The extent and significance in of cardiac remodeling in Fontan patients are unclear and were the subject of this study.This retrospective cohort study compared cardiovascular magnetic resonance (CMR) imaging markers of cardiac function, myocardial fibrosis, and hemodynamics in young Fontan patients to controls.2 vs 93 ml/m2, p < 0.001), and lower ejection fraction , circumferential , radial , and longitudinal strain . Compared to healthy controls, Fontan patients had higher extracellular volume fraction (ECV)  and native T1 . Patients with a dominant right ventricle demonstrated larger ventricles , lower EF , worse CS , and a trend towards higher ECV . Worse EF and CS correlated with longer cumulative bypass , cross-clamp  and circulatory arrest times . T1 correlated with aortopulmonary collateral (APC) flow  which, in the linear regression model, was independent of ventricular morphology (p = 0.9) and EDVI (p = 0.2). The composite outcome  was associated with increased native T1  and EDVI , as well as decreased EF  and worse CS . APC flow  was independently associated with the composite outcome, independent of ventricular morphology (HR 0.71 CI 0.30–1.69 p = 0.44) and T1 .Fifty-five Fontan patients and 44 healthy controls were included (median age 14 years (range 7–17 years) vs 13 years (range 4–14 years), p = 0.057). Fontan patients had a higher indexed end-diastolic ventricular volume (EDVI 129 ml/mPediatric Fontan patients have ventricular dysfunction, altered myocardial mechanics and increased fibrotic remodeling. Cumulative exposure to cardiopulmonary bypass and increased aortopulmonary collateral flow are associated with myocardial dysfunction and fibrosis. Cardiac dysfunction, fibrosis, and collateral flow are associated with adverse outcomes.The online version contains supplementary material available at 10.1186/s12968-021-00782-9. By early adulthood, up to 50% of patients with a Fontan circulation are in heart failure, , 2 a comThe aims of the present study were to investigate the degree of functional and fibrotic myocardial remodeling present in young Fontan patients, to unveil candidate etiologies for alterations in structure and contractility, and to describe their potential functional impact. We hypothesized that ventricular dysfunction and fibrotic myocardial remodelling are enhanced in the subgroup of Fontan patients with a dominant right ventricle (DRV).Fontan patients and control subjects who had received cardiovascular magnetic resonance (CMR) imaging between April 2014 and March 2018 were included in this retrospective cohort study. The study setting is a tertiary care hospital. If more than one CMR had been performed in the same patient, only the last CMR was included. Scans were excluded if non-diagnostic. Further, Fontan patients with two nearly equal sized ventricles were excluded to allow for a comparison between patients with DRV and dominant left ventricle (DLV). Patients referred for coronary artery imaging because of non-specific chest pain or for a family history of arrhythmogenic right ventricular cardiomyopathy were included as controls if their work-up, including CMR, was negative. Twenty-one Fontan patients and 24 healthy control subjects were previously reported . The priDemographic, surgical, and clinical data were retrieved from the patients’ medical records. Results from cardiac catheterization, echocardiography, and cardiopulmonary exercise testing were collected if the test had occurred within 12 months of CMR, without an interim intervention. ‘Failing Fontan circulation’ was defined by the presence of plastic bronchitis, protein-losing enteropathy, anasarca, or surgical take-down of the Fontan within 6 months of CMR. A composite outcome was used to assess the predictive value of CMR and included any of the following at or after the CMR, up until study completion: readmission for cardiac indication, cardiac reintervention, Fontan failure or any clinically significant arrhythmia. Of note, the former patients with Fontan failure were also included in the composite outcome. Significant atrioventricular valvar regurgitation (AVVR) was defined as at least moderate insufficiency by echocardiography.All examinations were conducted on a 1.5 T CMR system . The protocol included balanced steady-state free precession short-axis cine stacks for ventricular volumetry, as previously described . Phase cA single observer (MR) with several months of focused training and under the supervision of a single senior reader (LGW) for each case analyzed short axis cine stacks for ventricular volumes using commercially available software Qmass . In patients with a ventricular septal defect or a communication of the hypoplastic ventricle with the neo-aorta, the smaller chamber was included in the single ventricular contours. Blood flow volumes were quantified using Qflow . Aortopulmonary collateral (APC) flow was calculated as the difference between pulmonary venous and arterial flows, indexed to body surface area.T1 relaxometry analysis was performed by a single observer (CdL) with 10 years of CMR experience on the online motion-corrected source images, using’Qmap 2.2.36’ . Regions of interest (ROIs) were drawn in the freewall of the dominant ventricle, propagated to each of the eight source images and adjusted for motion , as previously described , 17. SegContinuous variables are presented as means ± standard deviations if normally distributed, otherwise as medians and ranges. For normally distributed continuous variables, two-tailed independent sample t-tests were used in conjunction with Levene’s Test of Equality of Variances. For non-normally distributed continuous variables Wilcox rank sum test was used. Univariate and multivariable regressions were performed to explore presence of linear relationships between measurements in the overall cohort. Correlations were assessed using Pearson’s correlation coefficient analysis for normally distributed continuous variables and Spearman rank-order test for non-normally distributed data. Intra- and interobserver variability of T1 and ECV was analyzed using Bland–Altman plots. P-values < 0.05 were considered statistically significant  had undergone cardiac catheterization within one year of CMR with a median interval of zero days (range 0–206 days). There was no difference in the central venous, atrial or ventricular end-diastolic pressure between DLV and DRV patients Table . EchocarCardiopulmonary exercise test results were available within one year of CMR in 28 of the 55 Fontan patients (51%), at a median of 87 days (range 0–365 days). There were no differences in the maximum oxygen consumption, anaerobic threshold or maximum work-load between DLV and DRV patients or between those with and without significant AVVR. There was no correlation between exercise parameters and myocardial strain, ejection fraction (EF), ventricular volumes, fibrosis markers, ESFS, or ESWS.2 versus 122 ± 36 ml/m2, p = 0.08).Ventricular volumes and myocardial function indices are provided in Table Worse EF and CS correlated with cumulative bypass , aortic cross clamp  and circulatory arrest  times. EDVI correlated with APC flow . There was a correlation between ECV and EDVI  that, in linear regression, was independent of APC flow  and ventricular morphology . As compared to controls, the Fontan cohort demonstrated evidence of intraventricular dyssynchrony with an increased standard deviation of the times to peak CS (p = 0.04).The VAC ratio was higher in Fontan patients as compared to controls subjects (p < 0.001) with a greater range and standard deviation. The DRV group demonstrated a higher VAC ratio (p = 0.015), Ees (p = 0.019) and ESWS (p = 0.006) as compared to the DLV group Table . PatientThere were no correlations between the haemodynamic or exercise data and wall or fibre stress, or VAC. Ees correlated with cumulative bypass , aortic cross clamp (R − 0.44 p = 0.001) and circulatory arrest times (R − 0.43 p = 0.001). VAC, ESWS and ESFS correlated inversely with EF  and correlated positively with CS .LGE was present in five if the 55 Fontan patients 10%) and in no controls. Myocardial T1 and ECV measurements demonstrated adequate reproducibility  which, in a linear regression model, was independent of ventricular morphology  and EDVi .We investigated the following two outcome surrogates: clinical Fontan failure concurrent to CMR and the composite outcome. Within six months of CMR, six of 55 Fontan patients (9%), four with a DRV and two with a DLV, had a failing Fontan physiology. The six patients with Fontan failure had decreased EF , global CS  and RS  and higher ECV  and T1 . Twenty-two of the 55 Fontan patients (40%) experienced the composite outcome . This model showed that APC flow  was independently associated with the composite outcome, independent of ventricular morphology (HR 0.71 CI 0.30–1.69 p = 0.44) and T1 .Fibrotic myocardial remodeling has been identified as an important culprit in the pathogenesis of ventricular dysfunction in acquired heart disease, but only preliminary results on its prevalence, extent, and impact on myocardial function are available for the single ventricle population –12. We pLGE has been associated with adverse outcomes in Fontan patients . In our In the present cohort, patients who experienced the composite outcome had increased fibrosis and decreased myocardial function, evidenced by worse EF and CS. In a series of older Fontan patients, Rathod et al. demonstrated that volume loading was independently associated with death or transplantation . SimilarOur morphological and functional results add to the growing body of evidence that patients with DRVs have worse ventricular function and, according to some studies, less favorable outcomes , 23, 24.The Fontan cohort had larger ventricular volumes as well as lower EF and strain in all domains as compared to controls, corroborating previous findings by both echocardiography and CMR , 9, 13. More recently, elevated ESWS and ESFS, which result in an imbalance between myocardial oxygen consumption and supply , have emVAC ratio is a novel parameter that can be measured invasively, using pressure volume loops , or non-Several study limitations warrant discussion. First, T1 relaxometry was consistently feasible only in the free wall and our assumption that the values are representative of the entire ventricular myocardium may be erroneous. Second, the control cohort was older than the Fontan group; for ethical reasons, younger children could not be recruited for a contrast-enhanced CMR study under general anaesthesia. However, one would expect a greater degree of myocardial fibrosis with increasing age and thus the older age of the control subjects would underestimate the significance of the fibrosis found in the Fontan population. Furthermore, the subgroup of patients with Fontan failure was small and related results need to be substantiated in a larger cohort. Lastly, two different gadolinium agents had been used, with potential impact on ECV. However, the influence of the type of gadolinium has been shown to be small .Our results point towards a preclinical adverse cardiac remodeling process which is evident even in young and comparatively healthy Fontan patients. A greater degree of aortopulmonary collateral flow is associated with higher myocardial fibrosis markers and adverse outcomes.Additional file 1: Figure S1. Bland-Altman plot for interobserver variability of ECV, with limits of agreement. There was good agreement and no statistically significant bias. Figure S2. Bland-Altman plot for interobserver variability of T1 with limits of agreement. There was good agreement and no statistically significant bias. Figure S3. Bland-Altman plot for intraobserver variability of T1 with limits of agreement. There was good agreement and no statistically significant bias. Figure S4. Bland and Altman plot for intraobserver variability of ECV with limits of agreement. There was good agreement and no statistically significant bias.


