With the advent of intensive combination regimens, an increasing number of patients with unresectable pancreatic cancer (UPC) have regained the opportunity for surgery. We investigated the clinical benefits and prognostic factors of conversion surgery (CS) in UPC patients.We retrospectively enrolled patients with UPC who had received CS following first-line systemic treatment in our center between 2014 to 2022. Treatment response, safety of the surgical procedure and clinicopathological data were collected. We analyzed the prognostic factors for postoperative survival among UPC patients who had CS.Sixty-seven patients with UPC were enrolled  and 14 with metastatic pancreatic cancer (MPC)). The duration of preoperative systemic treatment was 4.17 months for LAPC patients and 6.52 months for MPC patients. All patients experienced a partial response (PR) or had stable disease (SD) preoperatively according to imaging. Tumor resection was unsuccessful in four patients and, finally, R0 resection was obtained in 81% of cases. Downstaging was determined pathologically in 87% of cases; four patients achieved a complete pathological response. Median postoperative-progression-free survival (PO-PFS) was 9.77 months and postoperative overall survival (PO-OS) was 31.2 months. Multivariate logistic regression analyses revealed that the resection margin and postoperative changes in levels of tumor markers were significant prognostic factors for PO-PFS. No factors were associated significantly with PO-OS according to multivariate analyses.CS is a promising strategy for improving the prognosis of UPC patients. The resection margin and postoperative change in levels of tumor markers are the most important prognostic factors for prolonged PFS. Multidisciplinary treatment in high-volume centers is strongly recommended. Prospective studies must be undertaken to resolve the various problems regarding optimal regimens, the duration of treatment, and detailed criteria for CS.The online version contains supplementary material available at 10.1186/s12885-023-10529-7. Pancreatic cancer (PC) is an extremely malignant tumor type with high lethality in China . In the For this intractable disease, surgery is considered to the only way to take the tumor under permanent control. However, due to its characteristics of hidden onset, rapid progress and metastasis, ~ 80% of PC patients lose the opportunity to undergo surgery because invasion to vessels or distal metastasis have usually occurred at the time of the initial diagnosis .The treatment plan for PC patients is based mainly on the classification of resectability rather than the stage. According to the recommendation from the National Comprehensive Cancer Network (NCCN), systemic treatment is strongly recommended for patients with locally advanced pancreatic cancer (LAPC) or metastatic pancreatic cancer (MPC).Recently, reports have shown the advantages of multidisciplinary treatment for transforming unresectable disease to disease eligible for radical surgery, which has resulted in increased resectability and longer survival. FOLFIRINOX (FFX), modified-FOLFIRINOX (mFFX) and gemcitabine plus nab-paclitaxel (GnP) are the main regimens for all stages of PC for patients with good performance status, and enable downstaging before radical surgery , 6. SeveWe evaluated 67 UPC patients who had recieved downstaging surgery following first-line systemic treatment in our center between 2014 to 2022. We wished to discover the postoperative outcome and potential factors strongly associated with survival for LAPC patients and MPC patients.In this retrospective study, we enrolled 67 patients with UPC (53 LAPC and 14 MPC) who underwent CS after systemic treatment between April 2014 and March 2022 at our center. The protocol for this retrospective study was approved by the Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine . Patients were diagnosed with PC by histology. LAPC or MPC was verified based on the resectability definition set by the NCCN. LAPC was defined as encasement of the superior mesenteric artery (SMA) or celiac axis (CA) > 180°, abutment of the inferior vena cava, unreconstructable superior mesenteric vein/portal vein (SMV/PV) encasement/occlusion, and aortic invasion/encasement. In addition, all patients were reviewed by a multidisciplinary board for cancer treatment to finalize the treatment strategy, evaluate the response to treatment and make decisions regarding the surgical procedure.2 bodyweight), leucovorin (400 mg/m2), irinotecan (135 mg/m2) and subsequent intravenous infusion of fluorouracil (2400 mg/m2) over 46 h. The GnP regimen consisted of gemcitabine (1000 mg/m2) and nab-paclitaxel (125 mg/m2) administered on days 1, 8, and 15, every 28 days. Sequential use of GnP and mFFX was set as a 28-day treatment cycle of GnP followed by an additional two doses of mFFX. During each treatment session, patients were treated symptomatically and monitored closely. Adverse effects were recorded according to Common Terminology Criteria for Adverse Events (CTCAE) 4.0. High-resolution computed tomography (CT) of the lungs, contrast-enhanced magnetic resonance imaging (MRI) of the liver and contrast-enhanced CT of the abdomen were undertaken every 2 months to evaluate the responseto treatment.Some enrolled patients were selected from a prospective clinical study conducted previously in our clinical center. The regimens for these patients strictly followed the standard of clinical research (NCT03977272 and NCT03983057). mFFX (2-weekly schedule) was administered using oxaliplatin  no deterioration of performance status with willingness for surgery from the patient; (ii) radiologic response to systemic treatment according to Response Evaluation Criteria in Solid Tumours (RECIST) criteria; (iii) CR, PR, or SD with the possibility of R0 resection; (iv) improvement of vessel involvement was not essential; (v) marked decrease in cancer antigen (CA)19–9 level in patients with CA19-9 > 200 U/mL at the diagnosis; (vi) metastases were limited to a solitary organ (MPC); (vi) ≤ 3 active metastatic lesions irrespective of their distribution within the liver according to preoperative imaging studies (MPC) and hepatic lesions were technically resectable (MPC); (vii) intensive control of pulmonary metastasis by systemic treatment and suitable for further stereotactic body radiotherapy (SBRT)-mediated local control or resection (MPC). Surgical procedures were selected based on the location of and invasion by the tumor, but vessel resection/reconstruction were considered (if necessary) intraoperatively. Data regarding pathology grade, resection margin, lymph-node invasion and the pathologic response were collected. The College of American Pathologists (CAP) scoring system was employed to evaluate the pathological response to tumor resection : grade 0P < 0.05 upon univariate analysis without potential confounding were included in multivariate logistic regression analysis. P < 0.05 was considered significant.The primary outcomes were postoperative-progression-free survival (PO-PFS) and PO-OS. We followed up patients by telephone communication every 3 months. Statistical analyses were done with Prism 9  and SPSS 26.0 . Results are given as the median (range) or mean (standard deviation). PFS and OS were calculated using the Kaplan–Meier method and compared using the log‐rank test. Factors with Sixty-seven patients with UPC received systemic treatment and CS between April 2014 and March 2022. The median age of the study cohort was 63 years, and male patients accounted for 61% of cases  was done in 31 cases, distal pancreatectomy (DP) in 20 patients, distal pancreatectomy with celiac axis resection (DP-CAR) in 11 cases and total pancreatectomy (TP) in one patient. Reconstruction of the PV or SMV was conducted in 23 cases (22 LAPC and one MPC). Five patients with LAPC underwent arterial construction during resection. Lung metastasis and hepatic metastasis were observed in three and 11 patients, respectively. Synchronous metastasectomy was undertaken in nine patients with hepatic lesions and radiologic CR was obtained in an additional two patients. For patients with pulmonary metastasis, a single lesion disappeared in one patient. In another two patients with multiple lesions, most pulmonary metastasis disappeared after systemic treatment, and SBRT was carried out later.The median duration (in minutes) of PD, DP and DP-CAR was 360, 253, and 270, respectively, whereas TP took 748 min . Patients who underwent TP suffered from peritoneal infection and bleeding died within 90 days. The details of CS classified by resectability are demonstrated in Supplementary Table The mean diameter of 63 resected local tumors was 26.98 mm, and 41% of patients had positive lymph nodes. According to postoperative pathology . Patients with hepatic lesions experienced PO-OS of 25.47 months and PO-OS was not reached in patients with lung lesions. Through univariate and multivariate logistic regression analyses (Table P = 0.079), and the postoperative change in the level of tumor markers was related to PFS (P = 0.001) and OS (P = 0.013) in the univariate analysis . No factors were associated markedly with PO-OS according to multivariate analyses.Median PFS of the 67 patients was 14.27 months, and median OS was 32.13 months  or postoperative surgical complications  compared with that using a palliative procedure [Preoperative systemic treatment can reduce the accuracy of imaging in the evaluation of resectability after treatment owing to treatment-related fibrosis , 16. Morrocedure . Rangelorocedure . The prerocedure . Thereforocedure . In our Several reports have affirmed the survival benefit of CS for UPC patients . In the CS was carried out only for an extremely select group of patients who were super-responders to systemic treatment. An important indication for surgery in UPC patients is that radical excision of the tumor can be achieved. Imaging is the most widely used method to evaluate resectability, but the accuracy of imaging decreases markedly after systemic treatment. White et al. demonstrated that restaging unresectability based on CT findings deprived ~ 20% of patients the opportunity for curative resection . Hence, vs. 6.13 months, P < 0.05) in our study.The clinical response must also be taken into consideration. Changes in tumor marker levels likely represent a tumor-specific response to systemic treatment. Tsai et al. found that patients with normal preoperative or postoperative CA19-9 levels experienced a doubling in OS compared with patients who did not have normal levels . SimilarThe pathological response is the “gold standard” for assessing tumor degeneration/necrosis, and is a prognostic factor. Chatterjee et al. postulated that the major pathological response (CAP grade = 0–1) in tumor specimens was strongly correlated with longer survival in PC patients . In our Our study had three main limitations. First, there was a selection bias for patients. Second, the cohort size was small. Third, international consensus on CS for UPC is not available, so when and how to carry out a surgical procedure is controversial. CS may be the most effective way to lengthen survival in UPC patients, and systemic treatment is the best way to select surgical candidates at this stage. CS prevalence varies among regimens and treatment protocols, but GnP and mFFX are recommended. Several types of systemic treatment, including chemo-immunotherapy, have been applied recently, but the role of immunotherapy is not clear. The usefulness of adjuvant therapy in patients who have completed preoperative systemic treatment and radical excision is not clear.Additional file 1:Supplementary Figures 1-Figure 3.Additional file 2:Supplementary Table 1.Additional file 3:Supplementary Table 2.Additional file 4:Supplementary Table 3.


Caenorhabditis elegans’ by Qiang Luo et al., RSC Adv., 2020, 10, 39485–39494, https://doi.org/10.1039/D0RA06093C.Retraction of ‘Structural characterization of ginseng oligopeptides and anti-aging potency evaluation in  RSC Advances article due to a significant amount of unattributed text overlap with articles by different author groups that were not cited, including articles published in Phytochemistry by Shan Su et al.1 and Journal of Functional Foods by Elena M. Vayndorf et al.2The Royal Society of Chemistry hereby wholly retracts this Zhigang Liu agrees to the retraction. The other authors have been informed but have not responded to any correspondence regarding the retraction.RSC AdvancesSigned: Laura Fisher, Executive Editor, th April 2022Date: 25


Sympotthastia remained unstable. Our study revealed that the mitogenomes of Diamesinae are highly conserved, and they are practically useful for phylogenetic inference.Mitogenomes have been widely used for phylogenetic reconstruction of various Dipteran groups, but specifically for chironomid, they have not been carried out to resolve the relationships. Diamesinae (Diptera: Chironomidae) are important bioindicators for freshwater ecosystem monitoring, but its evolutionary history remains uncertain for lack of information. Here, coupled with one previously published and 30 new mitogenomes of Diamesinae, we carried out comparative mitogenomic analysis and phylogenetic analysis. Mitogenomes of Diamesinae were conserved in structure, and all genes arranged in the same order as the ancestral insect mitogenome. All protein‐coding genes in Diamesinae were under stronger purifying selection than those of other nonbiting midge species, which may exhibit signs of adaptation to life at cold living conditions. Phylogenetic analyses strongly supported the monophyly of Diamesinae, with Boreheptagyiini deeply nested within Diamesini. In addition, phylogenetic relationship of selected six genera was resolved, except  Here, coupled with one previously published and 30 new mitogenomes of Diamesinae, we carried out comparative mitogenomic analysis and phylogenetic analysis. Mitogenomes of Diamesinae were conserved in structure, and all genes arranged in the same order as the ancestral insect mitogenome. Phylogenetic analyses strongly supported the monophyly of Diamesinae, with Boreheptagyiini deeply nested within Diamesini. Since iIn this study, we provide 30 newly sequenced (nearly) complete mitogenomes from 30 species representing Boreheptagyiini (four species of one genus) and Diamesini (26 species of five genera) using next‐generation sequencing. We analyzed the genomic structure, base composition, substitution, and evolutionary rates among Diamesinae, expanding our knowledge of its diversity of mitogenomes. Coupled with published data, we carried out phylogenetic analysis of Boreheptagyiini and Diamesini based on 31 mitogenomes.22.1Field collection of 30 species were conducted in China during 2014–2020, using classical insect collection techniques such light traps, sweep traps, Malaise traps, and D‐nets. Specimens were preserved in ethanol , and stored at dark at −20°C before morphological and molecular analyses. The total genomic DNA was extracted from thorax of adult and middle larval bodies using a Qiagen DNA Blood and Tissue Kit (Qiagen) following the manufacturer's protocol. After DNA extraction, the cleared exoskeleton of thorax was mounted in Euparal on microscopy slides together with the corresponding wings, legs, and antennae following the procedures outlined by Sæther . The DNABoreoheptagyia species) and 26 species of Diamesini . Since mitogenomes of another four tribes were not available for current molecular study, we could not reconstruct the phylogeny of the whole subfamily Diamesinae. Therefore, by integrating one public Potthastia species (GenBank accession: MW373523), a total of 31 species of Boreheptagyiini and Diamesini were selected as in‐groups. In addition, we selected one Prodiamesinae species  and one Orthocladiinae species  as outgroups for phylogenetic analyses. Detailed information could be found in Table Thirty mitogenomes were newly sequenced in this study, representing four species of Boreheptagyiini . The raw sequencing reads were trimmed with Trimmomatic  barcode sequence for each species was obtained by Sanger sequencing herein and from previous study . Ribosomal RNA (rRNA) genes and protein‐coding genes (PCGs) were annotated by aligning with homologous genes of Potthastia sp. in Geneious. Newly sequenced mitogenomes were submitted to GenBank (accession numbers: pending). The mitogenome maps were drawn by the CG View server V 1.0   Figure . The sta) Figure .Boreoheptagyia, ND1 and ND4L showed a lower Ka/Ks value in Boreoheptagyia and in Pagastia, and the remaining ten showed a lower Ka/Ks value in Diamesa and in Pagastia  of all the 13 PCGs is less than 0.35, and the ATP8 exhibits the largest Ka/Ks value while the COI has the lowest Ka/Ks value Figure . To betta Figure . We alsoEach mitogenome of Diamesinae contains 22 typical tRNA genes, with A+T content ranging from 74.0% to 77%. The nucleotide skew of tRNA genes among Diamesinae is consistent, exhibiting positive AT‐skew and negative GC‐skew Figure . Both 123.2Sympotthastia was unstable  + (Diamesa + (Pagastia +Pseudodiamesa)))) was inferred from the PCG12 and PCG12R datasets; (ii) (Potthastia + (Boreoheptagyia + (Sympotthastia + (Diamesa + (Pagastia +Pseudodiamesa))))) was inferred from the AA dataset. The topology inferred from the AA dataset had the strongest nodal support. Based on three different datasets, Boreheptagyiini was deeply nested within Diamesini. The monophyly of Boreoheptagyia, Diamesa, Pagastia, Potthastia, and Pseudodiamesa was well supported by mitogenomes.Generally, six phylogenetic trees constructed by BI and ML analyses are similar in topology, only with the position of e Figure . The mone Figure . Within 44.1A total of 31 mitogenomes of Diamesinae are included in this study, of which 10 mitogenomes have incomplete control region by the highly gene duplication Cameron, . The len4.2Stenochironomus  + (Pseudokiefferiella + (Parapotthastia + (Onychodiamesa + (Diamesa +Lappodiamesa)))). Willassen  are sister to the remaining Diamesini. In our study, Potthastia is placed as the oldest of all Diamesinae genera studied here. Our result corresponds very well by Willassen ; Investigation ; Methodology ; Software ; Writing – original draft (lead). Zheng Liu: Funding acquisition (lead); Writing – review & editing . Li‐Ping Yan: Investigation ; Methodology ; Writing – review & editing . Xin Duan: Formal analysis ; Software . Wen‐Jun Bu: Supervision ; Validation . Xin‐Hua Wang: Investigation ; Supervision ; Validation . Chen‐Guang Zheng: Formal analysis ; Investigation ; Writing – original draft ; Writing – review & editing .The authors declare that they have no conflicts of interest.Supplementary MaterialClick here for additional data file.


