=0.030) were found in the control group, which presented reduction in thevalues \u200b\u200bof these variables. NCT02455128.There was no reduction in the levels of the variables state anxiety andsurgical fears resulting from the therapeutic listening intervention, eitherthrough the physiological or psychological indicators. However, the contactwith the researcher during data collection, without stimulus to reflect onthe situation, may have generated the results of the control group. ClinicalTrial Registration:  There areindications that psychotherapeutic interventions can contribute to the reduction ofemotional distress and to the improvement of the quality of life of people withcancerTherapeutic listening is a communication resource that can be valuable in the carerelationshipDespite of the recognized therapeutic value of listeningThis is a prospective, parallel, open-label randomized controlled trial with equalallocation rate (1:1). The study participants were patients admitted for surgical treatment of colorectalcancer in the surgical clinic of a teaching hospital located in a city in the stateof S\u00e3o Paulo (Brazil). For the calculation of the sample size, the State-TraitAnxiety Inventory (STAI) was used. Considering a difference of 10 points (\u03b4) in theState-Trait Anxiety Inventory - Sate (STAI-S) score, a significance level of 5% and a power of 80% (z1-\u03b2 = 1.96), the result was 25 individuals foreach group. The data related to the group variances was obtained by a proceduretest, with correlation of 0.5.Participants were eligible for inclusion if they: (a) were 18 years old or older, (b)were hospitalized for colorectal cancer surgery, (c) were not undergoing any othercancer treatment, (d) were not participating in another research (e) had a level ofeducation that allowed reading and interpreting the instruments used in the study,which were self-reporting (f) were clinically well and/or stable , and (g)presented a state anxiety score equal to or greater than 25 in the STAI-S in thefirst approach, which was performed previously and independently from thepre-intervention data collection. The cut-off point of 25 in the STAI-S was based onthe findings of a study on the effects of complementary therapies on clinicaloutcomes in patients being treated with radiation therapy for prostate cancerThe exclusion criteria were: (a) having psychiatric disorders , (b) presenting metastasis and (c) using medicationcontaining corticosteroids.The discontinuity criterion adopted for this study was related to patients who werereceiving procedures necessary for the surgery during the data collection process,such as the preparation of the intestinal tract. These patients were discontinuedfrom the study.In the IG, the patients were informed that they would have 30 minutes to talk to theresearcher about their experience with hospitalization for cancer treatment. Theinteraction was initiated with the following guiding question: \u201cHow has yourexperience been in the hospitalization for the treatment of your disease?\u201d Beforethe end of the therapeutic listening intervention, the patient was asked thefollowing question: \u201cIs there anything else you would like to talk about?\u201d In theCG, patients were told they would have some data collected. Subsequently, theresearcher would be absent for 30 minutes and, after this interval, would return forthe conclusion of the research.The data were collected from August 2014 to October 2015. Data collection scheduleswere previously set according to the hospital routine. Participants were admitted toa preoperative unit the morning of the day before surgery. Data collection occurredon the day of admission of the patients, who were invited to participate in thestudy after being informed about the purpose of the research. The data collection occurred in three moments: first approach, pre-interventionmoment and post-intervention moment. In the first approach, carried out at 8a.m.,when patients had already been assessed for eligibility according to the inclusionand exclusion criteria, the STAI and a questionnaire to characterize theparticipants were applied. After two and a half hours, in the second moment of thestudy (pre-intervention) the following data were collected: saliva samples foranalysis of salivary cortisol and salivary alpha-amylase (SAA), heart rate (HR) andrespiratory rate (RR), systolic blood pressure (SBP) and diastolic blood pressure(DBP), state anxiety, and surgical fears. One hour after the pre-intervention stageand shortly after the IG intervention and the CG, in the third and last moment ofthe study (post-intervention), the same variables were collected. In order to verifythe presence of circadian rhythm of cortisol in the participants, two samples ofsaliva were collected, one at 8p.m. and the other at 11p.m., on the day before thesurgery. The objective of the verification of the circadian rhythm was to identifyif the participants of this study, patients with cancer, would present differencesin this rhythm.Participants were randomized into two groups: control and intervention. For this, aperson who was not part of the activities developed in this research generated arandomized list in Excel 2007, which contemplated the CG and the IG. The sheetscontaining the descriptions \u201cIntervention Group\u201d and \u201cControl Group\u201d were eachplaced in opaque envelopes, sealed and opened by the main researcher after the datacollection in the pre-intervention moment, when it was decided on which group thepatient would be allocated. The instruments used in this study were answered by thepatients themselves and the data referring to the physiological variables werecollected by the researcher.During the data collection period, two participants declined to participate and fivewere discontinued due to routine hospital care activities during data collection.Thus, 50 participants reached the end of the study .The following instruments were used for data collection:Socio-demographic questionnaire: the socio-demographic variables collected were age,gender, level of education, marital status, monthly family income and religion;State-Trait Anxiety Inventory: the anxiety was evaluated through the STAISurgical Fear Questionnaire (SFQ): Fears related to surgery were measured using theSFQ, which was validated by the researchers for use in this study, with the author\u2019sauthorization. The scale assesses surgical fears in 8 descriptive statements dividedinto two subscales: \u201cfear of the short-term consequences of surgery\u201d (5 items) and\u201cfears of the long-term consequences of surgery\u201d (3 items). The score for each itemranges from 0 to 10. To calculate the overall score, the sum of the scores for eachitem should be divided by the number of items in the instrument. Thus, higher valuesare associated with higher levels of fear\u00ae (Sarstedt - Alemanha) swab in cotton was used to collectsaliva for salivary cortisol identification. Samples were analyzed using the HighSensitivity Salivary Cortisol Ezyme Imunoassay Kit , method ELISA/EIA. A system consisting of a disposable test strip and aportable analyzer, Cocoro Meter\u00ae (Nipro Corporation - Japan) was used to collect andanalyze saliva for SAA identification.Regarding the physiological variables, HR and SBP/DBP were measured using the Omron\u00aeblood pressure and heart rate portable monitor (Japan). RR was identified bycounting thoracic breathing movements for a period of 1 minute.SalivetteThe data obtained from the questionnaires were typed and organized in a spreadsheetusing Microsoft Office Excel 2007. This spreadsheet was exported to the statisticalprogram IBM - SPSS version 22, in which all the statistical analyzes of this studywere conducted.The variables for sociodemographic characterization of the sample were analyzed usingdescriptive statistics, with analyzes of distributions and frequencies. Thedistribution of the data was verified by the Shapiro-Wilk test. All variablespresented non-normal distribution; therefore, non-parametric statistics were chosento perform the other analyzes, considering p-values 0.05 as significant.The non-parametric Mann-Whitney U test was used for the intragroup comparison of thephysiological variables, state anxiety and surgical fears in the pre- andpost-intervention moments. For the intergroup comparison, the non-parametricWilcoxon test was used with repeated measures in the two moments.NCT02455128).After being informed about the study, participants were also informed about theanonymity and confidentiality of the data, and then signed the Informed ConsentForm.The research was approved by the Research Ethics Committees of the Ribeir\u00e3o PretoSchool of Nursing and of the Hospital das Cl\u00ednicas of the Medical School of Ribeir\u00e3oPreto (CAAE 11683313.9.0000.5393) and registered in the Clinical Trials platform( The mean age of the participants was 58 years in the IG (SD = 11) and 57 years inthe CG (SD = 15). Most of the participants had low level of education (incompleteand complete primary education), were married, Catholic and had a monthly familyincome between one and three minimum wages . The equivalence of the groups regarding the variables AAS, cortisol, HR, RR, SBP,DBP, state anxiety and surgical fears was verified in the pre-intervention moment.It is worth noting that the equivalence between the intervention and control groupsregarding the variables of interest  was verified in the pre-intervention moment was verified throughthe Mann-Whitney U test and there were no significant differences between groups.The means of the variables analyzed in the pre- and post-intervention are presentedin There were no significant differences between the groups after the intervention, soat that moment the groups were equivalent in relation to the studied variables.Thus, the therapeutic listening intervention did not cause differences between thetwo groups under the conditions in which it was applied to the participants .Two patients in the IG and three in the CG did not have enough saliva for the SAAanalysis. The same occurred for cortisol with one participant in the IG and one inthe CG. According to the results of Of the 50 participants in the study, 36 (72%) had their complete samples collected,in the morning and at night, with the volume of saliva necessary for the respectiveanalyzes. For fourteen individuals, it was not possible to evaluate the circadianrhythm of salivary cortisol for different reasons: the amount of saliva collectedwas not enough in 3 patients (6%), the collection of the nocturnal period was notperformed in 10 patients (20%) and one patient (2%) presented contamination of thesample. According to the specifications of the kit used for cortisol analyzes, thereference value for salivary cortisol at 11p.m. in normal subjects is between 0.007and 0.115 \u03bcg/dL. Thus, of the 36 subjects evaluated, 15 (41.6%) had a circadianrhythm and 21 (58.3%) had no circadian rhythm.In this study, we investigated the efficacy of therapeutic listening on state anxietyand surgical fears in preoperative colorectal cancer patients. Othernon-pharmacological interventions have also been tested for their effectiveness inreducing anxiety in cancer patients and, in the circumstances in which they wereperformed, presented results that corroborate this study, since they also had noinfluence in reducing this feeling. A randomized controlled trial was conducted withthe objective of testing the hypothesis that a multidisciplinary approach couldimprove understanding of the information provided by the anaesthesiologist and inturn, reduce anxiety in women undergoing surgery for breast cancerIt is worth noting that in the present study and in the aforementioned studyOn the topic of surgical fears, a study that aimed to identify the most commonconcerns about general anesthesia during the preoperative anesthetic clinic indifferent healthcare settings found that 88% of the patients experiencedpreoperative fear. The main causes were fear of postoperative pain (77.3%), fear ofintraoperative awareness (73.7%) and fear of being sleepy postoperatively(69.5%)According to the results of this study, in the pre-intervention moment the patientspresented moderate levels of state anxiety and low levels of surgical fears . It is pThere are reports that the communication between researcher and patient during thedata collection moments can contribute to decrease the anxiety levels of thepatients, even in individuals of the control groupsA possible justification for the changes found in the CG not being observed in the IGis that, in the latter, the issues discussed raised reflections about the experienceof hospitalization for the treatment of the disease, which may have made thepatients remember their concerns about such a situation. In order to assist thepatient in the management of feelings such as anxiety and fear, there may be a needfor more time for therapeutic listening, with a greater number of meetings betweennurse and patient. In adittion, the 30-minute time for the evaluation of theseparameters may have been small for processing the emotions raised in thenurse-patient interaction, affecting the parameters observed in the secondevaluation.-Regarding the circadian rhythm of cortisol, it should be considered that tumorpatients may exhibit nearly normal or markedly altered circadian rhythmsThe objective of this study was to evaluate the effect of therapeutic listening onstate anxiety and surgical fears preoperative colorectal cancer patients. In theconditions under which the intervention was conducted and considering the stateanxiety and surgical fears found in the pre-intervention moment, it was not possibleto observe a reduction in the levels of the physiological and psychologicalvariables related to the therapeutic listening.However, the meeting between the researcher and the patients of the CG for datacollection, when there was only the contact without stimulus to reflect on thesituation they were experiencing (intervention), may have allowed the reduction ofsalivary cortisol, HR and surgerical fears. Thus, on the day before the surgicalprocedure, the care and attitudes offered by the nurse to the patient in this studywere efficient in reducing the variables assessed.One factor that must be taken into account was the time of interaction with theresearcher for the therapeutic listening, which was the period between the twomoments of data collection (pre-intervention and post-intervention), when thepatients were invited to reflect on their situation and to expose their feelings andthoughts. The 30-minute time may have been insufficient for the patient to performthis task and then return to a regular emotional state. This may have interfered inthe results of the studied variables and, consequently, in their measurements.Possibly, a greater number of sessions could produce different results. However, inthe preoperative context in which this study was performed this would not bepossible, since the patients were admitted as close as possible to the time ofsurgery. In another context, such as in an outpatient service, it would have beenpossible to have more encounters with the patients.In addition, it is possible that a period longer than 30 minutes between theintervention and the collection of the post-intervention data can provide smallerscores of the studied variables, since, with a longer time after the therapeuticlistening, the patients would have time to process their emotions and, thus, couldpossibly present emotional conditions closer to the desirable. These aspects deservefurther study.The present study highlights the use of therapeutic listening as a nursingintervention in patients with colorectal cancer in the preoperative period,considering that the use of this intervention may enable a patient-centeredinformation collection, since therapeutic listening puts the patient, and not thedisease, as the center of the actions."}
+{"text": "Incomplete fracture healing may lead to chronic nonunion; thus, determining fracture healing is the primary issue in the clinical treatment. However, there are no validated early diagnostic biomarkers for assessing chronic nonunion. In this study, bioinformatics analysis combined with an experimental verification strategy was used to identify blood biomarkers for chronic nonunion.First, differentially expressed genes in chronic nonunion were identified by microarray data analysis. Second, Dipsaci Radix (DR), a traditional Chinese medicine for fracture treatment,\u00a0was used to screen the drug target genes. Third, the drug-disease network was determined, and biomarker genes were obtained. Finally, the potential blood biomarkers were verified by ELISA and qPCR methods.Fifty-five patients with open long bone fractures  were enrolled in this study, and urgent surgical debridement and the severity of soft tissue injury had a significant effect on the prognosis of fracture. After the systems pharmacology analysis, six genes, including QPCT, CA1, LDHB, MMP9, UGCG, and HCAR2, were chosen for experimental validation. We found that all six genes in peripheral blood mononuclear cells (PBMCs) and serum were differentially expressed after injury, and five genes  were significantly lower in nonunion patients. Further, CA1, MMP9, and QPCT were markedly increased after DR treatment.CA1, MMP9, and QPCT are biomarkers of nonunion patients and DR treatment targets. However, HCAR2 and UGCG are biomarkers of nonunion patients but not DR treatment targets. Therefore, our findings may provide valuable information for nonunion diagnosis and DR treatment.ISRCTN13271153. Registered 05 April 2020\u2014Retrospectively registered.ISRCTN,  Fracture healing is a complex process and is dependent on multiple factors . LimitedIn fracture healing, monocytes are involved in angiogenesis  and diffDipsaci Radix (DR) is derived from Dipsacus asperoides, a classical, traditional Chinese medicine with a long history of safe use for the treatment of bone fractures , 10. DR In this study, bioinformatics analysis methods, including microarray data analysis and an integrated systems pharmacology approach, were used to predict the potential biomarkers of chronic nonunion and mechanisms of DR treatment. First, the differentially expressed chronic nonunion genes in PBMCs were identified using microarray datasets. Second, the potential active ingredients of DR were used to screen the possible target genes. Third, the DR target genes that were also potential biomarkers of chronic nonunion were obtained. Finally, the predicted biomarkers of chronic nonunion were verified by experimental methods, such as enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR), with blood samples from healed and nonunion patients. Our study provides a more specific and effective way to investigate blood biomarkers for chronic nonunion and provides new insight into the mechanisms of DR in the treatment of chronic nonunion.p < 0.05 and |logFC| > 1 as cutoff values for screening differentially expressed genes (DEGs). The DEGs of each comparison group are shown as volcano plots. The interaction of the DEG sets was obtained by Venn diagram.Microarray datasets GSE93138, GSE93213, and GSE93215 were downloaded from the Gene Expression Omnibus (GEO) database  and collThe bioactive ingredients of DR were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database . We set The STRING database  was usedThe GO enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) , which iPatients admitted to the Department of Orthopedics of Xuanwu Hospital from August 2018 to July 2019 were enrolled in this study. Participants were skeletally mature, and all were diagnosed with only one open fracture of a long bone, including the humerus, radius/ulna, femur, and tibia/fibula. Exclusion criteria included patients who had a severe head injury, renal insufficiency, liver disease, systemic inflammation (CRP > 0.5\u2009mg/dL), and osteoporosis. Patients whose initial surgical debridement occurred within 8\u2009h and those whose surgical debridement occurred after 8\u2009h were treated with internal fixation according to the type of fracture by well-trained orthopedic surgeons. Ultimately, 55 participants were enrolled in the study and set as the acute injury (AI) group. Then, the AI group was categorized into healed and nonunion groups according to the amendment from the Food and Drug Administration (FDA) on the diagnostic criteria for nonunion. The FDA defines nonunion as a fractured bone that has not completely healed within 9\u2009months of the initial injury and without signs of healing for at least 3\u2009months . In thisg, and serum was isolated and stored at \u2212 80\u2009\u00b0C. The serum concentrations of the biomarkers were measured using separate ELISA kits  according to the manufacturer\u2019s instructions.Whole blood samples were taken from all participants and centrifuged for 5\u2009min at 4000\u00d7Five milliliters of peripheral blood was taken from all participants. Ficoll-Paque density gradient centrifugation was used to isolate the PBMCs, and the cells were washed with 4\u2009\u00b0C phosphate-buffered saline (PBS). Then, the RNeasy Mini Kit  was used to extract the total RNA, which was reverse transcribed by the ThermoScript\u2122 RT-PCR System . mRNA expression was detected by a Talent qPCR kit . The primers used in this study are shown in Table t tests or one-way or two-way ANOVA. Categorical data were analyzed using the chi-squared (\u03c72) test. Values of p < 0.05 were considered significant.Statistical analysis was performed by the GraphPad Prism software version 7.0 . All data are displayed as the mean \u00b1 SEM. The comparisons between the two groups were analyzed using two-tailed unpaired Student\u2019s p > 0.05). Eleven of nonunion patients were smokers, compared to 19 healed patients (p > 0.05). The majority of fractures occurred in healthy patients, and 5 nonunion patients had comorbidities, compared to 9 healed patients (p > 0.05). Most fractures were the result of a traffic accident, and 13 of the nonunion fractures were caused by traffic injury, compared to 27 of the healed fractures (p > 0.05). The larger proportion of injuries resulted from multi-trauma, and 11 of the nonunion fractures were from multi-trauma, compared to 23 of the healed fractures (p > 0.05). The severity of the soft tissue injury was assessed using the Gustilo classification, and 5 nonunion fractures were Gustilo grade III, compared to 3 healed fractures (p < 0.05). The initial surgical debridement times of 6 nonunion and 27 healed fractures were within 8\u2009h, and 10 nonunion and 12 healed fractures received the initial surgical debridement after 8\u2009h (p < 0.05). The results above indicate that urgent surgical debridement and the severity of soft tissue injury have a significant effect on the nonunion of open long bone fractures.The clinical characteristics of the nonunion group and the healed group are shown in Table Three PBMC gene expression datasets  of healed and nonunion patients were downloaded from the GEO database. Gene comparison analysis was performed on these two groups, and there were a total of 258 differentially expressed genes between healed and nonunion patients , a classical medicine for the treatment of bone fractures, to predict biomarkers for nonunion patients. Ten potential active compounds were retrieved from the TCMSP database, and a total of 443 target genes were obtained using the PharmMapper and Swiss Target Prediction databases. Finally, the interaction of drug target genes and nonunion-related genes was determined using a Venn diagram. As shown in Fig. p < 0.05). The results showed that after bone fracture, the six genes were all significantly increased, which may be closely related to the fracture healing process. However, the serum concentrations of all six potential biomarkers were not significantly different between fracture-only and multi-trauma patients  and acute injury (AI) patients. As shown in Fig. nts Fig. . To furtp < 0.05). DR treatment significantly increased CA1, MMP9, and QPCT, PBMC mRNA expression levels in both the healed and nonunion groups  were significantly lower in nonunion patients. Meanwhile, CA1, MMP9, and QPCT were markedly increased in patients using DR treatment, suggesting that the proteins might serve as potential blood biomarkers for nonunion and may be potential DR treatment targets. It is worth noting that the six potential biomarkers were related to the acute phase, shock, acidosis, multiorgan failure, and inflammation, but the specific changes of the biomarkers in these manifestations require further study.Matrix metalloproteases (MMPs) are a family of 23 zinc-dependent proteolytic enzymes that can cleave the extracellular matrix (ECM). MMPs play an essential role in tissue regeneration and bone remodeling processes . Previou2/H2CO3 [32\u2212 ions is also regarded as an essential part of bone fracture healing [Carbonic anhydrase I (CA1) is a member of the carbonic anhydrase (CA) family, which catalyzes the reversible hydration and dehydration reactions of CO2/H2CO3 . A study2/H2CO3 . Moreove healing . In the Pituitary glutaminyl cyclotransferase (QPCT), also known as pituitary glutaminyl cyclase, can convert active forms of gonadotropin-releasing hormone (GnRH) peptides to protected forms . A previHydroxycarboxylic acid receptor 2 (HCAR2) is an endogenous ketone produced by fatty acid oxidation in liver mitochondria during carbohydrate deficiency . HCAR2 hUDP-glucose ceramide glycosyltransferase (UGCG) is the only enzyme responsible for the de novo production of glucosylceramide (GlcCer), which is essential for proper cell function . LDHB isOur study provides valuable information to investigate blood biomarkers for chronic nonunion and the potential mechanisms of DR in the treatment of bone fracture. Despite the large patient cohort, only a small number of patients could be included in the study due to a lack of objective tools to assess nonunion. Although we attempted to increase the accuracy of the results, there was inevitable interference from other factors, and the predicted genes need to be validated on large-scale blood samples further.This bioinformatics analysis combined with the experimental verification strategy provides five potential blood biomarkers for nonunion patients and three DR treatment targets and is the first study to use such an approach for predicting nonunion blood biomarkers. Early diagnosis of chronic nonunion will help clinicians take timely countermeasures to improve bone healing, which will result in better clinical management of patients. In addition, further prospective clinical studies will evaluate the predictive power of these biomarkers for the prognosis of bone fractures. Changes in the potential biomarkers also need to be further studied in nonunion of other fractures in addition to long bone fractures.Additional file 1: Figure S1. Validation of the six potential biomarkers between fracture only and multi-trauma patients by ELISA. Concentration of CA1 (A), MMP9 (B), QPCT (C), HCAR2 (D), UGCG (E), and LDHB (F) in serum samples from the fracture only  and multi-trauma patients . The error bars represent means \u00b1 SEM. There has no significant difference between fracture only and multi-trauma patients. Table S1. Functional analysis of the six potential blood biomarkers."}
+{"text": "The aim of this study was to evaluate the clinical use of tumor abnormal protein (TAP) in the diagnosis of different cancers.Totally 394 patients were divided into 4 groups, namely 100 healthy volunteers, 167 patients with cancer, 20 subjects with precancerous lesions, and 107 subjects with benign lesions. TAP was detected in 4 groups of research subjects using a TAP testing kit and examination system. We correlated TAP levels with a wide variety of clinical indicators as well as established cancer markers, including alpha fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9). Besides, the changes of TAP level in 51 patients with liver cancer before and after surgery, and overall survival of patients with high or low TAP expression in pancreatic, gallbladder, bile duct, and liver cancers were analyzed.\u03c72\u200a=\u200a2.886, P\u200a=\u200a.410), and TAP expression was shown to be correlated with AFP in liver cancer (P\u200a=\u200a.034) but not with CA19-9 in pancreatic cancer (P\u200a=\u200a.241). Moreover, the overall survival of patients with low expression of TAP in pancreatic, gallbladder, bile duct, and liver cancers were significantly higher than of patients with high expression of TAP. Compared with the preoperative patients with cancer, TAP levels decreased dramatically among postoperative subjects (P\u200a<\u200a.001).Statistically significant difference was observed in the TAP-positive ratio among subjects with cancer (79.6%) and precancerous lesions (45.0%) compared to the healthy volunteers (4.0%). TAP expression in different cancers was characterized by high sensitivity (79.64%), specificity (89.87%), positive and negative predictive value (85.25% and 85.71%), overall compliance rate (85.53%) but low omission and mistake diagnostic rate (20.36% and 10.13%), Youden index (0.6951). In addition, there was no significant difference among patients with different types of cancer (In summary, TAP might hold promise in serving as universal indicator for the diagnosis of different cancers. As diagnostic indicator, it is supposed to distinguish healthy candidates from diseased patients within a wide variety of different cancers. Recently, accumulating studies have explored the universal usefulness of some biomarkers in human tumors.\u20135Cancer, as a multifactorial disease, remains the greatest challenge in the modern health care all over the world. It has been recognized that the majority of patients with cancer were diagnosed at advanced stage, which made radical surgery impossible and treatment efficacy poor. It occurs in different cancers and has been identified to be involved in cancer progression, metastasis, and the survival rate of patients.,8 Numerous tumor-associated glycans are found at low level in normal tissues and at high level in tumors.Glycoproteins with abnormal sugar chains, which exist in the cell membrane, have been implicated in the carcinogenesis.,11 TAP could be easily detected in the peripheral blood once their levels achieve a certain threshold, which makes little damage to the patients and renders great convenience in diagnosis. Lan et al have clarified that most patients with early stage gastric cancer were TAP-positive. Similarly, TAP expression has also been shown increased in bladder cancer and colorectal cancer. However, there is little report on the comprehensive study of TAP role among different cancers.Tumor abnormal protein (TAP), as a collective term for glycoproteins, is regarded as the common feature of malignant tumors.This study was designed to clarify the potential of TAP as cancer marker with comprehensive study of clinical patients. On this basis, we attempted to make further step by investigating the potential role of TAP in other cancers, for example, liver, pancreatic, gallbladder, and bile duct, which to our knowledge have not been clarified; the performance of TAP compared with other established cancer marker, such as alpha fetoprotein (AFP), and carbohydrate antigen 19-9 (CA19-9); the potential of combining TAP with other marker in cancer prognosis.22.1A total of 394 patients  who were treated in the Department of Hepatobiliary Surgery from May to September in 2016 were enrolled in the present study. Among them, there were 100 healthy volunteers, 167 patients with cancer, 20 precancerous lesions, and 107 benign lesions. TAP was detected in 4 groups of research subjects. For the cancer group, there were 34 cases with pancreatic cancer, 73 liver cancer, 30 gallbladder cancer, and 30 bile duct cancer. For the precancerous lesion group, there were 4 cases of gallbladder intraepithelial neoplasia, 13 gallbladder polyps, and 3 pancreatic intraductal papillary mucinous tumor. For the benign lesion group, there were 82 bile duct stone, 5 hepatic cyst, 10 hepatic hemangioma, and 10 splenomegaly. Exclusion criteria: patients with immunodeficiency, hepatitis, diabetes, tuberculosis, and other diseases. All patients were pathologically diagnosed after surgery. All patients signed an informed consent before treatment, and this study was approved by the ethics committee. The clinical data of the patients are shown in Table 2.2 Samples were regarded as TAP positive only if the condensed particles meet the criteria: having a single condensate with an area of \u2265225\u200a\u03bcm2 or having 3 or more condensates with an area of 121 to 225\u200a\u03bcm2, having 2 condensates with an area of 121 to 225\u200a\u03bcm2 or having 3 or more condensates with an area of 81 to 121\u200a\u03bcm2. Samples were confirmed as TAP-negative when there was no condensate, or condensates with an area of <81\u200a\u03bcm2 or 2 or less condensates with an area of 81 to 121\u200a\u03bcm2.Peripheral blood was collected from healthy volunteers, all patients (1 day before surgery) and patients with cancer (1 week after surgery). TAP was detected using a TAP testing kit and examination system  as previously reported.2.3The serum AFP and CA19-9 level that analyzed in a gamma counter  were collected from medical record.2.4x \u00b1 s) and compared with paired t test. For all comparisons, P\u200a<\u200a.05 was considered with statistical significance.Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 23.0 . The Pearson Chi-squared test and the Fisher exact test, if appropriate, were used to compare gender and the positive rate of TAP among groups. Analysis of variance was used to compare the age among groups. The correlation between TAP and established markers, for example, AFP and CA19-9 was analyzed. Preoperative and postoperative measurements were presented with mean \u00b1 standard deviation . Furthermore, the positive rate of TAP expression in 4 different groups were shown to be 4.0%, 9.3%, 45.0%, and 79.6% for healthy volunteers, benign lesions, precancerous lesions, and patients with cancer, respectively  , specificity (89.87%), positive and negative predictive value (85.25% and 85.71%), and overall compliance rate 85.53%) but low omission and mistake diagnostic rate (20.36% and 10.13%), and Youden index (0.6951)   (Table P\u200a=\u200a.241)  Table .https://cancergenome.nih.gov/)   Fig. . Results3.5P\u200a<\u200a.001), suggesting the correlation between TAP-positive rate with surgical intervention. Moreover, the TAP value was more closely related to effectiveness of surgery for patients with cancer . Moreover, there was no significant difference among four different cancers, including liver cancer, pancreatic cancer, gallbladder cancer, and bile duct cancer (P\u200a>\u200a.05). These results were consistent with previous studies that the positive rate of TAP in selected patients with cancer is significantly higher than that of nontumor patients. TAP is reported implicated in the early stage of cancer development, and is able to reflect the quantity and degree of cancerous cells. On these bases, it was suggested that TAP could possibly serve as a universal diagnostic indicator for cancers.In current work, we found consistently high expression of TAP in selected patients with cancer but not healthy ones  but not other cancers . In contrast, consistent performance has been observed for TAP in a variety of different cancers, including gastric, bladder, and colorectal cancers. Besides, this study indicated that high expression of TAP resulted in a significant augment of overall survival among patients with pancreatic, gallbladder, bile duct, and liver cancers. More importantly, TAP was detected more frequently among patients with cancer than other known markers, including AFP and CA19-9, in liver and pancreatic cancer, suggesting the promise of TAP as universal cancer marker. It was also suggested that combination with established markers and comprehensive judgment could improve the accuracy of tumor auxiliary diagnosis.,26 Consistently, our results proved that the expression of TAP in postoperative patients with cancer was significantly lower than that in patients before operation, suggesting the sensitivity of TAP in monitoring the therapeutic effects of surgery. Similarly, Liu and Huang also found TAP is sensitive in monitoring the responsiveness to chemotherapy in patients with advanced gastric or colorectal cancer.Surgery is the most effective and common way of treatment of cancer in the clinic, especially in the early stage which was characterized by satisfying therapeutic effect. TAP examination, as simple and nonexpensive means, can be not only used in early diagnosis of cancer but also in postoperative monitoring of the treatment's efficacy. This could be partially evidenced by the observations that the abnormal surface glycosylation is correlated with cancer invasion and metastasis.In conclusion, for patients with cancer, it is extremely crucial to detect cancer at the very early stage and treat disease as soon as possible. Our findings indicate that TAP detection represents a promising diagnostic tool. However, more extensive studies are in great demand to elucidate the potential role of TAP in cancers.Conceptualization: Lu-Xi Li, Ruizhi Gong.Data curation: Bin Zhang.Formal analysis: Lu-Xi Li.Investigation: Bin Zhang.Methodology: Lu-Xi Li, Bin Zhang.Project administration: Ruizhi Gong.Supervision: Ruizhi Gong.Validation: Lu-Xi Li, Bin Zhang.Writing \u2013 original draft: Lu-Xi Li."}