Loop-mediated isothermal amplification (LAMP) is a molecular diagnosis technology with the advantages of rapid results, isothermal reaction conditions, and high sensitivity. However, this diagnostic system often produces false positive results due to a high rate of non-specific reactions caused by formation of hairpin structures, self-dimers, and mismatched hybridization. The non-specific signals can be due to primers used in the methods because the utilization of multiple LAMP primers increases the possibility of self-annealing of primers or mismatches between primers and templates. In this study, we report a nanomaterial-assisted LAMP method that uses a graphene oxide–gold nanoparticles (AuNPs@GO) nanocomposite to enable the detection of foot-and-mouth disease virus (FMDV) with high sensitivity and specificity. Foot-and-mouth disease (FMD) is a highly contagious and deadly disease in cloven-hoofed animals; hence, a rapid, sensitive, and specific detection method is necessary. The proposed approach exhibited high sensitivity and successful reduction of non-specific signals compared to the traditionally established LAMP assays. Additionally, a mechanism study revealed that these results arose from the adsorption of single-stranded DNA on AuNPs@GO nanocomposite. Thus, AuNPs@GO nanocomposite is demonstrated to be a promising additive in the LAMP system to achieve highly sensitive and specific detection of diverse diseases, including FMD. Aphthovirus of the family Picornaviridae that causes a highly contagious and often fatal disease in cloven-hoofed animals, including cattle, sheep, goats, and swine [Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the genus nd swine . The disnd swine . FMDV hand swine . FMDV send swine . Thus, tFor in vitro detection of FMDV, molecular biological methods, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), have been widely employed due to their high sensitivity, achieved through gene amplification of the target nucleic acids ,6,7. PCRBst DNA polymerase, GO, and AuNPs@GO nanocomposite. The strong interaction between AuNPs@GO and primers was precisely identified, verifying that this remarkable technique would be useful in practice for rapid and sensitive virus detection in early stages of various infectious diseases, including FMDV.In this study, we developed a nanomaterial-assisted LAMP system for highly sensitive and specific detection of FMDV using a GO–AuNPs (AuNPs@GO) nanocomposite. In order to demonstrate the effect of AuNPs@GO as a LAMP additive, FMDV serotypes O and A were chosen as target viruses; we found that AuNPs@GO nanocomposite can successfully inhibit false amplification caused by wrong hybridization. Additionally, the sensitivity of our LAMP system using AuNPs@GO was increased by ~100-fold for FMDV serotype A genes, and its speed was faster by 7 min for FMDV serotype O genes, compared to that using GO. To verify how AuNPs@GO nanocomposite works in the LAMP system, we performed a mechanism study through analysis of the interaction between LAMP reagents, including primers, 4 3H2O, Sigma-Aldrich, St. Louis, MO, USA), trisodium citrate , and GO  were used as purchased. Bst DNA polymerase, 10X of ThermoPol buffer, 100 mM magnesium sulfate (MgSO4), and 10 mM dNTP were obtained from New England BioLabs . SYBR green I fluorescent nucleic acid was purchased from LONZA . Ultrapure water was used throughout this study.Tetrachloroauric (III) acid trihydrate (HAuCl4·3H2O (0.1 mg/mL) was added and the mixture was stirred for 30 min at 400 rpm. This procedure allows gold ions to adsorb onto the GO sheets. Next, the mixture was heated to 90 °C, followed by adding 1 mL trisodium citrate (0.035 g/mL) as a reducing agent. When the temperature was above 90 °C, the mixture was not well-dispersed. After heating for 10 min, the mixture was stirred for 1 h to complete the reaction. The prepared solution was cooled at ambient temperature, presenting a deep red color. Finally, the resultant solution was purified three times by centrifugation at 3940× g (15 min) with distilled water to remove free AuNPs.The AuNPs@GO nanocomposite with an elemental composition ratio (carbon: gold) of 0.84: 11.7 (wt %) was prepared through in situ growth of AuNPs on GO sheets ,31,32. BThe characterization of the prepared AuNPs@GO nanocomposite was conducted using UV-vis spectroscopy, field-emission transmission electron microscopy (FE-TEM), and zeta potential. The optical spectra of AuNPs@GO nanocomposite were collected using a UV-vis spectrophotometer . Morphology and zeta potentials were determined using a Talos F200X FE-TEM  at 200 kV and dynamic light scattering , respectively.Bst DNA polymerase, large fragment, MgSO4 , and dNTP mix (1.4 mM each); FIP/BIP primers (1.6 µM), F3/B3 primers (0.2 µM), LoopF/BoopF (0.4 µM), and Bst DNA polymerase, large fragment (320 U/mL); graphene oxide (5 µL), AuNPs (5 µL), or AuNPs@GO nanocomposite (5 µL) solutions; 1 µL target or ultrapure water (no target); 0.4X SYBR green I; and ultra-pure water to a final volume of 25 µL. FMDV genes used in this study were prepared according to a previous report [E. coli cells . The transformed cells were grown in a LB medium containing ampicillin (100 μg/mL) at 37 °C overnight. Plasmids were prepared from cultured and harvested cells using extraction kits . All LAMP primers  were designed using PrimerExplorer V5, primer design software that specializes in LAMP (http://primerexplorer.jp/lampv5e/index.html (accessed on 10 January 2022), Eiken Chemicals Corporation, Tokyo, Japan). The prepared primer sets of FMDV are shown in The protocol of LAMP assays was followed using a basic LAMP working system (New England BioLabs) at 65 °C for 60 min. The LAMP mixture  consisted of the following reagents: 1X ThermoPol buffer for s report . Briefly3+ ion seed-mediated growth on GO sheets, and its successful preparation was demonstrated using UV-vis spectroscopy, zeta potentials, and TEM . We designed the primers for FMDV serotype O and A genes and conducted LAMP assays by applying various amounts of GO and AuNPs@GO nanocomposite from 0 to 70 ng.As shown in In contrast, when more than 20 ng of GO or AuNPs@GO nanocomposite was added for FMDV target genes, the positive signals were appreciably delayed as well. Notably, we found that the appropriate amount of GO and AuNPs@GO nanocomposite had a significant effect on improvement of the LAMP reaction. Further, comparing the LAMP efficiency of GO and AuNPs@GO nanocomposite for FMDV genes, the use of AuNPs@GO nanocomposite generated the positive signals approximately 9 and 5 min faster than that of GO for FMDV serotype O and A genes, respectively  of LAMP assays. As shown in These findings suggest that both GO and AuNPs@GO nanocomposite served as efficient components for reduction of non-specific amplifications, but AuNPs@GO nanocomposite have less effect on the LAMP reaction with templates than GO. It might be considered that AuNPs@GO nanocomposite has strong affinity for certain components composing the LAMP mixture, especially primers, reducing the probability of non-specific reactions. These results demonstrate that AuNPs@GO nanocomposite could be useful as a nanomaterial additive for sensitive detection of FMDV genes as well as reduction of non-specific amplification in the LAMP system.E. coli genomic DNA as controls to determine the effect of AuNPs@GO nanocomposite on the specificity.FMDV has been classified into seven serotypes, of which serotypes O and A have recently spread around the world, especially in South Korea. Therefore, we designed a strategy to obtain high specificity through the cross-reaction between serotypes. The experiments were conducted using Lambda DNA and E. coli DNA. Meanwhile, we found that without AuNPs@GO nanocomposite, the target DNA produced fluorescence signals at 24 and 25 min, and Lambda DNA and E. coli DNA produced signals at 22 and 24 min, respectively. These results indicate that AuNPs@GO nanocomposite specifically yielded positive detection with serotype O and A primers, not allowing amplification of other DNA templates. These significant differences demonstrate that our proposed method could identify different types of FMDV by specifically detecting serotypes O and A DNA, showing an excellent specificity for target DNA.As shown in Recently, various nanomaterials including GO and AuNPs have been actively studied as an enhancer in the PCR system. GO and AuNPs have been known to interact with components such as primers and DNA polymerase in the PCR mixture through π–π stacking and electrostatic interaction, resulting in elimination of non-specific amplification and enhancement of detection performance. Bst DNA polymerase using UV-vis spectroscopy. In UV-vis spectra of GO and AuNPs@GO nanocomposite, a strong absorption peak observed at 225 nm arises from the π–π* transition of the C=C bond (Bst DNA polymerase with each solution containing GO and AuNPs@GO nanocomposite, an absorption peak at 280 nm was observed, which represents absorption peak of a protein (Bst DNA polymerase. Additionally, when LAMP primers were included in each solution, the appearance of the absorption peak at 260 nm presents the adsorption of primers on the surface of GO and AuNPs@GO nanocomposite, in which a difference in increase of the intensity of the absorption peaks was clearly observed. Whereas AuNPs@GO nanocomposite increased by 2.11 times the absorption peak around 260 nm, GO showed a slight increase of 1.32 times after addition of primers (To study how AuNPs@GO nanocomposite works in the LAMP system, we investigated the binding mode of AuNPs@GO nanocomposite with primers and C=C bond a. Upon m protein b,c. This primers b,c. Bst DNA polymerase, which indicates an interaction between AuNPs and Bst DNA polymerase. These results demonstrate that AuNPs@GO nanocomposite has a stronger interaction with primers than GO. Additionally, as shown in Bst DNA polymerase were added, the absorption peak was similar to that with AuNPs@GO nanocomposite and primers. This indicates that AuNPs are susceptible to binding to primers rather than Bst DNA polymerase. As a result, it revealed that AuNPs@GO nanocomposite have stronger binding affinity to primers than GO, as well as preferring interaction with primers rather than Bst DNA polymerase, as presented in a previous report [In contrast, when both primers and s report . Our results suggest that AuNPs@GO nanocomposite has a larger surface area than GO because AuNPs are incorporated onto GO sheets, inducing an enhancement in interaction with the LAMP mixture, especially primers, through synergistic effects of GO and AuNPs. Taken together, these findings demonstrate that AuNPs@GO could provide a valuable feasibility for efficient performance of a LAMP assay system.The LAMP platform has been spotlighted as an alternative to PCR due to its fast, convenient, and highly specific molecular diagnostic ability for certain diseases ,40,41. AWe aimed to address these issues by using AuNPs@GO nanocomposite in the LAMP reaction to reliably detect FMDV. As depicted in One solution is to reduce the primer concentration; however, this could reduce sensitivity and increase detection time. Therefore, the use of nanomaterials as components in the LAMP mixture could be considered as a simple and convenient approach. For instance, it was reported that AuNPs could enhance the specificity in LAMP assays by inserting a pre-incubation step before initiation of the LAMP reaction, which is called the hot-start LAMP technique . CitrateBst DNA polymerase through π–π stacking and electrostatic interactions [In addition, GO has various functional groups, such as hydroxyl, carboxyl, and epoxy groups, that can interact with primers and ractions . In thisWe developed a LAMP assay for detection of FMDV with reduced non-specific amplification using AuNPs@GO nanocomposite. AuNPs@GO nanocomposite was successfully synthesized and the optimal concentration for the LAMP assay was determined. Compared with GO, AuNPs@GO nanocomposite exhibited inhibition of non-specific amplification, 7 min faster reaction for FMDV serotype O, and 100-fold higher sensitivity for FMDV serotype A, enabling precise detection of FMDV genes. We also demonstrated the binding mode of AuNPs@GO nanocomposite with the LAMP mixture; the AuNPs@GO nanocomposite has a strong affinity for primers. Therefore, our system based on AuNPs@GO nanocomposite could provide the opportunity for detection of FMDV using other gene amplification tools.