The association between vitamin D and total and colorectal cancer risk was inconsistent in observational studies. We conducted Mendelian randomization approach in which the effect of confounding might be reduced. 110 single nucleotide polymorphisms (SNPs) associated with 25-hydroxyvitamin D concentrations were systematically selected according to the “GWAS Catalog” from all ethnic populations. For the SNP-vitamin D concentration association, 3978 individuals from two Japanese cohorts were included. Regarding SNP-total and colorectal cancer association, 4543 cancer cases and 14,224 controls and 7936 colorectal cancer cases and 38,042 controls, respectively were included from the Japanese Consortium of Genetic Epidemiology and other studies in Japan. There was no significant association between the genetically predicted plasma 25-hydroxyvitamin D concentration and total or colorectal cancer in any of the MR analyses. Odds ratios per doubling in vitamin D concentration were 0.83  for total cancer and 1.00 (95% CI 0.80–1.24) for colorectal cancer in inverse variance weighted method, 0.83 (95% CI 0.57–1.19) for total cancer and 1.01 (95% CI 0.75–1.37) for colorectal cancer in MR-Egger method. Consistent with previous MR analyses among European ancestries, there was no significant association identified between 25-hydroxyvitamin D levels and total or colorectal cancer among Asians. Cancer is a leading cause of death, and research on its preventive factors has been in focus. Dietary factors and related nutrients are modifiable factors, and the associations between these factors and cancer have been investigated.8. However, the results of these studies are controversial. A recent meta-analysis that included high-dose vitamin D supplementation RCTs did not find a significant reduction in cancer incidence : 0.93– 1.03, p = 0.42]7, while a meta-analysis of eight prospective studies showed a marginal association between 25(OH)D (a biomarker of vitamin D status in humans) and a lower risk of cancer when the highest with the lowest categories of 25(OH)D were compared 8. An umbrella review of diet and cancer showed that four meta-analyses revealed statistically significant results (p < 0.05) among 18 meta-analyses of observational studies on vitamin D and cancer, while heterogeneity was > 50% in nine meta-analyses9. Nine out of 18 vitamin D studies showed heterogeneity of > 50%, which was the highest percentage among nutrients included in the umbrella review.The association between vitamin D and total and site-specific cancer incidence/mortality has been investigated in many studies, including randomized controlled trials (RCTs), observational studies, and meta-analyses, as vitamin D may play a role in carcinogenesis10. This ranking is similar in Japan, and an increasing trend has been observed11, while a decline in the incidence of colorectal cancer was found in some countries, including the US12. Since the incidence of colorectal cancer is high and anti-tumor effect of vitamin D against colorectal cancer was shown14, the association between vitamin D concentration and colorectal cancer has been investigated and the present study was conducted under the same hypothesis. However, the previous results remain controversial17. A meta-analysis showed a significant reduction in colorectal cancer risk in Asians16, but not in the latest meta-analysis, which was based on prospective studies15. Therefore, in addition to the classical epidemiological method, a new approach is required to investigate the association between vitamin D and cancer.Colorectal cancer is the third most common incident cancer in men and second in women worldwide18. Since observational studies have faced controversy regarding the association between vitamin D and cancer, this MR method, in which the effect of confounding factors might be reduced, has been applied to examine these associations. A study from the global network showed that there was not a significant association between genetically determined 25(OH)D and colorectal cancer (the odds ratios [ORs] per 25 nmol/L increment were 0.92 [95% CI 0.76–1.10])19, and another study from the UK Biobank also showed that genetically low 25(OH)D levels were not significantly associated with overall cancer risk20. Considering the differences in results in a meta-analysis of Asians and ethnic differences in genetic variants, MR studies in Asian populations are required.Mendelian randomization (MR) is an analytical method in which genetic variants are recognized as instrumental variables, and the random allocation of genotypes is likened to randomized trials at conceptionTo examine the effect of genetically predicted vitamin D concentrations on total and colorectal cancer risk, we conducted an MR analysis in a large-scale case-cohort of 4,543 cancers with a mean follow-up of 15 years among Japanese and 7936 colorectal cancer cases in the Japanese consortium and a combination of several other studies in Japan.The basic characteristics of the included studies are presented in Table Power calculations under the power of 80% and Type I error rate of 5% were conducted. Minimum detectable ORs per 1 SD increment calculated with the percentage of our explained variance and the number of cases and controls were 0.81 for total cancer and 0.86 for colorectal cancer.p-value < 5.0 × 10−8), and 14 SNPs were statistically significant   and CYP24A1  which were related to vitamin D metabolism, were detected as nearby genes. D prime values of selected SNPs, rs10832254 (chromosome 11) and rs12803256 (chromosome 11), were both over 0.9 with SNPs near CYP2R1 and DHCR7/NADSYN1, respectively. Regardless of significance or type of nearby gene, we included selected 110 SNPs in MR analyses. The explained variance of 25(OH)D levels by the 110 SNPs was 7.0%. Associations between SNPs and total cancer, or SNPs and colorectal cancer are shown in Supplementary Table 110 single nucleotide polymorphisms (SNPs) were selected from previous studies and associations between SNPs and vitamin D concentrations are shown in Supplemental Table 5) Table . In thes2-transformed vitamin D concentration (95% CI) were 0.83 (0.63–1.09) for total cancer and 1.00 (0.80–1.24) for colorectal cancer in random-effect inverse-variance weighted (IVW) method. Results were not significant in MR-Egger method  and weighted median method . When we only included significant 14 SNPs in Table p = 0.05) for colorectal cancer in the heterogeneity test, the random-effect IVW method was used. MR-PRESSO method was used to detect horizontal pleiotropic outliers, however, there were no outliers for total and colorectal cancer assessment. No single SNP changed the result according to the leave-one-out analysis. When we included strong instruments  only in MR analysis to avoid weak instrument bias, heterogeneity became non-significant, however, non-significant results were not changed  from the UK biobank, including 46,155 cancer cases22. Although this study used six SNPs that explained 3.5% of the variation in vitamin D concentration, the authors conducted a reassessment for various types of cancer using 74 SNPs, which explained up to 4% of the variation. Nevertheless, they did not find a significant association between most types of cancer other than ovarian cancer21. Although we selected 110 SNPs, these were based on studies mainly from European ancestries and no study for East Asian only genome-wide association study (GWAS) was published so far. Because of this, a few instrumental variables were significantly associated with Vitamin D concentration. Explained variance was 7.0% in our samples, however, both analyses with all 110 SNPs and with significant SNPs only showed null associations. Considering the null results for most types of cancer in the large MR studies21, our null findings for total cancer were agreeable, although we included a relatively small sample size compared with European ancestry studies, which might be judged from the sample size calculation result.Although controversial results have been proposed in observational studies and some previous MR studies focused on vitamin D and cancer19 and bidirectional MR analysis with 26,397 cases also did not show significant results for vitamin D and colorectal cancer risk23. Our results are consistent with these findings. Among observational studies, a study in Japan that showed a significant reduction in overall cancer showed null results in the case of colorectal cancer 4. Inconsistent with this, a pooled analysis of 17 cohorts showed a significant reduction in the high vitamin D concentration (87.5– < 100 nmol/L) group24 and the