+{"text": "In this work, by applying a transfer method simultaneously with a solution doping process for graphene as top electrodes, we demonstrate a solution-processed semitransparent organic photovoltaics (OPV). The work function of doped graphene under various doping conditions was investigated via photoemission spectroscopy. The transparent device was fabricated using PEDOT-doped graphene as electrodes, which provide an energetically favorable band alignment for carrier extractions. The solution-processed semitransparent organic photovoltaics exhibit the power conversion efficiency (PCE) of 4.2%, which is 85.7% of the PCE of control devices based on metallic reflecting electrodes, while maintaining good transparency at most visible wavelengths. In particular, numerous state-of-the-art achievements employing organic conductive thin films offer the distinguishing feature of semitransparency for organic optoelectronics, enabling novel applications such as wearable electronics, transparent display screens, synthetic skin, electrochromic windows and solar shingles to be integrated into everyday life10.Since the beginning of the rapid development of organic semiconductors, extensive study of photoactive compounds and conductive polymers has led to promising applications in electronic devices such as sensors, memory, displays, lighting and solar cells with large processing areas and incorporating flexible technology11. However, most conventional organic photovoltaics (OPVs) consist a nontransparent metal electrode, which can reharvest the reflected photons not absorbed in the first pass and thereby to improve the power conversion efficiency (PCE)12. The use of metal electrodes limits the transparency of the whole device and hence the feasibility of window-integrated applications is hindered.He intrinsic semitransparency and flexibility of films formed by organic molecules facilitates the development of organic optoelectronics that are transparent and can be integrated with curved surfaces. For instance, photoactive bulk heterojunctions (BHJs) consisting of poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM) are intrinsically deep purple in color since the optical bandgap of P3HT is approximately 1.9\u00a0eV, which makes the heterojunction almost transparent to near-infrared radiation (>\u2009650\u00a0nm)15. Of additional interest are graphene transparent electrodes, which have been recognized as a next-generation substitute for indium tin oxide (ITO) due to their flexibility, high transparency, superior carrier mobility and excellent mechanical strength20. A great number of reports therefore suggest that graphene is an ideal candidate for achieving both adaptability and transparency for transparent facade integration24.To overcome obstacles to adapting innovative transparent elements to daily life, alternatives to transparent conductive electrodes have been intensively studied, including conductive polymers, various metal oxides, ultrathin metal layers or metal nanowires, carbon nanotubes, and photonic crystals26. In one study, stacked graphene layers were used as the anode for an inverted P3HT:PC60BM solar cell structure, where 10 layers of graphene were needed to achieve a PCE of 2.5%27. Thus, it has yet to be reported that graphene-based OPVs have simultaneously achieved semitransparency while yielding a PCE comparable to that of OPVs whose electrodes are deposited directly on the active layer.Nevertheless, neither tape detachment nor conventional polymer-assisted transfer is suitable for transferring a graphene top electrode onto organics, since these processes involve mechanical strains, solvent treatments, and residue issues that inevitably damage the underlying organic thin filmIn the present work, by using a polymer-free transfer method with an in situ doping process for graphene top electrodes, we demonstrate the fabrication of a promising graphene top electrode; the optimum performance of the device was examined by tuning the number of graphene layers as well as the active layer thickness. A systematic study of doped graphene under various doping conditions was completed via photoemission spectroscopy. The device we propose uses a PEDOT-doped graphene sandwich structure as a top electrode, which provides an energetically favorable band alignment to attain optimal efficiency while maintaining high transparency at most visible wavelengths. A solution-based process for fabricating semitransparent OPVs was therefore developed to facilitate vacuum-free technologies such as roll-to-roll processing. This result allows the development of semitransparent OPVs that exhibit approximately 50% visible light transmission and approximately 90% of the PCE of control devices based on metal reflecting electrodes, thereby providing a feasible green energy source and the additional advantages of saved time, less energy consumption and mass production for the solar industry.28. Moreover, graphene transferred by this method can be easily developed into stacked films in a layer-by-layer (LBL) structure to enhance the extrinsic conductivity, whereby the sheet resistance can be tuned to the requirements for functioning as an electrode. BHJ solar cells incorporating such graphene stacks as bottom transparent electrodes were first devised in the standard structure of glass/graphene/PEDOT:PSS/active layer/BCP/Al to investigate the adaptability of a polymer-free transferred graphene anode substituted for an ITO anode. Two common types of polymer donors, including P3HT and poly{dithiophene-2,6-diyl]-alt-thiophen-4,6-diyl]} (PBDTTT-C-T), were selected to ensure that the obtained PCE was not unique to a specific type of high-performance semitransparent OPVs. The fabrication details are described in the experimental section, and all device performance parameters presented throughout the work were characterized in a nitrogen-filled glovebox.To make a graphene top electrode capable of overcoming the aforementioned challenges, the transfer of unblemished high-quality graphene sheets is needed\u2014this process was recently unveiled in our preceding work, offering excellent electrical properties using a polymer-free transfer method29. Nevertheless, the optimal PCEs of 2.45% (for the P3HT device) and 4.64% (for PBDTTT-C-T) for graphene-based OPVs indicate that the strategy proposed for fabricating graphene bottom anodes is feasible without polymer assistance or additional processes. Moreover, for both the devices using two different polymers as the active layer, the series resistance of the devices with graphene as the bottom anode are larger than that with an ITO anode. The series resistance arises from bulk resistance of the organic material, contact resistance at the metal\u2013semiconductor, and resistance of the cathode. Since the sheet resistance of a graphene is normally larger than that of an ITO conducting thin film, which will result in a larger series resistance value in both polymer devices.Figure\u00a0Using the transfer process we developed, transferring graphene stacks onto delicate organic thin films is feasible since polymer-free transfer is free from chemical and mechanical treatments such as solvent cleaning or the use of a stamping press. We further experimentally established an inverted OPV fabrication process to obtain the rarely achieved result of a graphene-based device with a graphene top electrode. Devices with a structure of ITO glass/AZO/P3HT:PCBM/MoOx were prepared as target substrates, after which polymer-free graphene transfer was performed to complete the inverted OPV Fig.\u00a0a.Figure 31. Here, a PEDOT doping process was introduced in situ by adding PEDOT aqueous solution during transfer to modify the Fermi energy of the graphene stacks, achieving an energetically favorable band alignment  and FF .To gain insight into the doping effect, the doping-dependent evolution of the Raman spectral characteristics for the 2D-, G- and D-bands of doped graphene monolayers was studied, as shown in Fig.\u00a0To realize the energy alignment, ultraviolet photoemission spectroscopy (UPS) is used to measure the work function of the various doped graphene. The photon energy of 40.8\u00a0eV were utilized to excite the electrons in the materials and photoelectrons were collected with a monochrometer. All the binding energies in Fig.\u00a0The doping-dependent photovoltaic parameters for graphene-based devices are further demonstrated in Fig.\u00a03 was developed as a surface modification to achieve high-conductivity graphene sheets37. Since AuCl3 degrades graphene very quickly, it was spin-cast onto the prepared graphene stacks immediately before the J\u2013V and external quantum efficiency (EQE) measurements to prevent degradation. The OPVs using AuCl3-decorated G:PEDOT stacks for the top transparent anode provided a better Voc with incremental improvements corresponding to the number of graphene stacks, as shown in Fig.\u00a0To further improve the performance of the semitransparent OPVs, the incorporation of AuCl3 film further hindered the transparency of the G:PEDOT sheets, and the transmittance of AuCl3-decorated quadrilayer G:PEDOT sheets was less than 60% at \u03bb\u2009>\u2009650\u00a0nm (not shown here). Hence, opaque quadrilayer (or more) G:PEDOT is not applicable to transparent electrode applications, although its conductivity improved with increasing numbers of layers.Moreover, Fig.\u00a060BM , FF (59%), and PCE (over 2.6%)\u2014while the values for the PBDTTT-C-T:PC70BM devices were\u2009~\u20090.73\u00a0V, 54% and over 3.9%, respectively. Only a small variation in Jsc is associated with the side where the light entered, leading to a\u2009<\u20096% difference in PCE when the devices are illuminated from the AuCl3-decorated G:PEDOT and ITO electrodes. The slightly worse of PCE of semi-transparent solar cells, as compared to regular non-transparent solar cells, are because of lack of back reflection from reflective electrodes and higher resistance of doped graphene. Nonetheless, this semi-transparent solar cell, with average transmittance at visible light of 40%, gains the flexibility when using it to window-integrated application.For a better understanding of doped graphene in electrode applications, the semitransparent OPVs were illuminated on each side under an AM1.5G solar light to demonstrate semitransparency with green power generation. From Fig.\u00a03-decorated G:PEDOT and ITO; the device illuminated from the graphene side reached a higher EQE at short wavelengths (>\u2009450\u00a0nm) than that of the device illuminated from the ITO side, but the relative trend was reversed at long wavelengths regardless of which photoactive blends were used. This behavior not only agrees well with the decreased transmittance at long wavelengths for the G:PEDOT transmission spectrum , such as PEDOT:PSS or a transition metal oxide40, could replace thermally deposited MoOx to achieve vacuum-free fabrication. Here, the hydrophobic photoactive film was illuminated with UV light for 30\u00a0s before spin-coating with IPA-diluted PEDOT:PSS HTL to match surface energies and achieve better contact at the junctions. Additionally, a UV-resistant P3HT:ICBA photoactive blend was used to minimize polymer degradation during UV treatment. Here, various blend ratios of P3HT:ICBA in o-dichlorobenzene (o-DCB) were tested in solution-processed OPVs, as shown in Table 2 and FF of 42.4%, leading to a decent PCE of 2.60% for a solution processed device.With the development of AuCl70BMPCE based solar cells of 4.2%, using p-doped graphene. The proposed doping process allows graphene to be integrated with arbitrary substrates, including delicate organic thin films. The energy-band alignment and electronic structure of the doped graphene were investigated in detail via Raman and photoemission spectroscopy to assess the doping effect, showing a tunable work function from 4.5\u00a0eV to 5.1\u00a0eV. Optimally doped graphene is therefore a very good alternative for a transparent conducting electrode and was successfully integrated with OPVs as the top anode, achieving semitransparency. By engineering the interface between graphene layers and beneath the HTL, we probed the doping-dependent characteristics of graphene-based OPVs systematically to verify that an energetically favorable band alignment is essential to performance improvement. As a result, the optimized PBDTTT-C-T:PC70BM OPVs not only featured semitransparency but also exhibited high stability with remarkable performance in Voc (0.73\u00a0V), FF (54%), and PCE (4.2%) compared to that of conventional electrodes using metal electrodes. Above all, a solution-processed OPV with decent power conversion efficiency has been demonstrated, indicating its potential as a time-saving, low-cost and lightweight technology. These results suggest that graphene electrodes are practical for transparent facade integration and thus feasible for application to electrical power generation sources in highly populated urban areas.We have demonstrated the fabrication and function of a semitransparent OPV with the PCE of PBDTTT-C-T:PC41. The graphene/copper sheets of the desired size were placed into graphite confinement floating on an ammonium persulfate etchant  to remove the copper carrier. After the undesired copper foil was thoroughly etched, a blended aqueous solution (mixture of DI water and IPA at a ratio of 10:1) was subsequently substituted for the etchant42. At the same time, PEDOT:PSS solution was gradually introduced into the floating graphene film by liquid-phase diffusion. Then, the doped solution was rinsed off with the IPA-DI water mixture just before setting an arbitrary target beneath the doped graphene, ensuring that the immersed target substrate was free of PEDOT solution. Finally, the IPA-DI water mixture was pumped out to lower the doped graphene onto the target substrate, and the sample was heated at 70\u00a0\u00b0C for 10\u00a0min to improve adhesion. Note that graphene stacking in an LBL structure can be achieved by repeating this transfer process. The quality of the as-transferred graphene films was characterized by Raman spectroscopy, showing flawless planarity with almost no Raman D-band28.Commercial monolayer graphene films synthesized by CVD on copper foils were purchased from Graphene Supermarket60BM/MoOx/graphene or ITO/AZO/PBDTTT-C-T:PC70BM/MoOx/graphene in an inverted structure. First, sol\u2013gel AZO layers deposited on precleaned ITO substrates were prepared from a Zn precursor consisting of 0.5\u00a0M zinc acetate and monoethanolamine (MEA) in IPA with 1.35% aluminum nitrate in a molar ratio and subsequently spin on top of ITO substrate baked at 280\u00a0\u00b0C for 10\u00a0min to form the electron transport layer for an inverted BHJ solar cell32. Next, the following photoactive blends  were spin-cast in a nitrogen-filled glovebox from 600 to 900\u00a0rpm for 40\u00a0s. Then, a 15-\u00c5 MoOx interlayer was thermally deposited at a pressure below 10\u22126\u00a0Torr to form the HTL for the inverted solar cell. Moreover, a solution of PEDOT:PSS mixed with IPA (1:1 v/v) was spin-cast onto the sample at 4000\u00a0rpm for 40\u00a0s to complete the HTL for the vacuum-free process. Finally, the doped graphene sheet was transferred onto the as-fabricated ITO/AZO/active blend/HTL substrate using the polymer-free method with in situ PEDOT doping as mentioned above. Note that only the top surface of the graphene electrode was doped with AuCl3  before completion of the 10-mm2 graphene-based semitransparent OPVs.The BHJ solar cells in this study were either ITO/AZO/P3HT:PC\u22122. The photoemission experiments were performed in a Phi5400 ultrahigh vacuum system with a base pressure of 10\u221210\u00a0Torr for XPS and UPS analyses. The photoelectrons excited by He II (hv\u2009=\u200940.8\u00a0eV) or Al K\u03b1 sources were collected using a hemispherical analyzer with an overall resolution of 0.05\u00a0eV.In this study, the J\u2013V characteristics of the OPVs were measured in a glovebox under AM1.5G illumination with an irradiation intensity of 100 mW cm"}
+{"text": "Spike is one of the crop yield organs in wheat plants. Determination of the phenological stages, including heading time point (HTP), and area of spike from non-invasive phenotyping images provides the necessary information for the inference of growth-related traits. The algorithm previously developed by Qiongyan et al. for spike detection in 2-D images turns out to be less accurate when applied to the European cultivars that produce many more leaves. Therefore, we here present an improved and extended method where (i) wavelet amplitude is used as an input to the Laws texture energy-based neural network instead of original grayscale images and (ii) non-spike structures  are subsequently suppressed by combining the result of the neural network prediction with a Frangi-filtered image. Using this two-step approach, a 98.6% overall accuracy of neural network segmentation based on direct comparison with ground-truth data could be achieved. Moreover, the comparative error rate in spike HTP detection and growth correlation among the ground truth, the algorithm developed by Qiongyan et al., and the proposed algorithm are discussed in this paper. The proposed algorithm was also capable of significantly reducing the error rate of the HTP detection by 75% and improving the accuracy of spike area estimation by 50% in comparison with the Qionagyan et al. method. With these algorithmic improvements, HTP detection on a diverse set of 369 plants was performed in a high-throughput manner. This analysis demonstrated that the HTP of 104 plants (comprises of 57 genotypes) with lower biomass and tillering range  were correctly determined. However, fine-tuning or extension of the developed method is required for high biomass plants where spike emerges within green bushes. In conclusion, our proposed method allows significantly more reliable results for HTP detection and spike growth analysis to be achieved in application to European cultivars with earlier-heading types. Wheat is one of the major crop species in the world, with 762 million tons of grain produced annually (FAOSTAT 2018) and providing \u2265 20% of the world's calorie and protein demand , we draw conclusions regarding the performance of our algorithm and discuss its future improvements.We used images from one experiment with 260 diverse winter wheat cultivars of mainly Central European origin. Of these lines, 220 correspond to the collection described in Voss-Fels et al.  and reprm, while the side view camera had a resolution of 6,576 \u00d7 4,384 pixels and a pixel size of 5.5 \u00d7 5.5 \u03bcm. Plant images were later visually inspected to determine the time point of heading when the ear was half out of the flag leaf (BBCH55). Here, top view images turned out not to be suitable as, from the top, an emerging ear has a very low visible area and might be easily hidden under a bending leaf. Moreover, it is hard to define how much of the ear is above the flag leaves. Therefore, this determination was done on inspecting the three side view images. In this case, only the pots were rotated; the camera is stable. Out of all 520 plants, 369 reached BBCH55 during the imaging period belonging to 202 different cultivars. These 369 plants from 202 genotypes were available for testing our spike detection algorithm. These plants exhibit strong differences in plant architecture and are challenging for this kind of analysis, for example, spikes with or without awns, short and tall plants (plant height range at harvest time from 34 to 119 cm), and especially low and high tillering genotypes ranging from 1 to 38 tillers per plant counted at 125 DAS during the imaging period. Further, the data set exhibits differences in BBCH55 timing of 29 days.Images were taken from three side view angles  and one top view using RGB cameras. The top view camera (a Manta G-504) had a resolution of 2,452 \u00d7 2,056 pixels with a pixel size of 3.45 \u00d7 3.45 \u03bcThe workflow for spike detection following image acquisition is shown in rgb2gray routine. To enhance the separability between the plant and background pixels, discrete wavelet transform (DWT) is applied in the preprocessing step using the Haar basis function  it decorrelates the data Fan,  by stretwhere * denotes the convolution operator, and  and  represent the downscaling along rows and columns, respectively. L and G are the low- and high-pass filters, and I is the original image. The DWT decomposes an image into four sub-bands called approximation coefficients (A), horizontal (H), vertical (V), and diagonal (D), as shown in Laws' texture energy method Laws,  is a claThe 2-D masks are generated from the 1-D masks by convolving the vertical 1-D mask with the horizontal 1-D mask. For example, mask S3L3 is calculated by convolving vertical mask S3 with horizontal mask L3 and is a zero-sum mask. In contrast, mask L3L3 is a non-zero-sum mask, which is not considered for the textural analysis. The list of 2-D masks used for the textural analysis is as follows:The textural features are calculated in two steps  are segmented from the background pixels using the CIS method , see The neural network is used to perform the classification of spike and non-spike pixels in the study. In practice, the neural network is trained with a large quantity of spike and non-spike pixels from the different wheat plants. The trained neural network parameters are then adapted to perform the spike detection in an automated manner. Here, a total of 218282 spike and 731054 non-spike pixels were extracted from 150 manually segmented images and subsequently used for training, testing, and validation of a network model in the sample proportion 70:15:15. The performance of the network model, with eight input nodes, one hidden layer with 10 hidden nodes, and 2 output nodes, was assessed using the conventional confusion matrix , components of which indicate the total number of correctly and incorrectly classified spike and non-spike pixels, respectively. The true positive (TP) and true negative (TN) rates, as well as the overall accuracy (TP+TN)/(TP+FP+FN+TN), are summarized in The Frangi filter is a multi-scale second-order vessel enhancement method developed by Frangi et al.  that is Frangi-based vessel enhancement is achieved based on Hessian and eigenvalues. The Hessian matrix of image I is computed as follows:h11, h12, h21, h22 are the second-order partial derivatives of the image and \u03c3 denotes a variable scaling factor.where 1 and \u03bb2 are calculated, while \u03c3 is used for the enhancement of structures at different scales, see 1 and a very high magnitude of \u03bb2; see Equation (6). Furthermore, the bright vessel-like structures emerge with negative \u03bb2, and the filter response of the corresponding pixel with \u03bb2 > 0 is considered to be zero in the enhanced image.To extract information on structural patterns from the Hessian matrix, the eigenvalues \u03bbThe enhanced image is defined as follows:IE is obtained at various scales, i.e., \u03c3 = 1, 3, 5, 7, 9. Since the maximum scale approximately matches the size of the vessel to detect, the final enhanced image IFE can be obtained according to Frangi et al.  using the Frangi filter is shown in Consequently, the result of the neural network segmentation is subsequently filtered under consideration of leaf-crossing regions detected by the Frangi filter . This isAs shown in The above-described algorithm was applied to calculate the yield-related features at the transition from the tillering to flowering growth stages of wheat plants with an age of more than 90 DAS. Accordingly, the results of this study are presented in two sections dedicated to (i) detection of the time point of spike emergence and (ii) spike growth analysis from RGB images acquired using visible light cameras during an experiment with diverse winter wheat varieties. In the first section, the spike emergence was tested on 369 wheat plants from 202 different genotypes. Here, the HTP was defined as the first time in the imaging time course when the detected spike satisfied the minimum area constraint of 500 pixels. The spike area was then measured in a time series from the HTP to perform real-time growth analysis for a few selected plants.Image analysis was performed on an Intel Xeon CPU E5-2640-based workstation running under the Linux OS. The algorithms were implemented under MATLAB 2019a (MathWorks Inc.). Training of a neural network on 949336 manually segmented spike and non-spike pixels using ten 2.40GHz CPUs with a total of 20 cores in parallel mode took approximately 80 s. The spike detection algorithm takes approximately 2.5 s to process an 8-megapixel test image. However, the processing time might vary depending on the test image size.The root mean square error (RMSE) is used for quantification of the deviations of predictions from our model and Qiongyan et al.'s model from ground truth data,:y is the ground truth value and \u0177 is the model-predicted value. For consistent comparison of performance, the Qiongyan et al. model was retrained with the European cultivars.where The time-series images of a single plant described in Section 2 have three orientations. Accordingly, two factors are considered to estimate the HTP from multiple orientations: the spike should (1) appear in at least two orientations and (2) remain present in all days of the experiment. This means the spike or spikes should be continuously detected until the last day to avoid false emerging time points.On the other hand, the proposed method resulted in high HTP error rates of 4 days more and 3 days less for plant ID 1817KN373 and 1817KN412, respectively. For plant ID 1817KN373, this was because the spikes were narrow and the pixel-wise textural energy was similar to that of the leaves, as shown in R2 value of 0.776 compared to the R2 value (0.193) of the Qiongyan et al. method. Since the European elite cultivars possess more leaves, overlay artifacts result in too early HTP detection using the Qiongyan et al. method on 90% of our data. In the remaining 265 plants, the spike emerged in the final days according to the ground truth data, and they have early-stage spike textural features that are similar to the leaves. This resulted in the proposed algorithm failing to detect the spikes in the final days with a day number 0 in the output. This leads to a low value of the correlation coefficient R2 0.0586 for the remaining 265 plants.The advantages and significance of the results with the proposed method showed that it is feasible for high-throughput analysis of HTP detection. Consequently, we applied the method to all 369 diverse plants in our data set that reached heading within the imaging period. As expected, 104 plants corresponding to the supposedly earlier-heading genotypes obtained a good and reliable estimation of the true heading time point. We compared the general plant architecture features of all 369 plants tested and classified them into three categories: (i) both plants of the genotype were classified correctly by our algorithm  only one out the two plants of a genotype were classified correctly by our algorithm (20 plants from 10 genotypes), and (iii) none of the two plants of a genotype were classified correctly by our algorithm . It turnR2. The RMSE quantifies the difference between the ground truth area and the predicted area for all days from the ear-emergence day. The R2 value compares the goodness of our proposed models and of the Qiongyan et al. model compared to the ground truth data.Spike area is one of the key yield measures in wheat plants, so we have examined the total spike growth of a single wheat plant in three orientations from the spike emergence day for all images. In section 3.1, nine wheat plants were considered for HTP detection. Among those, only three plants  with a single spike and two with multiple spikes are considered for the spike growth analysis. Here, the spike area of a plant per day is calculated by taking the maximum area among the three orientations. The measured area of both algorithms is validated by the RMSE and https://www.gimp.org). The number of non-zero pixels in the segmented image represents the actual spike area or the ground-truth spike area of the image. This figure shows that the proposed method outperforms the Qiongyan et al. method overall, with a low RMSE and a high value of R2. Moreover, the RMSE is profoundly improved by more than 50% and the R2 value is significantly improved for plant ID 1817KN373 , height, tiller number, biomass, and heading time. Such a data set is very challenging as it is easier to find an algorithm for identifying the plant organs in a small genotype set with much more uniform morphology. However, the biological truth is that many studies employ non-invasive phenotyping to screen genotype collections that exhibit a high phenotypic diversity  to further improve the algorithm and to deliver more sophisticated solutions for plant breeders and cereal crop researchers.The datasets generated for this study are available on request to the corresponding author.NN and EG conceived, designed, and performed the computational experiments, analyzed the data, wrote the paper, prepared figures and tables, and reviewed drafts of the paper. KN and MR executed the laboratory experiments, acquired image data, wrote, and reviewed drafts of the paper.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": "Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers (PSs) with different charges through different linking strategies; \u201cClick\u201d conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and \u201cLysine\u201d conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells. Antibody-based therapies have become important clinical tools in treating chronic diseases. Different immunotherapeutic strategies, with limited success, have been investigated to target and eliminate viral-infected cells by using armed antibodies specific to viral proteins . This caIn this paper, we propose HIV immunotherapy via arming HIV monoclonal antibodies with photosensitizers (PSs). Anti-HIV immunoconjugates must be targeted to the HIV envelope spike (Env) that consists of gp160 ,15, gp12Recently, we have employed pyridazinediones (PDs) to functionally re-bridge the interchain disulfide bonds antibodies (PDs) bearing orthogonal \u2018clickable\u2019 handles into the native inter-strand disulfide bonds of trastuzumab full antibody ,33,34,35In this study, we utilized lysine and disulfide modifications to identify an appropriate photo-immunoconjugate (PIC) for our suggested PIT. Briefly, a human anti-gp41 antibody (7B2)  was conjTwo generations of PICs were produced; Porphyrin-7B2 PIC was produced through the use of the strain-promoted azide\u2212alkyne cycloaddition (SPAAC) methodology in two steps: firstly, the insertion of a strained alkyne harboring pyridazinediones (PD) into native interchain disulfide bonds in humanized IgG1 MAb (7B2), then, the antibody-alkyne reacted with azide-bearing water-soluble porphyrin.N-hydroxysuccinimide (NHS) ester of the photosensitizer  and polydispersity (%Pd), indicating the presence of some high molecular weight species in this region , the light and heavy chains of either 7B2 or 7B2-IR700 were observed, due to the reduction of all available interchain disulfide bridges. The reduced 7B2-porphyrin did not show the separation of chains, confirming the accuracy of click chemistry with DAR4 C.In total, the characterization of PICs showed that the shape, solubility and binding ability of IR700-PICs was affected by increasing the drug-to-antibody ratio (DAR), while they were preserved for water-soluble homogeneous porphyrin-7B2 with a constant DAR of 4, due to the retention of rigid structural bridges on the antibody. By using a Cary Eclipse UV-Vis-NIR spectroscopy, we analyzed the excitation and emission spectra before flow cytometry study without using a secondary antibody. We showed the excitation of porphyrin by blue laser at 488 nm would not interfere with the emission intensity. The excitation by violet laser at 405 nm caused red emission at 725 nm with 1.163 (a. u.) intensity, while the maximum red fluorescent intensity (3.779 a. u.) can be observed by the excitation of Soret band of porphyrin at 432 nm. The results were in agreement with the theoretical analysis of UV-Vis spectra . Specific cell binding to native Env was examined by flow cytometry. Without using a secondary antibody, the red fluorescent emission from IR700-PICs and porphyrin-PIC were directly detected on the Env-transfected cells, by filter Qdot 705 and filter Qdot 655, respectively A. By usiInterestingly, IR700-PICs with different DARs A and difIn cellular studies, to have an equal irradiation for both porphyrin-PIC and IR700-PIC, for a side by side comparison, a custom-made LED device with a broad spectrum of light (380\u2212780 nm) was utilized, as the blue spectra can excite the Soret band of porphyrin at 432 nm, meanwhile, red spectra excites IR700-7B2 at 689 nm A. 2. H2DCF-DA fluorescent probe was applied to detect ROS production in PIT-treated cells by using flow cytometry. The Env-transfected cells incubated with PICs exhibited a significant increase in ROS levels, whereas control 293T cells did not show ROS production  and control 293T cells were incubated with a serial dilution of PICs starting at 500 nM in the presence of sCD4 (anti-gp120) for 4 h then were assayed by photoimmunotherapy (PIT) with 20 J/cm2, depending on the selected wavelength, however, visible light irradiation is unlikely to be toxic [As a prospect of this approach in an in vivo study, the doses of irradiation can be increased to maximum energy fluence of 100 J/cmbe toxic . OpticalTo study how the combination of both PICs may affect the cytotoxicity, an equal molarity of porphyrin-PIC was mixed with IR700-PIC. Interestingly, this combination showed a significant increase in the cytotoxicity in compare to the average cytotoxicity of both PICs, due to the synergic effect of two PICs with two different mechanism A,B.Furthermore, the photo-cytotoxicity of porphyrin-7B2 increased twice by increasing the time of incubation from 1 h to 4 h, indicating the internalization-dependency of porphyrin-mAb. In contrast, the photo-cytotoxicity of IR700-7B2 did not change significantly on increasing the time of incubation at 37 \u00b0C C. The cells were treated with IR700-7B2 or 7B2-porphyrin for 1 h, then washed and irradiated by 2-photons, 800 nm, 2.2 mW/cm. Red fluorescence emission from IR700-7B2 was directly detected . As 7B2-In conclusion, we compared the mechanism of two photosensitizers in the formation of PICs regarding the induction of cell death in HIV Env-transfected cells. We also addressed some of the main issues associated with conventional PDT including low selectivity, controlled drug dosimetry, dark toxicity and low water solubility of photosensitizer. Finally, we studied the possibility of PIT application to directly killing HIV-1 Env-expressing cells. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the antigen on the cell membrane or HIV Envelope, whilst being independent of the PS type. Considering the fundamental restriction of light penetration, we suggest optimizing the excitation of PSs in deep tissue that may give us a highly flexible theranostic platform for HIV researches. This inevitably issue dictates the use of high-power irradiation . The othApproaches that focus on the combination of the PIT strategy with \u201cshock and kill\u201d technique could be a cornerstone of future research efforts towards an HIV cure . The phoAll reagents are from ThermoFisher Scientific  unless a statement to the contrary is made. 2 in DMEM medium with 10% fetal calf serum . 293T/92UG cell lines stably express clade A clinical isolate 92UG037.8 gp160 as native trimers of HIV gp120/gp41. In this paper, Env-transfected cells refer to 293T/92UG cell lines , and HEKMAb 7B2 (GenBank accession numbers JX188438 and JX188439) is an Anti-gp41 hIgG1 binding at amino acids 598\u2013604 (CSGKLIC) in the helix-loop-helix region . HY  was covalently linked to purified MAb 7B2 through an ructions . The pro280 = 250,440 M\u22121 cm\u22121 for antibodies, \u03b5335 = 9100 M\u22121 cm\u22121 for pyridazinedione scaffolds, \u03b5422 = 165,175 M\u22121 cm\u22121 for porphyrin and \u03b5689 = 165,000 M\u22121 cm\u22121 for IRDye 700DX. A Correction Factor at 280 nm of 0.25 (at A335) was applied for pyridazinedione scaffolds, as described elsewhere [UV-Vis spectroscopy was used to measure protein concentrations and photosensitizer-antibody ratios (DAR), firstly by using Nanodrop 1000 UV-Visible spectrophotometer (ThermoFisher Scientific) and then a Cary 100 Bio UV-Visible spectrophotometer  operating at 21 \u00b0C. Blank was the sample buffer to correct base line with extinction coefficients; \u03b5lsewhere . Molecular size, purity and accuracy of the conjugation of products were determined using Non/reducing glycine-SDS-PAGE and then confirmed by microcapillary electrophoresis in the presence or absence of TCEP\u00b7HCl , as a reductive agent , following standard lab procedures.Purified MAbs and photo-immunoconjugates (PICs) were analyzed for antigen-binding specificity and titration by using ELISA, in wells coated with gp41 antigen (1 \u03bcg/mL), as described elsewhere . The gp4Hydrodynamic radii, electrophoretic mobility, zeta potential, and polydispersity of naked antibody and PICs were measured. Samples with 70 \u03bcL volume at 1 mg/mL in UV-transparent 96-well plates were measured by using a DLS Wyatt M\u00f6bius  with incident light at 532 nm, at an angle of 163.5\u00b0. Samples were equilibrated at 25 \u00b1 0.1 \u00b0C for 600 s before the measurements, with the constant temperature during the experiments. All samples represent triplicate values with 10 acquisitions and a 5 s acquisition time. The change in cumulant fitted hydrodynamic radius in nanometers was tracked throughout the storage period. Results were analyzed using the Dynamics software version 7.1.7 . By using an Agilent Technologies Cary 5000 Cary Series UV-VIS-NIR spectrofluorimeter, we showed the incident laser beam at 532 nm is not within the fluorescent sample\u2019s (IR700-7B2 and porphyrin-7B2) band of excitation; as the analysis would not be disrupted or tainted.4 cells were incubated with PBS/BSA/0.01% sodium azide (PBA) for 30 min in RT, and then PICs added to reach the final concentrations of 10 \u00b5g/mL in the presence of 5 \u00b5g/mL soluble CD4. Cells were incubated with PICs for 1 h at room temperature. For indirect assay, half of the samples were washed, and then stained with FITC conjugated goat anti-human IgG secondary antibody (2 \u03bcg/mL) for 1 h. All the samples, for direct and indirect assays, were washed twice and fixed in 100 \u03bcL of 2% paraformaldehyde. After 4 h, 150 \u03bcL of PBS was added to the samples. Cells with 10,000 events were evaluated on BD LSRFortessa , analyzed by Flow-Jo software version 7.5 . Mean fluorescence of the gated cell population labelled with immunoconjugate was calculated in relation to the mean fluorescence of cells labelled with MAb 7B2. Direct and indirect immunofluorescence were applied using flow cytometry to analyze the binding of photo-immunoconjugates (PICs) to Env-transfected 293T cells. 8 \u00d7 10Direct immunofluorescence without adding FITC conjugated secondary antibody was applied to analyze the binding of PICs to Env-transfected 293T cells. For IR700-PICs, the cells were excited by either violet laser at 405 nm or red laser at 640 nm, and the red fluorescence emission was detected by 710 \u00b1 50 band-pass filter (Qdot 705). For porphyrin-PICs, the cells were excited by violet laser at 405 nm, and the red fluorescence emission was detected by 660 \u00b1 20 band-pass filter (Qdot 655).5 cells were incubated with 1 \u00b5M of native antibody, porphyrin-PIC, or IR700-PIC in 96 well plates in phenol-free DMEM for 45 min. The cells were washed and incubated with 1 \u03bcM H2DCF-DA solution prepared in PBS, and washed again and incubated with phenol-free DMEM before irradiation with 50 J/cm2. A mirror dark-plate, submitted to the same procedure, except for light exposure, contained the dark control groups. The cells incubated with 40 mM H2O2, without irradiation, were used as positive control. Fluorescence intensity (Ex/Em = 485/535 nm) was measured with the flow cytometry, with the procedure as mentioned above.ROS generation in the cell lines was detected with the fluorescent probe H2DCF-DA . 2 \u00d7 102; 140 mM NaCl) and stained with recombinant annexin V conjugated to green-fluorescent FITC dye  for 15 min. After three washes, cells were incubated with 2 \u03bcg/mL of propidium iodide (PI)  for 15 min. The mean fluorescence intensity was measured for 10,000 events. The data were collected using linear amplification for two optical detectors, FSC and SSC, and log amplification for related filters, and analyzed by associated software as mentioned above.Annexin V-FITC/PI cell staining was performed following 6 h of standard photoimmunotherapy. Briefly, cells were incubated with Annexin V binding buffer  by a homemade LED array (30 mW/cm2) in two equal doses separated by 10 min, as described elsewhere [490 with a subtraction from background. Data are represented as % of cell viability versus PIC concentration. Naked 7B2 antibody, IR700-7B2, porphyrin-7B2 and a mixture of both PICs with equal molarity were diluted in the phenol-free DMEM medium, but without FCS, to reach a range of five final concentrations of 500 nM, 250 nM, 125 nM, 62.5 nM and 31.2 nM into 5 wells of a 96-well plate. HIV Env-transfected cells and 293T cells were adjusted to a concentration of 1 \u00d7 10lsewhere ,52. AfteLive cell imaging was performed using an inverted LSM 780 multiphoton laser scanning confocal microscope  equipped with a Chameleon laser  as a source for two-photons (2P) excitation at 800 nm. The images were obtained by the average of 2 scans and no appreciate variation was observed. The spatial resolution was approximately 350 nm considering the numerical aperture and the excitation wavelength, as described previously . The occStatistical analyses were performed with GraphPad Prism version 8.0 . Data are shown as mean and standard error of mean (SEM) of the indicated number of replicate values. If no error bar appears signifying that the error bars are smaller than, and obscured by, the symbol. The unpaired two-tailed Student\u2019s t-test was applied for statistical comparison, unless specifically stated otherwise.https://docs.google.com/forms/d/e/1FAIpQLSdOC3q1uWNIpVj_iPcz5G8rLrQRjuffNowE2U-DcUrKI3CAw/viewform?usp=sf_link.Password:USP4030.All data generated or analyzed during this research are included in this published article and its"}
+{"text": "GEMR cells (GEM-resistant cells). Remarkably, convincing evidence was established by RAGE small interfering RNA transfection. Taken together, our study demonstrated that PTE promoted chemosensitivity by inhibiting cell proliferation and MDR1 expression via the RAGE/PI3K/Akt axis in PDAC cells. The observations in these experiments indicate that PTE may play a crucial role in MDR1 modulation for PDAC treatment.Gemcitabine (GEM) drug resistance causes high mortality rates and poor outcomes in pancreatic ductal adenocarcinoma (PDAC) patients. Receptor for advanced glycation end products (RAGE) involvement in the GEM resistance process has been demonstrated. Therefore, finding a safe and effective way to inhibit receptors for RAGE-initiated GEM resistance is urgent. Pterostilbene (PTE), a natural methoxylated analogue derived from resveratrol and found in grapes and blueberries, has diverse bioactivities, such as antioxidative, anti-inflammatory, and anticancer qualities. The overall research objective was to determine the potential of PTE to enhance tumor cytotoxicity and chemosensitivity in PDAC cells. Our results have demonstrated that PTE induced S-phase cell cycle arrest, apoptosis, and autophagic cell death and inhibited multidrug resistance protein 1 (MDR1) expression by downregulating RAGE/PI3K/Akt signaling in both MIA PaCa-2 and MIA PaCa-2  Over 90% of pancreatic ductal adenocarcinomas (PDACs) are exocrine pancreatic cancer, which is a highly malignant tumor that with the growing cause of cancer-related mortality worldwide. In the early stages of PDAC, the signs or symptoms are rarely noticeable, and patients often have local invasion or distant metastasis at diagnosis. Unfortunately, less than 15%\u201320% of stage I-II PDAC patients are resectable due to most PDAC patients have carcinoma in-situ intravasation, perineural invasion, and distant metastasis at diagnosis . Thus, PGemcitabine (GEM), a deoxycytidine nucleoside analogue for cancer DNA synthesis obstruction, is the most common chemotherapeutic drug for PDAC treatment. However, approximately 75% of PDAC patients have GEM resistance, which greatly contributes to treatment failure . In addiPterostilbene (PTE), a natural methoxylated analogue, is derived from resveratrol, which is produced by Pterocarpus plants, grapes, and blueberries. A previous report showed that PTE has diverse bioactivities, including antioxidant, anti-inflammatory, and anticancer activities . PTE treGEMR) [Moreover, receptor for advanced glycation end products (RAGE) is highly expressed in pancreatic tumor tissue relative to normal adjacent tissue . RecentlGEMR) . NotablyGEMR) . PTE, a GEMR) ,19. AccoDMEM, horse serum, and Opti-MEM were bought from Thermo Fisher Scientific . FBS was purchased from NEQQ International Biological Corporation . RIPA lysis buffer, ECL substrates, and polyvinylidene difluoride (PVDF) membranes were obtained from Millipore . The PTE was kindly provided by professor Chi-Tang Ho . The GEM was purchased from Sigma-Aldrich . \u03b2-actin, beclin-1, and autophagy gene 5 (ATG5) antibodies were obtained from Novus Biologicals . Bcl2-associated x protein (Bax), B-cell lymphoma-extra large (Bcl-xL), PI3K, Phospho-PI3K, Akt, Phospho-Akt, and MDR1 antibodies were bought from Cell signaling technology . RAGE antibody was obtained from Invitrogen . Microtubule-associated protein light chain 3 II (LC3 II) antibody, peroxidase goat anti-mouse and peroxidase goat anti-rabbit were purchased from GeneTex . RAGE siRNA and Dharmacon transfection reagent were purchased from GE Healthcare .GEMR) was established as reported previously [MIA PaCa-2 cells (BCRC NO. 60139) were obtained from the Bioresource Collection and Research Center . A stable GEM-resistant PDAC cell line  for 48 or 72 h, an MTT-based viability assay was performed as reported previously [Approximately 2 \u00d7 10eviously .The cells were incubated with different concentrations of PTE for 72 h. The cell cycle was measured by propidium iodide (PI) staining and analyzed by FACScan flow cytometry  .5 cells/mL were cultured on 6-well plates overnight and then incubated with 25 nM non-targeting control siRNA or RAGE siRNA for 24 h. The knockdown efficiency was verified by western blot analysis.The procedure was performed as reported previously . BrieflyWestern blotting was performed as reported previously . Bcl2-ast test using SPSS 16.0 statistical software .Experimental results were repeated in three independent operations and quantified as the mean \u00b1 standard deviation (SD). Data were analyzed by Student\u2019s GEMR cell line that can resist 0.5 \u03bcM GEM-induced cytotoxicity was established  for 48 or 72 h. Cell proliferation suppressed by PTE treatment in a time- and dose-response manner was observed by MTT analysis , respectively. The spindle-shaped morphology and loss of viability by PTE treatment for 72 h compared with untreated cells are shown in A stable GEM-tolerant MIA PaCa-2 ablished A,B. To aanalysis C, D. TheGEMR cells  drug resistance causes high mortality rates and poor outcomes in PDAC patients. RAGE has been demonstrated to be involved in the GEM resistance process. Therefore, finding a safe and effective way to inhibit RAGE-initiated GEM resistance is urgent. Pterostilbene (PTE), a natural methoxylated analogue derived from resveratrol and found in grapes and blueberries, has diverse bioactivities such as antioxidative, anti-inflammatory, and anticancer characteristics. Based on our results, it was demonstrated that PTE induced S-phase cell cycle arrest and apoptotic and autophagic cell death and inhibited MDR1 expression by inhibiting the RAGE/PI3K/Akt axis, which consequently enhanced chemosensitivity in both MIA PaCa-2 cells. Remarkably, convincing evidence was established by RAGE small interfering RNA transfection.Taken together, our study demonstrates that PTE promotes chemosensitivity by inhibiting cell proliferation and MDR1 expression via the RAGE/PI3K/Akt signaling pathway in PDAC cells. The observations in these experiments indicate that PTE may play a pivotal role in MDR1 modulation for PDAC chemoprevention."}
