Eclipta prostrata is an herbal medicinal plant belonging to the Asteraceae family. In this study, complete chloroplast genome sequence of the E. prostrata was characterized by de novo assembly using whole genome sequence data. The genome of E. prostrata was 151,757 bp in length, which was composed of large single copy region of 83,285 bp, small single copy region of 18,346 bp and a pair of inverted repeat regions of 25,063 bp. The genome harboured 80 protein coding sequences, 30 tRNA genes and 4 rRNA genes. We confirmed close taxonomic relationship between E. prostrata and Helianthus annuus through phylogenetic analysis with chloroplast protein-coding genes. Eclipta prostrata is an annual herbal plant species belonging to the Asteraceae family, which is mainly distributed in tropical and subtropical regions in the worlds . The extracted DNA was used to construct Ilumina paired-end (PE) genomic library and sequenced using an Illumina MiSeq platform  in Lab Genomics Co.  . High quality PE reads of ∼1.4 Gb were assembled by CLC genome assembler  according to the previous study . The draft sequence was manually corrected by PE read mapping. The genes in the chloroplast genome were annotated using the DOGMA program  was 151,757 bp in length, which was composed of large single copy region of 83,285 bp, small single copy region of 18,346 bp and a pair of inverted repeat regions of 25,063 bp, as typical chloroplast structure. A total of 114 genes were predicted in the genome, including 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes.The complete chloroplast genome of E. prostrata and related 10 species belonging to the Asteraceae family. The phylogenetic tree showed that E. prostrata was grouped with other two species in Heliantheae tribe, in which E. prostrata was placed more closely to Helianthus annuus (sunflower)  .


The epidemic of obesity is contributing to the increasing prevalence of people at high risk of cardiovascular disease (CVD), negating the medical advances in reducing CVD mortality. We compared the clinical and cost-effectiveness of an intensive lifestyle intervention consisting of enhanced motivational interviewing in reducing weight and increasing physical activity for patients at high risk of CVD.A three-arm, single-blind, parallel-group randomised controlled trial was conducted in consenting primary care centres in south London. We recruited patients aged 40–74 years with a QRisk2 score ≥20.0%, which indicates the probability of having a CVD event in the next 10 years. The intervention was enhanced motivational interviewing which included additional behaviour change techniques and was delivered by health trainers in 10 sessions over 1 year, in either group (n=697) or individual (n=523) format. The third arm received usual care . The primary outcomes were physical activity (mean steps/day) and weight (kg). Secondary outcomes were changes in low-density lipoprotein cholesterol and CVD risk score. We estimated the relative cost-effectiveness of each intervention.At 24 months, the group and individual interventions were not more effective than UC in increasing physical activity , reducing weight  or improving any secondary outcomes. The group and individual interventions were not cost-effective at conventional thresholds.Enhancing motivational interviewing with additional behaviour change techniques was not effective in reducing weight or increasing physical activity in those at high CVD risk. Cardiovascular disease (CVD) remains the leading cause of mortality.Psychological processes are important in initiating and maintaining change to healthier lifestyles. One approach is to use motivational interviewing, which is a collaborative, goal‐oriented behaviour change technique that encourages the language of change.The primary aim was to compare the effectiveness and cost-effectiveness of enhanced motivational interviewing delivered by health trainers in increasing physical activity, reducing weight in people at high risk of CVD over 24 months was greater in those who received it in a group format compared with individual format or with usual care (UC).This was a three-arm, parallel-group randomised controlled trial (RCT), called MOVE IT , for individuals at high risk for CVD using a partially clustered design followed-up at 12 and 24 months from baseline. The three arms were: enhanced motivational interviewing in a group format, enhanced motivational interviewing in an individual format and UC. As participants of the group arm, but not the other two arms, were clustered within groups, we had a partially clustered design. The protocol is published online.General practices with list sizes >5000 patients in 12 south London boroughs  representing a varied urban, socioeconomic and ethnically diverse population of approximately 3 million.Potentially eligible participants were identified from patient record databases and invited for screening. The inclusion criteria were: aged ≥40 and ≤74 years; CVD 10-year risk score of ≥20.0% calculated using QRisk2 , which is a validated predictive tool for identifying the percentage risk of having a fatal or non-fatal cardiovascular event in the next 10 years2); participating in a weight loss programme or another participant, already randomised, in the same household.The exclusion criteria were: medical diagnosis of CVD; having a pacemaker; diabetes, kidney disease, atrial fibrillation or stroke; chronic obstructive pulmonary disease; disabling neurological disorder; severe mental illness; registered blind; housebound or resident in nursing home; unable to move about independently; more than three falls in past year; pregnancy; advanced cancer; morbid obesity (body mass index (BMI) >50 kg/mSimple randomisation of participants was conducted by an independent Clinical Trials Unit (King’s College London) using computer-generated randomisation blocks. In each block, 10 subjects were randomised to group, individual or UC arms in a 4:3:3 ratio. The unequal allocation ratio ensured that the group arm had approximately 33% more patients to compensate for the loss of power from any clustering effect.We collected sociodemographic factors, such as age, gender, self-report ethnicity, occupational status, educational attainment and marital status. Biomedical data included weight , height (measured to 0.1), BMI (kg/m²), waist and hip circumferences (cm), blood pressure , glycated haemoglobin and fasting lipids. Lifestyle data collected were alcohol intake (Alcohol Use Disorders Identification Test (AUDIT)),For UC, this consisted of referrals to locally commissioned community-based weight loss, smoking cessation and/or exercise programmes.The theoretical framework for enhanced motivational interviewing was based on social cognitive theory, and the theory of planned behaviour which states that to change behaviour, people need to form an intention (cognition).The intervention was manualised and consisted of 10 sessions over 12 months delivered by health trainers. Participants received a workbook, key learning points for every session, action planning worksheets, case studies, self-monitoring diaries and a pedometer with instructions on its use. The intensive phase consisted of 6 weekly sessions during the first 3 months focused on physical activity and diet. The maintenance phase consisted of four sessions delivered at 3, 6, 9 and 12 months.The training consisted of 8 weeks of didactic learning, role-playing, group exercises and case discussions using standardised materials on motivational interviewing and behaviour change techniques drawn from cognitive behaviour therapy. Each health trainer’s competency was assessed at the end of training via a knowledge test and through observing delivery of two sessions.The primary outcomes were physical activity (average number of steps/day) and weight (kg) and the secondary outcomes were low-density lipoprotein (LDL) cholesterol and QRisk2 score, measured at 12-month and 24-month follow-ups and adjusted for baseline values.The EQ-5D-3L was used to generate quality-adjusted life years (QALY). Intervention costs were calculated taking into account staff time delivering the sessions and the unit costs included overheads and on-costs and accounted for the ratio of direct-to-indirect contact time. We assumed the unit cost/hour of a National Health Service band 3 clinician at £32.40. For the group intervention, the costs were apportioned over attendees. Other service costs were measured by combining service use data from the Client Service Receipt Inventory with unit costs.The primary analyses were aligned with Consolidated Standards for Reporting Trials. Baseline characteristics of those who did and did not provide follow-up data was described.Analyses based on an intention-to-treat principle using all available outcome data were used to estimate the differences 12 and 24 months using mixed-effects adjusted for the baseline value of the outcome. In the linear mixed model ‘treatment arm’, ‘time’ , the ‘interaction between treatment arm and time’, ‘borough’, ‘ethnicity’, ‘age’, ‘gender’ and the ‘baseline values’ of the outcome variable were fixed factors.The random parts of the models were ‘GP practice’ (patients are nested in practices) and ‘therapy group’. The study’s design was complex as it is partially clustered and cross-classified. In the preliminary analyses with blinded data, the model did not converge, therefore we removed the random effect for therapist from the analysis. In order to model the dependency of the repeated observations of the same subjects at 12 and 24 months, we model the covariance between the residuals within the lowest level group ‘patients’ to be correlated by using an unstructured covariance pattern model.A two-tailed α of 2.5% for the two main comparisons ‘group versus UC’ and ‘individual versus UC’ was used and 97.5% CIs are presented. All secondary hypotheses were assessed on a 5% α level with 95% CIs.Our analysis model assumes that data are missing at random with conditions for variables predictive of missingness. We compared baseline characteristics of those with and without complete physical activity and weight follow-up data. Models were rerun with predictors related to outcome missingness included as further covariates. Fourteen sensitivity analyses adjusting for the influence of missing data, protocol violations and potential model misspecifications were conducted for the primary outcomes. Group comparison of skewed data were performed using the median test. STATA V.14 was used for the primary and secondary analyses.We used bootstrapping methods to estimate 95% CIs around the mean cost differences. QALYs were calculated from the EQ-5D-3L. Area under the curve methods calculated the QALY gain over the follow-up period and QALY differences were analysed controlling for baseline EQ-5D-3L score. If costs were higher for one arm compared with another and QALY gains were greater, we constructed an incremental cost-effectiveness ratio (ICER) to show the cost per extra QALY gained. Uncertainty around cost and QALY estimates was explored using cost-effectiveness planes generated from 1000 bootstrapped resamples. Finally, we generated cost-effectiveness acceptability curves, using the net-benefit approach and bootstrapping, to assess which of the three approaches was the most