The incidence of ulcerative colitis (UC) is increasing steadily in developed countries, it is plaguing nearly 1 million people in the United States and European countries, while developing countries have had a rapidly increased incidence over the past decades. Curcuma is widely used in treating malaria, UC, Crohn's disease, and colon cancer, which lead to diarrhea and bloody stool. However, the systemic mechanism of curcuma in treating UC is still unclear. Our work was supposed to expound how does curcuma alleviate UC in a comprehensive and systematic way by network pharmacology, molecular docking, and experiment verification. α and STAT3 before and after the treatment of curcuma which was verified by immunoblotting.  Traditional Chinese Medicine System Pharmacology Database (TCMSP), Shanghai Chemistry & Chemical Industry Data Platform (SGST), and papers published in Chinese Network Knowledge Infrastructure (CNKI) and PubMed were used to collect the chemical constituents of curcuma based on ADME . And effective targets were predicted by Swiss Target Prediction to establish the curcuma-related database. The disease targets of UC were screened by GeneCards and DrugBank databases, and Wayne (Venn) analysis was carried out with curcuma targets to determine the intersection targets. AutoDock software and TCMNPAS system were used to dock the core chemical components of curcuma with key UC targets. Protein interaction (PPI) network was constructed based on the STRING database and Cytoscape software. Gene function GO analysis and KEGG pathway enrichment analysis were carried out by using Metascape database. Finally, HE staining was performed to identify the inflammatory infiltration and expression difference in TNF-α and STAT3.  Twelve active components containing 148 target genes were selected from curcuma. Potential therapeutic targets of curcuma in the treatment of UC were acquired from 54 overlapped targets from UC and curcuma. Molecular docking was used to filter the exact 24 core proteins interacting with compounds whose docking energy is lower than −5.5 and stronger than that of 5-aminosalicylic acid (5-ASA). GO and KEGG analyses showed that these targets were highly correlated with EGFR tyrosine kinase inhibitor resistance, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, MAPK signaling pathway, and inflammatory bowel disease (IBD). Experiments verified curcuma relieved pathological manifestation and decreased the expression of TNF-Helicobacter pylori infection pathway, probably by binding to STAT3 and TNF-α. Curcuma relieved the colon inflammation of ulcerative colitis via inactivating TNF pathway, inflammatory bowel disease pathway, and epithelial cell signaling in  UC is characterized by abdominal pain, diarrhea, and bloody stool . GloballTCM is gaining its popularity in the ameliorating subhealth state and treating disease. Much more attention has been focused on the treatment of UC by Chinese herb; curcuma is one of the popular ones. In ancient China, curcuma was widely used to invigorating the circulation of blood in clinical applications; nowadays, the function of the positive regulation of inflammatory cytokines in inflammatory diseases  and its It is well known that TCM is guided by the theory of TCM and characteristics of being multicomponents, multitargets, and multipathways in the treatment of diseases, which meets the requirements of systematically tackling complex diseases such as colorectal cancer. Guo et al. established the model of colorectal cancer and predicted the traditional Chinese medicine components of inhibiting inflammation-induced tumorigenesis by using network pharmacology method . Gupta ehttp://lsp.nwu.edu.cn/tcmsp.php), Shanghai Chemistry & Chemical Industry Data Platform , and papers published in Chinese Network Knowledge Infrastructure  and PubMed were used to collect the chemical constituents of curcuma. We screened curcuma compounds based on absorption, distribution, metabolism, and excretion (ADME) [https://pubchem.ncbi.nlm.nih.gov/) to finish Swiss ADME prediction [Traditional Chinese Medicine System Pharmacology Database   , and phan (ADME) . We furtediction , which wediction .http://www.swisstargetprediction.) [PubChem was used to search the chemical structures of the active compounds. Potential targets of curcuma were predicted by Swiss Target Prediction  . Probabihttps://www.genecards.org/) [https://www.drugbank.ca/) with the keyword “ulcerative colitis.” In addition, articles published in CNKI and PubMed about the known targets of its active compounds were counted [Data of UC-associated target genes were gathered from GeneCards (ds.org/)  and Drugds.org/)  (https:/ counted . Genes f counted .http://www.bioinformatics.com.cn/), and the overlapped targets of compound-disease were obtained as the potential targets for further analysis.The screened curcuma targets and UC targets were imported into Bioinformatics  (http://https://string-db.org/), which covered almost all functional interactions between the expressed proteins [Protein-protein interaction (PPI) network was derived based on the STRING database (proteins . Specieshttps://metascape.org/) [P value less than 0.05.The biological process (BP), molecular function (MF), cell component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out using Metascape system (pe.org/) . In thisA visual network was constructed through Cytoscape software to reflect the complex relationship between active compounds, filtrated targets , and patUsing TCMNPAS system  and Autoβ-actin, anti-STAT3 antibody, anti-TNF-α antibody , HE dyeing , and neutral gum  were used.Curcuma , DSS , absolute ethyl alcohol, Tween-20, xylene substitute , RIPA Lysis buffer, PMSF, BSA, BCA Protein Quantitation Kit (Beyotime), PAGE gel rapid preparation kit, Multicolor Restrained Protein Ladder , Homogenized tube, ceramic beads, frozen storage tube , centrifuge tube , PVDF membrane , and 96-well plate (Eppendorf Life Sciences Corporation) were used.H2050r high-speed refrigerated centrifuge (Hunan Xiangyi Company), MIX-S vortex mixer, shaker oscillator , TGear mini centrifuge (Tiangen), heating magnetic agitator , SIM-F140 ice maker , electronic balance , enzyme labeling instrument , tissue grinding homogenizer , electric constant temperature blast drying oven , electrophoretic system, transfer system, glue rack, ultralow-temperature freezer , microtome , and TKY-BMB, electrothermostatic water bath .Healthy male Sprague Dawley (SD) rats, weighing 180 ± 20 g, were provided by Charles River Experiment Technology Co., Ltd., and the certificate number is SCXK (Hu) 2017-0005. The rats were housed in the animal room of Shanghai University of Traditional Chinese Medicine.The native herb was selected, and standard decoction pieces were prepared with reference to the Chinese Pharmacopoeia (2015 edition). Extract was prepared by boiling the samples in 8 times amount of water for 30 min. The procedure was repeated 3 times.SD rats were accepted to the laboratory for 7 days before the experiments. According to the random number table, the rats were divided into 3 groups of 4 rats each: control, model, and curcuma. Except for the control group, the UC model was prepared with 5% DSS, and the intervention was given according to the group after 7 days.The curcuma group was administered continuously by gavage with 2 ml 0.1 g/ml curcuma suspension for 7 days. The other two groups were given saline 1.08 g/kg.The steps to stain the samples are as follows: xylene I and xylene II, 10 min for each; 100% alcohol I, 100% alcohol II, 95% alcohol, and 85% alcohol, 5 min for each; water washing for 20 s, hematoxylin for 7 min, water washing for 1 min, 1% hydrochloric acid alcohol for 10 s, 50°C water washing for 5 min, eosin stain for 2 min, and water washing for 10 s; 85% alcohol, 95% alcohol, 100% alcohol II, and 100% alcohol I, each for 2 min; and xylene II and xylene I, 3 min for each. Neutral balsam was added after xylene was passerillaged.μL of RIPA, and 5 μL PMSF were added to each tube. After five times' homogenization, the colon tissue was basically broken. The supernatant was extracted after centrifugation to test the protein concentration and to collocate protein solution. Equivalent amounts of protein (200 μl) were denatured at 98°C for 10 min in sample loading buffer, then separated by electrophoresis in 15% gel, and electrotransferred onto 0.45 μm polyvinylidene difluoride membranes for 60 min at 350 mA. Subsequently, the membranes were blocked in blocking buffer  at 25 ± 5°C, followed by incubation with primary antibodies against STAT3 (1 : 5000) and TNF-α (1 : 5000) at 4°C overnight. After being washed with Tris-buffered saline containing Tween-20 (TBST) for 5 min three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After the membranes were washed three times in TBST for 20 min each time, the bands were visualized on X-ray film using an enhanced chemiluminescence western blotting (WB) detection system. The Image Lab™ software was used for quantitative analysis.For protein extraction, tissues were placed in homogenized tubes; 5 porcelain beads, 500 The workflow of this study is summarized in TCMSP database, SGST, and articles published in CNKI and PubMed were used to gather the active components and targets, and 13 potential ingredients were discovered from curcuma . We exclWe obtained the curcuma-related targets from GeneCards (whose relevance score is above the median point) and the ones from DrugBank, CNKI, and PubMed . Then thhttp://www.rcsb.org/) to query their PDB ID [http://zinc.docking.org/). Molecular docking was progressed in TCMNPAS to calculate the docking score. The interaction strength between compounds and gene corresponding proteins can be expressed by docking fraction, and the lower the score is, the higher the interaction intensity is. Further, we docked the 54 potential targets with 5-ASA. As shown in We input 54 potential genes into the PDB database  was established through the STRING database. As shown in P < 0.05, such as cellular response to lipid, response to oxygen levels, and regulation of inflammatory response  are closely related to the inflammatory reaction process. p53 signaling pathway is included in the pathways in cancer. Moreover, MAPK signaling pathway and TGF-beta signaling pathway play a crucial role in immunological stress. In addition, we found some other pathways such as endocrine resistance, hepatitis B, serotonergic synapse, and longevity regulating pathway, which revealed that curcuma has a potential application in other related diseases. The KEGG pathways verified that curcuma cured UC by regulating gut microbiota, inflammatory process, immunization, and inflammatory reaction.To further reveal the potential mechanism of curcuma on the effect of UC, we conducted KEGG pathway enrichment analysis on 24 targets and screened out 95 pathways based on the threshold of P < 0.05. NumerouTo view the relationship between the components, common targets, and their corresponding pathways, a target-pathway network was constructed . Twenty According to the HE staining , DSS couα in the colon samples of the model group was significantly increased (P < 0.01). Compared with the model group, the curcuma group can reduce the expression of STAT3 and TNF-α (P < 0.05)  .α and STAT3 are two research hotspots. It was reported that curcumin and semibionic extraction of compound turmeric can inhibit the proinflammatory signaling by STAT3 and TNF-α in experimental colitis [κB expression by regulating tumor necrosis factor-α (TNF-α) in humans [UC is one of the autoimmune diseases affecting northern Europe, Canada, and Australia . The occ colitis , 31. Simn humans . Furthern humans .H. pylori later [α (TNF-α) through inhibiting NF-κB expression [The 12 found core compounds not only have anti-immune and anti-inflammation effects but also have the effect of regulating intestinal flora. Scholars verified that the extension of UC is important for the positivity rate of ri later . Moreovepression . Meanwhipression . Previoupression .α (degree = 35), STAT3 (degree = 33), and PTGS2 (degree = 32) might be the core targets of the PPI network, through whom curcuma may come into play.Molecular docking verified that the binding energy of 24 key targets was better than that of 5-ASA and lower than −5.5, meaning the interaction between the compounds and targets of UC can bind tighter than that of 5-ASA, indicating better therapeutic effects. Common target PPI network showed that the targets were cocontrolled by curcuma and UC, which revealed that curcuma could regulate the expression of UC-regulated targets and alleviate UC symptoms. AKT1 (degree = 43), EGFR (degree = 35), TNF-P < 0.05) indicated that the major hubs were significantly involved in multiple biological processes, including cellular response to lipid, cellular response to organic cyclic compound, and response to steroid hormone. Furthermore, molecular function enrichment analysis showed nuclear receptor activity, transcription factor activity, and steroid hormone receptor activity were contained in the healing process. The active targets involved were AKT1, EGFR, TNF, STAT3, and PTGS2, which mainly concentrate on the molecular processes of immunization, inflammatory reaction, gut microbiota, etc. Some of the genes and mechanisms have been verified in curcumin, while PTGS2 was proposed connecting with the treatment of UC by curcuma firstly as a gene with high possibility. Meanwhile, cellular components consist of RNA polymerase II transcription factor complex, transcription factor complex, nuclear transcription factor complex, and membrane raft, and EGFR, DPP4, ADAM17, BCL2, and CDK2 were contained in the cellular components. The verified impact of EGFR on UC by regulating epithelial barrier function could identify the reliability of this GO analysis [In order to predict the mechanism of curcuma in the treatment of UC, we analyzed the key candidate targets by performing GO enrichment results, such as biological processes, molecular functions, and cellular components. The GO terms  endocytosis the invading bacteria and secrete TGF-β and IL-6. Then, TGF-β and IL-6 stimulate Th17 activating STAT3 with RORγt and RORα for further inflammatory cytokines. The activated expression of STAT3 participates in the gene transcription and protein expression of a variety of inflammatory factors such as TNF-ammation –39. Besiammation . HoweverIn summary, consistent with clinical experience in the long history, experimental verification, and our HE staining, as well as our WB results, curcuma has significant advantages in the treatment of UC. At the same time, the uncovered targets and pathways were excavated for a better research of how curcuma relieves UC. It has a significant value to provide theoretical basis for clinical treatment of UC and a potent evidence for further study of the mechanism of curcuma in the treatment of UC.