meta-analyses from Asians showed significant dose–response reduction14. We observed a discrepancy between MR analysis and observational studies among Asians. A potential reason for this might be unknown or unmeasured confounders in the models used in observational studies or because our results were from a linear MR Further MR studies are required to confirm these results.Regarding colorectal cancer, MR analysis from the large consortium with 11,488 colorectal cancer cases showed that the OR was 0.92 (95% CI 0.76–1.10)13. The anti-tumor effects of vitamin D include pro-apoptosis, anti-proliferation, and pro-differentiation effects25. Based on these mechanisms of antitumor effects, epidemiological studies have shown the potential of vitamin D in cancer prevention. Genetic variations in vitamin D status, however, have not shown significant results, similar to those of MR studies27. As with our results, SNPs detected in GWAS studies explained a small percentage of vitamin D concentrations, and it might be difficult to detect the reduction in risk of cancer caused by the increased genetically predicted vitamin D levels. Other potential reasons that we did not find significant results were as follows: (i) the causal relationship between vitamin D level and cancer did not truly exist, and (ii) the power was was limited to detect an existing, but small effect. Because of the relatively low explained variance of vitamin D, GWAS of Vitamin D including more Japanese participants is required to allow MR with a larger sample size, and large randomized controlled trials to investigate the causal relationship between vitamin D and cancer in Japanese are needed.In vitro studies have shown that high concentrations of vitamin D inhibit tumor cell proliferation and induce differentiationA strength of this study is the application of a two-sample MR framework in a relatively large-scale Asian population to examine the association between vitamin D and cancer. The MR method can overcome the potential bias in observational findings. However, our study has few limitations. First, we assumed a linear association between vitamin D and colorectal cancer, and we could not investigate the nonlinear effect because we could not include individual-level data. Second, the sample size was small. In SNP exposure analyses, vitamin D was measured in a limited number of cohort studies because measurement of vitamin D concentration in many samples was not feasible in prospective cohorts. Based on the sample size calculation, a relatively weak association may not be detectable in this sample size. In addition, the measurement was conducted only once, and measurement errors could not be excluded. In the SNP outcome analysis, site-specific cancers other than colorectal cancer were not included because of the limited number of cases. Third, the selected SNPs were based on previously published studies that did not include the East Asian population. For this reason, we included both significant and non-significant instrumental variables in MR and this may cause weak instrument bias. Although sensitivity analysis with significant instruments showed similar results with main analyses, GWAS results for vitamin D in a Japanese population are required. Also, this study was conducted in Japan, so generalizability might be limited to Japanese. Fourth, because we could not collect individual genetic data and used summary results of GWAS as estimates, we did not assess the association of colorectal cancer by site. However, since the association between vitamin D concentration and cancer is controversial, and evidence from the Asian population is scarce, this study is worth reporting. Further studies with a larger sample size are required to confirm this hypothesis.In conclusion, consistent with MR studies in European ancestries, there was no statistically significant association between vitamin D concentrations and total and colorectal cancer risk from MR analysis among the Japanese population.22.We performed a two-sample MR analysis in which two types of estimates from two separate datasets were used to evaluate the objective association . One is the estimate between SNPs and the exposure of the objective association , and the other is the estimate between SNP and the outcome . MR analysis should be based on the following three assumptions: (i) SNPs  are associated with the exposure, (ii) SNPs  are not associated with confounding factors that are supposed to exist in the association between the exposure and the outcome, and (iii) SNPs as instrumental variables are not directly associated with the outcome and are related to the outcome only through exposurehttps://www.ebi.ac.uk/gwas/) to select SNPs. In September 2022, 713 SNPs, including duplicates, were shown to be associated with “vitamin D measurements” in the GWAS Catalog. We systematically chose SNPs for instrumental variables in the following criteria; (a) phenotype was vitamin D measurement (647 SNPs remained), (b) p-value of SNPs were < 5 × 10−8 (614 SNPs remained) (c) participants in the original papers were adults (609 SNPs remained), (d) duplicate of SNPs (475 SNPs remained), (e) minor allele frequency (MAF) > 0.01 in the East Asian populations based on 1000 genome project (325 SNPs remained), (f) clumping using “clump_data”  in “TwosampleMR (version 0.5.6)” library of R software. Finally, 110 SNPs were used for this analysis.  database named GWAS Catalog . A meta-analysis was performed for each target SNP using a fixed effects model.We calculated the estimates of SNP-vitamin D association from two Japanese cohorts: JPHC Study and J-MICC Study. The details of each study are provided in Supplemental Table For the SNP-outcome association, we examined two types of cancer . We selected colorectal cancer as a site-specific cancer because it is one of the most common cancers, and we could include a sufficient sample size of cancer cases. For total cancer, 3541 cases and 10,536 controls were identified from the participants who answered the baseline questionnaire of the JPHC (JPHC-base) and provided blood samples. A total of 1002 cases and 3688 controls were identified from participants who answered the 5-year questionnaire of JPHC, provided blood samples, and were not included in the baseline analysis (JPHC-5 year). For colorectal cancer, colorectal cancer cases and controls from the JPHC Study, NAGANO Study, Hospital-based Epidemiologic Research Program at Aichi Cancer Centre (HERPACC Study), J-MICC Study, and non-restricted published genome-wide association study (GWAS) analysis data from BioBank Japan (BBJ) were gathered and meta-analyzed for target SNPs. The descriptions of these studies are shown in Supplemental Table https://shiny.cnsgenomics.com/mRnd) and a type-I error rate of 5% and power of 80% were set.We performed power calculations with mRnd 2. After calculating the β coefficients for SNP-exposure and SNP-outcome associations in each study, we combined them with IVW in a fixed or random-effect model depending on the heterogeneity test. 110 SNPs identified from previous studies were regarded as instrumental variables. We selected them irrespective of the statistical significance of these SNPs in our data because overfitting or insufficient power in our data might cause bias in SNP selection.In this MR analysis, estimates (β coefficients and 95% CI) of selected SNPs from previous studies were calculated in our dataset (both SNP-exposure and SNP-outcome associations) and used in MR analysis to avoid overestimation of the association p-value < 0.05) 14 SNPs using “TwoSampleMR” (version 0.5.6) available as R package. All these procedures were conducted using the Statistical software R version 3.5.0 . To examine the association, the IVW, MR-Egger regression, and the weighted-median method were used. Because the IVW method is likely to be affected by horizontal pleiotropy, MR-Egger regression, in which the intercept reflects the pleiotropic condition, was conducted and MR-PRESSO method was used to detect and exclude horizontal pleiotropic outliers. The weighted-median method was used because consistent estimates were obtained from the weighted-median method when invalid instrumental variables were included. As sensitivity analysis, significant (p < 5 × 10−6) or strong (F-statistics > 10) instruments were selected and included in MR analysis. Moreover, we further conducted MR analysis without the result from JPHC base in SNP-outcome analysis to avoid sample overlapping. The significance of the association with MR was set at p < 0.05.We performed MR analysis with 110 SNPs and significant (Supplementary Tables.