cost-effective. The range of values used was £0–£100 000, including the guidance-based threshold of £20 000–£30 000.We selected a conservative difference of 0.25 pooled SD in the main outcomes, which translates to a mean clinical difference (MCD) between two groups of 675 steps/day, 1.25 kg and 0.25 mmol/L total cholesterol.On 18 October 2016, a university-wide IT network outage occurred leading to loss of 95 24 months accelerometer data files and 2651 audiotaped intervention sessions. We repeated the accelerometer data for 87 (91.6%) participants and retrieved 395 (14.9%) sessions from other computer drives.The funder of the study had no role in study design, data collection, analysis, interpretation or manuscript preparation. The corresponding author had full access to all the data and final responsibility for submitting for publication.Of the 455 general practices invited, 135 (29.7%) consented; there was no difference in the general practices which did and did not consent.Overall, 1220 participants were randomised to either the group or individual intervention; 28.2% did not start the intervention, 17.3% started but did not complete the intervention and 54.5% completed the intervention. The three most common reasons for participants not starting or completing the intervention were being too busy (27.4%), unable to contact (16.0%) or no longer interested in participating (15.5%). Participants in the individual arm attended more sessions  than those in the group arm . The online supplementary material provides further details of intervention delivery.Fidelity to the manual was high, with the majority of participants setting targets at each session (91.6%) and the majority of these targets were achieved =13.39, p=0.001). Data were collected for 91.6% of participants for at least one of the 12-month or 24-month follow-ups, and for 79.7% of participants at both follow-ups. Participants missing 24-month outcome data walked significantly less at baseline (6376.9 (2497.2) vs 6833.1 (2752.5) steps per day, t=−2.78, p=0.006), were more likely to be current smokers =6.4, p=0.041), to have no formal qualifications =12.3, p=0.002) and to be more depressed (PHQ-9 score of 2.47 (3.87) vs 1.89 (3.05), t=2.44, p=0.015). There were no other baseline differences between participants with missing data compared with those with data. The loss to follow-up for both primary outcomes at 24 months was 18.4%, 19.7% and 11.9% for the group, individual and UC arms, respectively. The differences in loss to follow-up between the treatment arms were significant at 24 months follow-up (χWe did not observe any treatment effects for the secondary outcomes at 12 or 24 months .Service costs (including zero costs for non-users) were similar for inpatient care, outpatient attendances and community contacts were similar between arms . The intThe group arm was less effective than UC and more expensive; as such it was dominated. Individual was more expensive and more effective. The ICER was £55 313 per QALY (£354 divided by 0.0064 QALYs). The ICER of the individual arm compared with the group arm was £8287 per QALY (£179 divided by 0.0216 QALYs) . At a vaF(2)=0.68, p=0.51)) or in the number of fatal or non-fatal cardiovascular events (Five hundred twenty-three adverse events (AEs) were reported between baseline and 24-month follow-up. There were no differences in mean (SD) number of AEs experienced by participants in the group, individual and UC arms (0.37 (0.61), 0.33 (0.54) and 0.35 (0.57), respectively,  because the medical records required for its algorithm were not always accurate resulting in high levels of ineligibility. Our sample may not have represented all those at higher CVD risk as the average QRisk2 score was at the lower end.One explanation for the negative finding is that our sample was skewed to non-modifiable risk factors in the QRisk2 algorithm , and on average not obese with a lower than expected CVD risk score.A second potential explanation is that the intervention potency was subtherapeutic. For this clinical group, namely patients with a high CVD risk but with few psychological or physical symptomatic distress, the motivational interviewing approach is inappropriate. For example, the prevalence for significant depressive symptoms was very low (1.4%), lower than the general population.Landmark studies have repeatedly shown that intensive lifestyle instruction, such as the diabetes prevention studiesFirst, this intervention might have been more successful in those with modifiable CVD risk factors rather than the QRisk2 score alone and if we had much shorter waiting lists. The potential of an enhanced motivational interviewing approach to a younger population, those living in deprived areas and of non-white ethnicity remains unknown. We used the same strategies for recruitment regardless of socioeconomic, ethnicity and inner city status and future studies could instead oversample in these subgroups to recruit those at higher CVD risk.Second, we may need to consider more intensive approaches to supporting lifestyle change in those most at risk of CVD. For instance, psychological constructs such as optimistic bias and habit formation are common patterns.An intensive lifestyle intervention using enhanced motivational interviewing skills was not associated with reduced weight or increased physical activity in people at high risk of CVD. Future interventions should focus on those at very high CVD risk and/or with modifiable risk factors.The expert-by-experience patient participants  contributed to the design, conduct, reporting, and dissemination of our research.Although mortality from cardiovascular disease (CVD) is falling, the epidemic of obesity and unhealthy lifestyles is increasing the risk of CVD.There is little evidence as to whether a primarily psychological approach that aims to change a person’s intentions (cognitions) can alter lifestyle behaviour.This study was a response to a National Institute of Health Research commissioned brief to test whether enhanced motivational interviewing  delivered by health trainers could improve lifestyles and thus reduce CVD risk.We found that enhanced motivational interviewing did not lead to a reduction in weight or increase in physical activity compared with usual care.We conclude that enhanced motivational interviewing have little impact for reducing CVD risk in people at high CVD risk when applied to the general population.This raises the question as to whether the administration of low intensity psychological techniques in lifestyle-related interventions are of any clinical benefit.


Laboratory strains, cell lines, and other genetic materials change hands frequently in the life sciences. Despite evidence that such materials are subject to mix-ups, contamination, and accumulation of secondary mutations, verification of strains and samples is not an established part of many experimental workflows. With the plummeting cost of next generation technologies, it is conceivable that whole genome sequencing (WGS) could be applied to routine strain and sample verification in the future. To demonstrate the need for strain validation by WGS, we sequenced haploid yeast segregants derived from a popular commercial mutant collection and identified several unexpected mutations. We determined that available bioinformatics tools may be ill-suited for verification and highlight the importance of finishing reference genomes for commonly used laboratory strains. The frequent transfer of genetic materials between life science organizations introduces opportunities for quality control issues. Genetic mutations accumulate naturally over time, and human errors in labeling and sample preparation are unavoidable. Anecdotally, it is not uncommon for researchers to complain of samples exhibiting unexpected behaviors, only to later discover that the genetic material they’re working with is not as expected.4. Despite a growing awareness, cell-line contamination and misidentification are persistent problems, particularly in mammalian cell research9. Comparably, much less attention has been paid to the potential for similar issues in non-mammalian samples. Yet even commonly used plasmids have been shown to vary dramatically from their published sequence10. The problem of plasmid verification has been addressed through the development of a web-based application for assembly of Sanger sequencing reads and alignment of the assembled plasmids with a reference11. Strain verification by a similar method will be orders of magnitude more challenging.Laboratory strains, cell lines, and mutant collections exhibit considerable nucleotide variation and background mutations even among lines thought to be isogenic13. These approaches are limited to particular regions of the genome or are insufficiently sensitive for capturing many types of sequence variations15.The methods currently used to verify samples/strains are biased towards a particular goal. For instance, diagnostic techniques such as PCR, targeted sequencing, or restriction enzyme-based methods are often used to identify whether or not a marker gene or known sequence variant is present, or for analysis of variable repeat regions such as in 16 s rRNA profiling16. Given reports that the life sciences are facing a reproducibility crisis17, it is more important than ever for researchers to verify the samples and strains they work with.In addition to wasted time and reagents, undetected genetic variation can lead to severe consequences including delays in publishing or patenting and misplaced conclusions that result in product recalls or retractions13. Many tools have been developed for assembling sequenced genomes and detecting variants by aligning sequencing reads to a reference genome18, but these tools have been largely developed and validated using human sequencing data. The same tools may not perform well when analyzing microbial sequencing data due to differing ploidy, genome size, and mutation rates19. The applicability of assembly and, especially, variant calling tools to microbial sample and strain verification has not been thoroughly explored.As the cost and turnaround time of next generation sequencing continues to decrease, sample and strain verification by whole genome sequencing (WGS) is becoming a more feasible approachIn order to identify the practical obstacles that must be overcome to ultimately implement WGS as a regular part of genetics workflows, we used haploid yeast strains with an unexpected phenotype derived from a mutant collection as a test case.20 to produce cln3∆ mbp1∆ double mutants in S. cerevisiae. mbp1 and cln3 knockouts are each individually known to result in an increased critical cell size at the start of S phase23. When the cln3∆::kanMX and mpb1∆::natMX mutant lines were crossed, half of the double mutant progeny had a wild-type G1 cell size. When we examined the cln3 mutant strain, it too had a wild-type-like cell size. Three of the cln3∆ mbp1∆ double mutant segregants were sequenced using an Illumina MiSeq sequencer: one segregant, 1691, exhibited the unexpected wild-type-like phenotype, the others, 1693 and 1694 (which exhibited the mutant phenotype) were used for comparison.As part of a series of yeast cell cycle experiments, we crossed two mutant lines from a knockout collectionde novo and