Postweaning offspring were maintained on a CD diet for the remainder of the experiment. Body weight, food intake, and water intake were measured weekly. Oral glucose tolerance tests (OGTT) were undertaken at 12 weeks, and the offspring were culled at week 14. Female, but not male, AS groups exhibited decreased glucose tolerance compared to Fr. There was an increase in gonadal fat adipocyte size in male offspring from AS and Fr groups compared to CD groups. In female offspring, adipocyte size was increased in the Fr group compared to the CD group. In female, but not male offspring, there was a trend toward increase in Fasn gene expression in AS group compared to the CD group. Maternal AS and Fr also negatively impacted upon female offspring estrus cycles and induced alterations to markers associated with ovulation. In summary, exposure to Ace-k via the maternal diet leads to impaired glucose tolerance and impacts adipocyte size in a sex-specific manner as well as significantly affecting estrus cycles and related gene markers in female offspring. This has implications in terms of providing tailored dietary advice for pregnant women and highlights the potential negative influence of artificial sweetener intake in the context of intergenerational impacts.Guidelines advising pregnant women to avoid food and beverages with high fat and sugar have led to an increase in the consumption of “diet” options sweetened by artificial sweeteners (AS). Yet, there is limited information regarding the impact of AS intake during pregnancy on the long-term risk of cardiometabolic and reproductive complications in adult offspring. This study examined the influence of maternal acesulfame-K (Ace-K) and fructose consumption on metabolic and reproductive outcomes in offspring. Pregnant C57BL/6 mice received standard chow  In However, despite growing evidence that suggests adverse impacts of AS consumption, the regulations and guidelines relating to AS intake, in particular during pregnancy, remain unclear . The AcaAll animal procedures were approved by the Animal Ethics Committee at the University of Auckland  in accordance with the New Zealand Animal Welfare Act, 1999. Breeding pairs of C57BL/6 mice were purchased from the Vernon Jansen Unit at the University of Auckland and housed under standard conditions .ad-libitum throughout the experiment. Age-matched female mice were housed with an unrelated C57BL/6 male for mating at 10 weeks of age. Confirmation of pregnancy by vaginal plug was recorded as gestational day (GD) 0.5. Pregnant dams were then randomly assigned onto one of the following three dietary groups (n = 8/group):All mice were maintained on a standard chow diet  Control Artificial sweetener Fructose ad-libitum from weaning until the end of the experiment (14 weeks). An oral glucose tolerance test (OGTT) was carried out 2 weeks prior to cull. At 14 weeks of age, mice were fasted for 6 h and tail blood and plasma samples were collected as detailed below. Mice were then weighed and culled by cervical dislocation. Female mice were staged prior to cull to ensure all female offspring were in diestrus. Gonadal adipose tissue and, in female offspring, ovaries were dissected, weighed, and snap-frozen, subcutaneous fat was collected and snap-frozen, and then stored at −80°C for later analysis.Artificial sweetener and Fr doses were calculated to be equivalent to a human dose of one standard can of soda a day. Diets were maintained throughout pregnancy and lactation. Date of birth was noted and marked as postnatal day 1 (P1). At postnatal day 2 (P2), litters were weighed, sex was determined using anogenital distance, and litters were randomly reduced to eight pups per litter  to standardize nutrition until weaning. Offspring were weaned at postnatal day 21 and one male and one female from each litter were assessed to represent true biological replicates across the eight litters generated per dietary group. Food and water intake were also measured weekly. All offspring were fed the standard control diet and water Onset of puberty in female offspring was determined by sighting of vaginal canalization. Screening for puberty onset began at day 25, occurring every morning at a consistent time (0900h), and continued until vaginal canalization was observed. The age and body weights of offspring at the time of puberty onset were recorded.Estrus cycle staging was carried out through vaginal smear cytology, as detailed previously . Smearinvia oral gavage. Blood glucose concentrations were measured from the tail tip at 0, 15, 30, 60, 90, and 120 min. Tail blood was also collected into EDTA microvettes  at 0, 15, and 60 min. Samples were centrifuged for 10 min/1500 x g/4°C, and the resulting plasma was stored at −20°C for plasma insulin analysis.At 12 weeks of age, mice were fasted for 6 h from 8 a.m. Following weighing, the tip of the tail was snipped (<1 mm) and the second drop of blood was read using a glucometer . Mice received 2 g/kg D-glucose Gonadal and subcutaneous adipose tissues were fixed in 10% neutral-buffered formalin, then paraffin embedded and sectioned (10 μm) using a Leica R 2,135 rotary microtome . The slides underwent H&E staining and were then mounted with DPX mountant. Slides were visualized at 20x and images were captured using a light microscope and NIS Elements-D software (Nikon 800). Four representative images of each section were taken and analyzed in a blinded manner with ImageJ software (NIH) to determine the mean adipocyte size and distribution.The mouse-specific insulin, leptin, and testosterone ELISA kits (Ultra Sensitive Mouse Insulin ELISA Kit (Cat. # 90080), the Mouse Leptin ELISA kit (Cat. # 90030), and the Mouse Testosterone ELISA Kit (Cat. # 80552), Crystal Chem Inc., IL, USA) were used according to the manufacturer's instructions.Female ovarian RNA was extracted using RNeasy Mini Kits  and a TissueLyser  as per the manufacturers' instructions.g/4°C, and the accumulated fat layer was removed to improve RNA yield. Following the addition of isopropanol, samples were incubated overnight at −20°C.Adipose tissue RNA was extracted using the trizol extraction method and a TissueLyser . Following homogenization, the lysate was centrifuged for 10 min/1200 x Rps29 and Rps13 expression. The comparative CT method was utilized to analyze the results . A High Capacity cDNA Reverse Transcription Kit  was used to generate cDNA as per the manufacturer's instructions. Polymerase chain reaction (PCR) was performed using the Applied Biosystems QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). Genes were normalized to the geomean of  results .post-hoc tests were performed as indicated for comparison testing between the groups. Significance between the groups was considered at P < 0.05. Unless otherwise stated, all data are presented as mean ± SEM with an n = 8 per sex per maternal dietary group.Statistical analysis was performed using GraphPad Prism and IBM SPSS Statistics Data Editor Version 27. The Shapiro–Wilk test was used to assess normality. Any data that was not normally distributed were transformed as appropriate. Female and male offspring data were analyzed separately. Repeated measures two-way ANOVA was performed for the OGTT data. Estrus cyclicity data were analyzed using nominal logistic regression. All other data were analyzed using the one-way ANOVA. The Holm–Sidak Maternal AS and Fr consumption had no impact on offspring birth, weaning, or final cull weight in either female or male offspring. Similarly, there was no difference in absolute gonadal fat weight or when expressed relative to body weight in either female or male offspring. There was no effect of maternal dietary group on ovary or testes weights. There was no difference between groups in relation to male/female ratios and litter size .Plasma leptin and insulin concentrations and HOMA-IR index were unchanged by either maternal AS or Fr consumption in female and male mice. There was no change in fasting plasma glucose concentrations at cull in female offspring. However, in male mice, AS reduced glucose concentrations compared to both CD and Fr groups. Maternal diet had no effect on the testosterone concentrations in female offspring. However, in male mice, maternal Fr intake significantly increased the plasma testosterone concentrations compared to CD and AS groups .In female offspring, glucose tolerance in Fr offspring was improved compared to the AS group, which was reflected in a significant difference at the 30 min time point in the OGTT graph and in a decreased OGTT area under the curve (AUC) . There wp = 0.067)  . AdipocyCD group .2, while AS and Fr distribution peaked at the larger size of 4–7,000 μm2. AS and Fr male mice had les adipocytes at <1,000, 1–2,000, and 2–3,000 μm2 compared to CD, but more adipocytes in the size range of 6–7,000, 7–8,000, and 9–10,000μm2 compared to CD mice. Further, a maternal Fr diet increased the number of adipocytes at 5–6,000 and 8–9,000 μm2 compared to CD , insulin receptor substrate 1 (Irs1), forkhead box O1 (Foxo1), SRY-box transcription factor 9 (Sox9), tumor necrosis factor alpha (Tnfα), delta-like non-canonical notch ligand 1 (Dlk1), toll-like receptor 4 (Tlr4), leptin receptor (Lepr), nuclear factor kappa-light-chain-enhancer of activated b cells (Nfkb), solute carrier family 2 member 4 (Slc2a4), peroxisome proliferator-activated receptor gamma (Pparγ), peroxisome proliferator-activated receptor gamma coactivator 1 (Ppargc1), or interleukin-1 (Il-1).There were no differences in Maternal diet had no impact on age of puberty onset or body weight at puberty onset in females . Female Pgr) compared to AS, inducing a significant overall dietary effect (Cyp17a1) similarly saw Fr expression reduced compared to AS, with a significant dietary effect (Fshr), leptin receptor (Lepr), growth differentiation factor 9 (Gdf9), forkhead Box O3 (Foxo3a), bone morphogenetic protein 15 (Bmp-15), estrogen Receptor 2 (Esr2), luteinizing hormone/choriogonadotropin receptor (Lhcgr), hydroxysteroid 17-beta dehydrogenase 1 (Hsd17a1), or Epiregulin (Ereg) across the different maternal dietary groups.Maternal consumption of Fr reduced the expression of progesterone receptor  in accordance with the New Zealand Animal Welfare Act, 1999.Conceptualization was done by CR. Methodology was carried out by CR, PB-C, and MV. Data collection was done by CR, AS, JR, JM-J, and PB-C. Data analysis was performed by CR, PB-C, and JR. Writing—original draft preparation was done by PB-C. Writing—review and editing was done by CR, PB-C, and MV. Supervision was carried out by CR and MV. Project administration, was done by CR and MV. Funding acquisition was taken care of by CR. All authors have read and agreed to the published version of the manuscript.This work was supported by funding from the Faculty Research Development Fund (#3710414) at the University of Auckland and The University of Auckland Foundation (#3721401). PB-C was supported by a Maori Health Research PhD Scholarship from the Health Research Council of New Zealand (#3714791). MV is supported by a James Cook Research Fellowship from the Royal Society of New Zealand. CR is supported by an Ad Astra fellowship from University College Dublin.