+{"text": "As the increased consumption of ready-to-eat meat alternatives is a fairly recent trend, little is known about the composition and dynamics of the microbiota present on such products. Such information is nonetheless valuable in view of spoilage and food safety prevention. Even though refrigeration and modified-atmosphere-packaging (MAP) can extend the shelf-life period, microbial spoilage can still occur in these products. In the present study, the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage, contrasting the use of a culture-dependent method to a culture-independent metagenetic method.Latilactobacillus sakei was the most abundant species. Metabarcoding analysis, in contrast, revealed that DNA of Xanthomonas was most prominently present, which likely was an artifact due to the presence of xanthan gum as an ingredient, followed by Streptococcus and Weissella.The former revealed that lactic acid bacteria (LAB) were the most abundant microbial group, specifically at the end of the shelf-life period, whereby Lb. sakei was likely the most prominent specific spoilage organisms (SSO) and, additionally, that the use of metagenetic analysis needs to be interpreted with care in this specific type of product. In order to improve the performance of metagenetics in food samples with a high DNA matrix but a low bacterial DNA load, selective depletion techniques for matrix DNA could be explored.Taken together, these results indicated that The online version contains supplementary material available at 10.1186/s12866-022-02446-9. Listeria monocytogenes, and the shelf-life period, e.g. Pseudomonas or LAB [Although meat is still a staple food in the diet of the average Belgian consumer, a market study commissioned by the Flanders\u2019 Agricultural Marketing Board (VLAM) has noted that, in 2019, 23% of the Belgian population consumed meat substitutes at least once a week [s or LAB , even ifs or LAB . Generals or LAB \u20138. TheseEvaluation of food spoilage is usually monitored by culture-dependent methods. These methods involve cultivation on selective agar media, followed by molecular techniques to further identify bacteria to genus or species level , 9. The In this study, the composition and dynamics of the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage using both culture-dependent and culture-independent methods. Samples were collected just before the products were sliced, shortly after slicing and at the end of the shelf-life period and identified microbiota were compared. The aim of the study was to uncover potential SSO\u2019s present in this product during its shelf-life period and to obtain novel information on the diversity and progression of the most abundant microorganisms during shelf life using the metagenetics method.2, 30% CO2), within the premises of a food business operator (FBO), as previously described [In this study, curry-flavored vegetarian slices, composed out of egg white (38%), water (37%), sunflower oil 15%), salt (2.7%) and 1.6% curry spices, and also containing <2% for each of the following individual ingredients: bamboo fibers, spices, sugar, thickeners, acidity regulators and colorings, were sampled. The product was pressed into a vegetarian product log which was subsequently cooked, reaching a core temperature of \u226572\u00a0\u00b0C. Sample collection was performed at the moment of slicing and packaging, under MAP conditions , moistened with 10 ml of maximum recovery diluent , were used to perform the surface sampling (approximately 625\u00a0cm\u00b2 each).Enterococcus spp., Slanetz and Bartley agar  was incubated at 37\u00a0\u00b0C for 48\u00a0h; and colonies were confirmed by testing for growth at 44\u00a0\u00b0C, in 40% bile and performing an additional catalase test. Streptomycin thallous acetate actidione agar  was incubated at 21\u00a0\u00b0C for 48\u00a0h for the enumeration of presumptive B. thermosphacta; and confirmation steps were carried out. For the enumeration of presumptive sulfite-reducing clostridia, tryptose sulphite cycloserine agar  was incubated at 37\u00a0\u00b0C for 24\u00a0h. For the enumeration of presumptive B. cereus, Mannitol egg yolk polymyxin agar  was incubated at 30\u00a0\u00b0C for 48\u00a0h; and confirmation steps were carried out. For the enumeration of presumptive Enterobacterales, Violet red bile glucose agar  was incubated at 37\u00a0\u00b0C for 24\u00a0h. One log colony forming units (CFU)/g was the lower limit for enumeration for all media.Ten grams of each sample were collected and analyzed, as previously described . Plate cSurface swab samples from the environment were analyzed for TAC, TANC and LAB counts on MRS, following the same method as described above.w) was measured for each food sample using the AQUALAB 4TE water activity meter .The pH was measured using a pHenomal pH 2100\u00a0L in combination with a SF113 electrode . Water activity 5 elements [Microbial DNA was extracted as previously described  from eacelements , 16. Usi5 fingerprint clusters, 43, 34 and 21 isolates  were selected as representative isolates of visually defined clusters grouping isolates with the same band patterns and were further identified. One representative isolate was selected for clusters containing up to three isolates, while a minimum of two isolates were selected for identification of clusters containing >4 isolates. As previously described [Based on the (GTG)escribed , identifescribed . Sanger escribed .From the food contact surfaces of the slicers, 56 isolates were identified .Sample preparation preceding metabarcoding analysis was performed as previously described . DNA extLactobacillus encompassing genera Companilactobacillus, Dellaglioa, Lacticaseibacillus, Lactiplantibacillus, Latilactobacillus, and Paucilactobacillus [Demultiplexing of the amplicon sequencing dataset was performed by the sequence provider and barcodes were clipped off. The processing pipeline was entirely performed in RStudio 1.3.1093. To remove primer sequences, the ShortRead package was used . Then, tbacillus . The \u201crabacillus .RStudio Version 1.3.1093  was used to perform analysis of variance  and Tukey\u2019s test, to verify potential significant differences between enumerations .Bacterial loads on the curry-flavored vegetarian meat alternative were estimated for TAC, TANC and LAB using samples obtained from the 1-week old vegetarian product log, the 3-weeks old vegetarian product log and after slicing and storage of these respective products  were retained for further analysis after rarefaction analysis, as a plateau phase was reached, meaning that sufficient sequence data was obtained for a reliable view on the bacterial ecosystem composition. The number of reads generated per remaining sample varied from 7,848 to 247,254 reads, with an average of 47,965 reads per sample  , 25\u201327. Latilactobacillus sakei has been reported before for vegetarian meat alternatives [B. thermosphacta, Leuconostoc mesenteroides and Listeria monocytogenes in charcuterie products [The high prevalence of rnatives . Selecteproducts \u201334. Howeproducts . Also, tEnterococcus faecium [E. avium, E. devriesei, E. gilvus, E. malodoratus and E. viikkiensis, but not E. faecium. The fact that these species were only found on PCA, RCA and MRS agar and not on S&B agar was probably due to the fact that most of them do not grow at 44\u00a0\u00b0C [In line with the fact that vegetarian meat alternatives may also contain enterococci such as  faecium , enteroc faecium . The isoat 44\u00a0\u00b0C \u201338 and wCarnobacterium spp. were consistently present throughout the shelf-life period, but were never able to reach a high abundance. Although Carnobacterium spp., in particular C. divergens and C. maltaromaticum, are known to be able to spoil meat in packaging with low traces of O2 [C. inhibens after slicing could be due to cross-contamination by a cooked chicken product [es of O2 \u201341, theyes of O2 . The low product , as bothB. thermosphacta was mostly not found despite being a common meat spoilage bacterium [Lb. sakei [Psychrobacter maritimus, Serratia myotis and Ralstonia mannitolilytica were recovered just after slicing but were marginalized towards the end of the shelf-life period, as is often the case in chilled MAP goods [With respect to bacteria other than LAB, acterium , 43. It acterium \u201346, and/b. sakei . Gram-neAP goods \u201350. ThisAP goods , 48, 51.To circumvent the problem of viable but non culturable microorganisms in microbiological mapping strategies , a complSerratia, the day-28 sample for VB3L3 (relatively high abundance of Leuconostoc and Psychrobacter) and both day-28 samples from batch 4: VB4L1 and VB4L3 (relatively high abundance of Lactobacillus). Of these 4 samples, the 2 samples that did not recover high relative abundances for Xanthomonas (product log VB5L1 and day 28 VB4L3) also had a low percentage of their ASVs identified as chloroplast and mitochondria. Another possible explanation for this variety could be differences in microbial loads: the product log for VB5L1 was below enumeration limit for TAC, TANC and LAB on both MRS and M17 medium, while both day-28 samples for batch 4 had the highest enumerations found for these parameters (e.g. 7.1 and 8.1 log CFU/g TAC). A first overall finding of the metabarcoding analysis was that there was not much variation observed in the recovered taxa and their individual relative abundances throughout the shelf-life period in contrast to the data from the culture-dependent analysis. Perhaps these limited observed variations were due to the mostly low bacterial loads on this type of product, even at the end of the shelf-life period. Exceptions in recovered microbiota included the unsliced vegetarian product log sample of VB5L1, with a high abundance of Xanthomonas through-out the shelf-life period with the metabarcoding approach, whereas this genus was not found using the culture-dependent method. This can potentially be explained, though, by the presence of xanthan gum (or E415) in the product, which is used as a stabilizing, thickening or emulsifying agent, industrially produced by Xanthomonas campestris strains [Xanthomonas reads were aligned against the EzBioCloud database, a hit for X. campestris was obtained with 99.77-100% identity. Although Xanthomonas can be a factor in food spoilage, this is usually the case for fruits and vegetables [Xanthomonas DNA was due to the presence of xanthan gum, no viable Xanthomonas cells were most likely present, which corroborates the inability to identify Xanthomonas using the culture-dependent method.Another contrast was the discovery of a potential strong presence of  strains . Indeed,getables . If recoWeissella and Streptococcus as the main LAB, which were not found as species using the culture-dependent method. The latter instead emphasized the presence of Latilactobacillus, Leuconostoc and Carnobacterium species, commonly found in chilled MAP charcuterie [Lactobacillus  was however recovered at a high relative abundance in two day-28 samples taken from the same batch , which could explain the high amount of isolates identified as Latilactobacillus sakei at the end of the shelf-life period. Coincidentally, both day-28 samples also had the highest total aerobic counts at 7.1 and 8.1 log CFU/g, while the average TAC for this product type was 4.7 log CFU/g. This lower TAC at the end of the shelf-life period was in accordance with earlier findings in vegetarian products that were also partly made up out of egg white powder . Weissella is known to occur on MAP meats and poultry and can cause slime and/or greening due to the formation of H2O2 [Streptococcus was more puzzling, as streptococci are not generally food-associated organisms or food spoilers, with the benign exception of S. thermophilus in certain fermented dairy products  [A third contrasting finding was that the metabarcoding data indicated rcuterie , 56, 57. of H2O2 , 56, 58. of H2O2 . The precheeses) . This spcheeses)  and thusLb. sakei and, to a lesser extent, C. divergens and C. maltaromaticum, though variation can be seen between batches (in the case of high relative abundances of Lactobacillus recovered by metabarcoding in both samples retrieved from batch 4 at the end of the shelf-life period) or individual samples . Also, reliable interpretation of the applied metabarcoding method proved difficult in this type of product, as there was too much interference against a low bacterial load from both chloroplast DNA and the presence of Xanthomonas DNA due to the use of xanthan gum as a stabilizing agent. A way to circumvent these issues might be the application of a bacteriological enrichment step before metabarcoding. This method also has its downsides, as it will select only a subset of culturable bacteria and hence will change the initial structure of the microbiota composition. The advantage of adding this step is that a rare species of interest might be detected where it was before missed [In conclusion, it can be stated that generally, the main SSOs in the type of product investigated were identified as e missed . Combinie missed .Additional file 1."}
+{"text": "Recent patent losses for antiretroviral drugs (ARV) have led to the debate of cost-saving through the replacement of patented drugs with generic drugs. The split of recommended single-tablet regimens (STR) into their single substance partners is one of the considerations mentioned in said debate. Particularly, generic tenofovir disoproxil/emtricitabine (TDF/FTC) is expected to hold untapped cost-saving potential, which may curb increasing overall expenditures for combined antiretroviral therapy (cART) within the statutory health insurance (SHI) of Germany.Data of ARV reimbursed by the SHI were used to describe the trends of defined daily doses (DDD) as well as the revenue within the German ARV market. They were also used to determine the cost-savings of moving to generic drugs. The time period observed was between January 2017 and June 2019. The potential cost-savings were determined with following assumption in mind: the maximum possible use of generic ARV, including 1) the split of STR and replacing all substance partners with generic ones, and 2) replacing patented tenofovir alafenamide/emtricitabine (TAF/FTC) with generic TDF/FTC.Throughout the observation period, the DDD of generic ARV increased nearly five-fold while their revenue increased more than four-fold. Total cost-saving showed a sharp increase over the same period, with generic TDF/FTC accounting for a share of around 70%. The largest potential cost-saving could have been achieved through replacing patented TAF/FTC with generic TDF/FTC, peaking at nearly 10% of total revenue, but showing decreasing trends in general., but consequently curbed the potential cost-savings. Unique price reductions of generic TDF/FTC have played a pivotal role for these effects. In any case, substituting with generic ARV should not fail to adhere to the treatment guidelines and continue to consider the medical requirements for the treatment.The progressive distribution of generic ARV ensured increasing cost-savingsThe online version contains supplementary material available at 10.1186/s12913-021-07390-4. Combined antiretroviral therapy (cART) has substantially reduced the morbidity and mortality of the growing number of people infected with human immunodeficiency virus (HIV) \u20133. HIV tImportant regulations have been introduced by the German legislation with the aim of curbing prices of patented drugs as well as the increasing expenditures on pharmaceuticals incurred by sickness funds. These include, firstly, the German Act of reinforcing SHI competition that was passed in 2007 in order to facilitate the use of generic drugs after the patent expiration of branded drugs which is usually 20\u2009years after the filings of patent applications \u201314. SecoRegardless of economic interests, the distribution of ARV must adhere to current treatment guidelines that recommend a combination of three drug classes for the initiation of the HIV therapy, preferably as a single-tablet regimen (STR) . GenericSince almost one-third of the available ARV lost their patent protections, the debate about cost-saving through generic replacements is still ongoing , 24\u201327. In this study, our aim was to firstly describe the current ARV market within the German SHI. Secondly, we aimed to determine the cost-savings achieved through the distribution of generic drugs, focusing particularly on TDF/FTC. Our third aim was to determine the potential cost-savings that could be achieved additionally when assuming the maximum possible use of generic ARV. This includes the splitting of STR, replacing all substance partners with generic ones and replacing the patented drug tenofovir alafenamide/emtricitabine (TAF/FTC) with generic TDF/FTC.Insight Health\u2122 provided the data of distributed ARV for the years 2013 to the end of the second quarter of 2019. These data were collected from the billing centers and included all prescriptions reimbursed to pharmacies by the German SHI. The data account for more than 99% of distributed ARV covered by the SHI.The dataset included all ARV by pharmaceutical registration numbers distributed on a monthly basis, regardless of whether the use was for prevention or treatment of HIV. Additional relevant items were product name, substance name, drug class, Anatomical Therapeutic Chemical (ATC) classification code which is unique for each ARV and thus will be used synonymously throughout the paper , patent The observational period was limited from January 2017 to June 2019 in order to account for market launches of generic alternatives. For all ARV, the DDD and corresponding revenues were determined in accordance with their respective patent status: patented, patent expired or generic. Additionally, the DDD and revenues were determined for a few ARV with an expired patent that do not have an equivalent generic substitute.To quantify cost-savings and potential cost-savings, the pharmacy retail price for each ARV was first calculated as a weighted average of patent status  in 1/2017 to 17.3 million \u20ac  in 2/2019  of total ARV revenue in mid-2019 (Supplementary Table SThis paper has several limitations. Firstly, the dataset used did not allow access to the private health insurance sector which accounted for approximately 11,5% of the total health insured community of Germany . This maSecondly, our assumption to determine the potential cost-savings was not applicable for ARV containing elvitegravir, bictegravir and lopinavir, since these substance partners are only available as a fixed combination preparation without generic substitute . Since sThirdly, we were informed that some pharmacies refused to transfer their data to the responsible billing centres in the health insurance regions of Berlin, North Rhine-Westphalia and Baden-Wuerttemberg at the end of 2018. This may have resulted in an additional decrease of total DDD, estimated at about 5\u20137%, in 2019.Generic drugs are progressively distributed in the German ARV market and ensure increasing cost-savings. Unique price reductions of generic TDF/FTC have played a pivotal role for both the cost-savings and potential cost-savings. However, as mentioned before, pursuing the maximum possible use of generic ARV should not fail to adhere to the treatment guidelines and consider the medical requirements for a change in treatment.Additional file 1.."}
+{"text": "Veno-venous extracorporeal membrane oxygenation (VV-ECMO) has been used successfully for the past decade in adult patients with acute respiratory distress syndrome (ARDS) refractory to conventional ventilatory support. However, knowledge of the health-related quality of life (HRQoL) in VV-ECMO patients is still limited. Thus, this study aimed to provide a comprehensive overview of the HRQoL following VV-ECMO support in ARDS patients.A systematic search was performed on PubMed and Web of Science databases from January 1st, 2009 to October 19th, 2020. Studies reporting on HRQoL following VV-ECMO for ARDS in adults were included. Two authors independently selected studies, extracted data, and assessed methodological quality.N\u2009=\u2009441). All eight studies had a quantitative design and reported 265 VV-ECMO survivors to have a reduced HRQoL compared to a generally healthy population. Follow-up time varied between six months to three years. Additionally, only four studies  compared the HRQoL of VV-ECMO (N\u2009=\u2009159) to conventionally treated survivors (N\u2009=\u2009176), with one study showing a significantly better HRQoL in VV-ECMO survivors, while three studies were stating comparable HRQoL across groups. Notably, most survivors in these studies appeared to experience varying degrees of anxiety, depression, and post-traumatic stress disorder (PTSD).Eight studies were eligible for inclusion, consisting of seven observational studies and one randomized controlled trial (total ARDS survivors supported by VV-ECMO have a decline in HRQoL and suffered from physical and psychological impairments. This HRQoL reduction is comparable or even better to the HRQoL in conventionally treated ARDS survivors. Acute respiratory distress syndrome (ARDS) is a frequent cause of respiratory failure in critical care patients. It is defined by the acute onset of non-cardiogenic pulmonary edema and hypoxemia, which might require mechanical ventilation . While tVeno-venous extracorporeal membrane oxygenation (VV-ECMO) has been successfully employed in adult patients with severe ARDS refractory to conventional ventilatory support , 7. The Health-related quality of life (HRQoL) is a multidimensional construct that describes the perceived impact of health status, including physical, psychological, and social domains of health . The resDespite the increasing number of reports describing the HRQoL of patients treated with ECMO, a study focusing specifically on HRQoL in ARDS patients supported by VV-ECMO is still lacking. Additionally, actual HRQoL scores are not always described or displayed in previous studies, which makes interpretation of the effect of VV-ECMO therapy on HRQoL in ARDS-related patients challenging. The present systematic review aims to describe HRQoL and long-term outcomes in adult ARDS patients supported by VV-ECMO.A systematic search was performed independently by two reviewers (EK and VR) utilizing the PubMed and Web of Science databases and was completed on October 19th, 2020. This search combined Medical Subject Headings (MeSH) and free search terms. The MeSH and free search terms related to VV-ECMO, HRQoL, and ARDS were used to optimize the database search output. The search string was computed as follows: \u201cExtracorporeal Membrane Oxygenation\u201d OR \u201cECMO\u201d OR \u201cExtracorporeal Life Support\u201d OR \u201cECLS\u201d OR \u201cVV-ECMO\u201d OR \u201cVV-ECLS\u201d OR \u201cvenovenous ECMO\u201d OR \u201cvenovenous ECLS\u201d AND \u201cquality of life\u201d OR QoL\u201d OR \u201cSF-36\u201d OR \u201cEuroQoL\u201d OR \u201cEQ-5D\u201d AND \u201cdisability\u201d OR \u201cphysical disability\u201d OR \u201chealth problem\u201d OR \u201cemotional problem\u201d OR \u201csocial problem\u201d OR \u201cgeneral health\u201d OR \u201clong-term outcome\u201d. The search was conducted in PubMed and Web of Science databases were conducted separately using the same MeSH and free terms. Search results were combined and reviewed to omit duplicate papers. Acquired articles were checked for relevancy step by step, as depicted in Fig.\u00a0The Population, Intervention, Comparison, Outcome and Study Design (PICOS) approach was used for the selection of studies included in the systematic search Table . StudiesTwo reviewers (EK and VR) independently assessed all studies for inclusion and extracted potentially relevant studies. The eligibility of the articles was determined by screening and reviewing the full-text article. Studies that did not answer the current research question were eliminated. Any potential disagreements regarding eligibility were resolved by consensus among three members of the research team . Agreement on study inclusion was examined using Cohen\u2019s kappa coefficient to assess inter-rater reliability . Next, iTwo researchers (EK and VR) performed the risk of bias assessment independently. Based on the study design, the Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies from the National Heart, Lung and Blood Institute  and the n\u2009=\u20096) or did not use self-reporting tools to evaluate HRQoL (n\u2009=\u20091). Ultimately, a total of eight studies [\u03ba\u2009=\u20090.87 (95% CI 0.62\u20131.12), p\u2009=\u20090.001.The initial search from PubMed and Web of Science databases yielded a total of 146 studies. Duplicate studies were removed after which 118 studies remained eligible. The initial screening of titles and abstracts excluded all studies that did not evaluate the HRQoL of ARDS patients supported with VV-ECMO. Studies that did not evaluate HRQoL using self-reporting tools were also removed. Two researchers (EK and VR) reviewed the remaining 15 studies for full manuscript review. As a result, seven studies were excluded because they did not specifically evaluate the HRQoL of VV-ECMO , Impact of Event Scale-Revised Score (IES-R), Hospital Anxiety and Depression Scale (HADS), mini-mental state examination, Centre for Epidemiologic Studies Depression (CES-D), the shortened Beck Depression Inventory, and the Beck Anxiety Inventory.Regardless of the variety of follow-up time, all included studies showed a decrement in the HRQoL score of ARDS patients following VV-ECMO. Mean SF-36 scores were significantly lower for VV-ECMO survivors compared to the matched general population. Mobility problems were reported by VV-ECMO survivors in two studies , 41. AddThe majority of the studies reported similar HRQoL between ARDS patients treated with VV-ECMO and conventional ventilatory support. Only one study reported better HRQoL at the follow-up time for VV-ECMO patients compared to survivors treated conventionally . Signs o\u03ba\u2009=\u20090.94 (95% CI 0.86\u20131.02), p\u2009<\u20090.001. Assessment of risk of bias across studies was not performed.Two researchers independently performed the risk of bias assessment using the Quality Assessment Tool for Observational Cohort and Cross-sectional studies from the National Heart, Lung and Blood Institute  and The Given the increased use of VV-ECMO to support refractory gas exchange in ARDS patients, efforts should be devoted to gain a better understanding of the HRQoL in ECMO survivors to ultimately improve patient care following ECMO support. While earlier systematic reviews lack focus specifically on the HRQoL of ARDS patients supported by VV-ECMO, the current review assessed the HRQoL of adult VV-ECMO survivors, indicating a lower HRQoL in these patients compared to the general healthy population.Eight studies were included in this review, which revealed that VV-ECMO survivors have lower SF-36 scores, i.e., reduced physical, mental, and social dimension scores compared to the general healthy population norms. This is consistent with previous studies showing reduced SF-36 scores in most SF-36 domains , 47. NevNotably, as compared to patients supported conventionally, selected studies suggested no reduction of HRQoL for VV-ECMO survivors , 45, 46.2 ventilation, allowing a minimum iatrogenic contribution to lung injury [Similarly, Peek et al. suggested that the comparable HRQoL outcomes between both groups might result from the fact that VV-ECMO protects the pulmonary system from high pressure and FiOg injury . Additiog injury . On the g injury . The autg injury . This mag injury . On the g injury .Hodgson et al.  reportedGalazzi et al. reported that early rehabilitation should be strived for ICU patients, especially for ECMO patients , to miniIn several studies, the prevalence of physical impairment in VV-ECMO survivors was higher than the prevalence of mental impairment at various follow-up points between 12\u00a0months and 3\u00a0years after discharge , 44, 46.It is well known that ICU survivors treated for ARDS, exposed to life-threatening circumstances, are prone to suffer from PTSD , 56. ThiSeveral limitations should be noted in this review. Of the eight included studies, seven were of observational design and thereby lacked in randomization and blinding. Due to this study\u2019s inclusion criteria, such as language and HRQoL reporting instruments, potentially valuable articles may not have been included. More importantly, since the included studies utilized self-reporting instruments, outcomes were highly subjective, implying heterogeneity in HRQoL outcomes, and thereby comparison of results and making specific conclusions and recommendations is hampered. The included studies offer valuable yet highly heterogeneous data, as there is variability in, e.g., reporting instruments and follow-up time. Additionally, most of the included studies were conducted in a single-center, had relatively small sample sizes, and thus may have lacked the power to adequately detect possible group differences. All included studies had a quantitative observational purpose, which does not capture detailed descriptions of patients\u2019 experiences during and after VV-ECMO support . Lastly,The present systematic review describes a reduced HRQoL in ARDS survivors supported by VV-ECMO and suggests this reduction to be similar to observations in conventionally treated ARDS survivors. Based on the quantitative design of the included studies and to gain further insight into the quality of life of VV-ECMO survivors, additional qualitative studies are warranted."}
+{"text": "Iftar) and one before dawn (Sohor). During this month, it is also an opportunity to share a meal with family and friends, a period of highly intensified socialization. In parallel with the nutritional changes brought about by this unique pattern of fasting in Ramadan, other metabolic and physiological changes may occur, such as fluctuations in body weight and/or disturbance in the quantity and quality of the sleep-wake circadian rhythm. In the verses of the Qur'an, the exemption from fasting in certain situations such as illness is clearly stated. Despite this religious tolerance, many faithful who are eligible for the exemption observe the fast of Ramadan either for the spiritual aspect it provides by performing it, by religious guilt or to mark a normalization in the Muslim community for fear of the gaze of others. The world is experiencing an increase in the emergence of non-communicable diseases (NCDs); leading cause of the global mortality. Environmental and behavioral risk factors related to lifestyle, such as smoking, excessive alcohol consumption, unhealthy diet, and sedentarity have a causal association with NCDs. Other factors, such as genetic and physiological factors may also be associated . Diabetes is one of the highest prevalent NCDs in the world and it continues increasing year by year. This chronic disease can lead to significant potential complications  to the patient's health. This requires an individual and appropriate care, both dietetic and therapeutic and over the long term will at best make it possible to sensitize the diabetic patient to the adverse effects related to his disease and thus improve its quality of life. Performing the Fast of Ramadan for a diabetic is a common situation. Diabetes is the only chronic disease widely studied in relation to Ramadan fasting. In the literature, many studies have investigated the effects of Ramadan intermittent fasting on diabetic patients. This article aims to provide a general overview and highlight if there are many effect of Ramadan fasting on diabetes, as an example of a NCDs.Although Ramadan lasts only for 1 month each year, it can be accompanied by significant changes in: both energy and nutritional intake; in the diet composition; in the working hours; and the usual way of life. The majority of practitioners consume two meals, one after sunset ( Ramadan is one of the five pillars of Islam. It constitutes the month of fasting, a holy month for Muslims who have the duty to fast from sunrise to sunset. Ramadan lasts between 29 and 30 days depending on the lunar cycle, shifts from year to year and gradually changes from one season to another. This religious rite concerns all healthy Muslims in good health, and only those who are at risk of harm from fasting are exempted from this obligation such as people with non-communicable diseases (NCDs) or other disabling diseases. In the Muslim community, there is an intense desire to participate in fasting, even among those who are eligible for the religious exemption . DespiteThe PubMed and Google Scholar databases were searched. We also used a national online documentation system of the algerian university (SNDL.cerist). The key words used were \u201cRamadan fasting,\u201d \u201cfasting\u201d and we have associated with each of these word \u201cdiabetes,\u201d \u201cNCDs,\u201d \u201ccircadian rhythm,\u201d \u201ccomplications,\u201d \u201cdietary habits,\u201d \u201cphysical activity,\u201d \u201cbody composition,\u201d \u201cnutritionnal education.\u201d We mainly took the recent and most relevant studies focused on human studies. The Ramadan comes without transition, and practitioners move from one way of life to another overnight. The whole rhythm of everyday life is upset, thus reflecting a reversal of activities characterizing day and night. Normally, people eat during the day, and most socializing activities take place during the day. During Ramadan, the schedule of meals and activities is changed. These changes vary according to the seasons, geographical and socio-economic situations as well specific traditions of each country. In these conditions, the fasting body tries to adapt twice in the space of a month: at the beginning and end of Ramadan . The manp < 0.05). The results of another study carried out on 1,266 diabetic patients showed an average sleep duration between 5 to 8 h in 70.3% of T2DM and 65.5% of T1DM. The same results both for the DMF and DMNF , followed by those treated with oral agents including sulfonylureas as compared to oral agents excluding sulfonylureas   . In para 0.2984) . The pat 0.2984) , 69, 70. 0.2984)  having r 0.2984)  between  0.2984) .Several international recommendations and consensus of experts have resulted in proposals to optimize diabetes management during the month of Ramadan:- The first international recommendations for a good practice of fasting were those of the Hassan II Foundation in Morocco in 1995. This consensus proposed criteria authorizing and prohibiting fasting and reinforced clinical-biological surveillance before, during and after Ramadan, a monitoring of glycemic control, adaptation of treatment, education of diabetics, and their families on the contraindications of fasting, the risk of acute complications and the means of prevention and treatment .- Other recommendations follow those of Morocco, including those of the American Diabetes Association (ADA) published in 2005 . In thes- In 2010 , researc- The third update to the recommendations of the ADA Working Group  was also- In the 2020 update , the res- The International Diabetes Federation (IDF) and the Diabetes and Ramadan (DAR) International Alliance met to provide comprehensive guidance on this topic. This guide  highligh- The update of the IDF-DAR International Alliance Practical Guidelines features new guidance based on a greater and more recent body of evidence . This inIftar) hyperglycemia and also prevent hypoglycaemia during the period of fast. With the use of analoges, these objectives may be met more easily.- In addition, the Asian recommendations of the South Asian Consensus Guideline, where researchers  indicateThe importance of the socio-cultural context of the holy month of Ramadan associated with the culturally-religious identity to which the diabetic patient is or wants to adhere are parameters to be included in the decision to fast. The advice of health professionals and members of religion will only reassure the diabetic in the accuracy of their decision. Each patient must be monitored individually, taking into account in particular the evolution of his disease, his therapeutic treatment, his socio-economic level, his level of education, his age, and his family status.Fasting is a very sensitive period for the glycemic control of DMF and DMNF patients, requiring multidisciplinary preparation and expert advice. This work gave a brief visualization of the effect of Ramadan fasting on a NCDs which is diabetes. The few studies cited reflecting the various points raised  showed heterogeneity of the results found probably due to the number of days of fasting, climatic conditions, cultural variations in eating habits, etc. Some slight advantages were noted. These particularities must be taken into account in the development of any study project on this topic. Effective management of diabetes with regular glycemic control will allow the patient to maintain an appropriate metabolic profile. Education on fasting and diabetes management is useful beyond Ramadan. It is important that recommendations have to be based on the gaps in existing data.MBenc: study design, literature searching, and article draft writing. IS, MBent, and FB: draft revision and literature searching. YB: draft revision. All authors agree to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Many Muslims with diabetes choose to fast against medical advice during Ramadan, potentially increasing their risk of acute complications. Patients are often reluctant to disclose fasting to their health care providers, and their needs regarding Ramadan are not met in consultations. For healthcare professionals to provide patient-centred care, it is important to gain more insight into patients\u2019 decision-making process. This study therefore aims to explore how Muslims with diabetes decide whether to fast during Ramadan.A qualitative study was conducted consisting of 15 focus groups with Muslims with diabetes within a constructivist paradigm. Convenience sampling was used. All focus groups were transcribed verbatim and analyzed using Braun and Clarke\u2019s reflexive thematic analysis.Four themes were found to be important in the decision on whether to fast: (1) values and beliefs concerning Ramadan, (2) experiences and emotions concerning Ramadan, (3) the perception of illness, and (4) advice from health care professionals, imams and family. Many participants indicated fasting against medical advice and trusting their subjective assessments on whether they could fast. Moreover, three main stages in the decision-making process for eventually refraining from fasting were identified: (1) the stage where positive experiences with fasting dominate, (2) the stage where one encounters challenges but their determination to fast prevails and (3) the stage where one decides to refrain from fasting after experiencing too many physical difficulties with fasting.Muslims with diabetes experience autonomy in their decisions on Ramadan fasting. The decision to refrain from fasting often resulted from a difficult and dynamic decision-making process and was often made after participants reached their physical limits. These findings highlight the importance of not only shared decision-making to empower patients to make well-informed decisions on Ramadan fasting but also pre-Ramadan diabetes education to help people with diabetes have a safe Ramadan. During the Islamic month of Ramadan, Muslims abstain from food, drink, oral medications, sexual activity and smoking from dawn to dusk . RamadanRamadan fasting is associated with an increased risk of developing acute complications in some people with diabetes, such as hypoglycaemia, hyperglycaemia, ketoacidosis and dehydration , 5. A reSeveral international Ramadan-related diabetes management recommendations and guidelines have been published , 11\u201313 tFurthermore, a survey study conducted in France  and one We conducted an explorative qualitative study with a phenomenological approach using 15 focus groups to explore and understand the perspectives of Muslims with diabetes on Ramadan fasting. A phenomenological approach aims to understand the essence of social phenomena from those who perceived it . Its undThe study is part of the Diabetes and Ramadan project, an educational counselling program that aims to improve diabetes care of people with T2D of Moroccan and Turkish descent who observe Ramadan. Therefore, this study is primarily focused on Muslims with T2D. The Medical Ethics Review Committee of VU University Medical Center (Amsterdam UMC) confirmed that the Diabetes and Ramadan project does not fall under the scope of the Medical Research Act Involving Human Subjects; approval from the committee was therefore not required (2018\u2013165).Convenience sampling was used to recruit participants at local mosques, general practices and community centres , primariIn 2019, we chose to conduct additional focus groups to include a more diverse sample, specifically more participants of Turkish descent, as we had conducted only one Turkish focus group in 2018. In addition, to secure input from both groups who would have experienced Ramadan in the same seasonal period, we also conducted two more focus groups with participants of Moroccan descent in 2019.Participants were initially included if they had T2D and were of Moroccan or Turkish descent. However, a small number of Muslims with T1D, LADA or of other ethnicities (Sudanese and Egyptian) were also interested in participating in the focus groups. Therefore, after discussion within the research team, we decided to expand our inclusion criteria and include those participants. Nevertheless, this study primarily aimed at Muslims with T2D. The focus groups took place in two large cities (Amsterdam and Den Haag) and a medium-sized city (Leiden) in the Netherlands that all have a relatively large population of individuals with a migration background from May to October 2018 and June to July 2019. We developed a topic guide  inspiredAll transcripts were analyzed according to Braun and Clarke\u2019s six phases of reflexive thematic analysis \u201330. In tTo conduct focus groups in participants\u2019 native language and facilitate the free sharing of experiences and perspectives, we chose homogenous groups in terms of ethnicity and sex , 34. TheDuring the focus groups, it became clear that deciding to refrain from fasting was often difficult for many of the participants. We identified factors related to the decision on whether to fast. These factors are described as four themes: (1) values and beliefs concerning Ramadan, (2) experiences and emotions concerning Ramadan, (3) the perception of illness and (4) advice from HCPs, imams and family. These four themes are clarified in the following section and illustrated with quotations from the respondents, who are identified by their age, sex, ethnicity and whether they fasted during Ramadan for context.We classified three main stages in the decision-making process for eventually refraining from fasting : (1) theMany participants clarified that people with an illness who cannot fast are exempted from fasting, whereas those who would harm their health through fasting would be committing \"suicide\" . A few participants were concerned about committing a sin if they unjustly refrained from fasting. They mentioned feeling uncertain about whether their conditions were severe enough to exempt them from fasting. Some participants who believed themselves incapable of fasting reported having accepted the exemption from fasting; it was considered Allah\u2019s will that they could not fast anymore:My doctor tells me every Ramadan not to fast\u2026 both my general practitioner (GP) as well as my dietician\u2026 but I want to fast, just like every Muslim\u2026 you want to fulfil your religious obligations\u2026 but now that fasting is really not possible anymore\u2026 it is the will of Allah\u201d .