against the S288C reference genome. (B) Variant finding tools were used to call single nucleotide polymorphisms (SNPs) that varied between strains 1691 and 1693. (C) Copy number variant tools were used to confirm the presence of strain-specific deletions and marker gene insertions (INDELs) and check for additional structural variations.We conducted three different sequence analyses Fig. : (A) Rea19, we tested both approaches. For each stage of the analysis, at least two different popular bioinformatic tools were tested. We selected tools partly based on Pabinger, et al.’s survey of 205 tools18. We tested additional tools to those described herein, but only results from the best performing tools were included in the manuscript. Known and unknown variants were confirmed visually .Because variant calling via genome alignment and variant calling via mapped reads can result in different types of errorsAn ideal approach to sample verification by WGS would be to sequence the sample, assemble the genome, and then compare the assembled genome to the exact reference genome (the genetic background plus any known variations). Unfortunately, there is not a finished reference genome available for the genetic background used in our analysis (BY4741), despite the fact that it is a commonly used laboratory strain. We thus conducted reference-based assemblies using the closely related S288C genome.de novo has a number of additional advantages. For instance, reads that do not align to the reference, such as those for a marker gene insertion or transgene, might be trimmed from the analysis. As such, we repeated the assemblies de novo.Analyzing a genome assembled 24 .Cost is a major barrier to using WGS for sample verification and cost directly relates to coverage. In this study, strains were sequenced on a MiSeq for a total of ~2 million, 250 bp paired-end reads, corresponding to a predicted 80x coverage of the 12 Mb yeast genome. Coverage metrics produced with Picard tools show that ~95% of genome was covered with at least 30 reads in all three samples , and by marker gene insertions at CLN3 and MBP1 , on the one hand, complicate analysis by WGS, but, on the other hand, emphasize the importance of frequently verifying strains, because the genome is a living dynamic structure, not a rigid set of permanent instructions.The main shortcoming in the described workflow was its inability to resolve repetitive regions such as transposons. Tools for finding transposons specifically have been developed, but are not routinely employedMET15 and LYS238. Both were derived from Saccharomyces cerevisiae strain FY2 which is a direct descendent of S288C. Both BY4741 and BY4742 are known to differ from S288C by the deletion of four auxotrophic markers. According to a recently prepared draft genome, BY4741 additionally differs from S288C by fewer than 5 SNPs per 100,000 bp27.The mutant collection from which the parental strains used in this study were obtained was generated using background strains BY4741 and BY4742, which differ only in their mating type and the auxotrophic markers CLN3 ORF replaced with a marker for G418 resistance (MATacln3∆::kanMX MBP1). The other has the MBP1 ORF replaced with a marker for nourseothricin resistance by marker switching of the commercial deletion strain (MATα CLN3 mbp1∆::natMX)47. These strains were crossed to yield the haploid progeny we analyzed by whole-genome sequencing. The list of strains used in this study is reported in Table One of the haploid parents obtained from the collection has the http://broadinstitute.github.io/picard/). In addition, the percentage of aligned reads versus total reads was in the range of 97–98% for all three samples.Each of the strains was sequenced on a Miseq for a total of ~2 million, 250 bp paired-end reads, corresponding to a predicted 80x coverage of the 12 Mb yeast genome. Coverage metrics produced with Picard tools show that ~95% of genome was covered with at least 30 reads in all three samples (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), Trimgalore! , cutadapt (https://cutadapt.readthedocs.io/en/stable/), bwa (http://bio-bwa.sourceforge.net), Picard (https://broadinstitute.github.io/picard/), ENSEMBL (http://ensembl.org), CNVnator (https://github.com/abyzovlab/CNVnator), Breakdancer (http://breakdancer.sourceforge.net/), cnv.kit (https://cnvkit.readthedocs.io/en/stable/), Samtools (http://www.htslib.org/doc/samtools.html), BCFtools (https://samtools.github.io/bcftools/bcftools.html), Genome Analysis Toolkit (GATK) (https://software.broadinstitute.org/gatk/), snpEFF (http://snpeff.sourceforge.net/), SOAPdenovo2 (http://soap.genomics.org.cn/soapdenovo.html), SPAdes (http://cab.spbu.ru/software/spades/), and QUAST (http://bioinf.spbau.ru/quast), BLAST (http://doi.org/10.1186/1471-2105-10-421). Default parameters were used unless otherwise noted.The following software tools were used in the described analysis: FastQC , corresponding to strain S288C (baker’s yeast), using BWA (v. 0.7.15) with default parameters. Alignment quality of the resulting bam files was assessed using Picard (v.2.9).CNVnator (v0.3.3) was used for structural variant calling with a bin size of 20 and 100. In addition, copy number variation was assessed with CNVkit (v0.9.3) and Breakdancer (v. 1.3.6).CNVkit was run with automatic binning and with p-value threshold of 0.000005 for accepting segments and their breakpoints. Copy numbers were called with log2 ratio thresholds of −1.0000000, 0.5849625, 1.3219281, 1.8073549, and 2.1699250 for copy numbers 0 to 4, with thresholds being the upper limits of log2 coverage ratio for each copy number. Thresholds were calculated by adding 0.5 to the integer copy number value for rounding, dividing by ploidy (1), and log2 transforming the result.Breakdancer was run with default parameters and configuration files generated using script bam2cfg.pl, included in its distribution.Variant calling was run both with the GATK pipeline and a pipeline consisting of Samtools (v1.8) and Bcftools (v.1.8).—sample_ploidy set to 1.The GATK pipeline was built on GATK version v4.beta.5, except for function CombineGVCFs, which was run with GATK version v3.8, as there was no working version of this function in GATK 4 at the time of setting up the analyses. GATK was run with default parameters and using GATK HaplotypeCaller for calling variants with —ploidy set to 1 and using multi-allelic calling mode (Bcftools flag -m).Variant calling with Samtools and Bcftools was run with 48.Variants called with GATK and Bcftools were annotated using snpEFF (v.4.3 T), a software for variant annotation and predicting effects. Samples aligned to R64-1-1 were annotated using a database for strain S288C provided by snpEFF authors. Samples aligned to BY4741 were annotated using snpEFF database custom built on annotation files downloaded from Saccharomyces Genome DatabaseDe novo assemblies were annotated by BLASTing assembled contigs against the reference genome of strain S288C. In addition, variant calling was performed using our de novo assembled genome as a reference. Called variants were annotated by BLASTing their flanking sequences against the reference genome of S288C to find corresponding gene annotations. Function blastn of BLAST toolbox (v2.7.1+) was run with following parameters: -outfmt 6 -max_target_seqs. 1 -max_hsps 1 -num_threads 10 -strand plus.De novo assemblies were performed with SOAPdenovo2 (v. 2.04) and SPAdes (v. 3.9.0), with SPAdes used for downstream analyses and assembly by subsampling. Reference-based de novo assembly, with S288C chromosome sequences used as trusted contigs for i.a. gap closure and repeat resolution, with and without subsampling was also performed with SPAdes (v. 3.9.0).Qualities of all assemblies were assessed using QUAST, with the reference genome of strain S288C used for benchmarking.Supplementary Information.


Various studies have excavated the potential prognostic biomarkers for cancer patients based on gene expression profiles. However, most of these reported biomarkers lack independent validation in multiple cohorts. Herein, we collected 35 datasets with long-term follow-up clinical information from TCGA (2 cohorts), GEO (32 cohorts), and Roepman study (1 cohort), and developed a web server named OSluca  to assess the prognostic value of genes in lung cancer. The input of OSluca is an official gene symbol, and the output web page of OSluca displays the survival analysis summary with a forest plot and a survival table from Cox proportional regression in each cohort and combined cohorts. To test the performance of OSluca, 104 previously reported prognostic biomarkers in lung carcinoma were evaluated in OSluca. In conclusion, OSluca is a highly valuable and interactive prognostic web server for lung cancer. It can be accessed at  Lung cancer (LUCA) is an aggressive disease with leading mortality and incidence in the world. Based on histology, there are two types of LUCA, including non-small cell lung cancer (NSCLC), which accounts for 80% of LUCA and small cell lung cancer (SCLC), which accounts for approximately 20% of LUCA . NSCLC cKRT8  allP-value and HR  with the built-in upper 25% cutoff. The R package “forestplot” was used to produce the forest plot for inputted gene in OSluca web server.Online consensus Survival for Lung Cancer is built in a tomcat server as previously described with minor modifications . BrieflyKeywords including “lung cancer,” “survival,” “biomarker,” and “prognosis” were used to search biomarkers of lung cancer in NCBI PubMed. We finally obtained 104 prognostic biomarkers using the following criteria : (1) immP ≤ 0.05 was considered statistically significant.The association of lung cancer clinical factors and survival outcomes was analyzed by GraphPad Prism 8.0 software. The Cox proportional hazards regression and Kaplan Meier plot functions from R package “survival” were used in the OSluca to determine the association between gene expression and survival. The P < 0.0001), stage (P < 0.0001), p-TNM stage (P < 0.0001), and smoking status (P < 0.0001) . TCGA comprises 513 lung adenocarcinoma cases and 499 squamous cell carcinoma cases , 2. GEO  0.0001) . Besides 0.0001) .TP53 (tumor protein p53) as an example and type “TP53” into the gene symbol box and click on “Survival analysis”  is significantly overexpressed in tumor tissue and could be as a poor prognostic biomarker for lung adenocarcinoma  (DDIT3 gene (KLF15 (Krüppel-like factor 15) had been proven to be higher in tumor tissue than that of adjacent non-tumor tissue and played an important role in promoting proliferation and carcinoma diversification in lung adenocarcinoma, associated with poor prognostic outcome  = 0.4 (0.28–0.58)]. In addition, the remaining 12 of 104 previously published prognostic biomarkers (11%) were not significant for prognostic analysis in the Top 9 cohorts in OSluca, but 8 of them (8/12) are significant in one or multiple datasets other than the Top 9 cohorts in OSluca (data not shown). All in all, the OSluca server is an interactive and free web server for researchers to develop potential prognostic biomarkers for lung cancer.A search for lung cancer biomarkers was performed using a set of keywords in NCBI PubMed, including “lung cancer,” “survival,” “biomarker,” and “prognosis.” In total, we collected 