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Feeding behavior traits are an essential part of livestock production. However, the genetic base of feeding behavior traits remains unclear in Pekin ducks. This study aimed to determine novel loci related to feeding behavior in Pekin ducks.P-value < 4.74E-06) SNPs for feeding behavior traits were discovered, and four of them reached the genome-wide significance level . One genome-wide significance locus associated with daily meal times was located in a 122.25 Mb region on chromosome 2, which was within the intron of gene ubiquitin-conjugating enzyme E2 E2 (UBE2E2), and could explain 2.64% of the phenotypic variation. This locus was also significantly associated with meal feed intake, and explained 2.72% of this phenotypic variation.In this study, the feeding information of 540 Pekin ducks was recorded, and individual genotype was evaluated using genotyping-by-sequencing methods. Genome-wide association analysis (GWAS) was conducted for feeding behavior traits. Overall, thirty significant (This study is the first GWAS for feeding behavior traits in ducks. Our results provide a list of candidate genes associated with feeding behavior, and also help to better understand the genetic mechanisms of feeding behavior patterns in ducks.The online version contains supplementary material available at 10.1186/s12864-021-07668-1. Animal behavior refers to the instant response of animals to changes in their living environment or variations in their internal physiological conditions. Therefore, feeding behavior is of great significance for maintaining individual survival and population development. Feeding behavior patterns have been studied intensively in different animals, including mice , zebra fFeeding behavior traits are also very vital in Pekin ducks. For one thing, feeding behavior can be an effective way to track animal performance and health status. For another thing, researchers had found that there were strong associations between feeding behavior and feed efficiency in broilers and ducks , 9. NowaThe goal of this study was to investigate the genetic markers associated with feeding behavior in Pekin ducks. Previous researchers had shown that genotyping-by-sequencing (GBS) could be an efficient approach to genotype in ducks –14. In tThe feeding behavior information was collected from 540 ducks. The descriptive statistics of phenotypic record are shown in Table P-value.A total of 1 TB clean reads were produced and 99.15% of these were aligned to the reference genome. Through imputation and quality control, 1,899,988 SNPs were attained for association analysis. The frequency of SNPs along the genome and estimated genomic inflation factors are illustrated in supplementary data , Fig. S2Estimation of genetic parameters using genomic information is summarized in Table P-value < 4.74E-06) among 9 different chromosomes were identified .The results of loci-based analysis are shown in Fig. ed Table , includiUBE2E2), which could explain 3.42 and 2.74% of phenotypic variation, respectively.For NM and MFI, a common peak of genome-wide significant SNPs was identified on chromosome 2 due to the strong genetic correlations between them Fig. b, c. TheUBE2E2 on chromosome 2 was also associated with MD, accounting for 2.38% phenotypic variance. The most significant SNP for DFI was in the upstream region of RB binding protein 8 (RBBP8), with a significance of 1.07E-06. Our results also revealed that the top SNP for TFT was suggested to be associated with eyes shut homolog (EYS).As for the other feeding traits, the SNP located in the intron of P-values of previous significant SNPs for NM on chromosome 2 were lower than the potential significance level. No significant association was found after conditional analysis. So we could infer that the other significant SNPs in this region gained the genome-wide significance due to their close linkage relationships with the top associated SNP.According to Fig. ARHGEF10L), which was associated with DFI in ducks in the current study, mapped to body weight QTL in chicken studies. UBE2E2, a common candidate gene for NM, MFI, and MD, is associated with Wattles weight QTL in chickens.A total of 25 candidate genes were detected by GWAS analysis. We performed QTL annotation of these 25 chicken ortholog genes using the chicken QTL database. Seventeen genes were annotated with QTL information in chickens. The QTL information for five genes was consistent with the feeding behavior traits collected in this study Table . Rho guaGenetic parameters play a vital role in designing the animal breeding plan, and genetic parameter estimation using the relationship matrix based on whole-genome genotypes can be an efficient way to achieve this goal . As showMoreover, NM had relatively strong negative genetic and phenotypic correlations (≤ − 0.63) with MFI and MD in this study. Similar intensely negative phenotypic and genetic relationships (ranging from − 0.49 to − 0.93) were obtained in our former work . It suggUBE2E2 is a common candidate gene for the top SNPs of NM, MFI, and MD. UBE2E2 belongs to the UBE2E enzymes family and can lead to the ubiquitination of specific proteins. Mizukami, et al. [UBE2E2 in rat liver cells could facilitate cell proliferation. Yaagoubi, et al. [UBE2E2 accounted for metabolic syndrome in the Moroccan population. It is also reported that UBE2E2 is involved in adipocyte development and type 2 diabetes in humans [Feeding behavior traits are complex and are controlled by a collaboration of various biological processes, including energy metabolism, neurological development, and muscle system movement. In this study, , et al.  found th, et al.  discovern humans , 21. ComRBBP8 and synaptosome associated protein 47 (SNAP47). Mumtaz, et al. [RBBP8 mutations led to microcephaly, intellectual disability, and short stature in Pakistani people. Li, et al. [RBBP8, which was closely associated with body weight at the age of 120 days in purebred Wengshang Barred chicken. Münster-Wandowski, et al. [SNAP47 had a distinctive location in glutamatergic neurons in the mouse. In comparison with these results, we inferred that these two genes might regulate body weight gain and neuron activity in Pekin ducks. Meanwhile, one significant locus located in the intron of CXADR like membrane protein (CLMP) was also obtained in this study, contributing to 1.46% of the phenotypic variation. CLMP encodes a type I transmembrane protein and takes part in cell-cell adhesion. Previous researchers had shown that this gene was essential for normal intestinal development, and mutations in the gene were associated with congenital short bowel syndrome [CLMP and feed intake in ducks.As for DFI, two significant SNPs were detected on chromosome 2, and the candidate genes were , et al.  revealed, et al.  found on, et al.  pointed syndrome –27. Our EYS. EYS was reported to play an important role in degeneration of retinitis photoreceptors in humans [EYS might be associated with visual perception in Pekin ducks. Additionally, EGF like domain multiple 6 (EGFL6), cadherin 2 (CDH2), and EH domain containing 4 (EHD4) were also closely associated with TFT in our results. EGFL6 is a member of the epidermal growth factor repeat superfamily, which coordinates the regulation of bone remodeling and glucose homeostasis in humans [CDH2 produces a classical cadherin that can be crucial for bone growth and osteoblast differentiation in mice [CDH2 is associated with knee osteoarthritis in children [EHD4 took part in the activation of ATPases in Caenorhabditis elegans. And EHD2, a paralog of EHD4, was identified to be differentially abundant at 4 and 24 h in postmortem normal breast and woody breast in commercial broilers (P < 0.05), which might imply that EHD2 took part in the breast muscle myopathies process [For TFT, the most significant SNP was located downstream of n humans , 29. Then humans , 31. CDH in mice . The mutchildren , 34. Melchildren  reported process . IntegraIL1RAPL1) was associated with feed conversion ratio (FCR) and the candidate gene for residual feed intake (RFI) was ring finger protein 17 (RNF17). Deng et al. [ATP11A) was closely associated with body weight, dressed weight, eviscerated weight, foot weight, and wing weight in Pekin ducks. Compared with their results, the 25 candidate genes for feeding behavior traits in this study didn’t have any overlap with them. This might due to the fact that the genetic correlations between feeding behavior traits  and feed efficiency traits (FCR and RFI) were relatively low, which ranged from − 0.03 to 0.20 [Moreover, as we had previously conducted a genome-wide association study for feed efficiency traits in a fat strain of Pekin ducks, Zhu et al.  found thg et al.  also ideIn this study, the genetic parameters of feeding behavior traits were estimated, and the related genomic variations were identified. We obtained 30 significant SNPs using mixed-linear models, which localized to 25 candidate genes for the five different traits studied. The results of this study contributed to the sparse knowledge of feeding behavior traits in Pekin ducks and helped to achieve balanced breeding in the future.All of the experimental ducks were reared under consistent conditions at Beijing Golden Star Duck Inc. Briefly, ducks were provided ad libitum water and commercial diets: a starter diet (from 1 day to 18 day of age) containing 19% crude protein (CP), 12.81 MJ/kg dietary metabolizable energy (ME), and a grower diet containing 17.1% CP and 11.95 MJ/kg ME during our observation period. A total of 540 19-day-old ducks  were divided into three batches , with an interval of 7 days between each batch. At six weeks of age, ducks were blood sampled via the caudal tibia vein after fasting for six hours.Combined with radio frequency identification (RFID) technology and electronic feeders, feeding information of each duck was collected during the test period. The observation period for feeding behavior started at the age of 19 days and ended at the age of 42 days. The details of the recording process and the summary of different feeding behavior traits followed the same methods in our previous research . OverallMseI . Fragments ranging from 550 to 580 bp, including adapter sequence, were sequenced using an Illumina HiSeq2500 instrument. The data were deposited in the NCBI sequence read archive (SRP155579).Genomic DNA was extracted using a phenol-chloroform-based method, and genotyping was performed with a GBS method, which was first applied in our previous research . Briefly2 > 0.98 for low-quality filtering. We identified a subset of tagging SNPs that passed the following thresholds: minor allele frequency (MAF) > 0.05, sample call rate ≥ 0.95, and SNP call rate ≥ 0.95 with PLINK.Overall, a total of 1 TB clean reads were generated, together with an average sequencing depth of 1.5X for each duck. Clean data was mapped to a reference genome using BWA (v 0.7.17) , and thehttps://mirrors.tuna.tsinghua.edu.cn/CRAN/, TUNA Team, Tsinghua University, Beijing, China), and covariates with P-value < 0.05 were included in a mixed linear regression model as the fixed effects  for principal component analysis (PCA) , and the top three eigenvectors were implemented in association analysis to account for the effect of population stratification. The effects of covariates, including sex and batch, on quantitative phenotypes were assessed with analysis of variance (ANOVA) using R software  algorithm in GCTA (v 1.26.0) , which iWhere y is a vector of the normalized phenotype, a is the general mean, X is the matrix of fixed effects , B is the vector of corresponding coefficients; g is the accumulated effect of all SNPs except those on the chromosome where the candidate SNP is located , e is the vector of random residuals.The SNP-based genetic parameters for feeding behavior and the contribution of the phenotypic variances for significant SNPs were estimated using the GREML algorithm in GCTA (v 1.92.2) . The linWhere y is a vector of phenotypic value, W is the matrix of fixed variables, and Z represents the matrix of random variables, which refers to the effects of all SNPs on phenotype; e stands for random residuals, I is an identity matrix, b and u are the corresponding coefficients of fixed and random effects, respectively; λ) was calculated with R package qqman (0.1.4) [The genomic inflation factor ( (0.1.4) . Bonferr (0.1.4) . TherefoP-value that passed the significance level. GWAS does not distinguish a genuine causal locus from those statistically significant loci within a strong linkage disequilibrium (LD) region. Therefore, in order to characterize potential candidate genes responsible for a trait, we conducted a LD analysis and inferred the haplotype blocks containing peak SNPs by LDBlockShow [The conditional analysis was conducted to examine the potential associated SNPs that might be masked by a strong signal. Briefly, the initial region was tested with the strongest SNP covariate. Then, association analysis conditioning on the selected SNP was implemented to search for the top SNP iteratively one by one via a stepwise model selection procedure until no SNP had a conditional lockShow .P-value < 4.74E-06), we looked for the nearest candidate genes using BEDTools [For all the SNPs that exceeded a potential significant threshold (BEDTools . Due to BEDTools  using chAdditional file 1 Table S1.Additional file 2 Fig. S1-Fig. S3.