Short bowel syndrome (SBS) with intestinal failure (SBS-IF) requires long-term parenteral nutrition (PN). This study investigated the real-world etiologies of SBS, treatment patterns, and PN-related outcomes among adult patients with SBS-IF in Japan.This retrospective, observational cohort study was based on data from April, 2008 to January, 2020 from one of the largest hospital-based claim databases in Japan. Analyzed patients were aged ≥ 16 years, had received continuous PN for ≥ 6 months, and had SBS or undergone SBS-related surgery with a diagnosis of a causative disease. The primary endpoint was PN weaning.We analyzed data for 393 patients. The most frequent causes of SBS-IF were ileus (31.8%), Crohn’s disease (20.1%), and mesenteric ischemia (16.0%). Of 144/393 (36.6%) patients who were weaned off their PN, 48 (33.3%) were subsequently restarted on PN. Of 276/393 (70.2%) patients whose PN was initiated in hospital, 156 (56.5%) transitioned to home management. The mean duration of initial PN was 450.4 and 675.5 days for patients who were able or unable to be weaned off PN, respectively. Sepsis (67.4%), catheter-related bloodstream infections (49.1%), and liver disorders (45.0%) were the most reported PN-related complications.Most patients with SBS-IF in Japan could not be weaned off PN and suffered life-threatening complications.The online version contains supplementary material available at 10.1007/s00595-022-02469-9. Short bowel syndrome (SBS) is a malabsorptive state caused by the physical or functional loss of portions of the small intestine. The leading cause of SBS in adults is extensive intestinal resection to manage Crohn’s disease (CD), cancer, mesenteric vascular disease, or traumatic injuries –4. Data SBS is the most frequent pathophysiological mechanism of chronic intestinal failure (IF), defined as the reduction of gut function below the minimum necessary for the absorption of macronutrients and/or water and electrolytes, necessitating intravenous supplementation to maintain health and/or growth . The maiComprehensive data on SBS-IF from analyses of large populations in Japan are lacking. A better understanding of the disease landscape in the real-world would contribute to the development of effective intestinal rehabilitation programs. This study used one of the largest hospital-based claim databases in Japan to reveal the etiologies of SBS, as well as the patterns of SBS-IF treatment in clinical practice and their outcomes.This study used anonymized electronic health insurance claims and diagnosis procedure combination data provided by Medical Data Vision Co. Ltd. This large-scale, acute-care hospital-based database covers inpatient and outpatient data of 30.15 million patients from over 400 hospitals  . The totPN was defined as PN administered in any setting and HPN was defined as PN administered at home. PN in hospital was defined as PN administration in either an inpatient or outpatient setting. A PN episode was defined as a record of continuous PN for ≥ 6 months; the index PN event was defined as the first PN episode recorded in the database; and the index date was the date of initiation of the index PN event.The time to first weaning was defined as days to first weaning from the index date, and total PN duration was the total duration of all PN episodes during the follow-up period. An index PN duration was defined as the duration of the index PN episode. Switching between PN at hospital and HPN was identified by two consecutive PN treatment records in the same month or in 2 consecutive months .Up to 12 months before the index date was considered as the ‘lookback’ period to identify the cause of SBS-IF. If patients received their first PN infusion as a matter of urgency, most likely following an emergency event, the enrollment date and the index date were the same without a lookback period. The duration of follow-up after the index date was not predetermined; eligible patients were followed in the database for as long as they could be tracked. Patients who were transferred to another hospital for treatment during their index PN episode were excluded.Patients eligible for inclusion in the study were aged ≥ 16 years at the index PN event, had received continuous PN treatment for ≥ 6 months, and had reimbursement claims records for either SBS, or a causative disease of SBS (Supplementary Table 1) and surgery related to SBS .The primary endpoint of this study was PN weaning. Successful weaning was marked by intervals of > 2 months between PN records or intervals of > 2 months between the date of the last PN record and the last date of follow-up. Secondary endpoints included: PN restart, time to first weaning, index PN duration, total PN duration, transition to HPN, complications related to PN, duration of hospitalization, and number of recurrent hospitalizations. Information on the causative diseases of SBS-IF and patient comorbidities was also collected.Ad hoc analyses were carried out to investigate patient characteristics, causes of SBS, PN-related complications (including deaths), discharge destination , and activities of daily living (ADLs).The study endpoints were analyzed using descriptive statistics. Continuous variables were reported using means and standard deviations (SDs) or medians and interquartile ranges, whereas categorical variables were reported using frequencies and percentages. All statistical analyses were conducted using SAS ver. 9.4 .Missing data were not imputed because of the descriptive analysis. Any patient with missing value(s) of outcome was included in the study population of baseline demographics; however, they were excluded from analyses that calculated the number and frequency of these outcomes with missing value(s).Overall, 393 patients were eligible for inclusion in this study Fig. . Of thes2 for men and 18.5 ± 3.8 kg/m2 for women. The most frequent comorbidities were diabetes (36.4%) and hepatopathy (34.6%). The most common clinical departments attended by patients during the index hospitalization (n = 272) were general surgery (39.2%), gastroenterological surgery (8.4%), or internal medicine (7.6%) (Table n = 272) had been referred from another hospital.A comparable proportion of men and women were included in the study population Table . At the The top three causative diseases of SBS-IF were ileus 31.8%), CD (20.1%), and mesenteric ischemia (16.0%) , small bowel obstruction resection 16.0%), and colostomy (12.2%)  patients were weaned off PN; however, 48 (33.3%) of these 144 patients subsequently were restarted on PN. Weaning rates declined with increasing age, with lower rates observed in patients aged over 80 years  than in younger age groups  and triple the mean total PN duration of patients without claims records for SBS  .n = 272), the mean duration of hospitalization was 170.9 ± 185.9 days. Forty-eight (17.6%) patients died during their index hospitalization. Patients who were transitioned from PN at hospital to HPN were generally younger than patients who were not . The mean number of recurrent hospitalizations was 4.3 ± 6.6.Of the 276 (70.2%) patients who received PN at hospital for their index PN episode, 156 (56.5%) transitioned to HPN management during that episode. Of patients who had been hospitalized . Ileus and acute pan-peritonitis were the most reported causative diseases  with the frequency of CD being low (2.5%). Ischemic heart disease (42.5%) and cerebrovascular disease (25.0%) were the leading illnesses potentially requiring continuous nursing care throughout the study period.n = 393), sepsis, catheter-related blood infections, and liver disorders were the most common complications related to PN. Sepsis developed in 265 (67.4%) patients, catheter-related blood infections developed in 193 (49.1%), and subsequent liver disorders developed in 177 (45.0%)  and 90% of patients were aged > 60 years at the time of death. The main causes of SBS in these patients were ileus (37.5%) and acute pan-peritonitis (20.8%); CD accounted for only 2.1%. Catheter infection was the leading complication relating to PN but occurred less frequently in patients who died than in the overall population (27.1% vs 49.1%). Conversely, liver disorders occurred more frequently in patients who died than in the overall population (56.3% vs 45.0%) (Table Two-thirds of patients 68.4%) were discharged from hospital