104 published lung cancer prognostic biomarkers verified by IHC or qPCR  to evaluarcinoma . The pro0 cases) , except 0 cases) . Next, e0 cases) . Meanwhi0 cases)  of publiIT3 gene . In contIT3 gene , suggest outcome . It was pression . The OSlis tools , such asOwing to tumor molecular heterogeneity, the prognosis of lung cancer patients is variable and difficult to predict. The prognosis of patients suffering from lung cancer had been demonstrated to be highly dependent on clinical factors of the patient, such as histological type, smoking status, and so on. However, it is also an imperative need to exploit novel prognostic biomarkers for determining the risk of cancerous lesions and predicting lung cancer patient outcomes by all available means, especially by high-throughput sequencing technologies. However, one major challenge to non-bioinformatics researchers is how to integrate the high-dimension profiling datasets of lung cancer and discover new biomarkers to potentially guide prognostic stratification. Previous studies had revealed that the online prognostic web servers of cancer  could suhttp://bioinfo.henu.edu.cn/LUCA/LUCAList.jsp.The advantage of OSluca over other online prognostic web servers is that the size of lung cancer samples in OSluca is large, and tens of independent cohorts are available, which is extremely valuable for the identification and validation of cancer prognostic biomarkers, since the most important part for the biomarker development is independent validation across different datasets/cohorts. The limitation of the current study is that OSluca can only test a single gene for outcome analysis. In summary, OSluca is a free web server for non-bioinformatics researchers to study potential lung cancer prognostic biomarkers, accessed at The datasets generated for this study can be found in the TCGA, NCBI GEO, and Roepman dataset.XG: research design. QW and XG: establish OSluca web server. ZY, ZL, and XS: deal with RNA sequencing with clinical data of lung cancer. ZY, LX, XS, LZ, YL, and XG: draft of the manuscript. YD, XS, LZ, PS, YL, TX, and JM: collect previously reported biomarkers of lung cancer. ZY, LX, LZ, WZ, YZ, and XG: critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterotrigona itama were analyzed for their plant sources, phenolic compositions, and antioxidant activities. The honey samples were acetolyzed and identified microscopically, and the phenolic compounds were identified by using HPLC-DAD. The antioxidant activities were evaluated using three different assays  by spectrophotometry. The melissopalynological analysis showed that 26 pollen types from 14 plant families were identified in the honey. Cocos nucifera and Rhizophora mucronata presented as predominant pollen. A total of 6 phenolic acids such as catechin, chlorogenic acid, epicatechin, protocatechuic acid, p-coumaric acid, and rutin were identified. Rhizophora mucronata honey possessed the highest antioxidant activity in all assays. The result showed the influence of plant sources on the phenolic compounds and the antioxidant properties of stingless bee honey. These findings could be significant contributions for the sustainability of stingless bee industry as well as to promote Malaysian stingless bee honey worldwide.Six honey samples produced by the stingless bee  Apis spp.) and stingless bees (Trigona spp.) from nectar and honeydew [Honey is the most popular and most recognized natural food produced by both honeybees  was expected to provide new alternative species that could supplement the current beekeeping projects from services . Among tApis spp. honey [The complexity of stingless bee honey composition is the major challenge in honey research, thus the profiling and understanding of honey produced from a different region are important. By doing so, the honey from predominant sources gained a lot of interest because of their specific phenolic profile related to the botanical origin . Honey'sp. honey . Thus, tH. itama) in Johor, Malaysia.Taking all these aspects into consideration, the present study was carried out to investigate the pollen compositions, phenolic compounds, and the antioxidant activity of honey produced by the stingless bee species  and a Shimadzu pre-column C-18 ODS. To analyze the phenolic acids, the elution method consisted of 5% formic acid (solvent A) and MeOH (solvent B). The conditions for elution were 0.01–15 min 20%–30% B, 15–20 min 30% B, 20–30 min 30%–40% B, and 40–50 min 100% B, at a flow rate of 0.8 mL/min. All solvents used were of the standard HPLC grade. The wavelength of 280 nm was employed for monitoring. The detection of phenolic compounds was carried out by comparing the retention time with reference levels to the analytes. Phenolic compounds  were obtained from Sigma (St. Louis) and used as standards of reference.Stingless bee honey samples were prepared and extracted using ethyl acetate, as stated by Aljadi and Yusoff . With so50 (mg/mL) and DPPH activity index (RSA), which was expressed as the measurement of the percent inhibition as follows:The free radical scavenging activity of the honey samples was determined with modification by the assay 1,1-diphenyl-2-picrylhydrazyl (DPPH), as defined by Abu Bakar et al. . Initial+ was synthesized by reaction of a solution of 7 mM ABTS with a solution of 2.45 mM potassium persulfate. The mixture was kept in the dark for 16 h at room temperature. The ABTS+ solution was then diluted with distilled water until an absorbance of 0.7 was reached at 734 nm. Aliquots of 2.0 mL from the ABTS+ solution were applied to the sample solutions diluted in methanol (MeOH) immediately after preparation to achieve final concentrations between 0 and 100 mg/mL. After 10 min, the percentage inhibition was calculated for each concentration at 734 nm and the ABTS+ radical scavenging potential (RAS) was determined using the following equation:The ABTS test was carried out according to the methods of Re et al. . The cat3+-TPTZ) in the presence of antioxidant to its ferrous, colored form (Fe2+-TPTZ). The FRAP reagent was prepared by mixing 2.5 mL of a solution of 10 mM TPTZ  in 40 mM HCL, 2.5 mL of 20 mM FeCl3, and 25 mL of a buffer of 0.3 M acetate at 3.6 pH. It was prepared daily and warmed before use until 37°C. An aliquot of 200 μL of honey solution was mixed with 1.5 mL of FRAP reagent, and reagent absorbance was measured spectrophotometrically at 593 nm after 10 min incubation. For the calibration curve, ferrous sulphate (Fe2+) was used, and the result were expressed as milligrams of Fe2+ equivalent per 100 grams of honey.The reduction in honey samples was calculated with slight modification based on the method defined by Benzie and Strain . The theP value of ≤0.05 was considered statistically significant, and level of confidence was 95%.Data were analyzed by using the Statistical Package for Social Sciences, version 22.0, software . The central tendency of variables was presented by the mean ± standard deviation (SD). One-way ANOVA was used to detect the differences between the continuous variables of the plant sources, phenolic compounds, and antioxidant activities. A Melissopalynological analysis was used to determine the possible botanical sources of honey, where pollen, spores, and hyphae found in honey were identified. This approach has revealed the plant origin and geographical origin of the foraged nectar in the honey and thus the plants that visited by stingless bees and the consistency of honey .Cocos nucifere (Arecaceae) in K02 honey and Rhizophora mucronata (Rhizophoraceae) in K03 honey were identified as predominant pollen, which are considered as unifloral honey. According to Agashe and Caulton [The present study showed a wide variability of pollen grains in honey, which represent their plant sources.  Caulton , uniflorRhizophora mucronata (Rhizophoraceae) and Cocos nucifera (Arecaceae) were the predominant pollen type represented 50.0% of all pollen in K03 honey and 66.7% in K02 honey, respectively. The secondary pollen types Acacia mangium (Fabaceae), Robinia pseudoacacia (Fabaceae), and Moringa pterygosperma (Moringaceae) were especially prevalent in K01 honey at 89.8% of all pollen, whereas in K04 and K06 honey, the pollen types Syzygium jambos (Myrtaceae), Cocos nucifera (Arecaceae), and Antigonon leptopus (Polygonaceae) comprised 19.0%, 21.0%, and 20.0% of pollen, respectively. The frequency of pollen in K05 honey was also poorly presented, which only contains the important and minor pollen. Moreover, the pollen types of Asystasia intrusa (Acanthaceae), Mikania cordata (Asteraceae), Acacia mangium (Fabaceae), Erythrina orientalis (Fabaceae), and Cocos nucifera (Arecaceae) found in more than one honey sample indicate that these pollen types are main attractive sources for bees either for nectar or pollen source.H. itama species within the foraging range of the hives. The Fabaceae family was represented by the seven species Acacia mangium, Robinia pseudoacacia, Peltophorum pterocarpum, Erythrina orientalis, Calliandra portoricensis, Mimosa scabrella, and Mimosa bimucronata, and these species were present in K01, K04, and K05 honey at high frequency. The diversity of Fabaceae as a largest plant family found in tropical rainforest such as Malaysia might attract the H. itama bees to visit them either for collecting nectar or pollen that contribute to the honey production. As compared with other countries, the abundance of pollen from Fabaceae in Brazil's pollen analysis proved the importance of this family for bees in the production of honey [of honey .Cocos nucifera, Elaeis guineensis, and Nypa fruticans, and these species were present as predominant pollen in K01 honey, important pollen in K03 honey, and minor pollen in K02 honey, respectively. Cocos nucifera is an important cultural, perennial plant known as the source of nectar and pollen for bees [Eleais guineensis does not produce nectar, the bees typically only visit it for their pollen [Cocos nucifera and Rhizophora mucronata from the family of Rhizophoraceae were predominant pollen types in K03 honey, which have been recognized as good honey plants [Bidens pilosa and Mikania cordata, which both belong to Asteraceae, were presented as important and minor pollen types. Moreover, Antigonan leptopus (Polygonaceae), which presented as secondary pollen in K06 honey, is a good source of both pollen and nectar for bees. It's a species of flowering plants that have a prolonged blooming period throughout the year [Besides Fabaceae, the Myrtaceae family also produced nectar and pollen, which played a good role in honey production, presented in four of the six honey samples. Arecaceae was represented by the three species for bees . While Er pollen . Pollen r pollen . Cocos ny plants . Bidens the year .H. itama bees. However, due to several factors, the bees can change their trophic niche during the year; examples involve floral abundance within a vegetative zone where hives are located, as well as climatic condition. Therefore, the variability of floral sources visited by bees might produce various types of honey, which influence the composition of phytochemical as well as biological activity such antioxidant capacity [The melissopalynological analysis of the present study revealed a variety of pollen grains collected by capacity .Phytochemicals are a wide class of nutraceuticals found in plants that are studied extensively by scientists for their potential for health promotion. Honey is known to have a large variety of phenolic compounds including phenolic acids and flavonoids, which act as antioxidant agents. The analysis of phenolic compounds was seen as a very promising way of investigating honey's floral and geographical origins. All these compounds are mostly the result of secondary plant metabolism and are characterized by several phenolic groups associated with more or less complex structures. The phenolic composition of honey depends mainly on its floral origin, which is suggested as a tool for classification and authentication, in particular for unifloral varieties . MoreoveH. itama honey. Chlorogenic acid (CLA) and p-coumaric acid (PCA) were present abundant in honey samples. These compounds have been previously reported in monofloral Malaysia honey from Apis spp. [o-coumaric acid (OCA) and quercetin (QH) did not appear in any of honey samples under investigation.High-performance liquid chromatography (HPLC) was performed to investigate the phenolic compounds in the honey samples. The HPLC chromatograms show the identification of six phenolic compounds in pis spp. . In contpis spp. . Therefopis spp. . In contApis spp. produced Tualang honey contains gallic acid, trans-cinnamic acid, p-coumaric acid, benzoic acid, and caffeic acid. Recently, Ranneh et al. [p-coumaric, apigenin, and 4-hydroxybenzoic as major compounds. Compared with others countries, phenolic compounds such as 3,4-dihydroxy, gallic, vanillic, and trans-cinnamic were detected in M. subnitida honey from Brazil by Silva et al. [C-pentosyl-C-hexosyl-apigenin, and kaempferol were identified in M. beecheii honey from Cuba [In Malaysia, there is very limited literature reporting the analysis of polyphenolic compounds in stingless bee honey. A study made by Ahmed and Othman  reportedh et al.  investiga et al. . There arom Cuba . The preIt may be noted based on the present analysis that the investigation of the phenolic compounds in stingless bee honey has recently been documented and has been growing, with a great variation in their composition. In fact, the location of the apiaries and the geographical origin in which the bees collect the nectar will influence the phenolic composition of the honey . OverallH. itama honey from different botanical origins in methanol extract ranged from 210.11 ± 0.11 to 413.94 ± 0.56 mg GAE/100 g and 179.36 ± 0.12 to 53.90 ± 1.30 mg RE/100 g, respectively [Because of several health benefits along with their crucial role in preventing oxidative rancidity of various foods, antioxidants are considered essential bioactive compounds. Three antioxidant assays  were used in this study to determine the antioxidant capacity of honey from different botanical origins. Each method has its own mechanism and uniqueness. DPPH and ABTS assays were performed to test the water-soluble antioxidant in different methods to determine heterogeneity in most techniques used, whereas FRAP assay was conducted to reflect the evaluation of lipid-soluble antioxidants . Basicalectively . These f50 value (mg/mL) in various concentration. Basically, the high DPPH scavenging behaviors are correlated with high antioxidant activity rates. Results obtained in our study showed that in methanolic extract, K03 honey from predominant pollen of Rhizophora mucronata and multifloral K04 honey had the lowest IC50 value (23.89 mg/mL) and the highest IC50 value (130.62 mg/mL), respectively  and expressed in ICectively . This suce versa .50 value of tested honey samples in water extract is lower as compared with methanolic extract, and the result showed the same trend. K03 honey displayed the highest level of antioxidant with an IC50 value of 34.45 mg/mL, while K04 honey remain the lowest with an IC50 value of 170.40 mg/mL. All findings were significantly different (P < 0.05). The methanolic extract displayed greater radical scavenging capacity than the water extract, which could be due to the reaction of methanol in various plants and tissues that produce different compound ranges than water. Moreover, this result shows that unifloral honey displayed a higher level of antioxidant potential than the multifloral honey. These findings are highly consistent with those stated by Chua et al. [50 value of Tualang, Acacia, and Gelam honey were 48.90 mg/mL, 29.85 mg/mL, and 15.68 mg/mL, respectively. Meanwhile, a higher IC50 value in the range of 3.17 to 8.79 mg/mL has been reported by Pontis et al. [In contrast, the ICa et al. , who fous et al.  for BrazP < 0.05) in the scavenger activity of ABTS free radicals. Like DPPH assay, K03 honey and K02 honey again showed the highest percentage of ABTS inhibition with a value of 65.02% and 67.77%, respectively, compared with the other samples, indicating that these unifloral honey types possess high antioxidant potential than multifloral honey. In terms of AEAC, all tested honey varied from 16.29 to 45.30 mg AEAC/100g and 10.94 to 37.47 mg AEAC/100g in methanol and water extract, respectively. Such values were comparable with those recorded in Malaysian raw honey that has 14.23 to 26.64 mg AEAC/100g and Czech honey that has14.15 to 40.71 mg AEAC/100 g [The principle of ABTS scavenging assay is similar to DPPH scavenging assay, which involve the scavenging activity of free radicals. The sample's antioxidant activity was determined based on the sample's capability to transfer an electron to ABTS radical cation, through the decolorization of the radical cation. The ABTS scavenging ability of the extracts were expressed as the percentages of ABTS scavenging (% RSA). The result showed that all tested honey samples had higher percentages of inhibition in methanolic extract than the water extract. This pattern of the result was in line with DPPH assay because both assays have the same concept, and the results for both assays should also be consistent with similar trend. AC/100 g . The difAC/100 g .3+) to a blue color solution containing ferrous ion (Fe2+). This method is a simple and direct test that is widely used in many different substances, including honey, for the determining antioxidant capacity [P < 0.05) in the scavenger activity of FRAP. The average FRAP value of honey samples were 665.23 ± 396.14 μM Fe (II)/100g in methanolic extract and 581.37 ± 313.47 μM Fe (II)/100g in water extract, respectively, with a significant difference between them (P < 0.05). As with DPPH and ABTS assays, the current result showed the same trend where K03 honey exhibited the highest FRAP value of 1401.80 ± 5.18 and 1175.80 ± 2.38 μM Fe (II)/100g in both of methanol and water extract, respectively. K07 honey displayed the lowest FRAP value in methanol extract, while in water extract, the lowest value was exhibited by K04 honey. These results showed a contradiction where K07 honey displayed the lowest value in both DPPH and ABTS assays. This inconsistency may indicate the existence of antioxidant lipid-soluble components, which should be taken into account when assessing the honey's antioxidant potential. Compared with previous research, the current findings were in line with Alves et al. [μM Fe (II)/100g), arbutus (1321.8 μM Fe (II)/100g), and eucalyptus (953.1 μM Fe (II)/100g). In another study, Kishore et al. [μM Fe (II)/100g, which is lower than that in our findings. These variations can occur in the honey due to different concentrations of phenolic compounds and flavonoids [The principle of FRAP assay involves the reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) containing ferric ion  and Rhizophora mucronata (Rhizophoraceae) pollen types, which classified them as unifloral honey. However, K01, K04, K05, and K06 are considered as multifloral honey, which contain multiple types of pollen grains at low density. Six phenolic compounds were detected in the tested honey samples, which were found in various combinations of chlorogenic acid, epicatechin, p-coumaric acid, rutin, catechin, and protocatechuic acid. When evaluating antioxidant function, the highest value of antioxidant assays  were observed for the Rhizophora mucronata honey. This finding has shown that the plant sources can influence honey's phytochemical composition and antioxidant activity. Thus, the present study provides a new insight into food preference of H. itama species, which is important for colony sustainability and promotes the meliponiculture industry in this region.Studies have demonstrated that K02 and K03 honey presented high frequency of the


The prognosis is very poor for lung cancer patients with bone metastasis. Unfortunately, a suitable method has yet to become available for the early diagnosis of bone metastasis in lung cancer patients. The present work describes an attempt to develop a novel model for the early identification of lung cancer patients with bone metastasis risk.As the test group, 205 primary lung cancer patients were recruited, of which 127 patients had bone metastasis; the other 78 patients without bone metastasis were set as the negative control. Additionally, 106 healthy volunteers were enrolled as the normal control. Serum levels of several cytokines in the bone microenvironment  and bone turnover markers  were detected in all samples by ECLIA or ELISA assay. Receiver operating characteristic (ROC) curves and multivariate analyses were performed to evaluate diagnostic abilities and to assess the attributable risk of bone metastasis for each of these indicators; the diagnostic model was established via logistic regression analysis. The prospective validation group consisted of 44 patients with stage IV primary lung cancer on whom a follow-up of at least 2 years was conducted, during which serum bone biochemical marker concentrations were monitored.P < 0.05).The serological molecular model for the diagnosis of bone metastasis was logit (p). ROC analysis showed that when logit (p) > 0.452, the area under curve of the model was 0.939 . Model validation demonstrated accuracy with a high degree of consistency . The average predictive time for bone metastasis occurrence of the model was 9.46 months earlier than that of the bone scan diagnosis. Serum OPG, PTHrP, tP1NP, β-CTx, and the diagnostic model logit (p) were all positively correlated with bone metastasis progression (This diagnostic model has the potential to be a simple, non-invasive, and sensitive tool for diagnosing the occurrence and monitoring the progression of bone metastasis in patients with lung cancer. Lung cancer patients are prone to developing bone metastasis accompanied by bone-related events such as bone pain, fracture, hypercalcemia, and nerve compression, which seriously affect patient quality of life , 2. ClinIn this study, the serum concentrations of BME cytokines  and bone turnover markers (tP1NP and β-CTx) were analysed in 205 lung cancer patients, and these bone biochemical markers were comprehensively analysed in relation to bone metastasis in lung cancer. The diagnostic values were assessed and a serological molecular model was established to provide an experimental basis for the early diagnosis and progression monitoring of lung cancer bone metastasis.This study was approved by the Ethics Committee of the East Campus of Shanghai Sixth People’s