Burkholderia pseudomallei, is associated with a high case fatality rate, due in part to difficulties in clinical recognition and diagnostic confirmation of the disease. Although head and neck involvement is common in children, specific disease manifestations differ between geographic regions. The aim of this study was to provide a detailed description of melioidosis of the head and neck among children in Sarawak, Malaysia, and determine if fine-needle aspiration of suspected head or neck lesions could improve melioidosis diagnosis.Melioidosis, the infection caused by We conducted a retrospective descriptive study of all children aged < 12 years with culture-confirmed melioidosis presenting with head and neck manifestations and admitted to Bintulu Hospital in Sarawak, Malaysia, from January 2011 until December 2020. Fine-needle aspiration of head and neck lesions suspected to be due to melioidosis with inoculation in blood culture bottles (FNA + BCB) was used from the beginning of 2016.B. pseudomallei lymph node infections were highly variable. Five (25%) children had salivary gland involvement. Lacrimal gland involvement (dacryocystitis) and skin or soft tissue infection  were less frequent. B. pseudomallei was isolated from the head or neck using FNA + BCB in 15 (75%) children and by standard culture methods of direct plating of pus on agar following incision and drainage in only 2 (10%) children. B. pseudomallei was isolated from non-head or neck specimens or blood in 3 (15%) children.Of 34 children with culture-confirmed melioidosis, 20 (59%) had an infection involving one or more sites in the head and neck. Of these, 17 (85%) were diagnosed in or after 2016. Cervical lymph nodes were the most common organ or site affected, involved in 19 (95%) children. Clinical presentations of B. pseudomallei detection and enables confirmation of melioidosis infections.Manifestations of pediatric head and neck melioidosis in Sarawak, Malaysia, differ from those of other regions. Fine-needle aspiration, mainly of affected cervical lymph nodes, facilitates The online version contains supplementary material available at 10.1186/s12879-021-06754-9. Burkholderia pseudomallei, is associated with a high case fatality rate [Melioidosis, the infection caused by the environmental saprophyte ity rate , 2. Thisity rate , but alsity rate , 5, espeity rate , 7.Difficulties in clinical recognition of melioidosis arise mainly due to the varied and non-specific presentation of patients. Patients from different regions can have different disease manifestations , and theIn view of and despite the limitations of the various diagnostic modalities, adequate sampling of the site of infection is key in diagnosing melioidosis . The useBintulu Hospital is a 302-bed hospital which provides primary and secondary health services to a total population of 256,000, including 72,000 children aged < 15 years, residing in Bintulu Division and Belaga district of Kapit Division in central Sarawak, Malaysian Borneo. Although adult general surgery and otorhinolaryngology services are available, specialized pediatric services are only available in a tertiary referral center located over 300 km away in southern Sarawak.B. pseudomallei and improve melioidosis diagnosis.In 2017, we reported a broad description of melioidosis infections among a total of 42 children presenting to three hospitals in central Sarawak, including Bintulu Hospital , 21. TheWe performed a retrospective descriptive study of all children with melioidosis of the head and neck presenting to Bintulu Hospital in Sarawak, Malaysian Borneo, over a 10-year period from January 2011 to December 2020.B. pseudomallei from any clinical sample, including from non-head or neck organ-sites. To identify cases of melioidosis of the head and neck, we first conducted a manual search of the microbiology laboratory logbooks and electronic database to identify children aged < 12 years with culture-confirmed melioidosis. Medical records were then retrieved and reviewed to identify those who had melioidosis of the head and neck. Details on demography, clinical, laboratory, and radiological findings, case management, and outcome were collected using a standardized case report form.A child was classified as having melioidosis of the head and neck if any structure or organ in the head or neck, with the exception of the nervous system organs, were involved  at presentation in a culture-confirmed melioidosis case. Culture-confirmed melioidosis was defined by isolation of 9/L in children < 2 years, > 12 × 109/L in children 2–9 years, and > 10.5 × 109/L in children > 9 years [9/L in children < 6 years and > 8.0 × 109/L in children 6 years or older. Monocytosis was defined as an absolute monocyte count > 1.0 × 109/L. Thrombocytosis was defined as a platelet count > 400 × 109/L. A melioidosis-active antibiotic regimen was defined as the use of either ceftazidime or a carbapenem during the intensive phase and oral trimethoprim–sulfamethoxazole during the eradication phase, with amoxicillin–clavulanate or doxycycline used as second-line oral agents when resistance, contraindications, or intolerance to trimethoprim–sulfamethoxazole was present [Based on admission body weight, poor nutritional status was diagnosed in children aged ≤ 10 years if weight-for-age was below − 2 z-score using the World Health Organization Child Growth Standards , while c 9 years . Neutrop present .Before 2016, pus and other samples from the head and neck were mainly collected during incision and drainage procedures using sterile bottles before being transported as quickly as possible to the microbiology laboratory. These specimens were then cultured directly on blood agar (BA), chocolate agar (CA), and MacConkey agar (MAC) (standard methods). From 2016, in an attempt to improve culture-confirmation of melioidosis infections, a fine-needle aspiration technique with inoculation in blood culture bottles (FNA + BCB) was used in addition to standard methods. With this method, fine-needle aspiration of affected lymph nodes and other lesions in the head and neck were performed using a 23-gauge needle, similar to fine-needle aspiration biopsy procedures previously described . AspiratB. pseudomallei was identified with either API®20NE  or BBL™ Crystal™ Identification Systems . Blood samples collected from patients were subjected to the BACTEC blood culture system.Statistical analysis was performed using SPSS Statistics 21. Continuous variables were presented using the median and interquartile range (IQR).Thirty-four children had culture-confirmed melioidosis during the 10-year study period. Medical records of all cases were available for review. Head or neck manifestations were present at admission in 20 (59%) children. Of these, only 3 (15%) were diagnosed between 2011 and 2015, while 17 (85%) were diagnosed between 2016 and 2020.The median age of children with head or neck melioidosis was 8.0 years   children. However, only 1 presented with symptoms/signs of salivary gland disease . In the remainder, the salivary gland involvement was detected on ultrasound examination, performed in children who had presented with cervical lymph node infection. Ultrasound findings in these four children showed a unilateral parotid abscess measuring between 2 and 3 cm (n = 2), multiple bilateral parotid abscesses smaller than 0.5 cm (n = 1), and unilateral enlarged submandibular gland with intra-glandular lymph nodes (n = 1), in addition to the enlarged cervical lymph nodes. Overall, 67% (12/18) of children who presented with cervical lymph node manifestations had head and neck ultrasonography; 33% (4/12) were found to have occult salivary gland involvement.Two children had lacrimal gland involvement. Both presented with cervical lymph node infection: one had bilateral lymphadenopathy and was found to have concomitant unilateral dacryocystitis; the other had unilateral lymphadenitis with ipsilateral dacryocystitis Fig. . Skin orOf the 20 children with head and neck melioidosis, 7 (35%) had a localized infection, whereas 13 (65%) had disseminated disease. In the 13 children with disseminated melioidosis, non-head or neck sites of infection included spleen (n = 11), lungs (n = 2), joints (n = 2), non-head or neck soft tissue (n = 2), axillary lymph node (n = 1), and brain (n = 1).Leukocytosis was present in 13 (65%) of the 20 children. Of 16 (80%) with differential counts, 13 (81%) had neutrophilia and 10 (63%) had monocytosis. Lymphocyte counts were normal in all but two children. The median hemoglobin was 11.0 g/dL (IQR 10.5–12.2 g/dL). Thrombocytosis was present in 13 (65%) patients. The median erythrocyte sedimentary rate was 89 mm/h (IQR 70–105 mm/h).Seven (39%) of the 18 children who presented with cervical lymph node infection had histologic examination. Five (71%) were reported to have epithelioid granulomas; among these, multinucleated giant cells and caseous necrosis were found in 4 and 1, respectively. Histology of the cervical lymph nodes in the remaining two cases showed neutrophils and lymphocytes in one, and sparsely cellular smears with poorly preserved histiocytes in the other.B. pseudomallei was isolated from blood in the two remaining cases.The source of positive isolates in the 20 cases included pus/tissue from cervical lymph nodes (n = 15), salivary gland (n = 1), head or neck skin/soft tissue abscess (n = 1), and non-head or neck skin/soft tissue abscess (n = 1). B. pseudomallei from the head or neck  . No significant difference was noted in the duration between FNA + BCB procedure and B. pseudomallei identification for cases with no/minimal aspirate (and no standard method performed) and those with more substantial volume of pus/tissue aspirate (as indicated by the availability of pus/tissue for standard culture methods) (P = 0.8).Of the three cases of melioidosis of the head and neck identified between 2011 and 2015 (before the initiation of FNA + BCB), 2 were diagnosed by isolation of All children had blood cultures