\u201cParticipants also expressed their trust in Allah for the ability and strength to fast:This is why I say if a person has imaan [faith in Allah] here [points to her heart] and intends to do something, Allah will not disappoint them. When I perform my dawn prayer, I ask Allah, \u2018One thing I ask from you is to help me fast during Ramadan\u2019\u201d .\u201cSome participants wanted to receive the reward of fasting, while others mentioned that those who refrained from fasting were also rewarded for accepting the exemption.Participants believed that fasting was beneficial to their health, especially that fasting purifies and heals the body. Their ideas on the health benefits of fasting originate from various sources, including the Quran.The participants shared their experiences and emotions of physical, mental and social wellbeing regarding Ramadan. Participants in the first stage shared positive experiences with fasting, which were described as the absence of physical symptoms, feeling fit, losing weight, having stable blood glucose levels and performing daily activities. A few participants expressed their wish for Ramadan fasting throughout the year; they felt good during Ramadan, and it allowed them to structure their diet habits and prevent overeating:(\u2026) I feel good during Ramadan. As far as I am concerned, it should be all-year-round Ramadan; that is what I want. As soon as Ramadan is over, we just keep eating. Food also plays an important role\u2026 we fill the stomach until it explodes [laughs]\u201d .\u201cNot all participants had positive experiences. Some mentioned feeling tired, weak and dizzy or suffering from objectified hypo- or hyperglycaemia. Some even continued fasting despite developing symptoms of objectified hypo- or hyperglycaemia:I nearly died. My children were also very shocked\u2026 I had symptoms just before iftar; there was one hour left. I lay down and did nothing else. In this way, I managed to remain fasting\u2026 but I regret it. I regret it! When I checked my sugar level, it was low\u201d.\u201cI: \u201cWhat was your sugar level at the time?\u201dIt was 4 mmol/l (72 mg/dl)\u201d .\u201cIn addition, participants who did not fast mentioned enduring challenges during Ramadan due to changes in their diets, sleep patterns and physical activity. For instance, some reported eating less during the day since their family was fasting:No, I do not fast. It is the third or fourth year that I have not fasted\u2026 but I spend Ramadan as if I am fasting a little bit because my family is fasting\u2026our rhythm also gets confused [laughs]\u201d.\u201cI: \u201cDo you minimize something?\u201dYes; you reduce somewhat, and you sleep less\u2026 you eat less during the day\u2026 [therefore] you still get too low sugar levels\u201d .\u201cAlthough some were worried about the possible health risks of fasting at times, most participants who could fast expressed feeling happy, grateful, proud and calm during Ramadan. They also reported enjoying the \"Ramadan feeling\" with their family and friends.In contrast, some participants expressed feeling sad, guilty or ashamed when they were not fasting. Some found it difficult to be seen eating by their fasting relatives, and therefore avoided eating during the day. Some indicated not eating in front of fasting individuals out of respect:It has been two years now that I have not fasted, and I find it very difficult when my children watch me eat, even though they know that I am exempt\u2026 nevertheless, it is still difficult to have to eat. When I am eating, I feel\u2026 sometimes my wife asks me whether I would like tajine; I say no, I do not feel like it. I only drink water or a cup of tea, and the rest of the day, I am basically fasting\u201d .\u201cNot everyone felt ashamed. Some indicated that the decision on whether to fast was between themselves and Allah. Nevertheless, participants reported that questions from individuals outside the family concerning whether they were fasting were experienced as unwanted interference, and some felt inferior in such a situation.R1: \u201cNo but look\u2026 they know you are ill\u2026 but when they run into you or they call you [high-pitched degrading voice], \u2018Hey, As-salaam alaikum [peace be upon you]\u2026 are you fasting or not?\u2019\u2026 If I fast, am I fasting for you or someone else? Good heavens! I fast for myself\u2026 it is between me and Allah\u2026 they make it worse\u201d.R2: \u201cThey make you feel more ill\u2026\u201dR1: \u201cThey make you feel\u2026 that you are inferior\u2026 do you understand?\u2026 as if you are no longer a complete person, as if you are less\u201d .Participants shared their opinions on conditions that were perceived to be severe enough to refrain from fasting. Feeling healthy or young, using only oral medication and the absence of comorbidity or diabetes-related complications were viewed as conditions under which fasting was permissible:Our diabetes is not in an advanced stage; we can still fast, but if you are in an advanced stage or if you have pain, you cannot fast\u201d .\u201cParticipants also stated that individuals should not fast if they are physically incapable of fasting, experiencing severe physical symptoms, suffering from hypo- or hyperglycaemia, using insulin or in a life-threatening situation. Those who refrained from fasting added that being diagnosed with cancer and fearing kidney problems or other health problems due to fasting were reasons for not participating in Ramadan fasting:I have been fasting for 14 or 15 years [with diabetes], but I was still healthy then. However, now that my glucose drops in the afternoon, I cannot stand up; it happens in the evening when it is almost iftar\u2013about 2 to 3 hours before iftar\u2013then it drops, but I do not faint. Now, I cannot fast anymore. I cannot remain fasting all day long. I ask Allah to forgive us\u201d .\u201cOne participant on hormone therapy for breast cancer reported struggling during Ramadan fasting but continuing to fast out of fear of Allah. She felt uncertain about whether her condition was severe enough to refrain from fasting and whether she would unjustly refrain from fasting. This participant became emotional when her fellow focus group members expressed support:R1: \u201cI find it difficult; I get tired\u2013no, not tired\u2013I lose a lot of weight. After Ramadan, I feel that I have no energy anymore\u201d [sounds sad and ill].R2: \u201cYou feel empty\u201d.R1: \u201cYes\u201d.R3: \u201cHer energy is gone; her body is out of energy\u201d.R4: \u201cBut why do you fast? Do you want to kill yourself? Allah gave you\u2026\u201dR1: \u201cI am afraid\u2013afraid of Allah\u201d.R4: \u201cWhy are you afraid?\u201dR5: \u201cWorship does not include only fasting\u201d.R4: \u201cDarling, Allah knows what you are going through. You have grounds\u2026 but why\u2013why? You have your children\u2026 you should not cry\u201d.R1: [emotional] .Those who refrained from fasting indicated that they attempted to fast again before eventually deciding to refrain from fasting permanently:No, I do not fast; I am not able to fast anymore\u201d.\u201cI: \u201cHow many years have you refrained from fasting?\u201dSince I have [been diagnosed with] diabetes\u2026 Every Ramadan I try to fast again\u2026 but I am not able to fast anymore\u201d.\u201cI: \u201cSo, as you said earlier, it has been 12 years since you last fasted?\u201dYes, but every Ramadan, I attempt to fast\u2026 but I also have other illnesses, not only diabetes\u201d.\u201cI: \u201cNevertheless, you still try to fast?\u201dYes, I always want to try\u2026 but I do not succeed\u201d .\u201cMany participants mentioned that they discussed fasting with their HCPs before Ramadan and indicated that their HCPs pro-actively initiated the conversation on Ramadan fasting. Those who did not discuss fasting with their HCPs self-adjusted their medication during Ramadan. One participant felt that HCPs should initiate the conversation on Ramadan fasting. Different attitudes towards HCPs\u2019 advice were expressed: Some respected their HCPs\u2019 advice against fasting, even though they often disregarded that advice, whereas others felt that HCPs advised patients with diabetes against fasting too quickly. A few participants preferred advice from an Islamic HCP, while others mentioned that the HCP\u2019s religious or ethnic background was insignificant. A few even mentioned that they would disregard a Muslim HCP\u2019s advice against fasting.Most participants whose HCPs advised them not to fast reported choosing to fast against medical advice, implying that they were their own doctors and relied on their own judgement on whether they could fast:The doctor tells you to eat\u2013that you cannot fast. He tells you to eat [and] that fasting is dangerous for your kidneys and eyes. But I say that I am my own doctor. I will try it; if I can, then I will fast, and if not, then it is the will of Allah\u201d .\u201cMost informed their HCPs that they wanted to try to fast and would break their fast if they felt ill. Others indicated that they had not informed their HCPs of their decision to fast against medical advice:Yes, indeed, I also fast. My previous GP was a Dutch physician, and he always advised me not to fast. I told him I would not fast, but I fasted anyway [laughs]\u201d.\u201cI: \u201cWe indeed hear that more often in Turkish patients with a Dutch GP\u201d.Yes, it could be because he does not understand me. He has different values, norms and traditions\u201d .\u201cSome participants mentioned consulting an imam when their HCPs advised them against fasting. The imam told participants that they were their own doctors :The imam said that I am my own doctor and that I could first try to fast. If I cannot handle it then it is fine, but I at least have tried it that month\u2026 From that moment on, I have been fasting\u2026\" \"Conversely, others were advised in a consultation or lecture to follow their doctors\u2019 advice and not harm their health due to fasting. Two participants mentioned refraining from fasting after the imam disclosed that they would sin if they fasted against medical advice.(\u2026) I requested her (a relative in Morocco) to ask a sheikh (Islamic scholar) in Saudi Arabia for advice\u2026 that I would like to fast, I do not dare to eat, I have never eaten during Ramadan\u2026 She (a relative in Morocco) asked me to tell her exactly what the doctor told me. I told her that the doctor said: \u2018do not fast, the days are long and fasting with diabetes is a \u201csilent killer\u201d\u2018. She told the sheikh this and sent me the audio-recording (of his reply). He said: \u2018you disobey Allah\u2019, \u2018you disobey Allah\u2019, \u2018you disobey Allah\u2019, he said it three times. The next day\u2026 my daughter argued with me that I should not fast\u2026 I was scared when he said \u2018you disobey Allah\u2019 [laughs]. So I have eaten/ refrained from fasting [the last 9 days of Ramadan]\u201d .\u201cWhile others did not follow the imams advice to refrain from fasting.He (imam) said: \u2018You people have diabetes, you must refrain from fasting, Allah has exempted you from fasting. We cannot tell him that we have strong faith in Allah. We make our own decision. If we get tired, then we will break our fast\u201d .\u201cSome participants also reported that their families advised them not to fast and sometimes argued with them about their decision to fast against medical advice.I always have a discussion with my husband about this. He does not want me to fast because I get sick.\u201c(\u2026) I thought to myself: \u2018I want to fast, do not interfere.\u2019If it (fasting) gets too hard, I will not fast\u201d .This qualitative study explores Muslims\u2019 decision-making process for whether to fast during Ramadan. We found that Muslims with diabetes experience a high degree of autonomy in their decisions on Ramadan fasting. Personal values, beliefs, emotions, perceptions of illness and previous experiences with Ramadan fasting were found to influence the decision-making process. Moreover, deciding to refrain from fasting is often difficult, and in many cases, only made after participants have reached their physical limits during fasting. Three main stages in the decision-making process for eventually refraining from fasting were identified: (1) the stage where individuals have positive experiences with fasting and therefore choose to fast, (2) the stage where individuals endure challenges but remain determined to fast and (3) the stage where individuals refrain from fasting after reaching their limits. Our results suggest that the decision-making process where one stage follows another during the course of diabetes does not move in one direction; patients may attempt to start fasting again before deciding to refrain from fasting permanently. We discuss these three stages in more detail below.Participants in the first stage reported positive effects of fasting on physical and mental well-being, and some perceived fasting as beneficial to their overall health. The reported positive effects are consistent with those of previous qualitative studies on diabetes and Ramadan , 24, 35.iftar). How some of our participants coped with their physical symptoms may be dangerous since small signs of discomfort can quickly develop into a hypoglycaemic coma. These efforts to overcome adverse effects without breaking the fast may also imply inadequate knowledge of diabetes self-management, which raises concerns and underlines the importance of pre-Ramadan diabetes education. Previous studies have suggested that Muslims with diabetes have poor knowledge of diabetes self-management during Ramadan [Participants in the second stage disclosed enduring physical challenges during fasting, such as symptoms of potential hypoglycaemia or objectified hypo- or hyperglycaemia. Nevertheless, most continued their fast as they wanted to try to fast for as long as possible. This finding is consistent with two observational studies, one in France  and one  Ramadan , 40. Par Ramadan , 24.Finally, participants in the third stage decided to refrain from fasting, mostly after reaching their physical limits, such as recurrent hypoglycaemia. The participants\u2019 search for their physical limits before refraining from fasting might be due to their uncertainty about whether their conditions are \"serious\" enough to exempt them from fasting. The Quran states that people with an illness are exempt from fasting but does not specify which illnesses are exempted . IslamicIn contrast to previous studies \u201321, 41, Consistent with previous studies, most participants fasted against medical advice , 17; theInterestingly, many participants reported fasting against medical advice without experiencing adverse effects. Several observational studies \u201347 have Our findings suggest that HCPs\u2019 and imams\u2019 advice on whether or not to fast sometimes differed. Some participants reported that imams advised them to follow their doctors\u2019 advice, while others were advised to assess whether they could fast themselves. As a few participants indicated, it would help in the decision-making process if advice from HCPs and religious leaders were consistent. Furthermore, participants who did not fast reported adverse emotions, such as sadness. Imams could reflect on this phenomenon and advocate the religious benefits of accepting the exemption, which might be helpful for some individuals who find it difficult to accept not participating in the Ramadan fast. Being aware of these emotions concerning not being able to fast and reflecting on them might help HCPs discuss Ramadan fasting with their patients.To the best of our knowledge, this is the first qualitative study that explores the decision-making process of Muslims with diabetes for Ramadan fasting. Here we discuss the trustworthiness of this qualitative study based on its credibility, dependability and transferability. First, we addressed its credibility by ensuring that our participants could express themselves and elaborate on their experiences freely by conducting the focus groups in Moroccan-Arabic, Berber (Tamazight) or Turkish. Although the moderators did their best to ensure that all participants felt free to share their perspectives and experiences, for example, by explicitly stating that there were no wrong answers and the importance of respecting each other\u2019s point of view. We cannot exclude that the more introverted participants have shied away from expressing their true opinions and therefore potentially leading to social-desirability bias. Another strength is that the focus groups were accessible since they were conducted in a familiar environment for the participants, such as a local mosque, and separate focus groups for both sexes and ethnic groups. The homogeneity of the focus groups could have resulted in the rich data as it empowers participants to express themselves freely , 34. It Muslims with diabetes feel autonomous in their decision to try to fast. This personal decision is primarily based on their beliefs, experiences and perception of illness. Consistent with previous studies , 23, we This study builds on previous research on the perspectives and experiences of Muslims with diabetes regarding Ramadan. We found that Ramadan fasting is a personal choice, and Muslims with diabetes experience a high degree of autonomy in their decisions on whether to fast. This personal decision is based on participants\u2019 beliefs, values, emotions, perceptions of illness and previous experiences with fasting. The decision to refrain from fasting is often a difficult one, mostly made after individuals have reached their physical limits. As to recommendations for clinical practice, our study advocates shared decision-making when counselling patients on Ramadan fasting. It is also crucial to educate fasting and non-fasting Muslims with diabetes on self-management during Ramadan.S1 Table(TIF)Click here for additional data file."}
+{"text": "As the kidney is involved in volume and blood pressure control through sodium handling, we set out to determine the impact of a low sodium diet on these parameters in WT and Nox4-/- mice. Nox4 expression in the murine kidney was restricted to the proximal tubule. Nevertheless, low-sodium-induced weight loss and sodium sparing function was similar in WT and Nox4-/- mice, disputing an important function of renal Nox4 in sodium handling. In contrast, a low sodium diet resulted in a reduction in systolic blood pressure in Nox4-/- as compared to WT mice. This was associated with a selectively lower pressure to heart-rate ratio, as well as heart to body weight ratio. In general, a low sodium diet leads to activation of sympathetic tone and the renin angiotensin system, which subsequently increases peripheral resistance. Our observations suggest that the control by this system is attenuated in Nox4-/- mice, resulting in lower blood pressure in response to low sodium.The NADPH oxidase Nox4 is a hydrogen peroxide (H Reactive oxygen species (ROS) and oxidative stress have been implicated in kidney disease . Numerou2O2, due to its ability to trap O2\u2022- in a pocket of its E-loop [2O2 is a relatively long-lived ROS, which can exert a signaling function through direct reaction with cysteines [The individual cells of the kidney exhibit a cell-specific expression pattern of the different Nox homologues, as well as a differential response to Nox enzyme-inducing and -activating stimuli. In models of angiotensin II infusion and a high salt diet, the renal expression of Nox2 and its accessory subunits is increased  and the s E-loop . H2O2 isysteines  and the ysteines . Despiteysteines ,15,16, tysteines ,19,20,21ysteines . In contysteines . Given t+) was obtained from Altromin (#C1036). Urine was collected in metabolic cages, and blood was collected in lithium heparin tubes. Plasma was obtained by 2000\u00d7 g centrifugation for 5 min at 4 \u00b0C.Tamoxifen-inducible Nox4-/- mice (Nox4flox/flox-ERT2-Cre+/0 mice) were generated by crossing Nox4flox/flox mice (backcrossed more than 10 generations into C57/Bl6J) with CreERT2+/0 mice, as described previously . KnockouAll experiments performed with animals were in accordance with German animal protection laws and were carried out after approval by the local authorities under the number FU1089. Animals were housed in groups with free access to chow and water in a specified pathogen-free facility with a 12/12 h day/night cycle. Given the impact of gender on ROS production, only male animals older than 8 weeks were used in this study.2O and incubated with Nox4 probe for 2 h at 40 \u00b0C. Peptidylprolyl isomerase and B. subtilis dihydrodipicolinate reductase were used as a positive and negative control, respectively. Following probe hybridization, the RNAscope 2.0 HD Detection Kit\u2013Brown (for detection of onlye RNA) or \u2013Red (to combine with immunofluorescence) was applied for visualizing hybridization signals.RNAscope in situ hybridization and protein detection by immunofluorescence were performed on paraffin embedded renal sections. Adult murine kidneys were fixed in 4% paraformaldehyde at room temperature overnight, dehydrated, then embedded in paraffin and cut into 5 \u00b5m thick sections. RNAscope was performed according to the manufacturer\u2019s protocol . Briefly, tissue sections were heated for 1 h at 60 \u00b0C and, subsequently, deparaffinized in xylene (2 \u00d7 5 min), isopropanol (2 \u00d7 5 min), ethanol (2 \u00d7 5 min) and then air-dried for 5 min. Samples were pretreated with hydrogen peroxide for 10 min and then with a target retrieval solution followed by Protease Plus reagent for 30 min at 40 \u00b0C. After Protease Plus, slides were washed in ddH3 solution for 1 h at 37 \u00b0C. After washing in PBS, probes were blocked a second time using a milk solution for 20 min at room temperature, and then incubated with primary antibodies (1:500 diluted in Roti) overnight at 4 \u00b0C. The primary antibodies used for immunofluorescence were anti-aquaporin 2 (AQP2)  and anti-megalin (Meg) . The slides were then washed in PBS and sections were incubated with the secondary antibody for 2 h at room temperature. Secondary antibody (1:500 diluted in Roti) was Alexa Fluor 488 goat anti-rabbit IgG . Slides were washed in PBS then treated with DAPI (1:500 diluted in PBS) and finally covered using Mounting Medium . RNAscope in situ hybridization and immunofluorescence images were captured on a confocal microscope .For immunofluorescence staining, slides were washed in PBS after performing RNAscope. Subsequently, specimens were blocked with a NaN2, 1.8 CaCl2, 5 glucose, 0.36 NaH2PO4, 10 HEPES) was added to 1 mL/mg tissue. The organs were then minced with a scalpel and H2O2-derived fluorescence was measured in the supernatant of 100 mg tissue in the presence of Amplex Red  and horseradish peroxidase (2 U/mL). Fluorescence was determined in the supernatant at 540 nm/580 nm excitation/emission. H2O2 concentration was estimated from a standard curve.The kidneys were freshly isolated, separated into cortex and medulla, and HT buffer . Measurements over 5 days were averaged per mouse.+, Cl\u2212, K+) in plasma and urine were analyzed with an AU480 clinical chemistry analyzer  with the integrated ion-selective electrodes unit (ISE) and reagent kits provided by Beckman Coulter, according to previous publications [Electrolytes  by plasma concentration.Unless otherwise indicated, data are given as means \u00b1 standard error of mean (SEM). Calculations were performed with Prism 5.0 or BiAS.10.12. The latter was also used to test for normal distribution and similarity of variance. In the case of multiple testing, Bonferroni correction was applied. For multiple group comparisons, ANOVA variance testing followed by post hoc testing was performed. Individual statistics of unpaired samples was performed by a t-test, and if found to be not normal, distributed by the Mann\u2013Whitney test. A pvalue of <0.05 was considered as significant. Unless otherwise indicated, n indicates the number of individual experiments or animals.\u00ae) to visualize Nox4 mRNA, which has been reported to strongly correlate with the protein level of Nox4 [\u00ae with immunofluorescence using megalin and aquaporin-2 as markers for proximal tubule and collecting duct cells, respectively. Nox4 staining was restricted to megalin-positive cells  release of H2O2, demonstrating that Nox4 is the source of H2O2 in the murine renal cortex (Freshly dissected kidneys from WT and Nox4-/- mice were separated into cortex and medulla and minced in HT (Hepes Tyrode) buffer and incubated with Amplex redl cortex C.The proximal tubule is the renal site of mass absorption. More than 65% of the water, a high proportion of bicarbonate and phosphate, and basically all amino acids, glucose, as well as numerous vitamins and trace elements, are recycled at this site. Almost all transport processes in the proximal tubule are coupled, directly or indirectly, to sodium reabsorption. The fine-tuning of sodium excretion occurs in the late distal tubule and collecting duct. A previous publication on the role of Nox4 in salt-induced hypertension in Dahl rats  suggestsTo study this aspect, mice were challenged with a low sodium diet: body weight, blood pressure and sodium excretion were first studied on regular chow (0.2 g/kg sodium), and subsequently on a low sodium diet (0.01 g/kg) applied for up to three weeks with prior tamoxifen-mediated knockout of Nox4 A.Under normal chow, the blood pressure and heart rate of WT and Nox4-/- mice were similar. A reduction in sodium intake slightly lowered systolic blood pressure in both strains; this effect was more pronounced in Nox4-/- than in WT mice (only significant at week 1 of low Na+). There was also a trend towards a higher heart rate in Nox4-/- vs. WT mice B\u2013D. The A reduction in food sodium content resulted in a loss of body weight of approx. 1 g within 2 weeks. Although this weight loss was, on average, slightly greater for Nox4-/- mice at any timepoint, the difference did not reach the level of significance A. As detIn order to directly determine the role of Nox4 for renal electrolyte handling, plasma and urine electrolytes were measured and their renal excretion and clearance was determined . Sodium In this study, we report that a low sodium diet results in an acute reduction in systolic blood pressure and a prolonged reduction in peripheral resistance in Nox4-/- mice. Despite the high expression of Nox4 in the kidney, this effect was unrelated to salt and water intake and renal sodium handling. Sodium restriction resulted in weight loss and renal sodium sparing, and this effect was similar between WT and Nox4-/- mice. Collectively, these data support the previous report of Nox4 in renal blood pressure control  but exclDidelphis virginiana) is a broadly used model to study ion transport and membrane trafficking mechanisms in the proximal tubule. Transcriptomics of these cells, compared with mammalian proximal tubule cells, however, reveal that Nox4 expression is also lost in this cultured cell line [In the present study, we observe a high expression of Nox4 in the proximal renal tubule. Our data are in line with the single-cell sequencing atlas of the murine kidney , where Nell line .Therefore, the search for a function of Nox4 has to rely on in vivo data. Given that the transport processes in the proximal tubule are largely sodium-coupled, studying sodium handling and its indirect consequence, plasma volume and blood pressure, can be seen as first approaches to dissect the function of Nox4 in the kidney. The proximal tubule reabsorbs two-thirds of filtered Na+  and, conInterestingly, cerebral knockdown of Nox4 resulted in attenuated sympathico-excitation in response to cardiac damage . Whether2O2 directly into the renal medulla increases mean arterial pressure [+ homeostasis because Na+ excretion and clearance were similar in WT and Nox4-/- mice. On the other hand, the effects might have been so subtle and transient that our study was not sensitive enough to detect them.Is there any evidence for a direct function of reactive oxygen species for renal hypertension? Superoxide anions lead to reduced medullary blood flow and increased sodium retention and, thus, hypertension , as showpressure . Nox4 hapressure . Our resThe current study has several limitations. Blood pressure was measured by tail cuff technology, which is less accurate than telemetry. Moreover, we only estimated cardiac output and peripheral resistance from the blood pressure to heart rate ratio. A true determination of cardiac output and peripheral resistance would have required indicator injection dilution methodology. Moreover, metabolic cages impose a considerable amount of stress on mice. Food and fluid intake in the cages is low, which is also documented in the present study by the substantial weight loss. A possible alternative would have been clearance measurements using radioactive isotopes, but this technology was not available to us.The present study demonstrates that the deletion of Nox4 potentiates lower arterial blood pressure in response to a low sodium diet in mice. The present data may suggest that this is a consequence of a change in peripheral resistance, rather than altered renal sodium handling in Nox4-/- mice."}
+{"text": "Frontotemporal dementia (FTD), hallmarked by antero-temporal degeneration in the human brain, is the second most common early onset dementia. FTD is a diverse disease with three main clinical presentations, four different identified proteinopathies and many disease-associated genes. The exact pathophysiology of FTD remains to be elucidated. One common characteristic all forms of FTD share is the dysregulation of glucose metabolism in patients\u2019 brains. The brain consumes around 20% of the body\u2019s energy supply and predominantly utilizes glucose as a fuel. Glucose metabolism dysregulation could therefore be extremely detrimental for neuronal health. Research into the association between glucose metabolism and dementias has recently gained interest in Alzheimer\u2019s disease. FTD also presents with glucose metabolism dysregulation, however, this remains largely an unexplored area. A better understanding of the link between FTD and glucose metabolism may yield further insight into FTD pathophysiology and aid the development of novel therapeutics. Here we review our current understanding of FTD and glucose metabolism in the brain and discuss the evidence of impaired glucose metabolism in FTD. Lastly, we review research potentially suggesting a causal relationship between FTD proteinopathies and impaired glucose metabolism in FTD. Frontotemporal dementia (FTD), indicated by neurodegeneration in the antero-temporal lobes of the brain, is the second most common early onset dementia affecting \u223c3/100,000 individuals under the age of 65 . ApproxiClinically there are two major forms of FTD, termed behavioral variant (bhv)-FTD and primary progressive aphasia (PPA)-FTD, which is further divided into semantic (sv)-FTD and non-fluent (nfv)-FTD . These tFTD is pathogenically diverse; different patients diagnosed with the same clinical form of FTD may present with distinct pathologies , charactTau, TDP-43 and FUS are all highly abundant in brains, comprising intrinsically disordered domains, and the latter two perform similar functions, however, it\u2019s not clear whether they all converge on a single pathogenic cascade.TARDP encodes TDP-43, a heterogeneous nuclear ribonucleoprotein (hnRNP), that possesses the ability to shuttle in-between the nucleus and the cytoplasm but primarily localizes to the nucleus . It bindPGRN) are found in 5\u201310% of all FTD cases, and PGRN mRNA depletion causes TDP-43 cleavage and subsequent inclusion formation (VCP) have been associated with FTD-TDP-43 pathology with VCP mutants preventing autophagosome maturation, which may lead to the increased inclusion of TDP-43 by preventing its degradation  in neurons and glia (Tauopathy)  have been identified  . Tau moduopathy) . Tau is uopathy) . Most FTf cases, . Over 50entified , with diocessing . Others,ocessing . Howeverocessing .CHMP2B; encoding a protein involved in protein sorting and trafficking), observed in familial FTD cases in Denmark and Belgium, leads to FTD-UPS   family. GLUT1 is responsible for glucose transport across the blood brain barrier and into glia, whilst GLUT3 mediates neuronal glucose uptake . Glucose2) . These prylation .via an astrocyte-neuron lactate shuttle (ANLS) (The brain\u2019s glucose metabolism is coupled between neurons and glia e (ANLS) . Astrocye (ANLS) . High exe (ANLS) . Furthere (ANLS) . Additioe (ANLS) .Neurons are hypothesized to preferentially metabolize glucose through the PPP, to maintain their redox homeostasis and prevent oxidative stress . NeuronsDrosophila melanogaster astrocytes\u2019 glycolytic enzymes  leads to neurodegeneration  in positron emission tomography (PET); a process termed FDG-PET . FDG-PETBrain glucose hypometabolism is observed across the FTD spectrum . In bhv-PGRN mutation carriers, displayed large heterogeneity in their glucose hypometabolism patterns in studies with 9\u201310 patients, aligning with the fact that PGRN mutations cause FTD across the clinical spectrum   .Collectively, these studies identify significant glucose hypometabolism in FTD patients, with identifiable patterns across subtypes indicating the potential of FDG-PET scans in aiding FTD diagnostics. Indeed, use of FDG-PET together with magnetic resonance imaging (MRI) scans can increase the ability to discriminate between Alzheimer\u2019s disease and FTD cases . These fn = 7)  . Familian = 7) . Studiesectively . These fMAPT carriers had intermediate levels of brain atrophy compared to symptomatic carriers but comparable glucose hypometabolism  to model disease. As these mutations lead to the formation of aggregates similar to those found in FTD patients, these studies are likely also relevant to FTD. However, few studies directly focus on glucose metabolism in TDP-43 proteinopathy .Many studies also focus on understanding TDP-43\u2019s physiological function using loss of function studies, which have highlighted the role of TDP-43 in glucose metabolism. Downregulation of TDP-43 in a variety of human cell lines leads to down-regulation of key glycolytic pathway components. TDP-43 depletion in SH-SY5Y cells leads to significant drops in expression of glycogen phosphorylase , and hexokinase-1 (which performs the first step in glycolysis) . In hepavia the PPP, which could affect redox homeostasis  of neurons and astrocytes derived from patients with FTD-TDP-43. Proteomic studies together with studies using labeled glucose could lead to further understanding of how the ANLS is modulated and how this has a pathogenic role.via a non-canonical form of translation called RAN (Repeat Associated Non-atg mediated translation) translation, occurring in all open reading frames, to generate five different dipeptide repeat (DPR) proteins, which also accumulate as aggregates in patients\u2019 brains (C9orf72 hexanucleotide repeat expansion (C9) is the most common genetic cause of sporadic and familial FTD and ALS in populations of European ancestry. The expansion is located in the first intron of the gene C9orf72, the general population has less than 30 copies of the repeat which, in patients, can expand to thousands of copies . C9 repe\u2019 brains . The pat\u2019 brains , and oth\u2019 brains . There i\u2019 brains .Increasingly, there is evidence that C9 expansion might also lead to alterations in glucose metabolism, independently of downstream TDP-43 aggregation, as most of the models used do not present with TDP-43 aggregates. Gene level network analysis of the transcriptome of MNs derived from C9 carrying ALS patients revealed that glucose metabolism associated genes\u2019 expression is almost halved . MoreoveTau proteinopathy is perhaps the most extensively studied among the FTD proteinopathies, primarily because of the link between hyperphosphorylated Tau and Alzheimer\u2019s disease (AD). Many AD studies for example utilize triple transgenic mice (3xTg) which, among two AD causing mutations in Amyloid Precursor Protein (APP) and Presenilin-1 (PSEN1) also incorporate the P301L Tau mutation, which is causal for familial cases of FTD Tauopathy . This me3xTg mouse-derived astrocytes show reduced glycolytic activity and glucose hypometabolism . This isMAPT mutations have a more significant reduction in ATP production compared to controls when treated with the glycolysis inhibitor iodoacetic acid, also suggesting impaired glucose metabolism  leading to higher membrane potentials and reduced complex I function  and increased Ser636 IRS-1 phosphorylation  leading to insulin resistance . Tau alsvia GSK-3\u03b2 (a Tau phosphorylating kinase)  experienced PP2A downregulation and Tau hyperphosphorylation\u2014a process reversed by insulin injections . In acco kinase) . A reduc kinase) , suggestvia the proposed ANLS  . NF-\u03baB ovia an interaction with HSP60 and, once there, FUS induces the mitochondrial unfolded protein response, promoting HSP60 expression in a potential negative feedback loop , can modulate glucose metabolism in the absence of downstream proteinopathy, and a reduction of insulin signalling, can exacerbate Tauopathies, suggesting glucose metabolism dysregulation may act to exacerbate proteinopathy as well as downstream toxicity.via a gain of toxicity or loss of function model, leads to divergent research models with studies using either overexpression or knockout models, sometimes resulting in opposing conclusions. Research also uses diverse organisms to model FTD proteinopathies ranging from insects to mammalian animal models or cell cultures, at different developmental stages, which are known to affect metabolism. Identifying the most relevant disease model can be challenging.Much, however, remains unknown. The lack of consensus on whether FTD proteinopathies act To further understand how FTD affects glucose metabolism, and in particular the interplay between astrocytes and neurons, research would greatly benefit from co-culture experiments with patient-derived cells.However, the finding that promoting glucose metabolism in FUS and TDP-43 proteinopathies ameliorates disease phenotypes indicates that glucose metabolism could be a driver of FTD pathophysiology and therefore could become a promising therapeutic target.LG and TN wrote the manuscript. Both 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": "The larger PAA thickness obtained for the same charge density strongly suggests that PAA formation efficiency increases in the 1:3 v/v EtOH:H2O mixture.Etidronic acid, used in aluminum anodization, has a great potential for the fabrication of porous anodic alumina (PAA) with large cell sizes (>540 nm). PAAs are particularly suited to applications in optics and photonics where large-scale periodicity corresponding to visible or infrared light is needed. Additionally, such PAAs should be characterized by long-range pore ordering. However, to obtain regular pore arrangement in an etidronic electrolyte, the anodization should be performed at high electric fields using relatively high temperatures, which makes the process challenging in terms of its stability. To stabilize the process, the electrolyte can be modified with ethanol. In this work, the impact of ethanol on pore geometry and a level of pore ordering is systematically analyzed. It is shown that the additive tends to reduce pore ordering. Moreover, by changing the anodizing temperature and the amount of ethanol, it is possible to tune the porosity of the PAA template. At 20 \u00b0C, porosity drops from 14% in PAA grown in a pure water-based electrolyte to ca. 8% in PAA fabricated in the 1: PAA templates with highly regular pores arranged into a close-packed hexagonal (hcp) structure, and with Dc (cell size) that correspond to visible-IR light wavelengths, are required for applications in fields such as photonics or plasmonics [a = \u2212log10Ka) of a given acid [a value . On the other hand, the applied voltage determines the interpore distance (Dc) in PAAs. In conventional anodization conditions, the Dc is linearly proportional to the anodizing potential (Dc = kU), with proportionality constants k ~ 2.5 nm/V for acidic