Hospital, affiliated with Shanghai Jiaotong University. All participants in the study provided written informed consent prior to recruitment.Patients with stage IV primary lung cancer that were admitted to the East Campus of Shanghai Sixth People’s Hospital from October 2015 to August 2016 were enrolled. After excluding patients who had received chemotherapy, radiotherapy, immunotherapy, bisphosphonate therapy, or other treatment within 3 months prior enrolment and those with endocrine disorders or immune or metabolic diseases, a total of 205 patients were enrolled in the study. Follow-up was conducted until October 2018, with a minimum follow-up period of 2 years. Of the enrolled patients, 127 (bone metastasis group) had bone metastasis, and the other 78 (non-bone metastasis group) did not have bone metastasis, as determined on the basis of pathology or bone scans. Bone scan examination was performed every 6 months to monitor the progression of bone metastasis in all patients. In addition, 106 healthy volunteers were recruited as normal controls. The control group was age- and gender-matched with the test group. The clinical characteristics of the patients and healthy volunteers are shown in Supplementary Table The prospective validation study included 44 patients with stage IV primary lung cancer who underwent a complete surgical resection between October 2015 and August 2016 and were followed until October 2018, for a minimum of 2 years. All patients had been diagnosed with non-bone metastasis by pathology or bone scan at the initial presentation. Bone scan examination was performed every 6 months to monitor bone metastasis in all patients. The clinical characteristics of 44 patients, including age, gender, histological type, and stage are shown in Supplementary Table g for 10 min, and then the serum samples from the individuals were transferred to new tubes in 500 μl aliquots and stored at − 80 °C.A total of 2–4 ml of fasting venous blood from patients and healthy volunteers was collected in the morning and left at 25 °C for 30 min. The supernatant serum was obtained by centrifugation at 4000 The serum concentrations of tP1NP (03141071190), β-CTx (11972308122), and IL-6 (05109442190) were quantified using a Diagnostics Cobas E601 fully automated electrochemiluminescence immunoassay (ECLIA) (Swiss Roche) and its original reagents. The inter-assay precision CVs of tP1NP, β-CTx, and IL-6 were less than 3.2, 4.7, and 3.9%, respectively. The intra-assay precision CVs of tP1NP, β-CTx and IL-6 were less than 8.7, 5.7, and 5.1%, respectively.The serum concentrations of CaN (BYS10821B), OPG (BYS10849B), and PTHRP (BYS10753B) were measured quantitatively by an enzyme-linked immunosorbent assay (ELISA) kit . All of the methods were performed in accordance with the manufacturer’s instructions. The optical densities (OD) at 450 nm and 622 nm were detected with a microplate reader , and the repeatability CV was less than 15%.P < 0.05 was considered statistically significant.MedCalc software  and GraphPad Prism  were used for statistical analysis and mapping. The serum levels of bone biochemical markers of the two groups were compared with the nonparametric Kruskal Wallis test. Spearman correlation analysis was used to assess the correlation between the serum concentrations of these indicators. An ROC curve analysis was used to evaluate the diagnostic value of each of these indicators for bone metastasis in lung cancer. The optimal Youden index method was used to determine the cut-off value of these indicators for the diagnosis of bone metastasis. In addition, univariate and multivariate logistic regression analyses were performed to assess the risk attributed by each of these markers for the development of bone metastasis in lung cancer and to establish a serological molecular model for the diagnosis of bone metastasis in lung cancer. The Hosmer-Lemeshow test and an ROC curve analysis were used to evaluate the model-fitting effect and diagnostic accuracy, and the Kappa test was used to evaluate the consistency of the follow-up bone scan diagnosis results with the model diagnosis results. In the follow-up study, the change in serum index was calculated with the following equation: [% year change (median)] = [ / ]. The nonparametric Mann-Whitney test was used to compare the initial levels, follow-up levels, % year change, and logit (p) between the bone metastasis group and the non-bone metastasis group. The nonparametric Wilcoxon test was used to compare differences within the bone metastasis group and the non-bone metastasis group, including initial levels, follow-up levels, and model logit (p). A value of Patients with lung cancer have high serum levels of bone biochemical markers (BME cytokines and bone turnover markers) and are prone to bone metastasis; there is no existing method that can diagnose bone metastasis early or monitor its occurrence in lung cancer patients.For patients with stage IV lung cancer, those with high serum concentrations of BME cytokines and bone turnover markers were significantly associated with the occurrence of bone metastasis.In addition to clinical pathological factors, this serum bone biochemical marker model could be a simple, non-invasive, and sensitive tool for the diagnosis and progression monitoring of bone metastasis in patients with lung cancer.P < 0.05). In non-bone the metastasis group, serum levels of all serum bone biochemical markers were slightly higher than those of the healthy controls (P < 0.05)   Fig.  A/B/D, e05) Fig.  C.Fig. P < 0.05)   Fig.  A/B/C/D.05) Fig.  A/B.FigP < 0.001, 95% CI (0.848–0.936)], with a sensitivity of 79.49% and a specificity of 89.76%  for these indicators were 0.808, 0.825, and 0.854, and the cut-off values were 1257.80 ng/ml, 1547.00 ng/ml, and 322.30 pg/ml, respectively. The diagnostic ability of serum IL-6 was limited; the AUC was 0.652, and the cut-off value was 14.36 pg/ml. The AUC for the four cytokines combined  was 0.898 , the sensitivity was 71.79%, and the specificity was 78.74%  (Supplementary Table P < 0.001)   Fig. A. Intere01) Fig. B.Fig. 3P < 0.05); age and sex were the only two excluded factors. All variables with P < 0.05 in the univariate analysis were analysed by multivariate logistic regression to determine whether these serum markers were independently related to the diagnosis of bone metastasis in lung cancer. Multivariate logistic regression analysis indicated that only OPG (P < 0.001), PTHrP (P < 0.001), tP1NP (P = 0.001), and β-CTx (P < 0.001) were independent diagnostic factors for bone metastasis in lung cancer, while CaN and IL-6 were not ]  > 0.452, the sensitivity and specificity of the predictive model were 85.8 and 89.7%, respectively . The % year changes of serum OPG (P = 0.001), PTHrP (P = 0.011), tP1NP (P = 0.002), and β-CTx (P = 0.027) in patients with bone metastasis were significantly higher than those in patients without bone metastasis, but there was no significant difference in serum CaN or IL-6 . The follow-up levels of these serum indicators were significantly higher than their initial levels, except for IL-6 Table . The othP < 0.001). In addition, the diagnostic logit (p) of bone metastasis in lung cancer patients increased over time (P < 0.001)  of patients with bone metastasis from lung cancer was significantly higher than that of lung cancer patients without bone metastasis (01) Fig. C.BME cytokines and bone turnover markers are involved in the early survival mechanisms of tumour cells in bone tissue, such as colonization, dormancy, micrometastasis, and metastasis formation . In thisPrevious studies have rarely reported the correlation between serum BME cytokine levels and bone metastasis in lung cancer. At present, there have been no reports on the application of BME cytokines combined with bone turnover markers in the diagnosis of bone metastasis in lung cancer. In a previous study, Zhou had established a molecular model which involved using four markers to perform immunohistochemical analysis on 105 stage III lung cancer patients, and the model was prospectively validated. It exhibited a prediction sensitivity of 85.7% and a specificity of 66.7% . HoweverBone biochemical markers that can be observed during bone metastasis include secretory cytokines in the BME, the collagen degradation product tP1NP and β-CTx, which are formed during bone resorption and bone formation. BME cytokines include CaN, OPG, PTHrP, and IL-6. CaN is a key regulator of gene transcription and differentiation in osteoclasts, and it can promote the differentiation and maturation of osteoclasts through the Ca-CaN-NFATc1 signalling pathway . OPG, a The bone turnover markers tP1NP and β-CTx are products of bone metabolism, and they are important serum biochemical markers of bone formation and bone resorption, respectively , 27. In P < 0.001)   Fig. A, indica01) Fig. B. The coMultivariate logistic regression analysis revealed that the serum BME cytokines OPG and PTHrP and the bone turnover markers tP1NP and β-CTx were independent diagnostic factors for bone metastasis. Furthermore, a mathematical model was established to combine these indicators, so as to predict the risk of bone metastasis in patients with advanced lung cancer more accurately: logit (p) = − 8.4988 + 0.0211 * tP1NP + 0.0079 * PTHrP + 0.0016* OPG + 0.0021*β-CTx. The AUC of this model reached as high as 0.940. When logit (p) > 0.452, the specificity and sensitivity of bone metastasis diagnosis in lung cancer are 85.8 and 89.7%. Prospective verification of this model revealed that its sensitivity and specificity for the diagnosis of bone metastasis in lung cancer were 87.5 and 85.7%, respectively, and the Kappa value was 0.770, which was highly consistent with the actual results. A follow-up study of 44 patients for a minimum of 2 years confirmed that the model could accurately predict the occurrence of bone metastasis in lung cancer, and the prediction of bone metastasis was 9.46 (5.00–17.63) months earlier than medical imaging evidence appeared  and bone turnover markers (tP1NP and β-CTx) can assist with the diagnosis and progression monitoring of bone metastasis in lung cancer. These bone biomarkers can be continuously  detected in the sera of lung cancer patients during disease progression, and a continuous, dynamic assessment of bone metastasis risk can be conducted based on the prediction model. If bone metastasis can be predicted early enough, then effective preventative measures could be taken, which may improve the life quality of the lung cancer patients. This model may provide a simple, non-invasive, and reliable method for the early screening of bone metastases in lung cancer patients.Additional file 1: Supplementary Table 1. The clinical characteristics of 205 lung cancer patients for establishment of the diagnostic model of bone metastasis analysis.Additional file 2: Supplementary Table 2. The basic information of 44 lung cancer patients for prospective validation analysis.Additional file 3: Supplementary Table 3. Correlations of serum levels for BME cytokines and bone turnover markers in 205 patients with primary lung cancer stage IV.Additional file 4: Supplementary Table 4. Univariate and multivariate analysis of diagnostic risk factors associated with bone metastasis diagnosis in 205 patients with lung cancer.


Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we examined paratuberculosis cases among 2–4-year-old dairy cows at farms in Shandong Province, China. Paratuberculosis cases were diagnosed based on clinical symptoms, pathological autopsy, and histopathological inspection. Characteristics of paratuberculosis in the affected dairy cattle included poor body condition, persistent diarrhea, subcutaneous edema, granulomatous ileitis (multibacillary), mesenteric lymphadenitis, and hepatitis. Acid-fast bacilli from fecal specimens and lymphocytes were putatively identified as MAP based on Ziehl-Neelsen staining, then confirmed using polymerase chain reaction-based testing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses. Overall, only one MAP strain was isolated from a herd with symptomatic diarrhea. However, analysis of 586 serum samples from nine herds in Tai'an City revealed that 66.7% of herds and 14.2% of animals were seropositive for MAP. Our findings suggest that paratuberculosis is widely prevalent and therefore a significant threat to the dairy industry in Tai'an City, Shandong Province, China.Paratuberculosis, a chronic and sometimes fatal disease of ruminants, is caused by  Mycobacterium avium subsp. paratuberculosis (MAP). Clinical symptoms of paratuberculosis include slowly progressive wasting and diarrhea, which are intermittent at first but become progressively more severe until they are constantly present. Affected animals become increasingly emaciated and usually die as a result of dehydration and severe cachexia. Paratuberculosis has also been reported in horses, pigs, rabbits, stoats, foxes, and weasels [Paratuberculosis, also known as Johne's disease, is a contagious, chronic, and sometimes fatal infection that primarily affects the small intestine of ruminants –4. It is weasels .MAP has been isolated from milk and colostrum from naturally infected dairy cows . HoweverShandong Province in China has a thriving cattle industry, with many of the largest dairy farms in China , 17. Howhttp://www.gov.cn/gongbao/content/2017/content_5219148.htm). The study was approved by the Committee on Animal Ethics of Shandong Agricultural University. Experiments were carried out in accordance with the approved guidelines (No. SDAUA-2016-004).All protocols and procedures were performed according to the Chinese Regulations for Laboratory Animals  or 3 (n = 3) years were prematurely culled at two separate farms in Tai'an City, Shandong Province, China. One herd consisted of 550 cows and the other had 536 cows. The diagnosis of the case and the isolation and identification of the pathogen were based on the methods reported in a previous study [During September and October 2016, five dairy cows aged 2  and P91 reverse (5′-GGCGTTGAGGTCGATCGCCCACGTGAC-3′) were used in amplifying a 413 bp fragment of multicopy insertion element ISescribed . Thermalw/v) hexadecylpyridinium chloride  and allowed to stand undisturbed for 12 h at room temperature for decontamination. Tubes were centrifuged at 1500 × g for 30 min, and the supernatants were decanted. The remaining cell pellets were resuspended in 1 ml of antibiotic mixture . Cell suspensions were then incubated for 12 h at 37°C before 0.2 ml volumes were inoculated on Herrold's egg yolk agar supplemented with mycobactin J and ANV . They were incubated in sealed 25 cm3 tissue culture flasks at 37°C for 6 weeks. Colonies that formed within 1 week were abandoned. Colonies with typical MAP morphology were checked by Ziehl-Neelsen staining and IS900 PCR as described above. For staining, single colonies were resuspended in 200 μl of sterile water and smeared onto clean glass slides.A modified centrifugation method was used to cultivate MAP as described previously , 22. BriTotal DNA was then extracted from putative MAP colonies using a TIANamp Bacteria DNA Kit  according to the manufacturer's instructions and used as a template for PCR-based MAP verification as described above.μl volume of 70% formic acid was spotted, air dried, and covered with 1 μl of HCCA matrix . MALDI-TOF MS identification was performed using a Microflex LT Smart MALDI-TOF mass spectrometer, MALDI Biotyper software version 4.0, and the MBT Compass Library (DB-7854). A log score of >2.0 was considered sufficient for identification at the species level.Ziehl-Neelsen stain- and PCR-positive colonies were then confirmed as MAP using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . Briefly1311 PCR restriction endonuclease analysis (REA) was performed as described previously, to identify the strain type of MAP [1311 PCR mix containing 1 μM of primers M-56 (5′-3′ GCGTGAGGCTCTGTGGTGAA) and M-119 (5′-3′ ATGACGACCGCTTGGGAGAC) and Taq Plus Master Mix II Dye Plus . 8 μl of the positive IS1311 PCR solution was digested for 2 h at 37°C in a 16 μl reaction, which contained 2 U of each endonuclease HinfI and MseI supplemented with buffers provided by the manufacturer . And the products were analyzed by gel electrophoresis with 2% agarose gel and observed under UV light .The ISe of MAP , 24–26. S/P ratios ≤ 0.45 were scored as MAP-negative, while ratios ≥ 0.55 were considered positive. Intermediate S/P values were scored as “suspect” and treated as negative for the data analysis.To determine the prevalence of MAP in dairy cows and on farms in Tai'an City, 586 serum samples were collected from cows from nine dairy farms, including the two farms mentioned above, in Tai'an City, Shandong Province. The samples were examined using paratuberculosis ELISA test kits  as per the manufacturer's instructions. Sera with Affected cows initially exhibited persistent diarrhea without other obvious symptoms. All cows had a normal body temperature, and their mental status was considered good. Diarrhea samples appeared homogeneous, nonhemorrhagic, and nonmucoid with no other inflammatory secretions. Diarrhea could not be controlled with antibiotics. From one to several months after the onset of persistent diarrhea, the predominant symptoms displayed by infected cattle included decreased milk production and chronic progressive weight loss with chronic or intermittent diarrhea. In the late stage of the disease, the affected cattle were emaciated, with obvious atrophy of skeletal muscle and fat .Necropsy of the euthanized cows revealed lesions in the gastrointestinal tract accompanied by swelling and thickening of the intestinal wall, with the most serious lesions occurring in the small intestine. The walls of the ileum and cecum showed thickening, with gyriform swelling and hemorrhaging of the mucosa. The intestinal mucosa was also corrugated and thickened Figures . Focal uMultiple acid-fast bacilli were observed in smears prepared from fecal samples and lymphocytes, as well as in pure cultures. While some scattered acid-fast bacilli were observed in the fecal samples , most baLymphopenia was observed in the mesenteric lymph nodes, although large numbers of epithelioid cells and multinuclear giant cells were observed . HyperemPCR analysis revealed that 5/28 fecal samples were positive for MAP. The amplified fragments were 413 bp in length. The suspected MAP colonies cultured on selective media were also subjected to PCR-based identification. The resulting positive colonies were then confirmed using MALDI-TOF MS. The resulting scores were all >2.0, confirming that the colonies were MAP.1311 PCR was conducted and followed by digestion by two restriction endonucleases (HinfI/MseI). IS1311 PCR-REA reaction products confirmed the presence of bands of 67, 218, 285, and 323 bp , 6.9% (4/58), 24.2% (32/132), 18.5% (5/27), 9.6% (9/94), and 42.2% (19/45) of samples tested positive for MAP, corresponding to a prevalence of 14.2% across all tested cows. All prevalence results are shown in There were very few reports on the characteristics and epidemiology of paratuberculosis in dairy cows in China, as well as a general lack of detailed descriptions of typical pathological changes resulting from MAP infection. Here, we provided a detailed description of the pathological findings from typical, naturally occurring paratuberculosis cases. The intestinal mucosa of infected animals, including the jejunum, ileum, cecum, and colon, was corrugated and thickened. Mesenteric lymph nodes showed medullary swelling, and consistent with previous reports, a large number of multinuclear giant cells, epithelioid cells, and macrophages were present in the diseased tissues. However, unlike other studies , we noteParatuberculosis is always difficult to control because of its chronic feature and the large percentage of subclinical infections . FurtherThe seroprevalence investigation indicated that 66.7% (6/9) of herds and 14.2% of animals were positive for MAP antibodies, which is higher than the findings from previous studies from Shandong  and otheBased on our findings from the epidemiology survey, we suspected that dairymen did not pay enough attention to the prevention and control of paratuberculosis in Shandong Province. Most of them did not have a clear idea about this disease. Even if some symptoms occurred, they did not expect it was caused by paratuberculosis. Normally, the potentially infected cows were not in quarantine, even new cattle were introduced into the farms. Lack of taking effective prevention and control measures against latent infection may be another cause of the outbreak. Due to the limited samples, these data cannot build the national picture. However, they would serve as a warning to the Chinese cattle industry because Shandong, as a traditional cattle-rearing area, was still short of measures to prevent paratuberculosis.This study provided a description of the pathological changes associated with paratuberculosis in naturally infected dairy cows in China. It indicated that paratuberculosis was an important infectious disease of dairy cattle in Shandong Province. Appropriate herd management practices, including separation and/or culling of infected animals, should be introduced in a timely manner as standard practice in the dairy industry. On-site veterinarians should pay more attention to this disease as well. Effective management practices are important to reduce both economic and stock losses associated with paratuberculosis and to reduce the risk of disease transmission to humans.


With a shift in the healthcare paradigm towards a more patient-centered approach, data on inflammatory bowel disease (IBD) needs to be further explored. This study aimed to determine patient perspectives on the effect of IBD and features of patients with lower satisfaction level and compare patient and physician perception of IBD-related Quality of Care (QoC).A previously developed pre-standardised set of questions regarding patient-centered outcome (PRO) measures for IBD, comprising 36 items, was administered in five centers, and a concomitant questionnaire for specialised physicians was adapted and administered.Overall, 1005 patients with IBD met the inclusion criteria. Sixty-five questionnaires were administered to specialised physicians. Both patients and physicians perceived the IBD-related QoC as being satisfactory. Furthermore, this study revealed areas of shortcomings where it comes to patient perceptions. Female sex and the presence of negatively impacting disease characteristics  were associated with lower satisfaction levels.Our findings can be used in establishing strategies aimed at improving patient QoC and defining strategic priorities. These data can aid in improving the communication of the pressing needs of IBD patients, to both the public payers and health authorities. Inflammatory bowel disease (IBD), comprising Crohn’s disease (CD), ulcerative colitis (UC) and IBD unclassified (IBD-U), is a chronic immune disease that frequently emerge early in life and requires chronic health care. The wide spectrum of IBD disease severity and frequency of complication occurrence impair patient physical and mental health, quality of life (QoL), and social functioning, resulting in the need for chronic intensive disease monitoring, regular outpatient appointments, hospitalization, and surgery . The preHealthcare is transitioning towards a value-based system , 12. TheThe aims of this study were to assess patient-centered outcomes, including patient perception of QoC, and investigate whether patient-reported outcomes are associated with demographic factors and clinical status. Subsequently, the differences between patient and physician perception of QoC were also determined.Patients in this multicenter, observational study were enrolled from three hospitals across different provinces of China, covering a population greater than 50,000,000 people. From March 2016 to November 2017, consecutive inpatients and outpatients with an IBD diagnosis were enrolled.age at least 18 years;verified IBD diagnosis ; andability to understand the questionnaire and provide written informed consent.Inclusion criteria were:follow-up duration at the center shorter than 12 months at the time of enrolment; orinclusion in other clinical trials at baseline.Exclusion criteria were:The questionnaire was delivered either on paper or online to each prospective outpatient while they were waiting for their clinic appointment. Most of the patients submitted a completed questionnaire to the investigators while still in the waiting room. Some patients completed the online version of the survey in the waiting room or shortly thereafter. Inpatients received the questionnaire during their hospital stay. IBD specialists were involved in the entire process of questionnaire admisnistration, and subsequently checked all answers for missing values or inconsistencies. Another concomitant electronic questionnaire for physicians was administered to IBD specialists from multiple hospitals across East China via email.n = 25), representing patients, patient associations, gastroenterologists, surgeons, specialist nurses, IBD registries