during the study period. Figure % were diThis real-world study in Japan revealed that the most frequent causative diseases of SBS were ileus, CD, and mesenteric ischemia; consistent with previous global studies , 4. The The prevalence of CD was lower in the subgroups of patients who died or who did not transition to HPN during the study period (2.1–2.5% vs 20.1%). CD is typically diagnosed at age 15–35 years, hence, the patient population with CD may have been younger than the overall population. Furthermore, surgeries for ileus and mesenteric ischemia are more likely to be performed as emergency procedures, than surgeries for CD, suggesting possible differences in the underlying health of the patients receiving the respective surgeries. Finally, the use of biologics may have allowed the patients with CD to preserve residual bowel and improve their function, facilitating earlier weaning from PN. The increase in the use of biologics observed in this study may reflect the increase in availability of new biologics.The top three complications associated with PN at hospital were sepsis, catheter-related blood infections, and liver disorders. Complications were common in patients who withdrew from PN, with approximately 50% experiencing life-threatening complications, such as sepsis and liver disorders. These results are consistent with those from a previous Japanese study on patients using central venous catheters (including patients with SBS), which found sepsis, phlebitis, infections, and inflammatory reactions to be common complications . Most ofForty-eight 12%) patients died during the study period. Other studies have reported a mortality rate of 12.8–50% % patient; howeverThe prevention and/or timely treatment of complications is a key strategy for both the survival and successful intestinal rehabilitation of patients with SBS . For thiFirst, the study cohort was identified from records of reimbursement claims for SBS and we were unable to validate the patients’ clinical outcomes such as by means of chart reviews. To increase the accuracy of patient identification, we defined patients with SBS-IF as not only having claims records for SBS (or related diseases/procedures), but also as receiving long-term PN. Second, the database used in the study covers 23% of acute care hospitals in Japan, which are mainly larger hospitals (≥ 200 beds). Although patients with SBS-IF often receive care from larger hospitals with specialists, the findings of this study may not be inclusive of patients receiving care from smaller hospitals in Japan. Third, the database does not contain information from clinical examinations, such as the length of the remaining small intestine, which may affect outcomes, or any surgical records of advanced procedure for bowel lengthening, such as serial transverse enteroplasty or small bowel transplantation. Lastly, the analysis does not take into account any changes and developments in the medical system, including improvements on diagnostic tests, policy, or treatment in Japan during the study period of 2008–2020. Further studies may clarify details, including the weaning rate by causative disease and improvements in future treatment strategies.To our knowledge, this is the first study to provide comprehensive data on the causes and outcomes of SBS-IF in Japanese adults. This study provides insights into the etiologies and treatment patterns of SBS-IF in Japanese adults. Overall, the characteristics of these Japanese adult patients with SBS-IF receiving PN were similar to those reported for patients with SBS-IF in other parts of the world. Successful weaning from PN was achieved for only a small proportion of patients. Moreover, because of the complexity of SBS-IF, we recommend that intestinal rehabilitation programs be overseen by a multidisciplinary team in a specialized center to improve the treatment outcomes of patients with this distressing condition.Supplementary file1 (PDF 231 KB)Below is the link to the electronic supplementary material.


Radiation exposure is inherently involved in endoscopic retrograde cholangiopancreatography (ERCP), which could cause radiation-induced injury to endoscopists with long-term exposure. Nonradiation ERCP has been applied to pregnant patients. Conceivably, the same techniques could be used to benefit endoscopists. This study was designed to evaluate the effectiveness and safety of nonradiation-to-endoscopist (NRE) ERCP, compared with standard ERCP.A retrospective, single-center study was conducted from August 2010 to December 2015. Patients aged 18–90 years and with choledocholithiasis (< 15 mm) or distal biliary stricture were eligible. Pre-ERCP evaluation with magnetic resonance cholangiopancreatography was mandatory. To overcome selection bias, we performed 1:2 match using propensity score matching (PSM) between NRE and standard groups. The primary endpoint was overall ERCP success rate. Secondary endpoints were cannulation success rate, stone clearance rate, complication rate, and duration of hospitalization.P = 0.70). There was no difference in cannulation success rates between the two groups . A total of 88.3% of patients in the NRE group and 93.9% of patients in the standard group had stones cleared at initial ERCP (P = 0.57). No difference in overall stone clearance rate between the two groups  was found after second ERCP. The complication rate  and hospital duration  were not different between the two groups.A total of 329 patients met inclusion criteria. After PSM, 73 patients were included in the NRE group and 146 in the standard group. The ERCP overall success rate for NRE and standard groups was equivalent (94.5% vs. 93.2%, Although technically demanding, NRE-ERCP is both safe and feasible in selected patients compared with standard ERCP. Endoscopic retrograde cholangiopancreatography (ERCP) is currently an established therapeutic technique for the treatment of lesions in the pancreatic or biliary duct and relies heavily on the use of fluoroscopy. ERCP endoscopists are therefore at risk for exposure to ionizing radiation over many years. While there is a general lack of data and of long-term follow-up data in particular, there is increasing awareness that accumulating doses of ionizing radiation from modern radiologic procedures may have harmful impact on health –3.ALARA is a term representing the use of radiation dose “as low as reasonably achievable , 5.” PreWe presented our experience of performing nonradiation-to-endoscopist (NRE) ERCP. Contrary to standard ERCP that routinely use fluoroscopy for real-time observation, radiation was not only applied at limited specific situations but also used while endoscopists left the operating room to avoid exposure. The non-use of radiation to endoscopists during ERCP stands out as our major modification to the standard approach.This is a retrospective, case–control, single-center study conducted at the No.174 Hospital of the People’s Liberation Army, from August 2010 to December 2015. To overcome selection bias, a propensity score matching (PSM) study protocol was used between NRE and standard ERCP groups. Informed consent was approved by the institutional review committee of the No.174 Hospital of the People’s Liberation Army. The study was registered at Clinical Trials.gov (NCT02697149).Patients aged 18–90 years and who were undergoing ERCP were enrolled. Exclusion criteria included: (1) common bile duct stone (CBDS) > 15 mm; (2) complicated stricture defined as Bismuth III and IV and multiple strictures; (3) distal biliary stricture combined with CBDS; (4) patients with altered anatomy or uncertain biliary stricture. Endpoints comparison was done between patients in the NRE group with those in the standard group.MRCP was mandatory for all patients enrolled. MRCP imaging facilitated the subsequent NRE-ERCP as a valuable anatomic guide. Informed consents for MRCP and ERCP were obtained after detailed counseling. In all cases, serum chemistry tests, including liver function  and amylase, and transabdominal ultrasound were conducted. Patients with suspected malignant biliary lesions received EUS-FNA or percutaneous biopsy, guided by ultrasound, before ERCP.All ERCP procedures were performed using a side-viewing duodenoscopy . An electrosurgical unit (ERBE IC-200) with a blended current and a power setting of 40 W was used. All ERCPs were performed