performed. Of the 18 without bacteremia, 7 (39%) had multiple blood cultures performed.One (5%) child had a fatal outcome. This was a 7-year 10-month old boy who presented in 2014 with bilateral cervical lymphadenitis, disseminated sites of infection , bacteremia, respiratory failure, and septic shock on admission.Twelve (60%) children were initially treated empirically for pyogenic infections other than melioidosis; antibiotics used included ampicillin, cloxacillin, cefuroxime, ampicillin/sulbactam, and metronidazole. Four (20%) were discharged home to continue oral antibiotics but returned with unresolved symptoms.All ultimately received melioidosis-active antibiotic regimens; surgical incision and drainage was required in 7 (35%) cases. Among survivors, the median duration of parenteral intensive phase treatment was 21 days (range 13–45 days). All had clinical resolution of the presenting manifestations. For eradication, 9 (47%) children received trimethoprim–sulfamethoxazole, 5 (26%) received amoxicillin–clavulanate, and 5 (26%) others received a combination of amoxicillin–clavulanate and doxycycline. The median duration of oral eradication therapy was 17 weeks (range 12–28 weeks). No relapses were recorded during the study period.No complications from the fine-needle aspiration procedure were recorded.This study reports the demography, clinical presentations, laboratory findings, treatment, and outcome of children with melioidosis of the head and neck in Sarawak, Malaysian Borneo. We show that children with head or neck melioidosis in Sarawak have distinct manifestations with involvement predominantly of cervical lymph nodes. In addition, we describe how the use of fine-needle aspiration improves diagnosis of melioidosis in these children.B. pseudomallei through the oral/nasal mucosa or conjunctiva, possibly via exposure to contaminated water sources, as opposed to inhalation and ingestion postulated in adults with pneumonia or bacteraemia and skin inoculation among children in northern Australia [The head and neck region was a major site of melioidosis infection in Sarawakian children, with melioidosis of the head and neck representing nearly 60% of the melioidosis cases diagnosed between 2011 and 2020. Head and neck infections are a well described feature of pediatric melioidosis; in Cambodia and Thailand, 65% and 33% of children, respectively, are reported to have head or neck involvement , 28. Intustralia , 32.B. pseudomallei infection from other common causes of cervical lymph node disease, including pyogenic or tuberculous (TB) infection, by clinical, routine laboratory, and even histologic examination was difficult. Cervical lymph node involvement was present in 19 (95%) of our 20 cases and was the primary reason for admission in 80% (16/20) of the cases. These results contrast with findings from Cambodia and Thailand, where salivary glands, and in particular parotid glands, are the most common site of infection [In our study, cervical lymph nodes were the most common organ-site involved in the head and neck, and differentiating nfection , 34. It nfection , the utiFine-needle aspiration, mainly of affected cervical lymph nodes, improved the diagnosis of melioidosis of the head and neck among children in this study, as it facilitated sampling of the site of infection and enabled microbiological confirmation of melioidosis  of children with disseminated disease and in 58% (11/19) overall. As previously reported, detection of splenic abscesses by abdominal ultrasonography offers a useful and rapid clue to melioidosis diagnosis . In addiB. pseudomallei were not performed. Throat swabs have been shown to be useful in the diagnosis of melioidosis and may have been especially beneficial in the present study, as some of the children could have had a pharyngo-cervical syndrome as reported in previous studies [B. pseudomallei requires the use of selective media, such as Ashdown’s agar. However, Ashdown’s agar cannot be used in Sarawak as the majority of cases are caused by gentamicin-susceptible B. pseudomallei [B. pseudomallei selective media and modified Ashdown (with colistin instead of gentamicin) are not readily available. Secondly, this study does not report cases of suspected head and neck melioidosis in which all cultures, including FNA + BCB, were negative and as such cannot inform on sensitivity of the method. This is mainly related to the study methodology and the lack of a gold standard for diagnosis. Another limitation of the study was that no cost-effectiveness calculations were undertaken. These may be important, as melioidosis is largely an affliction of resource-limited regions. Other limitations include the small sample size, the possibility of selection bias related to the monocentric nature of the study, and limitations inherent to the retrospective (bias of information) descriptive (bias of confusion) study design. Despite these limitations, the study manages to provide a fairly comprehensive characterization of pediatric head and neck melioidosis in Sarawak, of which very limited data was previously available.This study has several limitations. Firstly, throat swabs for culture of  studies , 47. Thrdomallei , while oNearly 60% of children with melioidosis in Sarawak had head or neck involvement. Cervical lymph nodes were the most common site of infection in the head and neck; salivary and lacrimal gland infections also occurred but were less frequently observed. Fine-needle aspiration, mainly of affected cervical lymph nodes, was extremely useful in enabling microbiological confirmation of melioidosis, allowing the diagnosis of 75% of our cases. Further studies are needed to confirm the sensitivity, specificity and cost-effectiveness of this simple and safe technique, which may be particularly useful in resource-constrained regions.Additional file 1: Figure S1. Source of positive B. pseudomallei isolate and method used to obtain and isolate the organism in 20 children with head and neck melioidosis in Bintulu Hospital  between 2011 and 2020.Additional file 2: Table S1. Detailed characteristics of the head or neck bacterial culture procedure in the 20 children with melioidosis of the head and neck in Bintulu Hospital  between 2011 and 2020.Additional file 3: Table S2. Patient, health personnel, and infrastructure characteristics present in Bintulu Hospital  related to the improved diagnosis of melioidosis of the head and neck in children with the use of fine-needle aspiration versus surgical biopsy.


Lower respiratory tract infection (LRTI) is an important cause of morbidity and mortality in infants and young children. However, the etiological role of viruses and the timing of developing LRTI are not well defined.We analyzed the data of a prospective cohort study in the Philippines as a birth cohort. We detected LRTI among children who visited healthcare facilities with respiratory symptom, and collected nasopharyngeal swabs for virus detection. We analyzed the incidence rates (IRs) and cumulative proportion of LRTI and severe LRTI by age group and each virus detected.A total of 350 LRTI episodes were observed from 473 child-years yielded from 419 children. The IRs of LRTI were 70.8, 70.7, and 80.8 per 100 child-years for 0–5, 6–11, and 12–23 months of age, respectively. By 12 months of age, 45% of children developed LRTI at least once. Rhinovirus and respiratory syncytial virus were the most frequently detected viruses in all age groups. However, the IRs of influenza virus were low especially at 0–5 months of age.We identified various patterns of age-specific IRs of LRTI and severe LRTI for different viruses, which should be considered to establish more effective interventions including vaccinations.The online version contains supplementary material available at 10.1186/s12879-022-07289-3. Streptococcus pneumoniae and Hemophilus influenzae type b (Hib) have been shown to be a major cause of LRTI deaths in children 24], EV , and reaBaseline characteristics were compared between age of follow-up using chi-square test. Incidence rates were calculated as the sum of episodes divided by the sum of the child-years observed in each age group. Poisson 95% confidential intervals (95% CIs) for IRs were computed. Incidence rate ratios (IRRs) were calculated by comparing the IRs between non-severe LRTI and severe LRTI and 95% CIs for IRRs were obtained using two-sided exact significance test. Cumulative proportions of children who developed LRTI were estimated using Kaplan–Meier survival analysis by the first and subsequent LRTI separately, and by detected viruses. When multiple viruses were detected in one LRTI episode, these LRTIs were included for each virus-specific survival curve. Stata version 15  was used for data analysis.Informed consent was obtained from the guardians of all participants at enrollment. The study protocol was approved by the Ethics Committee of Tohoku University Graduate School of Medicine, Japan, and the Institutional Review Board of the Research Institute for Tropical Medicine (RITM), Philippines. All methods were performed in accordance with the relevant guidelines and regulations.A total of 419 children, after excluding four children who withdrew from the study before age 28 days, were included in the birth cohort analysis .Among 419 cohort children, 350 LRTI episodes were identified  and 22.6 CI 18.5–27.3) per 100 child-years, respectively . Of those, at least one virus was detected from 228 episodes (69.3%). Among seven viruses, RV was the most frequently detected virus  followed by RSV 56/329, 17.0%)  . Lower IAt least one virus was detected in 69.3% of LRTI episodes, which indicate the importance of respiratory viruses in LRTI for children aged < 2 years. The most commonly identified virus was RV (26.4%) followed by RSV (17.0%). RSV has been recognized as the most important viral pathogens associated with hospitalized LRTI cases in infants and young children . In our In the present study, RV showed the highest IRs among detected viruses in all age groups. For 0–5 months of age, the IR of severe RV-LRTI was particularly high and incidence of RV-LRTI and severe RV-LRTI increased immediately after birth. However, the etiological importance of RV for LRTI in infants and young children is still controversial. A birth cohort study in Australia, which included healthy controls, showed that RV is more important than RSV as a cause of LRTI in infants < 1 year of age . On the The overall positivity rates of other viruses, including AdV, EV, MPV, PIV, and IFV were low ranging from 0.9% (AdV) to 8.5% (PIV). It has been shown that seasonal IFV has high disease burden particularly in infants aged < 1 year . TherefoThe study has some limitations. First, we conducted the study for only 2 years and 3 months by recruiting newborns throughout the study period. Therefore, the observed period for each age group varied  stratified by age group in the birth cohort, and number of virus positive samples observed in the original cohort study (among children aged < 5 years) in Biliran, Philippines, from March 2014 to June 2016.Additional file 2: Figure S2. Cumulative proportion of children who developed non-severe and severe virus-specific LRTI.Additional file 3: Table S1. Characteristics of children in the birth cohort study by age at the end of follow-up in Biliran, Philippines, from March 2014 to June 2016.Additional file 4: Table S2. Details of the detected viruses among cases with LRTI in which two viruses were detected in Biliran, Philippines, from March 2014 to June 2016.