and patient-reported outcome measure methodologists [A standard set of patient-centered outcome measures for IBD was previously developed by an international working group  was performed. Independent sample t-tests and one-way analysis of variance, as well as a chi-square test were performed to investigate differences in the clinical, epidemiological parameters, and patient-centered outcomes across the different subgroups. All tests were two-sided with a significance level of 5%, and were performed using R version 3.3.3.Overall, 1005 patients with IBD met the inclusion criteria; 925 of them agreed to participate and provided written informed consent. Finally, 891 of the questionnaires (response rate: 88.7%) for patients were found to be evaluable. Overall, 522 patients (58.6%) had CD, 363 (40.1%) had UC, and six (0.7%) had IBD-U. The socio-demographic and clinical data of the responders, by diagnostic group, are shown in Table p = 0.029). Among all the IBD patients with various current occupations, we found that 31 patients with “unemployment” and 58 with “long-term sick leave” shared the highest proportion of feeling anxious or depressed because of his/her IBD. In other words, 86.1% of the unemployed patients and 84.1% of the “long-term sick leave” patients felt anxious or depressed, which is much more frequent than those observed in other current occupations, such as “working (employee)” and “working (self-employed)” and “studying”, as well as “long-term sick leave for other reasons”, “early retirement due to IBD”, “early retirement for other reasons”, “retirement (because of age)” and “fulfillment of family role as parent or partner”. In the 12 months preceding questionnaire administration, the numbers of hospitalizations due to IBD-related symptoms in the CD group and UC group were 1.72 (1.8) and 0.71 (1.6), respectively (p = 0.001). The proportion of absenteeism from school or college due to IBD-related symptoms in the CD and UC cohorts were 65.6 (108.9) and 28.5 (76.4) days, respectively (p = 0.002). The durations of absence from work in the CD and UC groups were 71.1 (118.2) and 31.7 (84.3) days, respectively (p = 0.001). In the 24 months preceding questionnaire administration, the longest continuous durations of steroid use for IBD in the CD and UC groups was 2.1 (11.8) and 0.5 (4.2) months, respectively (p = 0.001). Detailed data on the patient-centered outcomes of the responders, by diagnostic group, are shown in Table In the 2 weeks preceding questionnaire administration, 61.7% of patients with CD and 41% of those with UC experienced anxiety or depression due to IBD (p < 0.001), and were positively correlated to age . There were no significant differences between male and female participants (p = 0.194). The average frequency of emergency room presentations for IBD-related symptoms before a definitive diagnosis was reached was 2.6 (5.4) times. The average frequency of emergency room presentation significantly differed between the CD and UC groups (3.4 (6.4) vs. 0.99 (1.4) months, p < 0.001). There was no significant difference between male and female participants in this regard (p = 0.070). In the whole cohort, the frequency of emergency room presentation was positively correlated to time to diagnosis . However, the time to diagnosis was not correlated to age (p = 0.294).The average duration from the time medical care was first sought for symptoms that were finally recognized as IBD-related to IBD diagnosis confirmation was 9.8 (19.8) months. The duration to diagnosis significantly differed between the CD and UC groups (13.3 (24.8) vs. 4.5 (5.8) months, p = 0.338), education level (p = 0.412), and age (p = 0.371) in this regard. The average duration of the last IBD consultation was 11.1 (8.3) minutes in the CD group and 12.2 (14.4) minutes in the UC group (p = 0.328). There was no significant difference between the sexes (p = 0.172) and between patients of different ages (p = 0.379). In total, 423 patients (47.5%) felt that the consultation time was sufficient for a satisfactory review of the IBD status, whereas 468 patients (52.5%) did not feel this to be the case. Eight-hundred-and-41 patients (94.4%) received specialist advice at a hospital or clinic during a flare-up. Few patients received information by telephone (11.2%) or email (0.6%). An interesting finding was that 55 patients (29.3%) felt that the number of toilets was sufficient for patients with IBD during hospitalization, whereas 133 patients (70.7%) did not.In total, 855 patients (96%) consulted gastroenterologists for IBD, 24 patients (2.7%) consulted general surgeons, 82 (9.2%) consulted colorectal surgeons, 13 (1.5%) consulted other specialists, seven (0.8%) consulted specialist nurses, and none of the patients consulted their family doctor. Overall, 615 patients (69%) had a regular IBD review appointment regardless of IBD activity status, whereas 276 patients (31%) did not. There was no significant difference based on sex . A larger number of specialists than patients believed that the level of communication was good; however, there was no difference between patients with CD and UC in this regard. A total of 347 (66.4%) patients with CD, 276 (76%) patients with UC, and 45 (81.8%) specialists regarded the quality of the IBD care received or provided in the past 12 months as being excellent or very good . There was no difference between patients with CD and UC, or between the specialists and patients in this regard. Female patients showed a higher tendency to feel that the quality of communication with specialists (p = 0.037) and quality of IBD care (p = 0.019) was less satisfactory than male patients. However, patients across different age groups (p = 0.231), disease duration groups (p = 0.307), education level groups (P = 0.549), and history of abdominal surgery for IBD groups (p = 0.113) did not show significant differences in terms of the level of satisfaction pertaining to communication with specialists. Similarly, patients across different age groups (p = 0.275), disease duration groups (p = 0.292), education level groups (p = 0.687), and history of abdominal surgery for IBD groups  did not show significant differences in the quality of IBD care. In the bivariate analyses conducted to further evaluate patient-centered outcomes, patients with significant pain or discomfort were less satisfied with the quality of IBD care than those without (P = 0.043). Patients who felt fatigued for the majority of the time were less satisfied with the quality of IBD care than those who did not (p = 0.028). Patients who had experienced anxiety or depression in the last 2 weeks were less satisfied with the quality of IBD care than those who had not (p = 0.003). There was no statistically significant difference in the perception of IBD management (p = 0.219).Overall, 295 (56.6%) patients with CD, 220 (60.6%) patients with UC, and 46 (83.6%) specialists felt that the level of communication between patients and health specialists was excellent or very good (CD group vs. UC group: p < 0.001), use of new/experimental treatments . Detailed data on the patient and specialist perception of satisfaction with healthcare are presented in Table Of all the topics discussed in the consultations, there were significant differences in regarding medical treatment options  and satisfaction with the level of communication with health specialists , but was not correlated to adequacy in the number of toilets during time of IBD hospitalisation .Using Spearman’s correlation, we found that patient level of satisfaction with the quality of IBD care was correlated with the presence of well-coordinated IBD management  in the order of “highest to lowest” were: education/studies, employment, practical daily living, nutrition/diet, general lifestyle issues, sexual relationships, formulation of a plan for daily life, personal relationships, main goals or priorities in caring for the current condition, current symptoms, and medical treatments. The corresponding topics in the UC group ranked from highest to lowest were: nutrition/diet, employment, practical daily living, medical treatments, education/studies, sexual relationships, formulation of a plan for daily life, personal relationships, general lifestyle issues, new/experimental treatments, main goals or priorities in caring for the current condition, and current symptoms Table .fewer expenses and better coverage (for drugs such as infliximab) by health insurance;provision of more efficient and better-coordinated management, shorter waiting times, and easier access to the purchase of medicines;better disease control and fewer hospital visits;better level of communication and easy access (such as telephone and Wechat), with a greater degree of information on diets and medicines, as well as disease control (more public lectures and popular science articles);demand for the combined use of traditional Chinese medicine with Western medicine;better inpatient facilities and a larger number of IBD healthcare providers.The two open-ended questions were: “What is the best thing about the care you currently receive?” and “What is the care that you would most like to be changed or improved?” The answers were as follows:In this study, we found that both patients and physicians perceived the IBD-related QoC as being satisfactory. Furthermore, we uncovered shortcomings according to the patient perception. Female sex and the presence of certain negatively impacting disease characteristics were associated with lower satisfaction levels.The importance of the systematic tracking and reporting of patient-centered outcomes is gradually being realized. Through the comparison of meaningful patient outcomes, providers can adopt strategies aimed atachieving the best “value” for participating stakeholders; this is defined as value-driven care . The preIn this study, 61.7% of the patients with CD and 41% of those with UC experienced anxiety or depression due to IBD in the 2 weeks preceding questionnaire administration. Unemployment and taking long-term sick leave due to IBD ranked the highest in terms of the causes of anxiety and depression, compared to other current occupations. A previous study showed that absence from work and the presence of active disease were the two main factors associated with a reduced health-related QoL (HRQoL) . AnotherNone of the patients consulted family doctor. It should be mentioned that there are very few family doctors or general practitioners in China, and that most patients consult specialists.There has been a tendency for patient perception of the quality of healthcare to be ignored as they are not adequately aware of what constitutes high care quality. As of 2018, no consensus on patient satisfaction or Quality of Care (QoC) measurement had been reached . PreviouIn our study, patient satisfaction with the quality of IBD care was related to the satisfaction with the level of communication with specialists. Patient-physician communication is vital in ensuring patient commitment and involvement in long-term disease self-management , and infThe bias and difference between specialists and patients requires investigation. Specialists should strive to improve the quality of doctor-patient communication. The differences in the scoring trends between physicians and patients could be related to the generally insufficient communication status due to high inpatient and outpatient department volumes and relative scarcity of healthcare providers, as well as the higher expectations of patients receiving IBD care at centers in first-tier cities. Furthermore, half of the patients considered the consultation time to be insufficient. Moreover, only two-thirds of the patients had a regular IBD review appointment regardless of the IBD activity status. Most patients were used to seeking specialist advice during a flare-up at hospitals or clinics; few received advice through telephone or email. The government should allocate a larger volume of funds aimed at improving the doctor-to-patient ratio to ensure better communication between physicians and patients.Furthermore, in this era of growing healthcare cost constraints, providers must also demonstrate a favorable cost:benefit ratio with patient-centered outcome-based intervention; this requires further evaluation beyond the scope of this study. However, making improvements to some of the patient-centered outcomes  may only result in small or negligible costs.This study has some limitations. Firstly, the participants were enrolled from care centers across the eastern parts of mainland China, and may not be representative of the entire Chinese IBD population. The regions included in our study are more developed and industrialized than other parts of China. Secondly, the current study had a cross-sectional observational design. We cannot infer causality from these results, although it was possible to identify measures for improvements in clinical practice and patient care. Last, but not least, this study included a high number of statistical tests, which may result in type I errors. Despite these limitations, the current study provides important insights into the factors associated with patient-centered outcomes, as well as broadens the scope of the discussion pertaining to the status of IBD care in China.In conclusion, we found that both patients and physicians perceived the IBD-related QoC as being satisfactory. We also identified areas of shortcomings and improvement where it comes to patient perception, and established that female sex and the presence of certain negatively impacting disease characteristics were associated with lower satisfaction levels. Therefore, patient concerns pertaining to their disease must be reassessed regularly, since there are certain aspects of IBD-care that may be improved, as evident from this study.Additional file 1:Table 1. DemographicsAdditional file 2: Table 2. Clinician Questionnaire