electrolytes [c > 380 nm\u2014an approximate lower limit of visible spectral range), anodization must by performed under high voltages using relatively weak acids. High-voltage anodization , in turn, is usually accompanied by intensive Joule heat evolution and large stress generation during the pore formation at the metal\u2013oxide interface. As an effect, it is difficult to maintain the process stability. Moreover, for applications in photonics or optics, other effects frequently encountered during anodization at high E, such as plastic deformation of aluminum substrate or non-uniform PAA thickness [Porous anodic alumina (PAA) is a well-known template for the synthesis of different nanostructures  for a vaasmonics ,11. Howeven acid ,13. Thertrolytes , or k ~ trolytes . Consequhickness ,17, needc. Anodization in etidronic acid was first demonstrated by Kikuchi et al. [a1 ~ 1.4 [c in the range 530\u2013670 nm was fabricated in a 0.3 M etidronic acid solution and various anodizing temperatures (20\u201360 \u00b0C). It was shown that the highest  anodizing voltage for the steady-state growth of anodic porous alumina decreased as the electrolyte temperature increased. Furthermore, the electrolyte concentration was explored in the range of 0.2\u20134.2 M to establish the self-ordering conditions, including the range of anodization voltage and temperature, for a given acid concentration [Etidronic acid offers a possibility of obtaining highly regular PAA with large Di et al. ,19,20. Aa1 ~ 1.4 ), the apa1 ~ 1.4 . In ordentration ,23. To s2O3 to the consumed Al) under a constant charge [c and pore arrangement [c and lower pore ordering was formed. Therefore, using ethanol as a stabilizer must be associated with detailed knowledge on other possible changes that can be induced by this modifier when introduced to the electrolyte. In particular, fully controllable electrochemical conditions to obtain PAA with precisely predicted cell size, porosity, and pore ordering are very desirable for designing 1D photonic crystals based on the PAA [Another way to prevent the burning phenomena is to use electrolyte modifiers, such as ethanol. Ethanol was used a cooling agent to prevent the catastrophic flow of current under high current anodization conditions and to enable the process to proceed at subzero temperatures ,24,25. Lt charge ,30,31. Tt charge ,30. In ct charge  have shot charge . Moreoveangement . For the the PAA ,36.v/v EtOH:H2O), and ethanol-modified solutions with 1:9 and 1:3 v/v EtOH:H2O proportion. The second anodization was conducted at different temperatures in the range of 0\u201320 \u00b0C. It is shown that the porosity of PAA increases with temperature; however, the dissolution strength of the electrolyte is weakened in the 1:3 v/v EtOH:H2O solution. Moreover, the PAA thickness vs. charge-density dependence suggests that the alumina formation efficiency starts to be larger above 10 \u00b0C for the latter electrolyte as compared to the other two electrolytes. The addition of ethanol stabilizes the PAA formation process, which is visible in the lower currents evolving during the anodization. However, a quantitative analysis of pore arrangement demonstrates that ethanol slightly impacts hcp pore ordering. The results presented in this work may be very useful for designing PAA with a large cell size (Dc > 540 nm) and good pore regularity for optical or photonic applications.In this work, we systematically analyzed the influence of ethanol (EtOH) on PAA growth in etidronic acid solution. Three different electrolytes were studied: pure water-based etidronic solution  were insulated at the back and the edges with acid-resistant tape, and served as the anode. A Pt grid (rectangular shape) was used as a cathode and the distance between both electrodes was kept constant (ca. 2 cm). Pt/Al electrode area ratio was about 25. Etidronic acid 60% aqueous solution was purchased from Merck KGaA . A large 1L electrochemical cell and cooling bath thermostat  were employed in the anodizing process. An adjustable DC power supply with voltage range of 0\u2013300 V and current range of 0\u20135 A, purchased from NDN, model GEN750_1500 TDK Lambda, TDK Co., Tokyo, Japan, was used to control the applied voltage. RIGOL DM 3058E digital multimeter  was used to measure and transfer the registered current to a computer. A scheme of the experimental setup is shown in High-purity Al foil  with a thickness of about 0.25 mm was cut into rectangular specimens (2 \u00d7 1 cm). Before the anodization process, the Al foils were degreased in acetone and ethanol and subsequently electropolished in a 1:4 mixture of 60% HClOv/v EtOH:H2O, 1:9 v/v EtOH:H2O, and 1:3 v/v EtOH:H2O.In anodization, 0.3 M etidronic acid solution was used. In the first anodization step, the voltage (U) of 80 V was applied at first, which was maintained for 3 min and then stepwise raised to 210 V. Next, a constant anodization at 210 V was performed for 3 h. A temperature of 38 \u00b0C was used in the first step. After the process, alumina was chemically removed using a mixture of 6 wt% phosphoric acid and 1.8 wt% chromic acid at 60 \u00b0C for 120 min. In the second step of anodization, temperature was varied between 0 and 20 \u00b0C. Moreover, in order to keep the starting voltage close to the target one, anodization was started at U = 150 V, which was at once linearly increased to 210 V. After reaching 210 V, anodization was continued for 3 h. The duration of the process was always the same in order to eliminate the effect of oxidation time on the PAA thickness. Three etidronic acid solutions were studied: 0:1 c) of the fabricated samples, Fast Fourier transforms (FFTs) were generated based on three SEM images taken at the same magnification for every sample, and were further used in calculations with WSxM software (version 5.0) [c was estimated as an inverse of the FFT\u2019s radial average abscissa from three FE-SEM images for each sample. Pore diameter (Dp) was calculated with the use of NIS-Elements image analysis software . Porosity (P) of the PAA membranes was calculated using the following formula: P = Morphological analysis was performed using a field-emission scanning electron microscope FE-SEM . To obtain the interpore distance (Dion 5.0) . The ave3(DpDc)2 . The porConductivity of the electrolytes was measured in a thermostatic cell with Elmetron CC 505 conductivity meter, Zabrze, Poland. At the end, average values from three measurements were given.Current density (j) vs. time (t) transients recorded during first anodization in 0.3 M etidronic acid solution, without and with different amounts of ethanol (EtOH), are shown in v/v EtOH:H2O). The addition of ethanol makes the arrangement of dimples slightly less ordered. In the sample produced in the 1:3 v/v EtOH:H2O solution, the domains with close-packed hexagonal pores were visibly smaller than those formed in the electrolyte without ethanol. At the same time, larger areas of pores with an irregular shape  that are not surrounded by exactly six near-neighbors were observed. The deterioration of pore arrangement upon addition of the additive was also observed during hard anodization in an oxalic electrolyte [In ctrolyte . The effctrolyte .v/v EtOH:H2O) possesses the highest level of hexagonal pore ordering (85% of relative frequency of pores with 6 nearest neighbors).The ordering quality of porous patterns in anodic oxide samples prepared in different solutions was also quantitatively determined by a method adapted from Hillebrand et al. . In the 3+, O2\u2212) and thus the steady currents [The j(t) transients recorded during II anodization at temperature (T) ranging between 0 and 20 \u00b0C are gathered in currents ,27. Morecurrents ,25. Lowev/v EtOH:H2O solution at 0 \u00b0C, the rise of current after the barrier layer formation is hardly visible, indicating early stages of pore nucleation and development. Additionally, because of the lower dielectric constant of ethanol compared to that of water, the dielectric constant of an ethanol\u2013water mixture decreases with the addition of ethanol . The lowv/v EtOH:H2O at 0 \u00b0C is characterized by narrow slit-like pores, which evidences the beginning of the pore development process and corresponds well with the j(t) behavior recorded for this PAA (c), pore diameter (Dp), and porosity of the PAA templates (except for the PAA produced in 1:3 v/v EtOH:H2O at 0 \u00b0C) were determined (p(T) dependence for PAA obtained in a pure water-based electrolyte was also studied in 0.3 M oxalic electrolyte [p in the etidronic electrolyte was more gentle (from around 150 to 220 nm in the same temperature range). The slightly less-pronounced effect of temperature on pore diameter in the etidronic electrolyte may be due to a slightly higher dissociation constant of etidronic acid (pKa1 = 1.35) compared to that of oxalic acid (pKa1 = 1.25) [v/v EtOH:H2O composition. However, the temperature effect is not so visible in the PAA produced in the 1:3 v/v EtOH:H2O mixture. It can be observed that at a temperature higher than 10 \u00b0C, ethanol demonstrates a suppression effect for the chemical dissolution of the oxide layer, while in the PAAs produced in the 1:9 v/v EtOH:H2O and 0:1 v/v EtOH:H2O solutions, porosity starts to sharply rise above 10 \u00b0C, and in the PAAs formed in 1:3 v/v EtOH:H2O solution the porosity remains more or less the same. Smaller porosity is directly related to a larger portion of Al3+ cations that is used to form Al2O3 instead of being rejected to electrolyte [In this PAA c. Based termined a\u2013c. The ctrolyte . In the  = 1.25) . Porositctrolyte . Therefov/v EtOH:H2O), the increase was insignificant (approx. 2%), whereas in the samples produced in the electrolytes with ethanol the increase was much greater (approx. 10%).Furthermore, the difference in pore regularity induced by ethanol seems to be weakened after the II anodization: the level of pore ordering looks comparable regardless of temperature applied . Pore-orv/v EtOH:H2O and 1:9 v/v EtOH:H2O etidronic electrolytes, the oxide thickness is exactly the same for the whole charge density range, whereas in the 1:3 v/v EtOH:H2O solution the PAA thickness increases above a certain charge density that corresponds to anodizing temperature of 10 \u00b0C. This result, together with the observed smaller porosity in the PAAs produced in the 1:3 v/v EtOH:H2O solution . In other words, upon addition of a certain amount of ethanol (1:3 v/v EtOH:H2O or more), the oxide growth at the aluminum/oxide interface occurs faster than the oxide dissolution at the oxide/electrolyte interface.In order to further analyze the efficiency of PAA formation in the etidronic solutions of different compositions, charge density, as well as the thickness of PAA formed in each process, were determined. In solution c, strong3+ motion and inward motion of various anions: O2\u2212, OH\u2212, electrolyte-anion contaminant species) and electronic parts, resulting from complex reactions to form anodic oxide driven by the external electric field [The current density is strictly related to the ion mobility in a solution. The conductivity (\u03c3) of the electrolytes measured in different temperature is shown in ic field . These ric field  and thusic field , furtheric field . Moreoveic field ,49. Thisv/v EtOH:H2O). This observation, together with the lower porosity determined for the corresponding PAA samples, strongly suggests that the PAA formation efficiency increased in the 1:3 v/v EtOH:H2O electrolyte above 10 \u00b0C. The higher alumina growth efficiency could be related to a suppression effect for the chemical dissolution of the oxide layer demonstrated by ethanol. The results show that depending on the amount of ethanol added to the electrolyte and anodizing temperature, the porosity of PAAs can be adjusted within a certain range of variability. At 20 \u00b0C, porosity dropped from about 14% in the pure water-based etidronic electrolyte to ca. 8% in the 1:3 v/v EtOH:H2O etidronic solution. Future studies will involve optimizing the process at higher voltages (even up to 300 V) using ethanol or other modifiers in order to increase the cell size while maintaining a high level of pore ordering. The work can be very helpful in designing photonic materials based on PAAs with large cell sizes (Dc > 540 nm). The production of porous anodic alumina (PAA) with large cell sizes that correspond to visible or infrared wavelengths remains a challenge to date because of the necessity of using high voltages, which make the process prone to burning and breakdown phenomena. Additionally, for applications in photonics or optics, PAAs should be characterized by high pore regularity. Self-ordered PAAs with a large cell size (>540 nm) were fabricated in etidronic acid solutions. To stabilize the process, different amounts of ethanol were added to the electrolyte. It is known that the additive can change the mechanism of pore formation and modify the PAA growth. Therefore, for the first time, the impact of ethanol on pore geometry and level of pore ordering was systematically analyzed in this study. It is shown using a quantitative method that the additive tends to lower pore ordering in PAA. The effect might be linked with lower electric field strength (lower current densities) and, consequently, lower stresses exerted on the neighboring pore, which, as a result, start to lose their ordering power. The second anodization was conducted at various temperatures ranging from 0 to 20 \u00b0C. The PAA growth rate increases exponentially with temperature and the PAA thickness is proportional to the charge passing during the process. It was, however, revealed that proportionality between PAA thickness and charge density was slightly different for the samples produced in the electrolyte with the largest amount of ethanol (1:3"}
+{"text": "Systemische Entz\u00fcndungsprozesse gehen mit unspezifischen k\u00f6rperlichen und psychischen Krankheitssymptomen einher, darunter Schmerz und affektbezogene Symptome. Diese immunvermittelten Symptome (\u201eSickness Behavior\u201c) beruhen auf der zentralnerv\u00f6sen Wirkung von Immunbotenstoffen wie proinflammatorischen Zytokinen und vermitteln bei akuten Entz\u00fcndungsreaktionen, etwa nach einer Impfung oder Verletzung, ein adaptives Schonverhalten. Bei chronischen Entz\u00fcndungsprozessen k\u00f6nnen die Symptome des Sickness Behavior jedoch zu Einschr\u00e4nkungen der Lebensqualit\u00e4t f\u00fchren und zur Komorbidit\u00e4t bei chronischen Schmerzerkrankungen beitragen. Trotz der hohen klinischen Relevanz des Sickness Behavior wurden bisher psychologische Ans\u00e4tze zur Modulation der immunvermittelten Sickness-Symptome kaum untersucht. Einen Ansatz k\u00f6nnte die Nutzung von Erwartungseffekten bieten, da positive und negative Erwartungen (Placebo- bzw. Nocebo-Effekte) nachweislich einen Einfluss auf Schmerz und affektbezogene Symptome haben.In dieser \u00dcbersichtsarbeit werden die immunologischen und psychobiologischen Faktoren, die zu Schmerz im Kontext des Sickness Behavior beitragen, zusammengefasst. Aufbauend wird diskutiert, wie durch positive und negative Erwartungen Sickness-Symptome beeinflusst werden k\u00f6nnen und welche biologischen und psychologischen Mechanismen dabei involviert sind. Ziel ist es, potenzielle Ansatzpunkte zur Optimierung von Erwartungen im Kontext immunvermittelter Sickness-Symptome zu identifizieren. Perspektivisch lassen sich darauf aufbauend Interventionen entwickeln, um diese Symptome zu reduzieren sowie die Wirkungen und Nebenwirkungen von immunassoziierten Therapien durch gezielte Erwartungsinduktionen im Rahmen der Kommunikation mit Patient:innen positiv zu beeinflussen. Systemische Entz\u00fcndungsprozesse gehen mit Krankheitssymptomen wie z.\u202fB. Schmerz einher. Insbesondere bei chronischen Entz\u00fcndungsprozessen k\u00f6nnen diese immunvermittelten Symptome eine erhebliche Belastung f\u00fcr Betroffene darstellen. Weitgehend unklar ist, inwieweit solche Symptome durch psychologische Interventionen gelindert werden k\u00f6nnen. Anhand von Befunden aus der Entz\u00fcndungs- und Placeboforschung soll hier diskutiert werden, wie sich immunvermittelte Krankheitssymptome und auch die Nebenwirkungen von immunassoziierten Therapien durch die Optimierung von Erwartungsprozessen und \u00e4rztliche Kommunikation beeinflussen lassen.Das Immunsystem und das zentrale Nervensystem stehen in einem engen und kontinuierlichen Informationsaustausch. Dadurch k\u00f6nnen einerseits Immunprozesse an Umweltbedingungen, physiologische Stressoren oder psychologische Belastungen angepasst werden. Auf der anderen Seite erh\u00e4lt das Gehirn \u00fcber afferente Kommunikationswege Informationen \u00fcber den Status des Immunsystems , 25. DieTrotz der weiten Verbreitung des Sickness Behavior und der resultierenden Belastung f\u00fcr Betroffene sind psychologische Interventionen zur Reduktion dieser immunvermittelten Symptome bisher kaum untersucht worden. Im Folgenden sollen daher die psychobiologischen Faktoren, die insbesondere zu Schmerz als Symptom des Sickness Behavior beitragen, zun\u00e4chst anhand experimenteller Befunde aus der Entz\u00fcndungsforschung zusammengefasst werden, um so potenzielle Ansatzpunkte f\u00fcr die Entwicklung und Nutzung von psychologisch orientierten Interventionen zur Optimierung von Erwartungsprozessen aufzuzeigen Abb.\u00a0.Entz\u00fcndungsmediatoren wie proinflammatorische Zytokine tragen auf lokaler Ebene zu einer Aktivierung von Nozizeptoren sowie auf Ebene des ZNS zu einer zentralen Sensitivierung bei , 23. KliDas Modell der experimentellen Endotox\u00e4mie wurde inzwischen in mehreren randomisierten, Placebo-kontrollierten Studien eingesetzt, um Ver\u00e4nderungen der Schmerzsensitivit\u00e4t w\u00e4hrend einer systemischen Entz\u00fcndungsreaktion bei gesunden Testpersonen zu untersuchen. Besonders gut dokumentiert ist hierbei eine Abnahme der Druckschmerzschwellen, welche f\u00fcr unterschiedliche Muskelgruppen des K\u00f6rpers gezeigt wurde , 31, 32.Eine ebenfalls gut dokumentierte Folge der LPS-induzierten systemischen Immunaktivierung ist das vor\u00fcbergehende Auftreten von Stimmungsbeeintr\u00e4chtigungen im Sinne einer negativen, depressions\u00e4hnlichen bzw. \u00e4ngstlich gef\u00e4rbten Stimmung . Vor demZusammenfassend unterst\u00fctzen die dargestellten experimentellen Befunde, dass systemische Entz\u00fcndungsprozesse nicht nur zu einer erh\u00f6hten Schmerzsensitivit\u00e4t beitragen, sondern auch zu einer Beeintr\u00e4chtigung der Stimmung, was von Bedeutung f\u00fcr komorbid auftretende affektive St\u00f6rungen bei chronischen Schmerzerkrankungen sein kann. Dar\u00fcber hinaus bieten die beschriebenen Prozesse einen Erkl\u00e4rungsansatz f\u00fcr das Auftreten unspezifischer Krankheitssymptome im Kontext von Entz\u00fcndungsprozessen, wie sie etwa nach akuten Verletzungen oder operativen Eingriffen sowie bei chronischen Schmerzerkrankungen beobachtet werden. Die experimentelle Endotox\u00e4mie bietet somit einen interessanten Ansatz, um unter kontrollierten laborexperimentellen Bedingungen immunvermittelte Sickness-Symptome zu analysieren und darauf aufbauend innovative Therapieans\u00e4tze zu entwickeln .Patient:innen entwickeln Erwartungen zum Verlauf von Erkrankungen und zum Erfolg von Therapien aufgrund von Informationen, die beispielsweise im \u00e4rztlichen Gespr\u00e4ch oder durch Medien vermittelt werden, sowie auf der Basis von Lernprozessen und Erfahrungen. Solche positiven (und auch negativen) Erwartungen beeinflussen die Wahrnehmung und Bewertung von Symptomen und k\u00f6nnen dar\u00fcber hinaus auch die Effekte von therapeutischen und pharmakologischen Interventionen beeinflussen. Erwartungseffekte wurden eindrucksvoll f\u00fcr akute und chronische Schmerzen (s.\u00a0die weiteren Beitr\u00e4ge in diesem Sonderheft) sowie auch im Kontext affektiver Erkrankungen beschrieben , 24, 29.Ein besseres Verst\u00e4ndnis dar\u00fcber, wie positive bzw. negative Erwartungen immunvermittelte Sickness-Symptome beeinflussen, kann dazu beitragen, psychologische Interventionen gezielt zur Erwartungsoptimierung, etwa im Kontext der Kommunikation mit Patient:innen, einzusetzen.Das einf\u00fchrend beschriebene Endotoxinmodell wurde in zwei Studien genutzt, um Erwartungseffekte auf die immunvermittelten Sickness-Symptome in einem kontrollierten experimentellen Setting zu untersuchen. In der Studie einer schwedischen Arbeitsgruppe wurden Testpersonen im Vorfeld der Entz\u00fcndungsinduktion nach ihren Erwartungen an den Verlauf der psychischen und k\u00f6rperlichen Krankheitssymptome nach LPS-Gabe gefragt . Dabei wIn einer weiteren Studie unserer Arbeitsgruppe wurden Daten aus den Kontrollgruppen von Endotoxinstudien analysiert . Bei allAus der Placebo- und Noceboforschung ist inzwischen gut belegt, dass die Effekte von positiven und negativen Erwartungen auf Schmerz und affektbezogene Symptome \u00fcber verschiedene biologische Systeme vermittelt werden, darunter das endogene Opioid- und Cholecystokininsystem sowie cannabinoide und dopaminerge Pfade . Somit s2-Konzentrationen assoziiert waren. Erhielten die Testpersonen eine wirkstofffreie Placebotablette, nahmen Prostaglandinspiegel und Kopfschmerzen ab.Hinweise, dass Erwartungsprozesse proinflammatorische Mediatoren beeinflussen k\u00f6nnen, lassen sich aus der \u201ePsyHeart-Studie\u201c ableiten, einer randomisierten kontrollierten Studie zu Erwartungseffekten im Kontext einer Bypass-Operation: Patient:innen, die pr\u00e4operativ eine psychologische Intervention zur Erwartungsoptimierung im Hinblick auf das Ergebnis der Operation erhielten, wiesen sechs Monate nach der Operation \u2013\u00a0neben h\u00f6herer psychischer Lebensqualit\u00e4t und subjektiver Belastbarkeit\u00a0\u2013 niedrigere Plasmaspiegel proinflammatorischer Zytokine gegen\u00fcber einer Vergleichsgruppe auf . Inwiewewww.treatment-expectation.de). Trotz der noch unvollst\u00e4ndigen Befundlage lassen sich aus den oben dargestellten Studien sowie aus der weiterf\u00fchrenden Literatur (s.\u00a0auch die weiteren Beitr\u00e4ge in diesem Heft) die nachfolgend dargestellten Hinweise ableiten, wie sich Erwartungseffekte in der Interaktion mit Patient:innen im Kontext des immunvermittelten Sickness Behavior ber\u00fccksichtigen und nutzen lassen, um die Effekte von immunmodulierenden Medikamenten zu verst\u00e4rken.W\u00e4hrend diese Befunde zumindest indirekt darauf schlie\u00dfen lassen, dass Erwartungsprozesse auch die immunbiologischen Mechanismen des Sickness Behavior beeinflussen k\u00f6nnten, konnte in den beiden zuvor vorgestellten Endotoxinstudien , 18 keinSystemische Entz\u00fcndungsprozesse gehen mit k\u00f6rperlichen und psychischen Krankheitssymptomen einher, darunter Schmerz und affektbezogene Symptome.Symptome des Sickness Behavior k\u00f6nnen im Kontext chronischer Entz\u00fcndungsprozesse und als Nebenwirkungen von Immuntherapien eine erhebliche Belastung f\u00fcr die Betroffenen bedeuten.Die Vermittlung von grundlegendem Wissen \u00fcber die Mechanismen und das Erscheinungsbild des immunvermittelten Sickness Behavior kann Betroffenen helfen, Symptome im Kontext systemischer Entz\u00fcndungsprozesse besser einzuordnen, zu verstehen und zu bew\u00e4ltigen.Dabei sollten m\u00f6glichst realistische Erwartungen an das Auftreten immunvermittelter Symptome entstehen, damit diese nicht durch ein \u201eMismatch\u201c verst\u00e4rkt werden.Die Effekte von positiven und negativen Erwartungen auf schmerz- und stimmungsassoziierte Symptome sowie auf Wirkungen (und Nebenwirkungen) von Therapien sind umfassend belegt.Erwartungsprozesse k\u00f6nnen vermutlich auch zur Modulation von Symptomen des immunvermittelten Sickness Behavior beitragen und sollten in der Kommunikation mit Patient:innen ber\u00fccksichtigt werden."}
+{"text": "In addition, chromatin regulators (CRs), as essential upstream regulators of epigenetics, play a significant role in tumorigenesis and cancer development.Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries and its prevalence is increasing. As an emerging therapy with a promising efficacy, immunotherapy has been extensively applied in the treatment of solid tumorsCRs and immune checkpoint-related genes (ICRGs) were obtained from the previous top research. The Genome Cancer Atlas (TCGA) was utilized to acquire the mRNA expression and clinical information of patients with EC. Correlation analysis was utilized for screen CRs-related ICRGs (CRRICRGs). By Cox regression and least absolute shrinkage and selection operator (LASSO) analysis, prognosis related CRRICRGs were screened out and risk model was constructed. The Kaplan\u2013Meier curve was used to estimate the prognosis between high- and low-risk group. By comparing the IC50 value, the drugs sensitivity difference was explored. We obtained small molecule drugs for the treatment of UCEC patients based on CAMP dataset.We successfully constructed a 9 CRRICRs-based prognostic signature for patients with UCEC and found the riskscore was an independent prognostic factor. The results of functional analysis suggested that CRRICRGs may be involved in immune processes associated with cancer. Immune characteristics analysis provided further evidence that the CRRICRGs-based model was correlated with immune cells infiltration and immune checkpoint. Eight small molecule drugs that may be effective for the treatment of UCEC patients were screened. Effective drugs identified by drug sensitivity profiling in high- and low-risk groups.In summary, our study provided novel insights into the function of CRRICRGs in UCEC. We also developed a reliable prognostic panel for the survival of patients with UCEC.The online version contains supplementary material available at 10.1186/s41065-022-00253-w. The differences between the two groups were compared by Wilcoxon signed-rank test. A A total of 165 CRs were shown to be differentially expressed in UCEC tissues when compared with normal tissues Fig.\u00a0A. These We constructed a signature containing 9 CRRICRGs  using LASSO Cox regression analysis and demonstrated its ability to predict the prognosis of UCEC patients Fig.\u00a0A and B. p\u2009<\u20090.001)  Fig.\u00a0E. Furthe01) Fig.\u00a0F. The re01) Fig.\u00a0. Then, a01) Fig.\u00a0.Fig. 3Thp\u2009<\u20090.001), stage (p\u2009<\u20090.001), grade (p\u2009<\u20090.001), and risk score (p\u2009<\u20090.001) independently predict the prognostic outcome of patients , and the tumors showed a greater frequency of a higher degree of differentiation (p\u2009<\u20090.001) and an earlier tumor stage (p\u2009<\u20090.001) , age\u2009<\u2009\u2009=\u200965 (p\u2009<\u20090.001), high grade (p\u2009<\u20090.001), low grade (p\u2009=\u20090.002), advanced-staging (p\u2009=\u20090.003) and early-staging (p\u2009=\u20090.015)  Fig.\u00a0D-F. In a15) Fig.\u00a0G-L.Fig. The above results suggest that the clinicopathological features and the CRRICRGs-based signature were associated with the prognostic outcome of UCEC patients. To graphically evaluate the survival probability of an individual, a nomogram that integrated clinical variables and prognostic signature was developed based on the TCGA-UCEC dataset to predict the 1-, 3- and 5-year survival time of UCEC patients , dasatinib, and cytarabine , was constructed.BTNL9 is a member of Butyrophilin (BTN) and Butyrophilin-like (BTNL) families, involved in inflammatory diseases and tumor development through the regulation of the T cell response , 56. TheThe results of the Kaplan\u2013Meier plotter analysis showed that patients in the low-risk group had a better prognosis compared with that in the high-risk group of patients. The AUC of ROC curves further illustrated the predictive value of our panel for OS in patients with UCEC. Using univariable and multivariable Cox analyses, we screened for 4 clinicopathological features , associated with prognosis of endometrial cancer. Moreover, we also found that patients in the low-risk group were younger, and that the tumors have a greater frequency of a higher degree of differentiation and earlier tumor stage. To visualize the 1-, 3- and 5-year survival of endometrial cancer patients, we built a nomogram with an integrated risk score and clinical characters, and the calibration curve also showed that the nomogram has good predictive efficacy.We next performed functional enrichment analysis on 9 CRRICRGs. The results of GO analysis suggested that regulation of immune cell activity, immune-related protein synthesis, and regulation of various receptor activities have the most frequent occurrences. The KEGG analysis indicated that CRRICRGs are mainly enriched in various immune- and metabolism-related pathways. The above results demonstrated that CRRICRGs can be involved in the development of UCEC through regulating multiple immune- and metabolism-related pathways and that they may also play important roles in regulating the TEM of UCEC. Meanwhile, the result of the analyses of TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, MCPCOUNTER, XCELL, and EPIC indicated the relationship between risk score and immune infiltration. By analyzing the distribution of immune cells among the high- and low-risk groups, we found that the expression of APCs, including myeloid dendritic cell, B cells and macrophage, negatively correlate with risk score. This illustrated that patients in the low-risk group may have a greater number of APCs, which can present tumor cells to T cells, and thus, enhance anti-tumor immunity. This could partially explain why patients in the low-risk group had longer OS compared with that in the high-risk group of patients. In addition, the differential expression of 7 key ICs between the two groups is demonstrated in Fig.\u00a0This study has some limitations. All analyses were conducted and validated based on TCGA-UCEC, GEO and GTEx database, and further validation should be done using clinical samples in the future. In addition, more experiments are required to investigate the molecular mechanisms associated with CRRICRGs influence on UCEC progression.In summary, we identified 9 prognosis-associated CRRICRGs . A panel was developed, and we proved that it could predict the outcome and immune microenvironment in UCEC patients. Furthermore, our findings also suggested a potential therapeutic value of CRRICRGs for UCEC.Additional file 1:\u00a0Supplementtable 1.\u00a0The list of 870 chromatinregulators.Additional file 2: Supplementtable 2. The list of 79 immunecheckpoint related genes.Additional file 3: Supplementtable 3. The list of 68 CRRICRGs.Additional file 4: Supplement Figure 1.\u00a0Additional file 5: Supplement Figure 2.\u00a0Additional file 6: Supplement Figure 3.\u00a0Additional file 7: Supplement Figure 4.\u00a0Additional file 8: Supplement Figure 5."}
+{"text": "This cohort study analyzes data for children from the population-based study Growing Up in Singapore Toward Healthy Outcomes (GUSTO) to examine the associations between infant screen time, electroencephalography markers, and school-age cognitive outcomes using a mediation analysis. To what extent is the association between infant screen use and cognitive impairments mediated by electroencephalography markers?In this birth cohort study involving 437 children, the use of a mediation analysis embedded in a structural education model provided evidence that electrocortical activity in the frontocentral and parietal brain regions mediated the association between infant screen use and later executive function impairments.Screen use during infancy may contribute to variations in neural activities implicated in the development of high-order cognitive skills. Research evidence is mounting for the association between infant screen use and negative cognitive outcomes related to attention and executive functions. The nature, timing, and persistence of screen time exposure on neural functions are currently unknown. Electroencephalography (EEG) permits elucidation of the neural correlates associated with cognitive impairments.To examine the associations between infant screen time, EEG markers, and school-age cognitive outcomes using mediation analysis with structural equation modeling.This prospective maternal-child dyad cohort study included participants from the population-based study Growing Up in Singapore Toward Healthy Outcomes (GUSTO). Pregnant mothers were enrolled in their first trimester from June 2009 through December 2010. A subset of children who completed neurodevelopmental visits at ages 12 months and 9 years had EEG performed at age 18 months. Data were reported from 3 time points at ages 12 months, 18 months, and 9 years. Mediation analyses were used to investigate how neural correlates were involved in the paths from infant screen time to the latent construct of attention and executive functioning. Data for this study were collected from November 2010 to March 2020 and were analyzed between October 2021 and May 2022.Parent-reported screen time at age 12 months.Power spectral density from EEG was collected at age 18 months. Child attention and executive functions were measured with teacher-reported questionnaires and objective laboratory-based tasks at age 9 years.2, 0.03-0.16; Cohen d, 0.35-0.87). A subset of 157 children had EEG performed at age 18 months; EEG relative theta power and theta/beta ratio at the frontocentral and parietal regions showed a graded correlation with 12-month screen use (r\u2009=\u20090.35-0.37). In the structural equation model accounting for household income, frontocentral and parietal theta/beta ratios partially mediated the association between infant screen time and executive functioning at school age , forming an indirect path that accounted for 39.4% of the association.In this sample of 437 children, the mean (SD) age at follow-up was 8.84 (0.07) years, and 227 children (51.9%) were male. The mean (SD) amount of daily screen time at age 12 months was 2.01 (1.86) hours. Screen time at age 12 months contributed to multiple 9-year attention and executive functioning measures (\u03b7In this study, infant screen use was associated with altered cortical EEG activity before age 2 years; the identified EEG markers mediated the association between infant screen time and executive functions. Further efforts are urgently needed to distinguish the direct association of infant screen use compared with family factors that predispose early screen use on executive function impairments. Of particular importance are findings of associations between screen use in early childhood (ages 6 months to 4 years) and impairments of attention and executive functions.11Since the advent of mobile electronic devices, infants aged between 6 and 18 months are exposed to 2 to 3 hours of screen time per day.13 These functions develop rapidly over the first years of life in concert with the prefrontal cortex and are highly susceptible to environmental influences.16 Infants exposed to screens are particularly vulnerable to executive function deficits due to their difficulty processing information on 2-dimensional screens, also called video deficit.17 The need to comprehend challenging screen content, particularly content designed for older children and adults that is unfamiliar and fantastical in nature, requires tremendous cognitive resources and processing.18 This kind of processing relies heavily on attention primarily through the sensory pathways of the brain , which leaves inadequate allocation of resources for prefrontal, top-down attention and typical development of executive functions.19Executive functions represent a collection of higher-order cognitive skills essential for self-regulation, learning, and academic achievement, as well as mental health.21 To date, little is known about potential neural correlates in infants who are most vulnerable to later executive function deficits due to the aforementioned video deficit. Furthermore, it is unclear whether these deficits persist into school age. The nature and timing of screen time on the underlying neural processes mediating cognitive changes are also unknown.Emerging neuroimaging studies in preschool-age children have demonstrated associations between exposure to screen-based media and alterations in white matter tracts important for executive functioning.23 Converging data reveal that an increase in low-frequency powers  and a higher relative ratio of theta to beta powers  are both neural correlates of poorer attentional control.25 A previous study found a correlation between screen exposure and higher functional connectivity in EEG theta waves; however, EEG was collected in only 14 children ages 4 to 6 years after a 9-hour screen-based storytelling intervention.26 We aimed to establish the association between screen use at age 12 months and known neural signals of poorer attentional control at age 18 months, particularly in the theta frequency band and in the theta/beta ratio, and to then examine whether these differences in early electrocortical activity contribute to the variances in attention and executive function outcomes at age 9 years.Electroencephalography (EEG) is a powerful and widely available tool that has been used extensively to identify neural correlates of various cognitive functions. Resting EEG data over the midfrontal and parietal regions have been used to delineate potential neural mechanisms of attention and executive function.27 Data were collected from November 2010 to March 2020. Race and ethnicity were reported by both parents. In Singapore, the 3 major ethnicities included Chinese, Indian, and Malay. Mother-child dyads were followed up throughout pregnancy and beyond. The study was approved by the SingHealth Centralised Institutional Review Board and the National Health Group Domain-Specific Review Board. Mothers provided written informed consent prior to enrollment in the cohort, and all children assented to the study at 7 years of age. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.Growing Up in Singapore Toward Healthy Outcomes (GUSTO) is a population-based, prospective cohort with the aim of understanding perinatal and early influences on long-term health outcomes. Women 18 years and older across all socioeconomic backgrounds were recruited at their first trimester of pregnancy from 2 main public hospitals in Singapore between June 2009 and December 2010.28This cohort consisted of 506 mother-child dyads who were invited to complete the study measures when children were ages 12 months and 9 years. Children who were born preterm (<37 weeks), born small for gestational age , part of a twin pregnancy, or those with major neurological conditions were excluded (n\u2009=\u200928). At age 9 years, 437 children (86.3%) had complete behavioral data for analyses. Reasons for the missing data (n\u2009=\u200941) are listed in eFigure 1 in 29 In Singapore, economic deprivation and psychological ill effects are often reported in families receiving subsidies, with estimates of 14% to 31% endorsing severe to extremely severe scores on the Depression, Anxiety and Stress Scale (DASS-21).30 When their child was aged 12 months, parents were asked to report the amount of time on average that the child spent on screens per day on weekdays, Saturdays, and Sundays in the past 1 month. We used the total hours of screen time watched over the entire week and divided it by 7 days to give the amount of screen time per day. Parents were asked the same question on their child\u2019s screen time at 5 time points between ages 12 months and 54 months. Screen time data across time points were moderately to strongly correlated (r\u2009=\u20090.40-0.51) in our cohort.31Monthly household income was stratified into 4 groups: less than SGD 2000 (US $1478), SGD 2000 to 3999 (US $1478-$2955), SGD 4000 to 5999 (US $2956-$4433), and SGD 6000 or greater (\u2265US $4434). The group with monthly income less than SGD 2000 represented those who likely received governmental financial subsidy based on absolute criterion in 2010.33 The processing involved 1-Hz high-pass and 100-Hz low-pass filtering, removal of 50-Hz line noise, bad channel detection, wavelet-enhanced independent component analysis (ICA), ICA with artifact removal through Multiple Artifact Rejection Algorithm, interpolation of bad channels, and re-referencing to the average reference.35 Data were segmented into contiguous 2-second segments, and segment rejection was carried out via HAPPE criteria.35 The eTable 1 in At age 18 months, a subset of the cohort underwent EEG, which was acquired in a dimly lit, electrically shielded room with 128-channel Geodesic Sensor Nets connected to a DC-coupled amplifier . Resting EEG was recorded continuously for 3 minutes while the child was seated on the lap of a caregiver and looking at bubbles being blown in the room. Data were processed using the Harvard Automated Processing Pipeline for EEG (HAPPE) embedded within the Batch EEG Automated Processing Platform.A fast Fourier transform with the multitaper method was used to calculate the power spectrum on each 2-second segment. Power was parsed into frequency bands . The summed absolute power across these frequencies represented the total power. The mean summed absolute power in each frequency band across all 2-second segments was calculated and normalized by a log-10 transformation. Relative power for each band was calculated as a percentage of the total power. The theta/beta ratio was calculated by dividing the power within theta frequencies by the power within beta frequencies. Given our attention and executive function outcomes, the regions of interest were the frontocentral and parietal regions, which included 16 and 4 electrodes, respectively  and the General Executive Control Problems scale from the Behavior Rating Inventory of Executive Function, second edition (BRIEF-2), were obtained from teachers.38 Higher scores in CBCL and BRIEF-2 reflected more problems in attention and executive functioning.Objective executive function assessments were administered using the Developmental Neuropsychological Assessment, second edition (NEPSY-II), which incorporated the 3 core executive function components, naming inhibition, shifting, and working memory.2 test and 1-way analysis of variance were conducted to compare categorical and linear variables across groups and to examine differences due to loss to follow-up between invited families, study sample, and EEG subsample. Linear regression models confirmed whether screen time at age 12 months was associated with attention and executive functions at age 9 years. Covariates in the model, including household income, birth weight, smoking exposure during pregnancy, child sex, and negative maternal mental health during pregnancy, were all associated with executive functioning in prior literature.40 We examined whether neural changes in the frequency bands responded in a dose-response manner based on the amount of screen time at age 12 months. We then tested for correlations between screen time duration and 18-month absolute and relative EEG power measures at the frontocentral and parietal regions. Mediation analyses using maximum likelihood estimation within a structural equation modeling framework were used to investigate the extent to which neural correlates were involved in the paths from infant screen time to the latent construct of attention and executive functioning. Data were analyzed between October 2021 and May 2022 using Stata version 15.1 (StataCorp) and Mplus version 12 (Muth\u00e9n & Muth\u00e9n). Topography maps were visualized using MNE Python.41Analyses using the \u03c7The mean (SD) amount of screen time was 2.01 (1.86) hours per day at 12 months. After adjustment for covariates, screen time at 12 months was independently associated with 9-year teacher-reported and objectively measured attention and executive functioning . HousehoWe examined the associations between screen time, relative theta, theta/beta ratio, and outcomes in a correlation matrix . All theRelative theta and theta/beta ratios of the frontocentral and parietal electrodes were associated with each of the laboratory-based executive function tasks . The hig2 Thus, we added household income as an exogenous variable in the structural equation model. We found a direct path from infant screen time to the latent outcome construct consisting of all 3 laboratory-based executive function tasks . Each of the 4 targeted mediators was tested individually, of which frontocentral and parietal theta/beta ratios provided indirect paths . In summary, frontocentral and parietal theta/beta ratios partially mediated the path from screen time at age 12 months to the latent executive function outcome at age 9 years.As confirmed in GUSTO and in the regression models, socioeconomic status was consistently associated with infant screen time.Our study provides evidence for the persisting longitudinal association between infant screen time at age 12 months and attention and executive functioning outcomes at 9 years of age. The outcome measures were teacher reports and objective laboratory tasks. Both corroborate real-world manifestations of observable impairments, although teacher reports may be limited by subjectivity or cultural interpretation of behaviors. In short, increased screen time in infancy is associated with impairments in cognitive processes critical for health, academic achievement, and future work success. However, the findings from this cohort study do not prove causation. Screen time likely represents a measurable contextual characteristic of a family or a proxy for the quality of parent-child interaction. Replication of this study\u2019s findings and randomized clinical trials are warranted.43We also document a positive \u201cdose-response\u201d association between infant screen time and cortical EEG correlates of attention and executive functioning. This association is detectable in a stepwise manner from 1 hour to more than 4 hours of screen time per day. The mediation analyses demonstrate cortical EEG activity, namely the theta/beta ratio, as a plausible frontoparietal-mediated pathway from infant screen time to poor executive function. The mapping of the EEG signals to the frontoparietal regions is of critical importance, as these regions are core neural substrates involved in working memory, orienting attention , and executive control subfunctions of attention.44 EEG activities associated with later outcomes offer a means of determining risks and facilitating earlier interventions. Because executive functions develop rapidly in conjunction with the prefrontal cortex and are highly trainable skills, timing interventions during this period of neuroplasticity and before neuronal circuits stabilize must be considered.45 While the clinical utility of these electrophysiological measures in the diagnosis of attention-deficit/ hyperactivity disorder is debated,46 this study is interested in using these measures to show a brain-behavior relationship that functions across a continuum in the context of infant screen time.47Our findings provide evidence for the potential value of EEG during early childhood in understanding executive function impairments later in childhood. Attention and executive functions are difficult to reliably assess in early childhood and may not be apparent until the academic demands increase during formal school years.48 The sensitive caregiving and reciprocal interactions between caregivers and infants remain crucial in regulating the physiology of the infant and in the building of cognitive, social, and affective competencies.16 On the other hand, there is reason to assume a direct effect of screen time on neurodevelopment. Very young media consumers have developmentally appropriate but incongruous response to stimuli presented on 2-dimensional screens, coupled with reduced ability to attend selectively to relevant stimuli via this medium.17 To interpret repeated and multiple novel streams of sensory inputs through the device, infants expend large amounts of mental effort, particularly through activating the orienting reflexes. They do so without the capacity to modulate such efforts, which impedes adequate development of the executive control system during the early years.49Our analyses underscore the association between infant screen time, cortical activity, and cognitive function. It is important to point out that screen use may be a proxy for cognitive impoverishment due to the displacement of social interactions in real life, which are \u201cexperience-expectant\u201d inputs needed to facilitate executive function development.2 While it was assumed that television viewing decreased with the advent of handheld devices, television viewing continued to account for a large proportion of screen time in children younger than 5 years.4 Although the amount of screen time of GUSTO infants was found to be comparable with other countries in this study, it was unclear whether infant screen time changed during the recent COVID-19 pandemic lockdowns. Older children in our Singapore cohorts were shown to use screens more often, particularly in online chats.50 Hence, generalizability may be limited because of these new trends in screen use.This study has limitations that warrant consideration. Screen time at 12 months of age was reported by parents and not an objective measure. At that point, precise recording of screen use via moment-to-moment capture and machine learning, now referred to as screenome, was still in development. Time spent on each type of electronic device was also not collected. In 2010, handheld devices were beginning to surface in Singapore, and 97% of families were using television alone as the main source of screen time.51 Lastly, families in the cohort reported household income in categories, which prevented us from examining this socioeconomic indicator with a greater level of granularity. Nevertheless, the arbitrary cutoffs of household income into 4 categories remained a strong correlate of infant screen time, which echoed prior literature on the need to address this issue with at-risk families.In addition, our study was not designed to inform on the issue of screen content. However, some argued whether the cognitive immaturity of infants rendered content less relevant compared with older children. This study was limited to identifying individual cognitive risks and lacked the inclusion of contextual influences, including parent-child interactions and language stimulation activities. We recognized the importance of ecological context of early media use and prioritized these topics in ongoing studies.We used a longitudinal cohort to define the enduring associations between screen time in infancy and cognitive skills in late childhood. Infant screen use was associated with altered cortical EEG activity before age 2 years, a time when real-life problems related to attention could not be reliably confirmed. Furthermore, the identified EEG markers mediated the association between infant screen time and executive functions. Given the pervasiveness of infant screen use, our findings have public health implications on a population level. Further efforts are urgently needed to distinguish the direct association of infant screen use vs family factors that predispose early screen use on executive function impairments."}
+{"text": "This cohort study analyzes data for young children to investigate the association of higher screen time with neurodevelopmental outcomes and whether this association is mediated by outdoor play. Is higher screen time in infancy associated with suboptimal neurodevelopment  at age 4 years, and are the associations mediated by frequency of outdoor play?In this cohort study, higher screen time (>1 hour a day) at age 2 years was associated with both lower communication and daily living skills at age 4 years. For daily living skills, 18% of the association was mediated and alleviated by the frequency of outdoor play at age 2 years 8 months.Frequent outdoor play may mitigate the connection between higher screen time and later suboptimal neurodevelopment, implying potential for intervention. Whether the association between higher screen time in infancy and later suboptimal neurodevelopment can be mitigated by frequency of outdoor play is unknown.To investigate whether higher screen time at age 2 years is associated with neurodevelopmental outcomes at age 4 years and whether this association is mediated by frequency of outdoor play at age 2 years 8 months.Participants were a subsample of the Hamamatsu Birth Cohort Study for Mothers and Children . Children were born between December 2007 and March 2012 and followed up from 1 year 6 months to 4 years. The analysis was conducted from April 2021 to June 2022.Screen time longer than 1 hour a day at age 2 years was coded as higher screen time.Standardized scores for communication, daily living skills, and socialization domains of the Vineland Adaptive Behavior Scale, second edition, at age 4 years were used . The mediating factor was frequency of outdoor play at age 2 years 8 months, with 6 or 7 days per week coded as frequent outdoor play.b\u2009=\u2009\u22122.32; 95% CI, \u22124.03 to \u22120.60), but the association was not mediated by frequency of outdoor play. Higher screen time was also associated with lower scores in daily living skills ; 18% of this association was mediated by frequency of outdoor play. Frequency of outdoor play was associated with socialization , whereas higher screen time was not .Of 885 participants, 445 children (50%) were female; mean (SD) screen time per day was 2.6 (2.0) hours. Causal mediation analyses revealed that higher screen time at age 2 years was associated with lower scores in communication at age 4 years (nonstandardized coefficient Higher screen time at age 2 years was directly associated with poorer communication at age 4 years. It was also associated with daily living skills, but frequency of outdoor play at age 2 years 8 months alleviated it, suggesting outdoor play mitigated the association between higher screen time and suboptimal neurodevelopment. Future research should specify the nature of the associations and intervention measures, enabling targeted interventions that reduce the potential risk in screen time. The inverse association of screen time with children\u2019s neurodevelopmental well-being has been investigated, with a recent meta-analysis revealing that 75% of children younger than 2 years use them.7 reported that screen time at ages 6 months and 1 year was moderately associated with cognitive dysfunction in school at age 10 years. However, another study found no association between screen time at ages 6 months, 1 year, or 2 years and cognitive skills at age 3 years.12 One issue is that neurodevelopmental domains are different between studies. Some focused on developmental milestones,13 language skills,14 or socioemotional problems,15 while recent studies have suggested that screen time is associated with developmental disorders, such as autism spectrum disorder (ASD).17 Further, the directionality of the association between high screen time and suboptimal neurodevelopment has been questioned,18 although recent studies support directionality of the association.15Findings on the association of screen time in young children with neurodevelopmental outcomes are still inconclusive. Pagani et al19 suggesting that the suboptimal neurodevelopmental outcomes of higher screen time in younger children may be mediated by the amount of outdoor play. Outdoor activities have been inversely associated with sedentary time20 and screen time21 and associated with a child\u2019s cognitive, social, and emotional skills later in life.22 The recent COVID-19 pandemic led to children having higher screen time, less outdoor play, and lower physical activity levels,25 putting them at a potentially increased risk for neurodevelopmental problems. What is concerning is that data show screen time has not decreased after seclusion measures were lifted.26 Given the data on the pandemic, its aftermath, and the existing cohort, it should be possible to consider how to minimize the effect of higher screen time in a future study.Further investigations on the association between screen time and neurodevelopment have examined how it may be related to third factors, including outdoor play. A recent study showed associations of outdoor play with both screen time and preschoolers\u2019 social behavior,27 we measured neurodevelopmental outcomes that could capture communication, daily living skills, and socialization domains.In this article, we examined whether (1) screen time at age 2 years is associated with neurodevelopmental outcomes at age 4 years; (2) frequency of outdoor play at age 2 years 8 months is associated with neurodevelopmental outcomes at age 4 years; and (3) frequency of outdoor play at age 2 years 8 months mediates the associations between screen time at age 2 years and neurodevelopmental outcomes. Using the second edition of the Vineland Adaptive Behavior Scale (VABS-II),29 This study was conducted under the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines.This study was conducted as a part of an ongoing birth cohort study, the Hamamatsu Birth Cohort Study for Mothers and Children . We invited all pregnant women who visited the 2 research sites and gave birth between December 2007 and March 2012 to join the study. Further details of the procedures are described elsewhere.We extracted data for a subsample of children participating in the study who completed observations at ages 1 year 6 months, 2 years, 2 years 8 months, and 4 years; children missing 1 or more observations were excluded. We compared the participants (n\u2009=\u2009885) and the excluded children (n\u2009=\u2009373)  and 0 representing 1 hour or less. The cutoff was consistent with the published guideline by the American Academy of Pediatrics,10 which recommended 1 hour a day of noneducational viewing for children aged 2 to 5 years.We interviewed the parents of the participating children about their children\u2019s lifestyle at age 2 years using 1 item from the ISAAC Phase 3 Environmental Questionnaire, developed for the International Study of Asthma and Allergies in Childhood (ISAAC)Parents were asked 1 question from the ISAAC questionnaire when children were aged 2 years 8 months to ascertain the frequency and duration of outdoor play. The original question, \u201cHow many days during a normal week does your child go and stay outside for 30 minutes or longer to make him/her breathe hard?\u201d was modified to specify the frequency of physical activity when outdoors. In the analyses, we dichotomized this variable, with 1 indicating fewer than 6 days with 30 minutes or longer of any type of outdoor play a week  and 0 for 6 or 7 days. We had to dichotomize this variable because the original value was heavily skewed, with a median of 6 days a week, and also because commands for causal mediation analyses available in the software, Stata version 17.0 (StataCorp), only allowed mediators regressed on exposure variables in linear or logistic functions.31 of VABS-II27 to measure the 3 domains of children\u2019s neurodevelopmental outcomes we analyzed: communication , daily living skills , and socialization . Assessment was made through a semistructured parental interview, which allowed us to obtain age-adjusted V scores, with a mean (SD) of 100 (15).We adopted the Japanese version32 Variables that could cause any 2 of 3  were treated as potential confounders and entered into the analyses. We treated child\u2019s sex, maternal and paternal education, and ASD symptoms at age 1 year 6 months as covariates in the adjusted model (33 (Japanese version34) at age 1 year 6 months. The cutoff for a possible early diagnosis of ASD was set at either 3 or more points for a total score or 2 or more points for 10 critical items as previously developed in Japan.35 We assigned a value of 1 if the child scored on or above the cutoff and 0 otherwise.To select appropriate covariates, we drew directed acyclic graphics using DAGitty software.ed model . SymptomFirst we independently tested the associations of screen time at age 2 years and outdoor play at age 2 years 8 months with communication, daily living skills, and socialization at age 4 years using linear regression in the crude and adjusted models.37 in Stata. Causal mediation analyses, originally proposed by Robins and Greenland,38 allow statistical models to have an interaction term for an exposure and mediator and decompose total effects into natural direct effect (NDE) and natural indirect effect (NIE) under a counterfactual framework. To achieve parsimony and ease model interpretation, we excluded the interaction terms from the statistical models in the mediation analyses if the exclusion did not meaningfully change either of the effect estimates (NDE and NIE) by more than 10%. When an interaction term is removed, the 2-way decomposition of controlled direct effect and NIE originally proposed by Baron and Kenny39 is mathematically equivalent to the 2-way decomposition of NDE and NIE proposed by Robins and Greenland.38 Accordingly, NDE is the difference in counterfactual outcomes between the presence and absence of the exposure if the mediator is held at the level in the absence of exposure. NIE is the difference in counterfactual outcomes between the presence and absence of the mediator if the exposure is held present. Total effect corresponds to a sum of NDE and NIE.In the causal mediation analyses, we investigated whether outdoor play mediated the association between screen time and neurodevelopmental outcomes (the 3 domains) using the paramed commandWe conducted sensitivity analyses to see if the finding was replicated in subsamples of children whose outdoor play was assessed during the warm months at our site  and during the cold months. If the analyzed results were inconsistent, we repeated the causal mediation analyses of the final model, adjusting for the available covariates and the season of the outdoor play measurement (warm vs cold months).P values was set at .05.Effect estimates were reported based on both unadjusted (crude) and adjusted models. Standard errors and corresponding 95% CIs (bias-corrected) were estimated using bootstrapping procedures with 100 replications. The cutoff for 2-sided b\u2009=\u2009\u22123.05; 95% CI, \u22124.91 to \u22121.19), daily living skills , and socialization  at age 4 years. The associations remained significant after covariate adjustment except for socialization . Infrequent outdoor play (<6 days a week) at age 2 years 8 months was not associated with communication but was significantly and inversely associated with daily living skills  and socialization . These associations remained significant in the adjusted models .38We first checked whether exposure-mediator interactions should be included by comparing the NDE and NIE estimates with and without the interaction term. Because the estimates did not show a change greater than 10%, we omitted the interaction terms and decomposed the associations.b\u2009=\u2009\u22122.32; 95% CI, \u22123.61 to \u22120.49) and NDE  and a nonsignificant, smaller NIE  were found , a nonsignificant NDE , and a significant NIE  were found, with 18.1% mediated. For socialization, no significant total effect was found.For the outcome communication, a large total effect , where the direction and significance of the associations remained the same as the results in In the sensitivity analyses, the direction and significance of the associations were supported in children whose outdoor play was assessed during warm months but not in those with outdoor play assessed during cold months (eTable 2 in This study showed that a higher screen time (>1 hour a day) at age 2 years was associated with poorer neurodevelopmental outcomes at age 4 years. Major findings were as follows: (1) Screen time at age 2 years was associated with communication and daily living skills; (2) the frequency of outdoor play did not mediate the association for communication, but it did mediate the association for daily living skills as indicated by a nonsignificant NDE but significant NIE and total effect, with the proportion of mediation 18%; (3) screen time at age 2 years was not significantly associated with socialization, but frequent outdoor play was. These associations were upheld after adjusting for potential confounders, including child\u2019s sex, parental education, and child\u2019s ASD symptoms at age 1 year 6 months.2 demonstrated that screen time in children as young as 6 months was associated with an increased risk for delay in language development. This observation is supported by recent studies.42 The direct link between early screen time and later language and communication difficulties in these studies is in line with the results of our causal mediation analysis for communication, where total effect and NDE were significant while NIE was nonsignificant, with the coefficient of NIE much smaller than that of NDE, indicating no mediation (Tomopoulos et alediation .Similarly, we found a significant total effect in the association between screen time and daily living skills . The coe44; others do not.42 In contrast, frequency of outdoor play is consistently associated with socialization. Similarly, consistent with the literature,24 higher screen time is associated with frequent outdoor play. Intriguingly, outdoor play is associated only with daily living skills and socialization and not communication (45 understanding the role of outdoor play in the link between early screen time and later neurodevelopment may offer leads for possible interventions.As expected, we found that the coefficient of total effect as a sign of association between screen time and socialization was negative, although nonsignificant. The literature offers limited support for an association between screen time and socialization: Some studies support a significant assciationnication . Because46 and are also determinants of neurodevelopmental outcomes.48 We observed a reduction in both coefficients after adjustment (50 we tested for exposure-outcome association and mediator-outcome association, both showing that confounding did not fully explain the associations.Parental education and ASD symptoms are potential cofounders that need to be considered. Both are associated with changes in screen timejustment . As they51 Another study supported this, suggesting that background television viewing might decrease parent-child interaction.52 These associations have been observed in young children with ASD, who are prone to delays in language skill development.53 Specifically, the amount of passive screen viewing has been inversely associated with the receptive language score of children with ASD at ages 1 to 3 years,42 suggesting that high screen time in young children is associated with suboptimal neurodevelopment in communication, possibly through reduced attention to adults and compromised comprehension in conversation, and that such a explanation stands irrespective of levels of language skills or ASD diagnosis. However, understanding the association of screen time with daily living skills and socialization is complex and needs to take account of cofounders such as the cumulative risk of socioeconomic disadvantage and genetics, dating back to birth or earlier.Different mechanisms that might explain a direct association between screen time and neurodevelopment in communication have been suggested. One study suggested that an increase in audible time to television decreased the magnitude of attention to parental vocalization, leading to a decrease in child vocalization.54 It is also possible that the time spent in outdoor activities could mediate the relationship between parenting attitude and screen time.57 Taken together, it is imperative to investigate the role of outdoor play in optimal child neurodevelopment, not limited to potential remedial and mitigating factors. For example, recent studies have indicated the relevance of \u201cgreen time\u201d58 and sleep59 when considering the role of outdoor play in the triangulation of screen time, parenting attitude, and socioemotional neurodevelopment. All these factors need to be reevaluated in the face of natural disasters such as a pandemic.A prospective study has suggested that screen time, together with parenting attitude, can function as a mediator between early socioeconomic disadvantage and distal outcomes, including socioemotional suboptimality.60 Concerning the generalizability of the findings, children raised in the early 2010s in this study might not have been exposed to screen time as much as those raised in the 2020s. Ownership of digital devices, including mobile phones and personal computers, was stable, at around 95%, during the 2010s, and watching YouTube movies had been increasingly prevalent in Japan since the 2011 Great East Japan Earthquake.61This study has several advantages. First, it is a longitudinal study, with a relatively large sample size. Second, causal mediation analysis allowed us to assess the mediating effect of outdoor play in the association between screen time and neurodevelopment, suggesting a potential point for intervention. However, it also has limitations. The use of parental reports to measure screen time may have resulted in underestimation. Information about the types of screen programs watched and played is lacking; this should have been collected because the effect of high screen time differs depending on the type of program.In this cohort study, higher screen time at age 2 years was associated with suboptimal neurodevelopment in communication at age 4 years, and this association was not confounded or mediated by factors examined in this study. Higher screen time at age 2 years was associated with suboptimal development in daily living skills at age 4 years and mediated by outdoor play at age 2 years 8 months. Future research should specify the nature of the associations and intervention measures to reduce the potential risk inherent in higher screen time and explore the mechanisms underlying the association between screen time and neurodevelopmental outcomes. Further, updating guidelines regarding media use is extremely important for parents, educators, researchers, and the children themselves."}
+{"text": "Gene-based association tests provide a useful alternative and complement to the usual single marker association tests, especially in genome-wide association studies (GWAS). The way of weighting for variants in a gene plays an important role in boosting the power of a gene-based association test. Appropriate weights can boost statistical power, especially when detecting genetic variants with weak effects on a trait. One major limitation of existing gene-based association tests lies in using weights that are predetermined biologically or empirically. This limitation often attenuates the power of a test. On another hand, effect sizes or directions of causal genetic variants in real data are usually unknown, driving a need for a flexible yet robust methodology of gene based association tests. Furthermore, access to individual-level data is often limited, while thousands of GWAS summary data are publicly and freely available.https://github.com/Xuexia-Wang/OWC-R-packageTo resolve these limitations, we propose a combination test named as OWC which is based on summary statistics from GWAS data. Several traditional methods including burden test, weighted sum of squared score test [SSU], weighted sum statistic [WSS], SNP-set Kernel Association Test [SKAT], and the score test are special cases of OWC. To evaluate the performance of OWC, we perform extensive simulation studies. Results of simulation studies demonstrate that OWC outperforms several existing popular methods. We further show that OWC outperforms comparison methods in real-world data analyses using schizophrenia GWAS summary data and a fasting glucose GWAS meta-analysis data. The proposed method is implemented in an R package available at Z) and includes several popular methods as its special cases. Results of the simulation studies and real data analyses illustrate that the proposed test, OWC, outperforms comparable methods in most scenarios. These results demonstrate that OWC is a useful tool that adapts to the underlying biological model for a disease by weighting appropriately genetic variants and combination of well-known gene-based tests.We propose a novel gene-based association test that incorporates four different weighting schemes (two constant weights and two weights proportional to normal statistic The online version contains supplementary material available at 10.1186/s12859-022-05114-x. The gene-based association test using GWAS summary statistics can be viewed as a complementary approach to the traditional single marker association test in GWAS.To date, genome-wide association studies (GWAS) have identified more than thousands of genetic variants associated with complex traits or diseases. However, these identified genetic variants only can explain a small to modest fraction of heritability . To idenWhen testing for genetic associations with a gene-based test, proper weights can boost power substantially. However, one major limitation of existing gene-based association tests lies in using weights predetermined biologically or empirically. This limitation often attenuates the power of a test. For example, both the burden test , 3 and tThe power of a gene-based test depends on the underlying genetic architecture of a complex trait. For different traits, the genetic architecture can differ in number, location, effect size, and direction of effect for causal genetic variants in different genes. To circumvent the difficulties in gene-based association test, we propose the combination method, which is a general, flexible, and powerful method. When testing for weak associations caused by small effect sizes or low frequency common genetic variants, the proposed method performs significantly better than several popular gene based tests such as sum test (ST) , squaredZ is the Z score summary statistics and R is the correlation matrix of genetic variants. Six existing methods can be considered as its special cases and are summarized in Table Testing association between a phenotype and a gene based on individual level data (i.e. genotypes) of genetic variants in the gene is the same as testing association between the phenotype and the gene based on summary statistics (i.e. Z-scores) in that gene , 11. In p-value promptly. sumSTAAR creates a frameworks to combine multiple gene based tests with ACAT method  of European ancestry. This GWAS using the UK Biobank Resource under Application Number 9161 (McCarthy) was restricted to HRC variants. We downloaded the GWAS summary data from 50 genes . The samp-value < 5 \u00d7 10\u22128) within 20 kb in the UKB data [The Venn diagram in Fig.\u00a0UKB data . Based oWeighting genetic variants in a gene appropriately plays an important role to boost the power of a gene based association test. In this paper, we propose a novel combination test - OWC. This is a general linear combination test incorporating four different weighting schemes: two constant weights and two weights proportional to normal statistics thSPU(2) . As a geFurthermore, we show that the general linear combination test statistic can reach its maximum when the weight is estimated as a certain value. For example, the score test putation . In realTrue disease model is usually unknown. Disease models underlying different diseases may be different: some of the disease models may include causal genetic variants with same directions while other disease models may include causal genetic variants with different directions. In addition, some diseases models may include some weakly associated genetic variants, while other disease models may include some strongly associated genetic variants. There is no uniformly most powerful test that is powerful in every situation. An association test may perform well in one dataset, but may perform less well in another dataset. For example, SCZ can be considered as a representative of complex disease. People have identified some common genetic variants with weak effects on SCZ. These variants may be working in tandem to produce SCZ. A robust, flexible method such as OWC can elucidate these weakly associated genetic variants better so that the roles of theses genetic variants in disease etiology can be understood more clearly. The proposed OWC method can be a useful tool as it adapts to the underlying biological disease model for a disease by selecting In summary, the novelty of the proposed method lies in two aspects: 1) proposing a new score test https://github.com/Xuexia-Wang/OWC-R-package.The proposed OWC method only needs the publicly available GWAS summary statistics as input, without the need to access raw genotype and phenotype data. Researchers will be able to identify more novel disease associated genes with OWC by utilizing publicly available GWAS summary data. Novel disease associated genes can shed more light onto underlying mechanism of diseases. In this paper, we focus on developing a powerful genetic associated test using single trait GWAS summary data. The proposed OWC method can be easily extended to analyze GWAS summary data for multiple traits. We have implemented OWC in an R package which is freely available at n individuals with both genotype and phenotype data available in a genomics region (gene or pathway) with M genetic variants (e.g. SNPs). For the Consider a sample including P is an idempotent matrix. That is, The generalized linear model was used to model the relationship between the trait and the genetic variants in the considered region:tatistic  to test mentclass2pt{minimTo test the association between a trait and a genetic variant, a Z test is usually employed. We can use the Z test below to test the main effect of the Denote the linkage disequilibrium (LD) matrix for the considered region as mentclass2pt{minimmentclass2pt{minimcument}R . This coIn the aforementioned weight function mentclasspt{minima(1) test . We knowM degrees of freedom   and the st (SSU) . Based ovariants . If the t method . Naturalp-value across the values of p-value of T can be obtained by a simple grid search across a range of We have proved and demonstrated that most of the gene-based associate tests can be expressed as a weighted combination of Z-scores. Thus, we can propose a new weighted combination method by utilizing the good properties of different weights. The statistics of t method .p-values for T in a single layer of simulations. Briefly, after obtaining p-value of the test is the proportion of the number of the null test statistic p-value.Monte Carlo simulations are used to obtain the ent}Tb\u2264T . A largeM elements where each element is independently generated from a standard univariate normal distribution with mean 0 and variance 1; that is, p-value in detail as follows: Generate independent T by searching across a range of Using asymptotic distribution of p-value for the T test, T test based on the observed data, T test based on the Finally, the If the Z statistic in the summary data is not provided, we need to first transform the p-value in the summary data into a Z statistic using p-value of the test T.The aforementioned vector wing way : we firsp-values, such as the above one, is the computational burden. Especially, when there are about twenty thousands genes in a GWAS and a small significance level is used to claim significant findings. We adopted a fast algorithm [p-values, which will dramatically reduce the computational time. This algorithm reduces computational time by scarifying the precision of the p-value estimation for those tests with large true p-values.One limitation of the Monte Carlo simulation to estimate lgorithm  to estimM = multiplying increment for the number of random sampling (e.g. 10)The fast algorithm works as follows:Step 0 Calculate the statistic T of OWC based on the observed dataStep 1 Set initial values: Step 2 Use Eqs.   Step 2 UStep 3 If We first define the following parameters for the algorithm:Current gene-based association tests, while providing greater interpretability and power over usual single variant association tests, still have many limitations such as weights predetermined biologically or empirically. In this paper, we propose a combination test OWC to overcome these limitations. OWC is a general linear combination test which uses GWAS summary statistics as its input and incorporates different weighting schemes, and includes traditional gene-based tests as its special cases. Simulation studies and real data analyses demonstrate that OWC is more powerful than comparable methods in many scenarios and can adapt to the  underlying genetic architecture of the trait of interest. While the focus of this paper was single-trait analysis, OWC can be easily extended to analyze GWAS summary data for multiple traits.Additional file 1. Linkage equilibrium matrix of gene EPB41.Additional file 2. Significant genes identified by OWC, aSPU, GATES, sumSTAAR, and GW in SCZ1 data, SCZ2 data and UKB data."}