by one endoscopist (ZW). Before start of NRE-ERCP study, Dr. Zeng has been engaged in standard ERCP operations for 7 years (2008–2015) and has completed more than 2000 cases in total. Therefore, he is skillful in the field of ERCP. Amylase levels were checked in all patients within 24 h after ERCP.All devices used for NRE-ERCP and standard ERCP groups were same. The specific cannulation device, papillotomy knife, balloons, and guidewires used were at the discretion of the endoscopist and could be changed interprocedurally. Commonly used devices were Ultratome XL Triple Lumen Sphincterotome , guidewire , marked balloon (5 cm with red and 10 cm with green tape), and distance-marked wire-guided retrieval basket .ERCP was performed with the patient placed in a left lateral to prone position. Moderate conscious sedation was achieved and maintained with intravenous propofol. Biliary cannulation was attempted using a triple lumen sphincterotome with a 0.035-inch super guidewire. Once deep cannulation was achieved, the guidewire was withdrawn and successful cannulation of bile duct was confirmed by visible bile in the catheter upon aspiration. The length of sphincterotomy was determined based on the diameter of the bile duct and the size of stones depicted at MRCP.The strategies and methods after cannulation are summarized. For common bile duct stones less than 10 mm, stones were removed with a basket after Endoscopic sphincterotomy (EST); for common bile duct stones larger than 10 mm, balloon dilation and balloon retrieval were performed following EST. Uncovered metal stents were placed for malignant stenosis, while fully covered metal stents or plastic stents were used for benign stenosis. In facing of difficulty to pass the stricture, the distance between the stenosis segment and the duodenal papilla was first determined with MRCP and then wires were manipulated with the goal of advancing the wire freely and without resistance at various angles until deep access was achieved. ERCP catheters were then advanced over the wire and into the duct. Successful deep cannulation was judged by visible bile in the catheter with aspiration.For NRE-ERCP, when fluoroscopy was used, it was only used under the following situations, all when endoscopist and assistants were out of the operation room: (1) if resistance was met when inserting guidewire; (2) after contrast is injected; (3) before all-in-one plastic stent is released. During the procedure, the endoscopist leaves the operating room to avoid exposure. An extension tube was used to connect the sphincterotome and syringe, and a micro pump was used for contrast agent injection. The injection was stopped once proper cholangiography was achieved.Statistical data and clinical characteristics of all patients were collected. Patient-related data included gallbladder condition , intrahepatic bile duct stones, previous surgical history, comorbidities, clinical diagnosis, blood examination pre-ERCP, and papilla . Procedure-related data included intubation techniques, stone extraction techniques , stone basket, balloon extraction), dilation techniques , endoscopic probes dilation), drainage techniques (endoscopic nasobiliary drainage (ENBD), stent placement), and other techniques . Other information, such as the maximum diameter of CBD, the number of stones, serum amylase within 24 h after ERCP, days of hospitalization after ERCP, and complications of ERCP, were all collected. All above data were assessed by one investigator (HJ).To overcome possible selection bias and potential confounders between NRE and standard groups, we performed one to two matching using PSM on Empower (R) software. This method tries to construct an RCT-like situation where observed outcomes can be compared between intervention groups. When performing PSM, six variables were included, such as gender, age, prior EST or not, gallbladder , total bilirubin (TBIL), and diseases .χ2 tests or Fisher’s exact test, as appropriate. Analyses were performed with SPSS software V.19.0 for Mac. P values of ≤ 0.05 were considered significant.Categorical variables were analyzed using From August 2010 to December 2015, a total of 388 patients underwent ERCP in our endoscopy center, with 96 patients undergoing NRE-ERCP and 292 patients undergoing standard ERCP. Thirteen patients from the NRE group and forty-six patients from the control group were excluded based on exclusion criteria. A total of 329 patients (83 in NRE and 246 in standard group) who were eligible for PSM. After PSM analysis, 73 patients in the NRE group were matched 1:2 to 146 patients in the standard group. Figure For the indications of ERCP, in the NRE group, 60 (82.2%) patients were diagnosed as common bile duct stone (CBDS), 12 (16.4%) were biliary stricture, and 1 (1.4%) was pancreatic cancer. In the standard group, 115 were CBDS (78.8%), 27 were biliary stricture (18.5%), and 4 were others (2.7%), 3 with acute pancreatitis and 1 with pancreatic pseudocyst.The primary endpoint was overall ERCP success rate. Secondary endpoints included cannulation success rate, stone clearance rate in the initial ERCP, overall stone clearance rate, complications after ERCP, and the duration of hospitalization.P = 0.70) in NRE and standard groups, respectively (Table P = 0.39). Although 12 and 27 patients were diagnosed stricture in NRE and standard groups, respectively, the successful cannulation rate through stricture was similar .The success and safety of an ERCP are heavily dependent on cannulation of the desired duct. There was no significant difference in the overall ERCP success rate . After second ERCP, the total rates of stone clearance were 95.0% and 93.9% of patients, respectively (P = 0.77). Comparing the clearance rate in relation to the size of stones, similar clearance rates were observed for both small stones (≤ 10 mm)  and large stones (11 mm–15 mm) , respectively.With respect to stone clearance, a total of 53 of 60 (88.3%) of patients in the NRE group and 98 of 115 (93.9%) of patients in the standard group had stones cleared at initial ERCP (P = 1.00). No difference of CBD size was observed between the two groups  (Table P < 0.01), while the usage of stone basket in the NRE group was less frequent than that in the standard group . There were no differences in use of extraction balloon  and mechanical lithotripsy (ML)  between the two groups. Stent placements was done in 58.3% patients in the NRE group and 44.4% patients in the standard group had (P = 0.43).For all procedures, no difference was observed in naive papilla between NRE and standard groups   compared with that in standard ERCP group. However, as the endoscopist did not perform the procedure under fluoroscopy, the radiation exposure time was significantly shortened in NRE group  (Table P = 0.07).Regarding complications after ERCP, there was no difference between the two groups , large stones (≥ 10 mm), and complicated stricture . Third, in the procedure of NRE-ERCP, the success of cannulation was confirmed with the technique of bile duct aspiration. Mistakes might be made in judging the color of aspirated liquid in case of severe CBD obstruction.In conclusion, from our retrospective study, we demonstrated that NRE-ERCP is a feasible and safe alternative technique compared with standard ERCP. It is designed to avoid radiation exposure to endoscopists and their assistances and patients when performing ERCP. Among selected patients, NRE-ERCP achieved similar overall success rate of cannulation and stone clearance, as that with standard ERCP. No additional complications or prolonged hospitalization duration were associated with NRE-ERCP compared to standard ERCP. For patients with choledocholithiasis (< 15 mm) and hilar bile duct stricture (stricture defined as Bismuth I and II), this technique has the potential to reduce radiation exposure to endoscopists, other medical staff, and patients. Nevertheless, although ERCP was safely completed without the use of fluoroscopy at our center, as the operator has extensive experience in the field of ERCP, patient safety cannot be assured when performing ERCP without the benefits of fluoroscopy at all ERCP centers. The current study warrants the conduct of future studies to specifically examine this. Moreover, studies from other centers are warranted to determine whether NRE-ERCP could be widely adopted.