Colorectal cancer (CRC) is the third most prevalent cancer in the world, which remains one of the leading causes of cancer-related deaths. Accurate prognosis prediction of CRC is pivotal to reduce the mortality and disease burden. Lymph node (LN) metastasis is one of the most commonly used criteria to predict prognosis in CRC patients. However, inaccurate surgical dissection and pathological evaluation may lead to inaccurate nodal staging, affecting the effectiveness of pathological N (pN) classification in survival prediction among patients with CRC. In this meta-analysis, we aimed to estimate the prognostic value of the log odds of positive lymph nodes (LODDS) in patients with CRC.PubMed, Medline, Embase, Web of Science and the Cochrane Library were systematically searched for relevant studies from inception to July 3, 2021.Statistical analyses were performed on Stata statistical software Version 16.0 software. To statistically assess the prognostic effects of LODDS, we extracted the hazard ratio (HR) and 95% confidence interval (CI) of overall survival (OS) and disease-free survival (DFS) from the included studies.Ten eligible articles published in English involving 3523 cases were analyzed in this study. The results showed that LODDS1 and LODDS2 in CRC patients was correlated with poor OS compared with LODDS0 ; LODDS2 vs. LODDS0: HR = 3.49, 95% CI ). Meanwhile, LODDS1 and LODDS2 in CRC patients was correlated with poor DFS compared with LODDS0 ; LODDS2 vs. LODDS0: HR =3.30, 95% CI ).The results demonstrated that the LODDS stage was associated with prognosis of CRC patients and could accurately predict the prognosis of patients with CRC.The online version contains supplementary material available at 10.1186/s12885-022-09390-x. Colorectal cancer (CRC) is one of the most common malignant tumors in the world, with high morbidity and mortality. It is estimated that there were over 1.8 million new cases in 2018, and at the same time, more than 881,000 deaths were estimated to have occurred . CurrentConsidering the current controversies regarding the significance of LODDS in the prognosis of CRC patients, we systematically analyzed data obtained in published literature and summed the prognostic significance of LODDS in CRC patients.We systematically searched PubMed, Medline, Embase, Web of Science and the Cochrane Library for relevant studies from inception to December 3, 2021. The following keywords were used: “log odds of positive lymph nodes”, “Colonic Neoplasms” [Mesh], and “Rectal Neoplasms” [Mesh], “Colorectal Neoplasms” [Mesh]. We used the following strategy: ((log odds of positive lymph nodes) OR (LODDS)) AND ((((((((((((“Colonic Neoplasms”[Mesh]) OR ) OR ) OR ) OR ) OR ) OR (Colonic Neoplasms)) OR (Colon Cancer)) OR (Colon Tumor)) OR ) OR ) OR ). For the meta-analysis, we followed PRISMA  guidelines .Studies fulfilling the following criteria were included: (i) the article reported at least one of the outcomes of interest or the outcome could be calculated according to data extracted from the published data; (ii) only articles published in English, focused on human, and reporting at least one outcome of interest were evaluated, or the outcome could be calculated according to data extracted from the published data; (iii) all CRC patients were diagnosed with the gold standard test; (iv) we included the studies which classified LODDS into three hierarchical levels because currently classification of LODDS has no uniform standard and we found that most of the studies classified LODDS into three categories during the study selection process.Articles were excluded based on the following criteria: (i) missed crucial information needed for detailed stratification; (ii) number of participants less than 20; (iii) the article was a review, case report, comment, letter, or meeting record; (iv) the article shared a study population with another article.Two reviewers independently used a standardized form to extract the data from the included articles: reference, published year, country, type of cancer, number of patients , age, gender, treatment and prognostic indicators  and disease-free survival (DFS)). Any disputes or differences were settled by a third independent investigator. For articles with multiple arms, each arm was considered an independent data set.Prognostic values (OS and DFS) were used to compare the different LODDS groups.Two investigators independently assessed the quality of the included articles according to the Newcastle-Ottawa scale (NOS) , on the P values were not directly provided in the original literature, the estimated HR was used to assess prognostic effects based on the method described by Tierney et al. [P values, the threshold of which was set to 0.05. Heterogeneity between articles was calculated using the Q test and I2 statistic [I2 statistic, heterogeneity was defined as low (25–50%), moderate (50–75%) or high (> 75%) [P ≤ 0.1 was considered to indicate significant heterogeneity. In addition, based on the differences in the data retrieved, subgroup analyses were performed. Then, we also conducted a sensitivity analysis in which each study was removed in turn to evaluate the undue influence of the study on the overall summary estimates including Duval and Tweedie’s trim-and-fill method [P values for pooled results were two-sided, and the inspection level was 0.05.We used Stata statistical software Version 16.0  to analyze the data in our meta-analysis. To statistically assess the prognostic effects of LODDS, we extracted the hazard ratio (HR) and 95% confidence interval (CI) of OS and DFS from the included studies. If HRs, 95% CIs, or y et al. , and HR tatistic . For the (> 75%) . For thel method , and Gall method . Publical method . P valueThe original search yielded 204 records in PubMed, Web of Science, Medline, the Cochrane Library and Embase. Of these, 128 duplicate articles were excluded. We excluded 46 records after reading the titles and abstracts. After reviewing the full texts, 10 articles , 35–38 wWe analyzed OS and DFS in different LODDS categories according to the data from the included articles , 35–38. I2 statistic = 18.3%, Pheterogeneity = 0.280). The pooled results indicated that LODDS2 CRC patients had a worse OS ) than LOODS0 CRC patients. Regarding the heterogeneity, there was no statistical significance , as shown in Fig. Compared with LOODS0 CRC patients, LODDS1 CRC patients had a worse OS ) where the heterogeneity was insignificant (.28) wherI2 statistic = 35.0%, P heterogeneity = 0.203). The result of pooled analysis using the random-effects model showed that LODDS2 CRC patients was also associated with poor DFS ) than LODDS0 CRC patients, and between-study heterogeneity was obvious , as shown in Fig. Compared with LOODS0 CRC patients, LODDS1 CRC patients had a worse DFS ). The heterogeneity was moderate insignificant . I2 statistic = 74.4%, P heterogeneity = 0.002 to I2 statistic = 0.0%, P heterogeneity = 0.948; Fig. To explore the potential sources of heterogeneity, we used Galbraith plot and Duval and Tweedie’s trim-and-fill method to further explore the source of heterogeneity in DFS, and the result showed that the training set of the study by Ogawa T et al.  might haWe performed subgroup analysis according to differences in the variables, including the publication year, country, and type of cancer. Consistent with above results, LODDS1 and LODDS2 CRC patients had a worse OS and DFS compared with LODDS0 CRC patients in most subsets. Although it is found that OS and DFS of non-Asian CRC patients were better than patients from Asian, high LODDS is a marker for poor prognosis both in non-Asian and Asian CRC patients. Meanwhile, although OS and DFS of rectal cancer patients were better than colon cancer patients, high LODDS is a marker for poor prognosis both in colon and rectal cancer patients, as shown in Table p = 0.729), LODDS2 versus LODDS0 (p = 0.265) in OS. Similarly, there was no evidence for significant publication bias in LODDS1 versus LODDS0 (p = 0.860), LODDS2 versus LODDS0 (p = 0.949) in DFS. The results with heterogeneity adjusted are listed in Table Publication bias was assessed by funnel plots and Egger’ s test, as shown in Fig.  p = 0.26 in OS. SI2 statistic = 74.4%, P heterogeneity = 0.002). To explore the potential sources of heterogeneity, we used Galbraith plot and Duval and Tweedie’s trim-and-fill method to further explore the source of heterogeneity in DFS, and the result showed that the training set of the study by Ogawa T et al. [I2 statistic = 74.4%, P heterogeneity = 0.002 to I2 statistic = 0.0%, P heterogeneity = 0.948; Fig. To our knowledge, this is the first meta-analysis that focused on the significance of LODDS in the prognosis of CRC patients. Arslan NC  suggesteT et al.  might haDespite recent advances in novel antitumor therapeutics, the overall survival is far from satisfactory, especially in patients with advanced CRC. To improve the quality of life of oncological patients, it is necessary to accurately estimate prognosis and adopt personalized therapeutics. Although the number-based UICC/AJCC pN classification in patients with radically resected CRC is currently considered as the most reliable predictor of poor prognosis , 3, the However, several limitations of the current meta-analysis should be emphasized. First, because several studies did not report HRs that were estimated based on the method described by Tierney et al. . Second,In conclusion, our systematic review demonstrated that LODDS is associated with the prognosis of CRC patients and accurately predicts survival of CRC patients. Our meta-analysis indicated that LODDS1 and LODDS2 patients have a poorer OS and DFS compared with LODDS0 patients. Moreover, the results of summary analysis demonstrated the significance of LODDS as a remarkable prognostic indicator of OS and DFS in most subgroups. Further high-quality, large-scale, international, well-designed multicenter prospective studies are required to obtain the optimal cutoff point of LODDS until the utilization of LODSS in the clinical practice.Additional file 1: Figure S1. Egger’s funnel plots on OS and DFS. a-b Egger’s Funnel plot analysis of studies on OS ((a) LODDS1 vs. LODDS0, (b) LODDS2 vs. LODDS0). c-d Egger’s funnel plot analysis of studies on DFS ((c) LODDS1 vs. LODDS0, (d) LODDS2 vs. LODDS0). HR, hazard ratio; OS, overall survival; DFS, disease-free survival; LODDS, log odds of positive lymph nodes; SE, standard error; SND, standard normal deviate.