+{"text": "MBT and MycoEx provided identification results in 97.0% and 95.0% of the cases, respectively. With both protocols, 100% of the provided results agreed with LPA with no registered mismatch. MBT achieved an elevated number of highly probable identifications (88.0% vs. 83.0%) and a higher reproducibility rate of correct results (86.6% vs. 75.8%) in comparison to MycoEx. This study provides results about MBT performance for liquid and solid media, underlining the strengths and weakness under different conditions. Our results suggest that MALDI-TOF MS could provide a great advantage for timely and cost-saving NTM identification with potential implications for patient outcome.Nontuberculous mycobacteria (NTM) identification is essential for establishing the relevance of the isolate and for appropriate antimicrobial therapy. Traditionally, NTM identification is performed by using Line Probe Assays (LPA), a costly and time-consuming technique requiring trained personnel. MALDI-TOF MS is a promising tool for NTM identification, and its use is rapidly growing. We evaluated the newly introduced MBT Mycobacteria kit (MBT) and the MycoEx preparation protocol  for NTM MALDI-TOF MS identification using LPA results as a reference. Fifty NTM grown on 7H11 agar and MGIT broth were analyzed with both protocols using the Bruker Microflex Mycobacterium includes a high number of species (more than 190) divided into the following three groups: Mycobacterium tuberculosis complex, Mycobacterium leprae and Nontuberculous mycobacteria (NTM), also named atypical or environmental mycobacteria [The genus bacteria . They ocbacteria . Mycobacbacteria .An early and accurate diagnosis of mycobacterial infections is pivotal in the clinical management of patients and for treatment options. An inappropriate antimicrobial therapy can negatively impact patient outcome due to exposure to toxic drugs and the selection of multi-resistant strains . Thus, NThe gold standard test for NTM identification is gene sequencing. Unfortunately, the access to this technique is currently confined to a limited number of laboratories due to its costs, the scant availability of qualified staff with dedicated professional roles and the long response times ,8.Clinical Microbiology Laboratories (CML) routinely perform NTM identification with Line Probe Assays (LPA), a genotyping method based on DNA sequences detection through hybridization with nucleotide probes fixed on nitrocellulose strips. However, even this technique is characterized by long turn-around times (TAT), high costs and the need for specifically trained personnel .Among new technologies, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), currently used in CML for bacteria and fungi identification, could represent a powerful tool for NTM detection. This proteomics-based technology facilitates the rapid recognition of microorganisms based on unique spectral fingerprints produced by extracted proteins derived from colonies grown in solid and liquid culture media . Given tThe new ready-to-use kit MBT Mycobacteria kit (MBT) , based on chemical cell-inactivation, could overcome the MycoEx protocol\u2019s disadvantages for rapid mycobacteria identification by MALDI-TOF-MS. The study aim was to compare MycoEx and MBT for MALDI-TOF MS identification of the principal NTM strains of clinical interest. Both techniques were tested from positive liquid broth and a solid medium in order to define the optimized identification solution. LPA results were used as reference.M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. fortuitum, and 4 slow growing mycobacteria (SGM), M. avium, M. chimaera, M. intracellulare, and M. gordonae were considered.The study was performed in the Microbiology and Virology Unit  between October 2021 and March 2022. Fifty NTM clinical isolates, previously collected from different types of specimens (mainly respiratory) and belonging to 6 different species were selected from laboratory strains stored at \u221280 \u00b0C. In particular, 3 rapid growing mycobacteria (RGM), In agreement with our laboratory Standard of Care (SoC), all NTM positive cultures were identified by molecular genotypic methods. The LPA commercial system, the most reliable tool for mycobacteria identification for labs not performing sequencing, was used. DNA extraction was carried out with the GenoLyse kit v. 1.0 . The amplification and hybridization for NTM species-level identification were performed with GenoType Mycobacterium CM v. 2.0 or GenoType Mycobacterium AS v. 1.0 and GenoType NTM-DR v. 2.0 (Hain Lifescience GmbH), if required, according to the manufacturer\u2019s instructions. Amplification and hybridization steps were performed with the Veriti\u2122 Thermal Cycler  and TwinCubator (Hain Lifescience GmbH) instruments, respectively. The estimated TAT could range from 4 to 6 h on the basis of the considered mycobacterium and of the group, species, and subspecies identification level.2 atmosphere at 37 \u00b0C up to NTM evidence as visible colonies. Therefore, positive samples from MGIT and 7H11 agar were processed in parallel with the new MBT Mycobacteria kit and the MycoEx protocol according to the manufacturer\u2019s instructions.The selected strains were thawed and cultured in BBL\u2122 MGIT broth (MGIT)  and incubated at 37 \u00b0C in BACTEC\u2122 MGIT\u2122 960  until positive signal detection; at the same time, they were seeded on Middlebrook 7H11 agar  and incubated in 5% COA mycobacterial aliquot was washed and inactivated with the provided reagents  for 30 min at room temperature. Then, each sample was combined with acetonitrile , and 70% formic acid  and centrifuged at 13.000 rpm for 2 min. Finally, 1 \u00b5L of supernatant was spotted in triplicate onto the MALDI plate. The estimated TAT both on MGIT and 7H11 agar was about 40 min.At the same time, the MycoEx protocol was performed as follows: after an initial heat-inactivation treatment (100 \u00b0C for 30 min), samples were processed with absolute ethanol (900 \u03bcL) and centrifuged at 13,000 rpm for 2 min. Then, the pellet was dried and resuspended with zirconia microsphere (40 mg), acetonitrile (25 \u03bcL) and 70% formic acid (25 \u03bcL) in order to break the mycobacterial cell wall. Finally, samples were centrifugated at 13,000 rpm for 2 min and, as mentioned above, 1 \u00b5L of supernatant was spotted in triplicate onto the MALDI-TOF MS plate. In this case, the estimated TAT both on MGIT and 7H11 agar was about 1 h.Escherichia coli proteins  was used as internal quality control by spotting two replicates onto the MALDI-TOF MS plate.An extract of \u00ae LT MALDI-TOF MS  instrument and analyzed with the latest release of IVD MBT Mycobacteria Library v.4.0 . Spectra were acquired in a linear positive ion mode at a laser frequency of 60 Hz across a mass/charge ratio (m/z) of 2000 to 20,000 Da.For each sample, spectra were acquired with the Bruker Microflexlog(score) \u2265 1.80, as probable with log(score) between 1.60 and 1.79, and not assigned with log(score) < 1.60. Mycobacterial identification was considered correct at the species/complex level if at least one of the three replicates agreed with the SoC results with log(score) \u2265 1.60. Identifications of species closely related inside the M. abscessus complex, M. fortuitum group and M. chimaera-intracellulare group were considered concordant.Interpretation of results was performed according to the manufacturer\u2019s instructions. NTM identification was considered highly probable with a log(score) distribution, and we compared the effects of the extraction methods on the frequencies of samples without identification using Fisher\u2019s exact test. As an exploratory analysis, we tested the effects of the type of culture media and of the rapid and slow mycobacteria growth rate on the frequencies of samples without identification. Moreover, the reproducibility rate of the triplicate NTM identification results for both testing protocols was assessed.For both extraction methods (MycoEx protocol and MBT Mycobacteria kit), we described log(score) \u2265 1.80, with an overall rate of 88.0% (95% CI 80.0\u201393.2) and 83.0% (95% CI 74.4\u201389.2), respectively. Considering the growing media, the MBT kit and MycoEx protocol reached 94.0% (95% CI 83.2\u201398.6) and 84.0% (95% CI 71.2\u201391.9) in 7H11 agar, respectively, and both reached 82.0% in MGIT broth  and 95.0% (95% CI 88.7\u201398.4) of the cases with the MBT kit and MycoEx protocol, respectively. Taking into account the type of culture media, the MBT kit provided identification results in 98.0% (95% CI 89.4\u201399.9), and in 96.0% (95% CI 86.3\u201399.5) on 7H11 agar and MGIT, respectively, while the MycoEx protocol provided identification results for 92.0% (95% CI 80.8\u201397.8) and 98.0% (95% CI 89.4\u201399.9) see .M. chimaera, M. intracellulare, and M. gordonae regardless of the starting growth media. In addition, the MBT kit correctly identified all the samples positive for the M. fortuitum group (see The MBT kit and MycoEx protocol reached 100% agreement in the samples positive for roup see .The MBT kit did not identify NTM in three (3%) cases and MycoEx protocol in five (5%). Two (2%) samples, 1 M. avium and 1 M. abscessus complex, were not identified by both extraction methods on the same media. The details of the unidentified NTM according to the extraction protocol and the culture media are reported in p-value = 0.721) and according to the growing media. In 7H11 agar, the number of unidentified samples was five  and in MGIT it was three .We did not observed differences in frequencies of samples without results both overall  \u2265 1.60) when compared with the MycoEx protocol : 94.8% (95% CI 88.2\u201398.1) vs. 91.6% (95% CI 84.0\u201395.9), respectively. Concerning 3 on 3 probable identifications, the MBT kit\u2019s analytical reproducibility rate was 86.6% (95% CI 78.3\u201392.1) vs. 75.8% (95% CI 66.2\u201383.4) for the MycoEx protocol .Focusing on the reproducibility of the triplicate tests, the MBT kit resulted in a higher rate of 2 on 3 and 3 on 3 probable identifications (log(score) and type of mycobacteria and growing media are summarized in Reproducibility results according to the preparation kit or protocol, NTM identification, usually based on commercial hybridization methods, is limited to less than 40 species. Moreover, LPA assays are expensive and time consuming and could require multiple additional sessions to distinguish between mycobacterial species, especially inside groups or subspecies . The impOur work focused on the evaluation of two different preparation methods, namely the recently introduced MBT Mycobacteria kit and the MycoEx protocol, for NTM spectral MALDI-TOF MS identification on Bruker MALDI-TOF MS platforms, from MGIT broth and 7H11 solid medium towards our SoC, LPA assays. To our knowledge, this is the first pilot study estimating the new MBT Mycobacteria kit\u2019s MALDI-TOF MS identification performance and defining its potential advantages and disadvantages for application in the clinical microbiological routine workflow.log(score) analysis highlights a better performance of the MBT kit in producing results with a high probability of correct identification. The difference in the achievements was independent from RGM or SGM groups but was linked to the growth on solid 7H11 medium.The M. chimaera, M. intracellulare, M. gordonae, M. fortuitum group) while the MycoEx protocol failed in achieving 100% success for the M. fortuitum group. Identifications were correct for all the examined clinically isolated strains of NTM with both methods and media, showing a good agreement with the LPA reference technique. Interestingly, no mismatches were observed for all tested samples, confirming MALDI-TOF MS as an effective and reliable method for NTM identification regardless of the adopted extraction protocol. Overall, these results are in line with those reported in the literature, even if a fair comparison could present bias due to the different instruments, libraries, identification criteria, number of replicates, extraction protocols, media and groups/species considered [The MBT kit, in comparison to the MycoEx protocol, also provided successful identifications in more cases with the major advantage in its application on positive 7H11 solid media. Moreover, the MBT kit achieved completely successful results in 4 of 6 of the clinically significant considered species and groups . In addition, the MBT kit\u2019s protocol process (five main steps) and TAT (about 40 min) were lower even than those for MycoEx (seven main steps and around 1 h), suggesting that its adoption could be less time-consuming and labor-intensive.MALDI-TOF MS application to NTM isolates identification could positively impact on CML with a reduction in terms of cost per test from EUR 42.00 with LPA to EUR 1.50 ,19,26. TMycobacterium tuberculosis complex, M. abscessus subsp. bolletii, M. kansasii, M. chelonae) and to assess its real-life TAT and cost-benefit impact.This work provides preliminary data about the recently introduced MBT Mycobacteria kit performance for both liquid and solid media, highlighting, for the first time, its strength and weakness in different conditions. Despite the promising MBT results, the study limitations include the number of analyzed NTM strains. Therefore, further studies with a higher number of NTM strains should be performed to confirm these preliminary results. Moreover, even if the analyzed NTM species are the principal aetiologic agents of mycobacteriosis, in-depth studies are needed to evaluate MBT in a wider group of mycobacteria species and subspecies (i.e., To date, NTM identification has been limited to a few specialized laboratories. The capacity of MALDI-TOF MS to detect clinically interesting NTM in a rapid, reliable and cost-effective manner could provide a significant improvement for all CML.The data collected in our study showed excellent identification performances for the new MBT Mycobacteria kit, especially when applied on NTM strains grown on solid media; however, it is important to perform an in-depth evaluation of a higher number of mycobacteria, in order to assess its effectiveness in mycobacteria identification and with CML workflows and workloads."}
+{"text": "Oryza sativa), FP-OsATG8 driven by Cauliflower mosaic virus (CaMV) 35S promoter had been used for imaging autophagosome and autophagic bodies. Here, we constructed three vectors carrying GFP-OsATG8a, driven by 35S, ubiquitin, and the endogenous ATG8a promoter, individually. Then, we compared them for their suitability in monitoring autophagy, by observing GFP-ATG8a puncta formation in transiently transformed rice protoplasts, and by tracking the autophagic flux with GFP-ATG8 cleavage assay in rice stable transgenic lines. GFP-Trap immunoprecipitation and mass spectrometry were also performed with the three marker lines to show that they can be used reliably for proteomic studies. We found out that the ubiquitin promoter is the best for protoplast imaging. Transgenic rice seedlings of the three marker lines showed comparable performance in autophagic flux measurement using the GFP-ATG8 cleavage assay. Surprisingly, the levels of GFP-ATG8a transcripts and protein contents were similar in all marker lines, indicating post-transcriptional regulation of the transgene expression by a yet unknown mechanism. These marker lines can serve as useful tools for autophagy studies in rice.Autophagy is a conserved intracellular trafficking pathway for bulk degradation and recycling of cellular components in eukaryotes. The hallmark of autophagy is the formation of double-membraned vesicles termed autophagosomes, which selectively or non-selectively pack up various macromolecules and organelles and deliver these cargoes into the vacuole/lysosome. Like all other membrane trafficking pathways, the observation of autophagy is largely dependent on marker lines. ATG8/LC3 is the only autophagy-related (ATG) protein that, through a covalent bond to phosphatidylethanolamine (PE), associates tightly with the isolation membrane/pre-autophagosomal structure (PAS), the growing phagophore, the mature autophagosome, and the autophagic bodies. Therefore, fluorescent protein (FP)-tagged ATG8 had been widely used for monitoring autophagosome formation and autophagic flux. In rice ( Plants are constantly in need of nutrient reallocation during growth and development, yet they are continuously challenged by nutrient limitation and stresses. To combat starvation, biotic, and abiotic stresses while maintaining growth, plants have to efficiently remobilize and reallocate nutrients and clear up pathogens, damaged proteins, and even organelles. Among the degradation/remobilization pathways employed by plants, an intracellular trafficking pathway termed autophagy is particularly important, and the defects in autophagy strongly compromise biomass production and yield .Autophagy is an evolutionarily conserved, bulk degradation pathway of eukaryotic cells that can eliminate and recycle damaged or obsolete proteins and organelles . In thisSaccharomyces cerevisiae), through the screen for autophagy /LC3-interacting region (LIR), ULK1/ATG1 and ATG13 bind ATG8 to regulate autophagosome formation . AutophaApart from the shared characteristics with the yeast and animal ATG8s, the plant ATG8s have unique properties . ArabidoThe way ATG8 is used as a marker for autophagy is also unique in plants. Firstly, the fact that plants have many ATG8s with different molecular weights prevented us from using one anti-ATG8 antibody to examine the autophagic flux, for it is futile to separate different ATG8 isoforms from the PE-conjugated ATG8s. Secondly, the plant vacuole (pH 5.4\u20135.8) is not as acidic as the lysosome (pH 4.5); hence, the acid-sensitive GFP tag is not promptly degraded in the vacuole. For this reason, it is not possible to use a double tagged ATG8, such as mCherry-GFP-LC3, and take the fluorescent color change as an indicator for autophagic flux . So far,35S:mRFP-OsATG8a and 35S:mRFP-OsATG8d lines were firstly generated and imaged for autophagosome accumulation upon ConA treatments  was amplified from rice cDNA and inserted at the Eco91I site through homologous recombination as described 2\u00b74H2O, 1\u2009mM KH2PO4, 0.05\u2009mM FeSO4\u00b77H2O, 0.05\u2009mM Na2EDTA, 46\u2009nM H2BO3, 9\u2009nM MnCl2\u00b74H2O, 0.3\u2009nM CuSO4\u00b75H2O, and 0.8\u2009nM ZnSO4\u00b77H2O) at 28\u00b0C/24\u00b0C, 14\u2009h light/10\u2009h dark in a growth chamber. Transgenic rice was cultivated in paddy fields in growth seasons of 2019 to 2021 in Tianjin, Jiangsu, and Hainan provinces of China.Transgenic rice lines were generated similarly to a previous report on OsATG8s . BrieflyGFP-OsATG8a driven by three different promoters were transiently expressed in tobacco leaves as described , vacuum infiltrated for 30\u2009min, and further digested in the dark for 4\u20135\u2009h with gentle shaking. After enzyme digestion, protoplasts were released by adding an equal volume of W5 solution  and gentle shaking by hand for 2\u2009min. Then, the protoplasts were filtered through a 40\u2009\u03bcm-gauge nylon mesh with 3\u20135 washes using W5 solution and collected by spinning at 100\u2009g for 3\u2009min. After protoplasts were re-suspended and washed once with W5 solution, MMG solution  was used to re-suspend the pellets at a concentration of 2\u2009\u00d7\u2009106 cells ml\u22121. For each transformation, 6\u20138\u2009\u03bcg of freshly prepared plasmid DNA and 200\u2009\u03bcl protoplasts (about 4\u2009\u00d7\u2009105 cells) were mixed with 220\u2009\u03bcl freshly prepared PEG solution  and were incubated at room temperature for 30\u2009min in the dark. After incubation, the protoplasts were mixed with 1\u2009ml\u2009W5 solution and incubated at 28\u00b0C for 12\u201315\u2009h in the dark before imaging.Sterilized rice seeds (japonica rice cultivar Jinjing 818) were germinated on 1/2 Murashige and Skoog (MS) medium with a photoperiod of 14\u2009h light and 10\u2009h dark at 26\u00b0C for 5\u20136\u2009days, then moved to the dark for another 5\u20136\u2009days. The etiolated stem and sheath of rice seedlings were cut into pieces of approximately 0.5\u2009mm with sharp razors. These pieces were immediately transferred into 20\u2009ml enzyme solution  and rice protoplasts were observed with a Ni-E A1 HD25 confocal microscope . Prior to image collection, the background auto-fluorescence was eliminated using untransformed tobacco leaves or rice protoplasts. The GFP fluorescence signal was exited at 488\u2009nm and emission was collected at 500\u2013550\u2009nm. The chlorophyll auto-fluorescence was exited with 561\u2009nm laser and emission was collected at 600\u2013700\u2009nm. For NaCl treatments, protoplasts were incubated in W5 solution with 250\u2009mM NaCl, or 50\u2009\u03bcM E-64d, or both NaCl and E-64d, for 30\u2009min at room temperature. For inhibitor treatments, transformed protoplasts were incubated in W5 solution containing 200\u2009nM AZD8055, or 1\u2009\u03bcM ConA, or both AZD8055 and ConA, for 3\u2009h at room temperature.GFP and OsATG8a were normalized to OsEF1a (LOC_Os03g08020) with three biological replicates consisting of four technical repeats. Specific amplification was verified by a melt curve analysis following the completion of the PCR cycles. Each PCR product generated a single peak in melt curve analysis, indicating specific amplification. The 2-\u0394\u0394CT method was used for relative quantification of qRT-PCR data. Primers used are listed in Leaves from 14-day-old seedlings were used for RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR (qRT-PCR) as described . TranscrTwo-week-old seedlings were used for autophagic flux measurement. Excised leaves were incubated in the Hoagland\u2019s solution with 0.01% Tween-20, and stirred and vacuumed to make sure the leaves were completely soaked in the solution. For NaCl treatments, excised leaves were incubated in the Hoagland\u2019s solution plus 150\u2009mM NaCl, or 100\u2009\u03bcM E-64d, or both NaCl and E-64d, for 1\u2009h at room temperature. Alternatively, 250\u2009mM NaCl was used. For inhibitor treatments, excised leaves were incubated in the Hoagland\u2019s solution containing 2\u2009\u03bcM AZD8055, or 1\u2009\u03bcM ConA, or both AZD8055 and ConA, for 6\u2009h at room temperature. In addition, 6\u2009h with 200\u2009\u03bcM BTH plus 100\u2009\u03bcM E-64d treatment and 4\u2009h with 2\u2009mM DTT plus 100\u2009\u03bcM E-64d treatment were used to induce autophagy. Protein extraction and immunoblotting were done as described . Semi-qu2, 0.2% NP-40, and 1% Protease Inhibitor Cocktail) was added at a proportion of 1:2 (m:v). Extracts were centrifuged at 12,000\u2009rpm for 15\u2009min at 4\u00b0C. Then, the supernatants were collected and centrifuged again at 12,000\u2009rpm for 5\u2009min at 4\u00b0C. The supernatants (1\u2009ml to 10\u2009\u03bcl bead volume) were incubated with GFP-Trap A beads pre-equilibrated in wash buffer  at 4\u00b0C for 2\u20133\u2009h. The beads were washed 3\u20134 times with wash buffer and were re-suspended in 100\u2009\u03bcl wash buffer. Samples were verified by Western blotting; the corresponding gels were cut and sent for mass spectrometry analysis at the Instrumental Analysis Center of Shanghai Jiao Tong University.Transgenic rice seedlings were frozen and ground in liquid nitrogen, and protein extraction buffer  promoters was firstly examined in transiently transformed N. benthamiana leaf epidermal cells. Pro35S:GFP gave typical, strong nuclear, and cytoplasmic GFP signals. Pro35S:GFP-OsATG8a gave relatively weak cytoplasmic signals and punctate signals that likely represent autophagosomes. Strong cytoplasmic/endoplasmic reticulum and punctate signals were observed from cells transformed with ProUBQ10:GFP-OsATG8a  can reliably induce autophagy, we treated the protoplasts with 200\u2009nM AZD8055, a potent TOR inhibitor , in the hibition .Inferring from the expression intensities, the ubiquitin promoter is a good choice for the transient expression of OsATG8a.After validating and comparing the three constructs in transient expression systems, transgenic rice carrying these constructs were generated. T1 transgenic lines regenerated from the transformed calli were firstly validated with genomic PCR and immunoblotting ,B. Then,Pro35S:GFP-OsATG8a, ProUBQ10:GFP-OsATG8a, and ProOsATG8a:GFP-OsATG8a were evaluated for their suitability in measuring autophagic flux. The autophagic flux was measured with the GFP-ATG8 cleavage assay, in which the flux is represented by the ratio of the amount of cleaved GFP (27 kD) to total GFP in the lane (40 kD plus 27 kD), i.e., GFP/(GFP\u2009+\u2009GFP-ATG8a). Firstly, GFP-OsATG8a (40kD) and free GFP (27kD) bands were readily detected in the transgenic lines  or treated with Hoagland\u2019s solution containing 150\u2009mM NaCl for 1\u2009h. The immuno-precipitated proteins were verified with SDS-PAGE and sent for mass spectrometry analyses.ATG8 is a star molecule in proteomic studies of autophagy. To see if the Pro35S:GFP-OsATG8a, ProUBQ10:GFP-OsATG8a, and ProOsATG8a:GFP-OsATG8a under control conditions  to QG.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": "Healthcare workers are susceptible to blood borne pathogens, such as human immunodeficiency virus (HIV). Occupational exposure to HIV infection among healthcare workers is becoming a global public health concern. However, there is limited evidence about occupational exposure of healthcare workers to HIV and utilization of post-exposure prophylaxis in Addis Ababa, Ethiopia. Accordingly, this study was conducted to assess the prevalence of occupational exposure to HIV and utilization of post exposure prophylaxis among healthcare workers at St. Peter\u2019s specialized hospital, Addis Ababa, Ethiopia. A health facility-based cross-sectional study was conducted among 308 randomly selected healthcare workers in April 2022. Structured and pretested self-administered questioner was used to collect data. Occupational exposure to HIV was taken as any percutaneous injury or blood or other body fluids exposure while administering medications, specimen collection, and other procedures with HIV confirmed patients. Multivariable binary logistic regression analysis was used to identify factors associated with occupational exposure to HIV and utilization of post-exposure prophylaxis. Statistically significant association was declared on the basis of adjusted odds ratio with 95% confidence interval and p-value less than 0.05. The study found that 42.3%  of the healthcare workers had occupational exposure to HIV during their career time, out of whom 16.1%  used post-exposure prophylaxis. Healthcare workers with lower-level education such as diploma  and BSc , and healthcare workers who received infection prevention training  had less risk of exposure to HIV. On the other hand, nurses , midwifes , and physicians  had high risk of exposure to HIV compared with other professionals. Moreover, healthcare workers with BSc degree compared with healthcare workers with masters degree , healthcare workers with long service year , and healthcare workers who are working in facilities where prophylaxis is available  had higher odds to utilize post-exposure prophylaxis. Significant proportion of healthcare workers included in the current study had occupational exposure to HIV and very few of them used post-exposure prophylaxis. Healthcare workers need to use appropriate personal protective equipment, safely manage contaminated equipment, and safely administered medications and collect specimen to protect themselves from exposure to HIV. Moreover, use of post-exposure prophylaxis should be promoted when exposure exists. An occupational exposure that may place a healthcare workers at risk of HIV infection is defined as a percutaneous injury (e.g. a needle stick or cut with a sharp object), contact of mucous membrane or contact of skin  with blood, tissues or other potentially infectious body fluids4. According to the World Health Organization (WHO), it is estimated that about 3 million HCWs are exposed to blood-borne pathogens each year and occupational exposure causes approximately 170,000 HIV infections5.Occupational exposure to infectious agents is one of the most important risk factors for HIV transmission among healthcare employees. Healthcare workers face a greater challenge as a result of their duties and responsibilities9. The prevalence of HIV infection among patients, frequency of incidents in which HCWs are exposed to HIV-infected fluids, and the likelihood of transmission following occupational HIV exposure all affect the occupational risk of HIV infection among healthcare workers10. It is believed that 56.2% of healthcare workers worldwide sustained needle-stick and sharp injuries during the course of their careers, making needle-stick injuries the most common form of HIV exposure in healthcare settings11. However, there is a less than 1% chance of contracting HIV through a needlestick wound, and there is a less than 0.1% chance of getting exposed through direct skin contact with the fluid12.The risk of infections from blood-borne pathogens is increased by a number of circumstances, including absence of fundamental personal protection equipment, poor adherence to safety procedures, excessive use of injectable therapy, and needle-stick or sharp injuries16.There are many ways to prevent occupational exposure to HIV. Healthcare workers should assume all body fluids are infectious and take precautions such as use of protective covering like gloves and goggles, wash hands and other skin areas right after contact with blood and body fluids, careful handling and disposal of needles and sharp instruments, use of available safety devices to prevent needle stick injuries. If an exposure does occur, induce bleeding at the site of a skin puncture by applying gentle pressure around the wound, rinse the area well with water for a skin or mucous splash, know the infected person\u2019s information, report to supervisor and coworkers, and seek immediate medical care17. Post-exposure prophylaxis for HIV is the use of antiretroviral medications for a brief period of time to lower the risk of HIV infection following potential occupational or sexual exposure. Post-exposure prophylaxis for HIV should be administered as part of a comprehensive universal precaution package in the health sector18. Occupational exposure to HIV and not using post-exposure prophylaxis cause occupational burnout among healthcare workers due to severe adverse psychological pressure, such as stress and anxiety. This could largely erode the quality of healthcare services and in turn increase the risk of injuries to healthcare workers. The healthcare system needs therefore identify the predisposing factors for high risk of exposure. Accordingly, this study was conducted to assess the prevalence of occupational exposure to HIV, post exposure prophylaxis uses and associated factors among healthcare workers at St. Peter\u2019s specialized hospital, Addis Ababa, Ethiopia.Post-exposure prophylaxis use is an important medical care to reduce the risk of HIV after occupational exposure occurred. Post-exposure prophylaxis provide 81% protection when started between 60\u00a0min and 72\u00a0h and followed for 28\u00a0daysA health facility-based cross-sectional study was employed in St. Peter\u2019s specialized hospital in Addis Ababa, Ethiopia. The hospital is located high in the Entoto mountain range, north of the city. The hospital was established in the era of Emperor Haile Selassie in 1948. Currently, the hospital has a total 936 healthcare workers .19, 95% confidence level, 5% level of significance, 5% margin of error.Sample size was calculated using single population proportion formula with the following assumptions: prevalence of occupational exposure to HIV in Dilla university referral hospital, southern Ethiopia\u2009=\u200976.1%n\u2009=\u200920. Post-exposure prophylaxis use is the administration of antiretroviral medication within 72\u00a0h of contact with potentially contaminated blood or other body fluids in order to reduce the risk of infection20. For low-risk HIV infections, a combination of Tenofovir (TDF)\u2009+\u2009Lamivudine (3TC) or Zidovudine (AZT)\u2009+\u2009Lamivudine (3TC) while for high-risk exposures triple therapy should be used i.e., Zidovudine (AZT)\u2009+\u2009Lamivudine (3TC)\u2009+\u2009Lopinavir (LPV), Zidovudine (AZT)\u2009+\u2009Lamivudine (3TC)\u2009+\u2009Atazanavir (Atv), Tenofovir (TDF)\u2009+\u2009Lamivudine (3TC)\u2009+\u2009Dolutegravir (DTG), Tenofovir (TDF)\u2009+\u2009Lamivudine (3TC)\u2009+\u2009Lopinavir (LPV) and Tenofovir (TDF)\u2009+\u2009Lamivudine (3TC)\u2009+\u2009Atazanavir (Atv)20.Occupational exposure to HIV, the primary outcome variable of this study was defined as any percutaneous injury and blood or other body fluids splash resulted while administering medications, specimen collection, and other procedures with HIV confirmed patients that may exposed healthcare workers to blood-borne pathogens24. The questionnaire was initially prepared in English and translated into Amharic version, and back translated to English language to check for consistency. The questionnaire consists of socio demographic characteristics and behavioral factors, exposure to HIV, and post-exposure prophylaxis use. The data collection process was facilitated by three BSc nurses. Data were collected after obtaining written consent from the study participants. Data collection facilitators checked completeness of the questionnaire upon return.Structured and pretested self-administered questionnaire was used to collect data. The questionnaire was adapted from published articlesp value (p\u2009<\u20090.25). Statistically significant associations in the multivariable model were identified on the basis of adjusted odds ratio (AOR) with 95% confidence interval (CI) and p values\u2009<\u20090.05. Model fitness was check using Hosmer and Lemeshow goodness-of-fit test.Data were entered to Epi-data version 3.1 epidemiological software and exported to Statistical Package for Social Sciences (SPSS) version 25 for further analysis. For most variables, data were presented by frequencies and percentages. We included predictors to the multivariable binary logistic regression model based on bivariate Ethical clearance was obtained from the Institutional Review Board of Addis Ababa Medical and Business College  and submitted to St. Peter\u2019s specialized hospital for permission. There were no risks due to participation and the collected data were used only for this research purpose with complete confidentiality. Written informed consent was obtained from the study participants. All the methods were carried out in accordance with relevant guidelines and regulations.From a total of 308 study participants, 298 of them returned the completed questioners with a response rate of 96.7%. The study participants were aged between 21 and 45\u00a0years and the mean (\u00b1\u2009SD) age was 30 (\u00b1\u20094.10) years. More than half, 158 (53%) of the study participants were married at the time of data collection and 151 (50.7%) of the study participants were Orthodox Christians by their religion. One hundred and nineteen (63.8%) of the study participants were male. Two-third, 198 (66.4%) of the study participants were Bachelor of Science degree holders and 108 (36.2%) of them were nurses. One hundred and fifty-three (51.3%) of the study participants had less than five years of work experience. One hundred and seventy-nine (60.1%) of the healthcare workers reported that they did not take on job infection prevention training (Table One hundred and twenty-six 42.3%)  of the healthcare workers included in the current study had occupational exposure to HIV during their career time, out of which 27.8% exposed once, 28.6% exposed twice, 36.5% exposed three times, and 7.1% exposed four times and above. Forty-four (35%) of the healthcare workers reported that they exposed to HIV at workplace in the last three months prior to the survey. Fifty-two (42.9%) and 39 (31%) of the healthcare workers experienced needle-stick injuries and blood splash exposures, respectively. Eighty-four (66.7%) of the healthcare workers exposed to HIV while giving injections and 67 (53.2%) of the healthcare workers exposed during recapping needles. Eighty-two (65.1%) of the healthcare workers exposed to HIV during the day-time work shift. Fifty-nine (46.8%) of the healthcare workers did not use personal protective equipment during exposures (Table .3% (95% Forty-eight (16.1%) (95% CI 11.9\u201320.3) of the healthcare workers used post-exposure prophylaxis. One hundred and sixty-seven (56.0%) of the healthcare workers reported that they did not take training on post-exposure prophylaxis and 99 (33.2%) of the healthcare workers reported that post-exposure prophylaxis is not available in their facility . Healthcare workers who received infection prevention training had 45% lower odds of occupational exposure to HIV risky conditions compared with healthcare workers who did not receive infection prevention training . Moreover, the odds of having occupational exposure to HIV risky conditions among nurses were 1.98 times higher compared with other healthcare workers  . Healthcare workers who had long work experience had higher odds to use post-exposure prophylaxis . Moreover, the odds of utilization of post-exposure prophylaxis was 3.41 times higher among healthcare workers who reported that post-exposure prophylaxis is available in their facility  (Table 25 and Nigeria (45.0%)26. The prevalence of occupational exposure to HIV in the current study is also lower than studies in Bule Hora General Hospital, Ethiopia (61.6%)20 and Tanzania (50.6%)27. On the other hand, this study finding is higher than studies in South Africa (10.6%)28 and Tanzania (35.1%)22. This high prevalence of occupational exposure to HIV risky conditions might be explained by poor safety system that includes lack of basic personal protective equipment, poor adherence to safety practices, and poor sharp waste management in the healthcare facilities. Furthermore, overuse of injectable therapy, suturing, recapping needles, bend or break needles, removing needles from syringes after injection, washing contaminated instruments, workload, working hastily, fatigue, crowded work environment may associate with needle-stick and sharp injuries that may result exposure to HIV. Moreover, 16.1%  of the healthcare workers who had occupational exposure to HIV risky conditions used post-exposure prophylaxis, which is in agreement with studies in Ethiopia (19.6%)29, Cameron (18.9%)17, and Tanzania (16.7%)27. However, this study finding is lower than findings of studies in west Guji zone of Ethiopia (24.3%)20, South Africa (58.8%)28, and Tanzania (26.4%)27. This low-level utilization of post-exposure prophylaxis might be explained by frequent stock-outs and continuous absence of post-exposure prophylaxis. In addition, some individuals who exposed to HIV risky conditions might perceived that their risks to HIV due to occupational exposure might be low.This hospital-based cross-sectional study was conducted to assess occupational exposure of healthcare workers to HIV and post-exposure prophylaxis use in St. Peter\u2019s specialized hospital, Addis Ababa, Ethiopia and found that 42.3%  of the healthcare workers exposed to HIV risky conditions during their career time and 16.1% (95% CI 11.9\u201320.3) of the exposed healthcare workers used post-exposure prophylaxis. The prevalence of occupational exposure to HIV in the current study is comparable with findings of studies in Gondar city, northwest Ethiopia (40.4%)30.This study revealed that occupational exposure to HIV risky conditions was associated with educational status. Healthcare workers with lower education level had lower odds of occupational exposure to HIV risky conditions. This might be due to healthcare workers who have higher educational status performing advanced surgical procedures which might increase their exposure to HIV risky conditions31. This might be due to the fact that infection prevention training is an effective strategy to develop knowledge and skills on safety measures and trained healthcare workers can protect themselves and other coworkers from work place injuries32.In the current study, occupational exposure to HIV risky conditions was significantly associated with infection prevention training. Healthcare workers who received infection prevention training had lower risks of occupational exposure to HIV. This study finding is in line with findings of other studies33. The possible explanations could be nurses, midwifes, and physicians had more frequent contact with patients during healthcare provision as well as they do invasive procedure like injection of medication, surgery, and delivery that increases their risk to healthcare associated infections34.This study found that occupational exposure to HIV risky conditions was statistically associated with field of study. For instance, nurses, midwifes, and physicians had higher odds of exposures compared with other healthcare workers such as public health officers, anesthetists, psychiatrists, radiologists, pharmacists, and optometrists). This finding is in agreement with a study in Serbia35. This could be due to the fact that healthcare workers at a higher educational level may have awareness about the mechanisms how post-exposure prophylaxis helps to prevent from HIV infection so that they may not have fear of side effects and could develop positive attitude. Moreover, healthcare workers at a higher educational level can identify risky conditions for acquiring HIV infection36.Furthermore, this study depicted that utilization of post-exposure prophylaxis among healthcare workers exposed to HIV risky conditions was significantly associated with educational status. Healthcare workers who had master\u2019s degree and above had higher odds of post-exposure prophylaxis use, which is in agreement with a study in Uganda35. This might be due to the fact that experienced healthcare workers might have information about advantage of post-exposure prophylaxis use over its side effects37. Moreover, as service year increases, the perceived vulnerability to infection might be high that may motivate healthcare workers to use prophylaxis when they are exposed to HIV risky conditions and long work experience might be associated with increased adherence to infection prevention strategies38.The current study reported that utilization of post-exposure prophylaxis was associated with work experience. Healthcare workers who had long work experience had higher odds to use post-exposure prophylaxis. This finding is in line with a study in Uganda41.This study also revealed that availability of post-exposure prophylaxis in the facility at the time when healthcare workers were exposed to HIV risky conditions was associated with higher odds of post-exposure prophylaxis utilization. The continuous availability of essential medicines within healthcare facilities plays an important role in promoting utilization of health services. On the other hand, frequent stock-outs of medicines have been shown to influence healthcare utilization. The continued absence of medicines in health facilities influences healthcare utilization and individual decisionsAs a limitation, the self-reported data may not be reliable since the study subjects may make the more socially acceptable answer rather than being truthful and they may not be able to assess themselves accurately. The study might be also affected by recall bias since we asked healthcare workers to recall occupational exposures during their career time. There might be also possibility of unmeasured confounders . Moreover, the variable of interest was not equally important for all departments. It might be more in some departments such as laboratory, emergency, delivery, etc.\u00a0However, we didn\u2019t address this variation in the analysis. The number of healthcare workers in each profession was not also proportional, even if random sampling was utilized. This study also included only one hospital data in Ethiopia. All these may affect the generalizability of study results.Significant proportion of healthcare workers included in the current study had occupational exposure to HIV and very few of them used post-exposure prophylaxis. This implies that occupational exposure to HIV in the studied healthcare facility is a great concern that may result exposure of healthcare workers to blood-borne pathogens. Healthcare workers need, therefore, use personal protective equipment , safely manage contaminated equipment and other items in the patient environment, and follow safety procedures during medication administration, specimen collection, and other procedures to protect themselves from HIV and other blood-borne pathogens including use of post-exposure prophylaxis when needed. Moreover, the health facility needs to strengthen the health and safety culture of the institution and availability of post-exposure prophylaxis."}
+{"text": "PD-L1 staining using long-stored paraffin sections may not be consistent with the true PD-L1 expression of patients. Therefore, it is necessary to explore the expression of PD-L1(SP142) in paraffin sections of invasive breast cancer with different storage times and the optimal storage temperature for unstained paraffin sections.The study included 71 cases of PD-L1(SP142) positive breast cancer. The unstained paraffin sections were stored at room temperature conditions (20\u201325\u00a0\u00b0C), 4\u00a0\u00b0C, -20\u00a0\u00b0C and \u2212\u200980\u00a0\u00b0C, respectively. PD-L1 staining was performed at 1, 2, 3, 4, 8, 12 and 24 weeks of storage. PD-L1 expression was assessed with a continuity score.The PD-L1 antigen was gradually lost as the storage time of paraffin sections increased. The PD-L1 positivity rate was 97.18% at 1 week for the sections stored at room temperature, and decreased from 83.10 to 71.83% for the sections stored for 2 weeks to 4 weeks, and 61.97%, 54.93%, and 32.93% for 8, 12, and 24 weeks, respectively. When stored at low temperatures of 4\u00a0\u00b0C, -20\u00a0\u00b0C and \u2212\u200980\u00a0\u00b0C, the positivity rate decreases with the same trend but more slowly compared to room temperature. The mean IC score of PD-L1 also showed a gradual decrease in all cases. In the consistency analysis, PD-L1 expression in slices stored at room temperature for 2 weeks was consistent with PD-L1 expression in fresh slices , and PD-L1 expression in slices stored at 4\u00a0\u00b0C or -20\u00a0\u00b0C for 4 weeks was consistent with PD-L1 expression in fresh slices . When stored under refrigeration at -80\u00a0\u00b0C, PD-L1 expression in slices stored for 3 weeks was consistent with that in fresh slices (ICC\u2009\u2265\u20090.9).To our knowledge, this is the first article on the effect of preservation time and preservation temperature of paraffin sections on PD-L1 expression in breast cancer. Long-term storage of paraffin sections of unstained invasive breast cancer can lead to antigen loss of PD-L1 (SP142). Refrigerated storage of paraffin sections can delay antigen loss, with best results at 4\u00a0\u00b0C or -20\u00a0\u00b0C, and a storage time of no more than 4 weeks is recommended.The online version contains supplementary material available at 10.1186/s13000-023-01423-8. Programmed death-1 (PD-1) inhibits T cell function and prevents tumor cell destruction through interaction with programmed death-ligand 1 (PD-L1) . Immune The results of immunohistochemistry (IHC) assays for biomarkers may be affected by a variety of factors. Earlier studies have shown that the use of long-stored paraffin sections for immunohistochemical staining can affect the accuracy of the test results , 11. ProThe degree of loss of antigenicity in paraffin sections can also be influenced by the conditions under which they are stored. Some studies suggest that the optimal storage conditions for unstained paraffin sections are not at room temperature \u201316. ManyFew articles have investigated the effects of preservation time and preservation temperature of paraffin sections on PD-L1 expression in breast cancer. Therefore, we aimed to explore PD-L1(SP142) expression in paraffin sections of invasive breast cancer stored for different times and the optimal storage conditions for paraffin sections. It is of significance for the precise diagnosis of invasive breast cancer patients and the quality control of PD-L1(SP142) IHC staining.Invasive breast cancer cases that underwent modified radical surgery from January 1 to February 29, 2020 were selected, totaling 211 cases. All underwent immunohistochemical staining for PD-L1 (SP142) and 71 positive cases were screened. All 71 cases were serially sectioned in 28 slices, and unstained paraffin sections were stored at room temperature conditions (20\u201325\u00a0\u00b0C), 4\u00a0\u00b0C, -20\u00a0\u00b0C, and \u2212\u200980\u00a0\u00b0C, respectively. PD-L1 (SP142) staining was performed at 1, 2, 3, 4, 8, 12 and 24 weeks of storage  using the OptiView DAB IHC detection kit, strictly following the manufacturer\u2019s instructions on benchmark XT automatic immunohistochemistry (IHC) .PD-L1(SP142) antibody was scored using immune cells (IC) positivity score, defined as the percentage of PD-L1 immune cells with any intensity of staining within the tumor area. Immune cells were cells in the intratumor and adjacent peritumor stroma, including lymphocytes, macrophages, dendritic cells and granulocytes, excluding IC staining in the vascular cavity. Tumor area is defined as the area occupied by tumor cells and their associated intratumoral and periatumoral stroma, excluding necrotic area and carcinoma in situ area . The conp\u2009<\u20090.05.Statistical analysis was performed using SPSS 23.0 and GraphPad Prism 8.0.1. Use two-way mixed consistency intragroup correlation coefficient (ICC) to evaluate the consistency of PD-L1 expression in paraffin sections at different times and conditions with fresh sections. The concordance was regarded as \u201cpoor\u201d, \u201cmoderate\u201d, \u201cgood\u201d and \u201cexcellent\u201d for the ICC values in , , , and , respectively . All tesA total of 71 patients were included in this study. The patients were all females with a mean age of 55 years (32\u201379 years). Histologic diagnosis was invasive ductal carcinoma in 67 cases (94.37%) and invasive lobular carcinoma in 4 cases (5.63%). Histologic grading was grade I in 2 cases, grade II in 49 cases, and grade III in 20 cases. There were 21 cases (29.58%) with tumor size less than 2\u00a0cm, 41 cases (57.75%) with tumor size between 2 and 3\u00a0cm, and 9 cases (12.67%) with tumor size greater than 3\u00a0cm. There were 42 cases of luminal type, 10 cases of HER-2 overexpression type, and 19 cases of triple-negative breast cancer. The clinical staging was 20 cases (28.17%) in stage I, 42 cases (59.15%) in stage II, 8 cases (11.27%) in stage III and 1 case (1.41%) in stage IV. PD-L1 was positively expressed in all cases. 43 cases (60.56%) had PD-L1 expression\u2009>\u20091 and \u2264\u20095, 16 cases (22.54%) had\u2009>\u20095 and \u2264\u200910, and 12 cases (16.9%) had\u2009>\u200910 . Storage of paraffin sections under refrigeration delays antigen loss.Our study found that storage at 4\u00a0\u00b0C or -20\u00a0\u00b0C worked best. However, in order to more accurately screen patients who can undergo PD-L1 immunotherapy, it is recommended that unstained paraffin sections be stained for PD-L1 (SP142) within 4 weeks.Long-term storage of paraffin sections of unstained invasive breast cancer can lead to antigen loss of PD-L1 (SP142). Refrigerated storage of paraffin sections can delay antigen loss, with best results at 4\u00a0\u00b0C or -20\u00a0\u00b0C, and a storage time of no more than 4 weeks is recommended.Below is the link to the electronic supplementary material.Supplementary Table 1: Clinicopathological Characteristics of patientsSupplementary Table 2: Consistency analysis of PD-L1 scores between sections stored at different temperatures and times and fresh sections"}
+{"text": "Morus alba leaf, MF) and Mori Cortex Radicis  have been studied for their anti-obesity effects by enhancing the browning process and inhibiting adipogenesis. However, important aspects of their protective mechanisms have not been thoroughly investigated, which could aid in developing functional food. Thus, this study aims to determine the synergistic effects of MF and MR against obesity and its associated mechanisms. In an in vitro cell culture model of brown adipocytes, a 1:1 mixture of MF and MR showed a synergistic effect on the expression of brown adipocyte-specific genes, including Ucp-1, Ppargc1a, Cbp/p300-interacting transactivator (Cited), Prdm16, Tbx1, and Fgf21 compared with either MF- or MR-treated conditions. Moreover, they demonstrated the involvement of cAMP and Ca2+ in induction of brown adipocyte-specific genes. In an in vivo model using HFD-fed mice, MF/MR significantly inhibited weight gain, plasma cholesterol, LDL, TG content, fat mass, and adipocyte size. Furthermore, MF/MR inhibited morphological alteration and the expressions of fatty acid synthesis genes such as Srebp1 and Fasn in the white adipose tissue. Thermogenesis genes were recovered in the brown adipose tissue with MF/MR supplementation, indicating that MF/MR regulated adipocytic dysmetabolism where AMPK signaling is involved. In conclusion, these results suggested that MF/MR regulates brown and beige adipocyte processes, providing one of the preventive functional food/herbal medicines against obesity and its associated metabolic diseases.Mori Folium ( The rising incidence of obesity is a global concern with profound implications for human health. Primarily caused by factors such as excessive food intake, lack of physical activity, and genetic susceptibility, obesity is linked to various metabolic diseases. These include cardiovascular disease, type 2 diabetes, hyperlipidemia, and hypertension, which not only compromise health but also increase the economic burden on patients. Beige/brown-like adipocytes have a thermogenic function. They express uncoupling protein 1 (UCP-1) in the inner mitochondrial membrane, which can dissipate the heat produced by the proton electrochemical gradient. Subcutaneous white adipocytes (WAT) are more responsive to browning agents than visceral WAT , show poMorus nigra L.), white , and red , followed by other species: Morus australis, Morus bomycis, Morus laevigata, Morus serrata, Morus macroura, Morus cathayana, Morus multicaulis, and Morus insignis [Morus alba L.  leaves and roots contain compounds with potential health benefits, such as antioxidants, vitamins, and minerals [Given these limitations, functional foods derived from natural products known for weight loss effects are gaining popularity due to their safety, low cost, and efficacy. Among these, the mulberry plant stands out. Its leaves, fruits, roots, and root barks are widely used in traditional Chinese medicine and cuisine for their excellent health benefits, particularly against obesity and associated metabolic disorders. Further, there are ecological reasons for mulberry use, including carbon sequestration and soil erosion prevention . There ainsignis . White mminerals .M. alba is rich in isoflavonoids, but the composition and concentration of isoflavonoids may vary with plant parts. The unique health benefits are influenced not by a single component of the natural product but by the combined effect of all of the components in the natural product. Naturally, all the constituents of natural products produce a biological effect that benefits health [M. alba has functional components, such as 1-deoxynojirimycin (1-DNJ), morin, moracin, and flavonoids, which contribute to its anti-obesity, antihyperglycemic and anti-inflammatory effects [Morus alba leaf, MF) and Mori Cortex Radicis  were evaluated separately. Additionally, the optimal combination of MF and MR at 1:1 was used to evaluate the in vivo effects on obesity in a high-fat diet (HFD) feeding animal model.s health . Althoug effects , each pl effects . TherefoMori Folium (MF) and Mori Cortex Radicis (MR) were provided by the Institute of Jinan Red Ginseng . The dried MF (5 kg) was pulverized with a blender and extracted with 50 L of distilled water at 80 \u00b0C under reflux for 3 h. Similarly, dried MR (5 kg) was pulverized with a blender and extracted with 50 L of 30% ethanol at 50 \u00b0C under reflux for 3 h. Filtered MF and MR were concentrated using a vacuum rotary evaporator  and lyophilized with a freeze dryer . The yield of MF and MR were 20.1% and 18.4%. The lyophilized MF and MR extracts were stored at \u221280 \u00b0C until use. Different solvents were selected for extraction based on preliminary tests and experience. The greater the number of sugar molecules attached to a compound, the greater its water solubility. The presence of sugar molecules in mulberry water led to its selection as one of the solvents. In addition, preliminary experiments demonstrated that 30% ethanol enhanced the extraction efficiency of mulberroside A. Consequently, 30% ethanol was used for this investigation.\u00ae HPLC system  equipped with a Zorbax Eclipse C18 analytical column with acetonitrile in solvent (A) and distilled water in solvent (B). The retention time of rutin , isoquercitrin , astragalin , mulberroside A , and cis-mulberroside A  were compared with those in MF and MR and their mixtures (MF/MR). Acetonitrile was used in solvent A and distilled water was used in solvent B. HPLC conditions used for the analysis of standards are shown in Standards and sample solutions were using Hitachi Lachrom EliteAntibodies against p-AMPK (#P-2535) and AMPK (#2532) were purchased from Cell Signaling Technology . Antibodies against UCP-1 (#Cat sc-293418), PGC-1\u03b1 (#Cat sc-518038), p-CREB (#Cat sc-81486), CREB (#Cat sc-271), PPAR-\u03b3 (#Cat sc-271392), C/EBP\u03b1 (#Cat sc-166258), p-HSL (#Cat sc-139656), HSL (#Cat sc-74489), FAS (#Cat sc-74540), SIRT-1 (#Cat sc-74540), SREBP-1c (#Cat sc-36553), and \u03b2-actin (#Cat sc-47778) were purchased from Santa Cruz Biotechnology . Compound C (CC), H-89, and BAPTA-AM were purchased from Sigma-Aldrich .2. 3T3-L1 preadipocytes were grown to 100% confluence under standard culture conditions (day 1). The cells were treated with differential medium containing 10% fetal bovine serum , 5 \u00b5g/mL insulin, and maintained in 10% FBS DMEM containing insulin for another 5 days (day 3\u20138). For induction of browning in 3T3-L1 adipocytes, they were treated with a medium containing 50 nM triiodothyronine  (day 1\u20133), and maturation medium was supplemented with 50 nM triiodothyronine and 1 \u03bcM rosiglitazone  (day 3\u20138), called browning media [3T3-L1 preadipocytes were purchased from the ATCC  and were cultured in DMEM , containing 1% penicillin/streptomycin, and 10% newborn calf serum , and cultured in a humidified incubator maintained at 37 \u00b0C with 5% COng media . ML and 3--2,5-diphenyltetrazolium bromide  assay was used to find out the effect of MF and MR on cell viability. Briefly, cells were treated with MF or MR (0\u2013100 \u03bcg/mL) for 24, 48, and 72 h in 96-well plates. Then, 100 \u03bcL MTT solution (5 mg/mL) was added to each well containing culture media and incubated for 4 h at 37 \u00b0C. Cells were then exposed to dimethyl sulfoxide (DMSO), and the absorbance was recorded at 540 nm using a microplate reader . The test was repeated three times.The animal investigations involved in the study were approved by the Institutional Animal Care and Use Committee (IACUC) of Jeonbuk National University Hospital (JBUH-IACUC-2021-14); 7-week old male C57BL/6 mice were procured from Orient Science Co. . Mice were housed and cared for by the following laboratory animal care guidelines . The micSerum insulin concentration was measured by using IDDM2 ELISA kit . Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, glucose, and lipids, including triglyceride (TG), total cholesterol (TC), and LDL-cholesterol (LDL-C) in the tissue, were analyzed using automated biochemical analyzer . The test was repeated three times.Histological and immunohistochemical analyses were performed as previously described . Briefly\u03b2-actin expression. The primers used in this study are listed in Total RNA from cells and tissue were isolated with TRIzol . Then, reverse transcription was performed with 2 \u00b5g of RNA with oligo dT primers. qRT-PCR was conducted with Power SYBR\u2122 Green PCR Master Mix . Quantification was performed by a comparative cycle threshold (Ct) method, and the resultant product was corrected for the amount of Immunoblotting was performed as reported earlier . The tisDXA scan was performed as described earlier . The perThe tissue sections were deparaffinized, xylene removed with ethanol, hydrated in graded alcohols, and placed in deionized water for 5 min. For immunostaining, antigen retrieval was conducted using 0.05% trypsin solution for 20 min at 37 \u00b0C, and the sections incubated in 3% hydroperoxide for 10 min and incubated overnight with primary antibodies against UCP-1  at 4 \u00b0C. After washing, sections were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody. The HRP-conjugated antibody was visualized with a 3,3\u2019-diaminobenzidine tetrahydrochloride (DAB) kit . The sections were counterstained with hematoxylin and imaged using an optical EVOS M5000 imaging system .p < 0.05 is considered significant. Data are shown as mean \u00b1 SEM.The statistical analyses were performed using GraphPad Prism version 8.0 . The data were determined using one-way analysis of variance (ANOVA) and followed by Tukey\u2019s multiple comparisons test, where To determine the non-toxic concentrations of MF and MR on 3T3-L1 cells, we treated the cells with various concentrations of MF and MR (0\u2013100 \u03bcg/mL) for 24, 48, and 72 h. Ucp1 and Ppargc1a , exhibited high expression under the browning condition  and browning cocktail agents such as 50 nM triiodothyronine and 1 \u00b5M rosiglitazone, referred to as browning medium (Browning M). We then treated the cells with MF, MR, or MF/MR at the ratios of 1:1, 1:2, and 1:3, followed by browning medium, shown by a scheme A. Thermoondition . Interesondition B,C. In pUcp1, Ppargc1a, Cited, PR domain containing 16 (Prdm16), T-box transcription factor (Tbx1), and Fibroblast growth factor 21 (Fgf21). The MF/MR mixture treatment showed a synergistic effect relative to the maximum concentration-treated MF or MR in a concentration-dependent manner (Considering that increased energy consumption and regulation of thermogenesis in mitochondria-rich brown adipose tissue (BAT) occur upon Ucp1 expression , we perft manner C. Furthet manner D. This pUCP-1 and PGC-1\u03b1 are known downstream effectors of AMPK-dependent metabolic signaling . Thus, A2+-based calcium/calmodulin-dependent protein kinase (CaMKK) are involved [To gain more insight into the activation mechanism of AMPK, in which cyclic AMP-linked PKA and Cainvolved , we appl2+ chelation. However, BAPTA-AM treatment did not influence the protein expressions in the presence of MF, indicating that the Ca2+ involvement is largely attributable to MR, not MF.Next, we assessed whether BAPTA-AM, a known cytosolic calcium chelator, would attenuate the effects of MF/MR on the expressions of p-AMPK, UCP1, and PGC1\u03b1. As expected, BAPTA-AM suppressed the MF/MR-induced activation of AMPK and its linked expressions of UCP1 and PGC1\u03b1 C. Similay = ax + b). The correlation coefficients (R2) of all compounds\u2019 standards were found to be greater than 0.99 . HPLC analysis revealed the patterns of rutin, isoquercitrin, and astragalin in MF and MF/MR . Furtherhan 0.99 . Using than 0.99 .To investigate the effect of MF/MR on obesity, we administered MF/MR to HFD-induced obese mice for nine weeks. After the dietary intervention, HFD-induced obese mice that had not received MF/MR showed a significantly higher weight gain than the NCD mice. However, obese mice supplemented with MF/MR had a lower weight than their counterparts without MF/MR supplementation A,B. In aIn eWAT, the diameter of adipocytes in HFD mice supplemented with MF/MR was lower than in HFD mice A,B. We t2+ and PKA, are suggested to contribute to the AMPK and UCP-1 signaling pathway, thereby facilitating adipocyte browning. These findings suggest that MF/MR modulates brown/beige adipocyte processes, potentially making it one of the functional foods that can regulate obesity and its associated metabolic disorders.Functional foods derived from natural products that promote health are safe, affordable, and have long-term health benefits. However, the selection of a natural product to treat a specific disorder is crucial. This study demonstrated that extracts of mulberry leaf, roots, and their mixture potentially protect against obesity by elevating thermogenesis. Additionally, the study showed that combining leaf and root extract was the most effective against obesity through adipocyte browning and thermogenesis. Specifically, MF/MR at a ratio of 1:1 significantly increased expressions of thermogenesis genes, and proteins, with CaUcp1 expression and its related signaling, reflecting its importance in the browning process in 3T3-L1 adipocytes  mice were not utilized to understand the mechanism completely. Second, no correlation study was conducted between MF/MR and endogenous components for in vitro and in vivo studies. In the near future, conducting a thorough investigation on the combined effect of MF and MR in knock-out mice is essential.In this study, different parts of the mulberry plant were used to produce extracts with various functional components, which were combined to exert a protective effect against obesity. Collectively, the study results suggest that a mixture of 1:1 MF and MR forms one of the functional foods that can prevent obesity and its associated metabolic diseases. Therefore, clinical trials are essential in the near future."}
+{"text": "Fungi are known to be older than plants, while plant-fungal interactions are as old as the evolutionary period of higher plants  in fungal tissues involves scavenging intracellular reactive oxygen species and sulfite assimilation. Tian et al. showed that Thioredoxin VdTrx1 is a virulence factor in Verticillium dahlia. The results further confirmed that VdTrx1 is necessary for the full virulence of V. dahliae on susceptible hosts. Mangrove-associated microorganisms have received increasing attention due to their important ecological roles and the wide range of services they provide to the environment and economy. Zhu et al. used high throughput sequencing of the internal transcribed spacer 2 (ITS2) to assess epiphytic and endophytic phyllosphere fungal communities of six true mangrove species and five mangrove associates. The results of this study highlight the important role of plant phylogeny in the assembly of epiphytic but not endophytic fungal communities in mangrove ecosystems. To understand the resistance mechanisms of the Ganoderma lingzhi response to Trichoderma hengshanicum infection, Wang T. et al. observed G. lingzhi transcript accumulation at 0, 12, and 24 h after T. hengshanicum inoculation. The results revealed that the down-regulation of differentially expressed genes led to the inhibition of heat shock protein (HSP) function, which compromises the HSP-mediated defense signaling transduction pathway, leading to the susceptibility of G. lingzhi. Miang, a traditional fermented food product produced from the leaves of Camellia sinensis var. assamica is found in the hill areas of Northern Thailand. Using the culture-dependent method, Kanpiengjai et al. investigated the yeast ecology of C. sinensis var. assamica tea flowers collected from six provinces in upper Northern Thailand. They characterized the tannin tolerance ability of the isolated yeasts. This study suggests that floral nectar supports the formation of yeast communities beneficial for Miang production. Paridis Rhizoma is a Chinese medicinal herb with strong anti-inflammatory and anti-tumor properties. Chen Y. et al. showed that the inhibitory effects of fermented Paridis Rhizoma extract (PRE) on liver cancer cells , cervical cancer cells (Hela), and lung cancer cells (A549) are stronger than that of the unfermented extract. Venturia carpophila, the causal agent of peach (Prunus persica) scab disease, mume (Prunus mume), and apricot (Prunus armeniaca), is widely distributed around the world. Zhou et al. carried out the genetic variation and population structure in V. carpophila isolated from peach, mume, and apricot in China. They found that the genetic identity of V. carpophila isolates depends on the host, not the geographic region. Morphological characteristics and multigene phylogenetic analyses are used as the latest techniques for fungal species identification. Tennakoon et al. introduced Montagnula acaciae, Paraconiothyrium zingiberacearum, and Paraphaeosphaeria brachiariae, as distinct new species from dead plant litter based on morphological differences and DNA sequence data. In addition, Montagnula jonesii, Paraconiothyrium fuckelii, Spegazzinia deightonii, and S. tessarthra were reported as new host records from Ficus benjamina, Dimocarpus longan, Hedychium coronarium, and Acacia auriculiformis respectively, for the first time. Furthermore, Paraconiothyrium archidendri and P. brasiliense were reported for the first time from Magnolia sp. in China, and Paraconiothyrium rosae was synonymized under Pa. fuckelii. Introducing exotic or non-native trees fails due to a lack of suitable fungal partners. Wang R. et al. showed that exotic P. radiata is a suitable tree capable of successfully getting established by interaction with the co-introduced L. deliciosus or local ectomycorrhizal fungi. However, care should be taken when large-scale planting of P. radiata is done. Plants of the genus Iris are widely cultivated because of their medicinal, ornamental, and economic values. However, it often suffers from Alternaria leaf spot or blight disease, leading to considerable losses for its commercial value. Gou et al. isolated 122 Alternaria strains in section Alternaria from diseased leaves of Iris spp. in 14 provinces or municipalities of China from 2014 to 2022. They introduced two novel and two known species that can induce leaf spots in Iris. In Sichuan province, Juglans regia, J. sigillata, and the hybrid J. regia \u00d7 J. sigillata are the commercially important walnut plants, while J. regia is the most widespread walnut plant. In order to update fungi associated with Sichuan walnuts, Xu et al. surveyed 15 representative regions in Sichuan. The survey revealed 10 fungi belonging to Dothideomycetes and Sordariomycetes that were described based on morpho-molecular analyses. Rhododendron, an essential ornamental plant, is abundant in Yunnan Province of China, and 61 species of Rhododendron have been reported from Cangshan Mountain in Yunnan. Gu et al. introduced six new fungal species isolated from fresh leaves of Rhododendron cyanocarpum, R. decorum, and R. delavayi at Cangshan Mountain for the first time. Ganoderma is a globally distributed genus covering ecological, medicinal, economic, and cultural species. He et al. used morphology and multigene phylogeny to identify 21 specimens of Ganoderma collected in the Yunnan Province of China, representing 18 species. In addition, a checklist and a key to Ganoderma in Yunnan Province were given in the paper. Drought stress is one of the major abiotic factors that limit plant growth and cause ecological degradation. To investigate the role of arbuscular mycorrhizal fungi (AMF) on reactive oxygen species (ROS) generation and ROS scavenging ability under drought stress in Bombax ceiba, Li et al. carried out an experiment. The results revealed that AMF inoculation could maintain ROS homeostasis by mitigating drought-induced ROS burst via decreasing ROS generation and enhancing the ROS scavenging ability of B. ceiba seedlings.Liu et al. used 86 Trichoderma isolates against Phytophthora nicotianae, Ph. capsici, Pythium vexans, Py. ultimum, and Py. dissotocum through dual culture assay and the results showed that the fungal strains exhibit strong antagonistic effects against oomycete pathogens, and those fungal strains can be integrated into disease management strategies. Multiple interactions happen between host plants and phyllosphere microbes, such as bacteria, oomycetes, and fungi. Wang K. et al. explored the interaction of Protomyces arabidopsidicola (isolated from phyllosphere), with Arabidopsis plant and found that Pr. arabidopsidicola strain C29 is pathogenic on Arabidopsis plant but can survive in its phyllosphere both in a controlled environment and under natural field conditions. Wood-associated fungi play a vital role in the degradation of wood and the recycling organic matter in forests. Luo and Zhao introduced a new fungal order Xenasmatales from Yunnan, China, based on morphology and multigene phylogeny to accommodate the family Xenasmataceae. In addition, Xenasmatella nigroidea and a key to Xenasmatella worldwide were provided. Colletotrichum, a widespread fungal pathogen, causes various plant diseases and Colletotrichum fructicola causes oil-tea (Camellia oleifera) anthracnose. Chen, Chen, Tan, He et al. selected eight candidate reference genes  from Co. fructicola. Camellia oleifera transcriptome data and evaluated and sequenced using geNorm, NormFinder, and BestKeeper algorithms. The results revealed that CfRrp has better stability in Co. fructicola, during the growth and invasion of different oil-tea leaves. Wheat (Triticum aestivum), an important cereal crop, is widely grown in temperate zones and higher elevations. Fusarium-head blight (FHB) is a critical wheat disease throughout the world. Tang et al. identified Fusarium avenaceum as the causative agent of FHB disease in Linzhi City, southeast of Tibet, China, based on morphology, multigene phylogeny, and pathogenicity test. As a result of an ongoing survey of microfungi associated with Magnolia grandiflora in Qijing, Yunnan, China, Wijayawardene et al. introduced three new species and five new records of saprobic fungi. Camellia oleifera (oil tea), mainly used for producing high-quality edible oil, is an important cash crop in China. Anthracnose of oil tea is a significant disease that limits the tea oil yield. Considering this fungal disease, Chen, Chen, Tan, Mo et al. reviewed the status of the harm, pathogen types, control measures, and pathogenic molecular mechanism of oil tea anthracnose, and this review provides essential information to control oil tea anthracnose. Ectomycorrhizal (ECM) fungi play a vital role in forest ecosystems. However, little is known about the bacterial and fungal communities associated with ECM roots. Zeng et al. surveyed the bacterial and fungal microbiome of ECM roots from stone oaks (Lithocarpus spp.) and Yunnan pines (Pinus yunnanensis) in the subtropical forests of the Ailao Mountains, Yunnan, China, and the results revealed that Rhizobiales and Acidobacteriales dominate bacterial community. In contrast, the fungal community is mainly composed of Russulales and Thelephorales. Microbial co-occurrence network analysis is commonly used for data exploration in plant microbiome research. To overcome oversimplified interpretation of the networks stemming from the stereotypical dichotomy between bacteria and fungi, Lee et al. recommend; understanding the dynamics and mechanisms of co-occurrence networks through generalized Lotka-Volterra and consumer-resource models, finding alternative ecological explanations for individual negative and positive fungal-bacterial edges, and connecting cross-kingdom networks to abiotic and biotic (host) environments. Fungi secrete various effectors to control host defense systems. Yang et al. identified necrosis- and ethylene-inducing protein 1 (Nep1)-like protein (NLP) effector gene, CgNLP1, which contributes to conidial germination, appressorium formation, invasive growth, and virulence of Co. gloeosporioides in rubber tree. A fairy ring is fungal fruiting bodies that occur as a ring on a spot. Wang Q. et al. showed the presence of abundant beneficial microbes driving the flourishing growth of plants in the fairy ring soil and provided bio-resources for agricultural growth-promoting agents.Plant diseases caused by oomycetes inflict severe damage to various crops; however, biocontrol of oomycete-related diseases is poorly done. In this regard, SK drafted the editorial. All authors contributed to editorial revision and approved the final paper."}
+{"text": "Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed.Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant E. cloacae complex\u201d by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR.Clinical isolates identified as \u201chsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies  and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-l-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10  exhibited similar CST susceptibility and PAP compared with mcr-negative isolates.Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens . arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of The online version contains supplementary material available at 10.1186/s12941-023-00610-1. Enterobacter cloacae complex (ECC) and Enterobacter aerogenes (currently as Klebsiella aerogenes) represent the most frequently isolated Enterobacter spp. in respiratory, urinary tract, and bloodstream infections  and the CST-heteroresistance  to comprehensively understand CST resistance in ECC clinical isolates.This study aimed to elucidate the relationship between the genetic features and CST resistance mechanism by using the integrated analysis with molecular epidemiology [E. cloacae complex\u201d by MALDI-TOF MS Biotyper Compass (software version 4.1.80.24), bacterial library version 8  in clinical laboratory testing. We also collected K. aerogenes  isolates (n\u2009=\u200930). These clinical specimens were respiratory , urine , blood , and others .ECC isolates (n\u2009=\u2009138) were derived from clinical specimens of different patients at Sapporo Medical University Hospital  from May 2017 to May 2018. These isolates were identified as \u201cCST, ampicillin, meropenem, gentamicin, and amikacin were purchased from FUJIFILM Wako Pure Chemical Corp. . Cefotaxime and ciprofloxacin were purchased from Tokyo Chemical Industry . Minimum inhibitory concentration (MIC)s were determined by the broth microdilution method using cation-adjusted Mueller\u2013Hinton II broth  according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) . Isolate600) after cultivation for 20\u00a0h under the same condition as used for the susceptibility test in the presence of CST (0\u2013128\u00a0mg/L). Turbidity was measured using an Infinite M200 PRO instrument . The skip-well phenomenon was defined as no growth (OD600\u2009<\u20090.1) at a certain CST concentration(s) and growth (OD600\u2009>\u20090.5) at higher CST concentrations. Data are expressed as the means\u2009\u00b1\u2009standard deviations from independent triplicate experiments.Bacterial growth was measured by assessing turbidity (OD9\u00a0CFU/mL) was centrifuged and resuspended in 1\u00a0mL of sterile 0.85% NaCl. One hundred microliters of tenfold serial dilutions (from 1\u2009\u00d7\u20091010 to 1\u2009\u00d7\u2009103\u00a0CFU/mL) of the suspension were spread onto cation-adjusted Mueller\u2013Hinton II agar  plates containing 0, 0.125, 1, or 8\u00a0mg/L CST and cultured for 20\u00a0h at 37\u00a0\u2103. The occurrence frequencies of the CST-resistant subpopulations were calculated by dividing the numbers of CFU on CST-containing MHII agar plates by those on plain MHII agar plates. CST heteroresistance was determined using a previously published method based on the following criteria [\u20137 to 1\u2009\u00d7\u200910\u20131) were at least eightfold higher (\u2265\u20091\u00a0mg/L CST) than the highest CST concentration that did not affect the growth of CST-susceptible subpopulations (0.125\u00a0mg/L CST). Data are expressed as the means\u2009\u00b1\u2009standard deviations from three independent experiments.Heteroresistance was determined by PAP as previously described . Brieflycriteria , 21: (1)hsp60 fragment was amplified by PCR with specific primers as described previously [hsp60 fragment were aligned in MAFFT [http://www.ebi.ac.uk/Tools/msa/mafft/). The phylogenetic tree was generated by MEGA X [The 273 nucleotides of eviously . The nucin MAFFT   and the Center for Genomic Epidemiology MLST 2.0 Web tool (https://cge.cbs.dtu.dk/services/MLST/).MLST was performed using ECC MLST databases in PubMLST.org , using bwa version 0.7.17. The core genome of the applied strain was extracted using mpileup of SAMtools version 1.9 and the \u201ccns\u201d option of VarScan version 2.3.9 [https://doi.org/10.1093/bioinformatics/btx713). DNA sequences were compared using Clustal W [https://www.genome.jp/tools-bin/clustalw). mcr genes were identified using assembled genome data by Resfinder (https://www.genomicepidemiology.org).Core genome SNPs-based phylogenetic analysis was performed with WGS data. MiSeq read data were mapped to the reference genome, on 2.3.9 , 28. A pon 2.3.9 . Genome lustal W  . Total RNA was extracted using a RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from total RNA (100\u00a0ng) using ReverTra Ace RT-qPCR Master Mix with Genomic DNA (gDNA) Remover (Toyobo). RT-qPCR was performed on LightCycler 480 II system  using KOD SYBR qPCR Mix (Toyobo) and primer pairs for phoPQ, eptA, and arnT were generated by the \u03bb red recombinase method using pKD46-hyg [Deletion mutants of KD46-hyg , 33. Min2-test. p\u2009<\u20090.05 was considered significant. Pearson\u2019s correlation coefficients were also calculated.Mann\u2013Whitney and a one-way ANOVA with the Kruskal\u2013Wallis tests were used to examine the significance of differences between two and multiple groups, respectively. Occurrence rates of phenotypes among groups were examined by \u03c7K. aerogenes isolates. Because K. aerogenes was formerly classified in genus Enterobacter, and CST-resistant K. aerogenes was reported [hsp60 clusters, and 30\u00a0K. aerogenes isolates were defined as unclassified  of ECC isolates were CST-resistant (MIC\u2009\u2265\u20098\u00a0mg/L), whereas none of CST-resistant isolates were observed in K. aerogenes.The MIC of CST was determined for the 138 ECC and 30\u00a0hsp60 cluster types significantly differed between CST-susceptible and -resistant isolates (p\u2009<\u20090.01)  Fig.\u00a0B. CST-re01) Fig.\u00a0. All iso600 of 0.2\u20130.6) in wells containing 0.5 or 1\u00a0mg/L CST and strong growth (OD600\u2009>\u20090.8) in wells containing higher CST concentrations (\u2265\u20092\u00a0mg/L)  (Table \u20137 (ranged from 1.6\u2009\u00d7\u200910\u20139 to 8.5\u2009\u00d7\u200910\u20138) in the presence of 1 and 8\u00a0mg/L CST. For all CST-resistant isolates, the occurrence frequency of CST-resistant subpopulations in the presence of 1 and 8\u00a0mg/L CST (more than eightfold higher than 0.125\u00a0mg/L) exceeded 1\u2009\u00d7\u200910\u20137 (ranged from 2.2\u2009\u00d7\u200910\u20136 to 7.1\u2009\u00d7\u200910\u20133 at 1\u00a0mg/L and from 1.3\u2009\u00d7\u200910\u20134 to 6.4\u2009\u00d7\u200910\u20132 at 8\u00a0mg/L). These results indicate that all CST-resistant ECC clinical isolates were CST heteroresistant.To analyze the CST-heteroresistant phenotype by PAP, we measured the occurrence frequencies of CST-resistant subpopulations in the presence of various CST concentrations Fig.\u00a0A. Growthp\u2009<\u20090.05)  and moderate 8\u201332\u00a0mg/L) MICs were significantly higher than those in CST-susceptible isolates  were found in certain hsp60 clusters  but not in other hsp60 clusters (p\u2009<\u20090.001)   Fig.\u00a0A, and in01) Fig.\u00a0B.Fig. 4Ehsp60 clusters I, II, IV, and XII, respectively. In antimicrobial susceptibility testing, 12 (31.6%) CST-heteroresistant isolates were resistant to cefotaxime and one was resistant to meropenem s, ST1939, ST125, ST997, and ST1953 were the dominant in hsp60 cluster were examined. Ten species were identified by ANI , E. kobei, and E. roggenkampii , E. kobei, E. ludwigii, E. mori, and E. soli  lacked the arn operon in their genomes according to annotation analysis, whereas other chromosomal CST resistance determinants  were present  and eight isolates , respectively  and the CST-susceptible strain SMEnt52  in the presence and absence of CST. CST induced arnT mRNA expression in SMEnt513, but not in SMEnt52  upon treatment with 16\u00a0mg/L CST for 45\u00a0min   in parent strains and eptA-deleted mutants in the presence of 8\u00a0mg/L CST, but were markedly decreased (<\u20091\u2009\u00d7\u200910\u20138) and below the criteria of heteroresistance (ranged from 1\u2009\u00d7\u200910\u20137 to 1\u2009\u00d7\u200910\u20131) in phoPQ- and arnT-deleted mutants  Fig.\u00a0A. All pharnT mRNA expression levels were increased more than eightfold (9.1\u201356.7-fold) in all CST-heteroresistant isolates, but less than fourfold in CST-susceptible isolates  , 11.3\u201313The CST-heteroresistant isolates were assigned to certain species and lineages similar to a previous French report . Howevermcr-9/10-positive isolates in various lineages regardless of CST susceptibility. Acquisition of the plasmid-mediated CST resistance genes mcr-9 and mcr-10 contributed little to CST heteroresistance in ECC because CST MICs and occurrence frequencies of CST-resistant subpopulations did not obviously differ between mcr-9/10-positive and -negative isolates. This observation strongly indicates that CST heteroresistance of ECC was mainly mediated by intrinsic genes, namely, CST-inducible arnT, not by exogenous genes.We found several E. asburiae ST1939 (hsp60 cluster I), E. kobei ST125 (hsp60 cluster II), and E. roggenkampii ST997 (hsp60 cluster IV) accounted for more than 40% of each species. E. kobei ST125 and E. roggenkampii ST997 have been clinically isolated in many countries [The STs of CST-heteroresistant isolates were diverse, but ountries \u201338. Thes\u20134 to 1\u2009\u00d7\u200910\u20132) by PAP did not display the skip-well phenomenon because this frequency exceeded the inoculating cell number used in the susceptibility test of 5\u2009\u00d7\u2009105\u00a0CFU/mL (5\u2009\u00d7\u2009104\u00a0CFU/100 \u00b5L in each well of a 96-well plate). Thus, monitoring the skip-well phenomenon was an unreliable method to detect CST heteroresistance in ECC clinical isolates. PAP is a gold standard for detecting heteroresistance [600) in the presence of various CST concentrations should be convenient method for screening CST-heteroresistant isolates in routine clinical laboratory. It did not require any additional experiments other than those performed to determine CST MICs by the broth microdilution method , E. kobei, and E. roggenkampii. Contribution of this pathway has been shown in a reference strain E. cloacae subsp. cloacae ATCC 13047 [arnT induction occurred in the presence of CST at a concentration around the MIC. Thus, CST-heteroresistant phenomenon, namely decreased bacterial growth in the presence of 0.5 or 1\u00a0mg/L CST, was attributed to inadequate (or unresponsive) arnT induction at lower CST concentrations (<\u20092\u00a0mg/L), while induced ArnT overcame the bactericidal activity of CST at higher CST concentrations (\u2265\u20092\u00a0mg/L). These findings suggest that the appearance of CST-resistant subpopulations in a strain displaying CST heteroresistance reflects the ability of arnT induction by CST.Why do CST-resistant subpopulations appear? The present study demonstrated that the PhoPQ-CC 13047 . In addiarnT pathway in CST-resistant isolates of certain ECC species, such as E. roggenkampii, E. kobei, E. chuandaensis, E. cloacae, and a part of E. asburiae. On the other hand, no CST-resistant isolates were found in E. hormaechei and E. ludwigii. Therefore, attention is required to be paid to CST heteroresistance at all infection sites of these ECC species, which have a common intrinsic gene background, such as species, hsp60 clustering, MLST, and/or core genomic features. Exogenous mcr genes, such as mcr-9 and mcr-10, found in ECC clinical isolates [This study demonstrates that CST-heteroresistant ECC is present in clinical specimens from various infection sites. Inducible CST heteroresistance is mediated via the PhoPQ-isolates , 39, 40 Additional file 1: Table S1. Sequences of primers used in this study. Table S2. Characteristics of ECC and K. aerogenes clinical isolates used in this study. Table S3. Characteristics of CST-heteroresisatnt ECC clinical isolates. Table S4. CST MICs of none-mcr, mcr-9-positive, and mcr-10-positive ECC isolates. Table S5. Comparison of CST susceptibility of phoPQ-, eptA-, and arnT-deleted mutants of different ECC species.Additional file 2: Fig. S1. Contribution of mcr-9 and mcr-10 to CST heteroresistance of ECC clinical isolates. A Comparison of the occurrence frequencies of CST-resistant subpopulations among none-mcr, mcr-9-positive, and mcr-10-positive ECC clinical isolates. There was no significant difference among the groups. B\u2013G Comparison of the occurrence frequencies of CST-resistant subpopulations between mcr-9-positive and -negative B\u2013D and between mcr-10-positive and -negative E\u2013G isolates. These isolates were selected from the same node of the core genome phylogeny. PAP was performed with the following CST concentrations: 0.125 (B and E), 1 (C and F), and 8 (D and G) mg/L. There were no significant differences among the groups. a, E. hormaechei subsp. xiangfangensis."
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