Acinetobacter baumannii can acquire and disseminate antimicrobial resistance (AMR) genes often associated with mobile genetic elements (MGEs). Analyzing the genetic environment of resistance genes often provides valuable information on the origin, emergence, evolution, and spread of resistance. Thus, we characterized the genomic features of some clinical isolates of carbapenem-resistant A. baumannii (CRAb) to understand the role of diverse MGEs and their genetic context responsible for disseminating carbapenem resistance genes. For this, 17 clinical isolates of A. baumannii obtained from multiple hospitals in India between 2018 and 2019 were analyzed. AMR determinants, the genetic context of resistance genes, and molecular epidemiology were studied using whole-genome sequencing. This study observed an increased prevalence of blaOXA–23 followed by dual carbapenemases, blaOXA–23, and blaNDM. This study identified three novel Oxford MLST sequence types. The majority of the isolates belonged to the dominant clone, IC2, followed by less prevalent clones such as IC7 and IC8. This study identified variations of AbaR4 and AbGRI belonging to the IC2 lineage. To the best of our knowledge, this is the first study that provides comprehensive profiling of resistance islands, their related MGEs, acquired AMR genes, and the distribution of clonal lineages of CRAb from India.With the excessive genome plasticity,  Acinetobacter baumannii is a member of the ESKAPE group of pathogens and is considered to be one of the major global causes of hospital-acquired infections (HAIs)  under “Urgent Threat” and as Priority 1: Critical pathogen, respectively. Recently, the WHO Country Office for India developed the Indian Priority Pathogen List (IPPL) and categorized carbapenem-resistant, colistin-resistant A. baumannii under “Critical Priority.”1s (HAIs) . A. baumpatients . This papatients . Carbapec region . A studyc region . Both thA. baumannii can acquire and disseminate antimicrobial resistance (AMR) genes that are often associated with various mobile genetic elements (MGEs)   phenotype of A. baumannii   . Carbape–58–like . The blads (RIs) . The ass earlier . Typicalansposon . Recent aumannii . Additioaumannii . RIs in y metals . The Aba (MARRs) . Similar islands . Table 1Although the endemic burden of CRAb is a significant public health problem within Indian hospitals, the lack of genomic information makes it difficult to track its persistence . StudyinWe aimed to characterize the prevalent genomic features of clinical isolates of CRAb in India. We also compared the structural configuration of RIs with the complete genetic information and observed structural variations within the genetic environment of resistance genes. We found that the backbone of MGEs and their associated AMR genes among this study isolates were similar to that of the global context.A. baumannii collected as a part of a surveillance study were used. Of the 17 isolates included in this study, 13 were from Christian Medical College (CMC), Vellore, three from All India Institute of Medical Sciences (AIIMS) Trauma Center, New Delhi, and one from Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh (n = 10), six from endotracheal aspirate , and one from pus . Phenotypic characterization of all the isolates as A. baumannii calcoaceticus (Acb) complex was determined using standard biochemical tests. Confirmation of the Acb complex at the species level was performed by Vitek-MS  as described earlier and by identifying chromosomally encoded blaOXA–51–like gene by PCR , cefepime (30 μg), piperacillin-tazobactam (100/10 μg), cefoperazone-sulbactam (75/30 μg), imipenem (10 μg), meropenem (10 μg), levofloxacin (5 μg), amikacin (30 μg), netilmicin (30 μg), tobramycin (10 μg), aztreonam (30 μg), tetracycline (30 μg), minocycline (30 μg), and tigecycline (15 μg) using the Kirby Bauer’s disk diffusion (DD) method.Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were included in every batch for quality control (QC). For colistin susceptibility testing, in addition to QC strains, an mcr-1 positive E. coli isolate and two Klebsiella pneumoniae strains (BA38416 and BA25425) were also included for QC.For colistin, broth microdilution (BMD) was performed. Isolates identified as carbapenem-resistant by DD were further subjected to BMD to determine the minimum inhibitory concentration (MIC) for imipenem and meropenem. The susceptibility was interpreted as per the criteria defined by CLSI guidelines , 2019. EThe isolates were recovered overnight on blood agar, and genomic DNA was extracted from pure cultures using a QIAamp DNA mini kit  following the manufacturers’ instructions. DNA was quantified by NanoDrop spectrophotometry  and Qubit 3.0 fluorometry  and stored at −20°C until further characterization.2 and assessed with FastQC version 0.11.4.3Short read sequencing of the 17 isolates was carried out using the Ion Torrent PGM™ platform using 400-bp chemistry  or 150-bp paired-end sequencing using HiSeq 2500 platform . The PHRED quality score was checked on the sequences, and reads with a score below 20 were discarded. Adapters were trimmed using cutadapt v. 1.8.14All genomic DNA was further subjected to Oxford Nanopore MinION sequencing  to obtain long-read sequences. For this, the DNA library was prepared as per the manufacturer’s protocol using the SQK-LSK108 ligation sequencing kit (version R9) and the ONT EXP-NBD103 Native Barcode Expansion Kit . The library was loaded onto the FLO-MIN106 R9 flow cell and run for 48 h using the standard MinKNOW software. The Fast5 files from MinION sequencing were subjected to base calling using Guppy.de novo hybrid assembly of Illumina and Oxford nanopore sequences as described earlier (5 with parameters set at min_length 1000 −90 %. The short reads generated using ion torrent were error-corrected using Ionhammer (CP040080–CP040083), AB02 (CP035672–CP035675), AB03 (CP050388–CP050389), AB06 (CP040050–CP040052), AB10 (CP040053–CP040055), AB11 (CP040056–CP040057), AB13 (CP040087–CP040088), AB14 (CP040259–CP040262), AB15 (CP050385–CP050387), AB16 (CP050523–CP050525), AB18 (CP050390–CP050391), AB19 (CP050410–CP050411), AB20 (CP050412–CP050414), AB23 (CP050432–CP050435), AB26 (CP050401–CP050402), AB27 (CP050421–CP050423), and AB28 (CP050403–CP050409).Complete circular genomes for the 17 isolates were obtained using the  earlier . The lononhammer  availablonhammer . The obtonhammer . The assonhammer . The gen6 AMR genes were identified from the genome sequences using the BLASTn-based ABRicate (version 0.8.10) program7 to query the ResFinder database.8 The Capsular Polysaccharide loci (KL) and the Outer Core Lipooligosaccharide loci (OCL) types were identified using the Kaptive database.9Further downstream analysis of the 17 complete genome sequences was performed using tools available at the Center for Genomic Epidemiology (CGE).10 Using BLAST analysis, the plasmid repAci types from the complete genomes were identified and characterized. The PHASTER server was used to determine the prophages.11 The prophage regions identified were analyzed for the presence of any AMR genes using the ABRicate (version 0.8.10) program(see text footnote 7). A BLAST similarity search was performed on the individual genomes to identify the comM region that flanks AbaR type genomic islands. Based on the known AbaR sequences collected from published literature, the precise boundary of AbaRs and the respective backbone were curated manually. The sequence types were identified with MLST Finder 2.0 using Oxford MLST and Pasteur MLST schemes.12The presence of ISs was identified using ISFinder.13 algorithm, and the variants were filtered with FreeBayes available in Snippy (The assembled genome sequences were mapped to the reference genome ATCC 17978 (CP012004) using the BWA MEMn Snippy . The corn Snippy . The maxn Snippy  using thn Snippy .Acb-complex and further reconfirmed as A. baumannii using Vitek-MS (Data not shown). Among the 17 isolates, AB01 was pan-susceptible (PSAB), AB23 was multidrug-resistant (MDRAB) but susceptible to carbapenem (CSAB), 12 isolates  were CRAb, and the remaining three isolates  were pan-drug resistant (PDRAB). blaOXA–51–like and blaADC–25–like genes. Whole-genome sequencing (WGS) identified seven different variants of blaOXA–51 , a single variant of blaOXA–23 (blaOXA–169), and a single variant of blaNDM–like gene (blaNDM–1). Among the 17 study isolates, ten carried blaOXA–23–like alone, one isolate carried blaNDM–1 alone, and four isolates co-harbored blaOXA–23–like and blaNDM–1. No acquired carbapenemase genes were identified in the remaining two isolates. More than one copy of blaOXA–23 was observed in thirteen isolates (blaOXA–24–like or blaOXA–58–like genes. The A. baumannii isolates in this study belonged to diverse sequence types (STs) representing four International clones, ICs , one clonal complex, i.e., CC862, and one singleton  were identified, ST195 (2), ST451 (2), ST848 (2), ST208 (1), ST218 (1), and ST349 (1), but there was a single Pasteur MLST ST2Pas. IC2 isolates predominantly carried either blaOXA–23 alone (7/9) or coproduced blaOXA–23 and blaNDM–1 (2/9). All had blaOXA–23 in Tn2006, an ISAba1-bounded composite transposon in the chromosome  was identified in AB13, where the genome harbors a single copy of ISAba125 and an incomplete transposase at the left-hand and right-hand extremities of Tn125, respectively  and aph(3)-I, mobilization gene; mobL, transposable element; CR2, phosphoglucosamine mutase; pgm, arsenic resistance encoding gene; arsR, tetracycline efflux protein; tet(B), and tetracycline resistance transcriptional repressor gene; tetR(B) along with △Tn6022 and Tn2006 . In AB20, one copy of Tn2006 was observed between the Tn6022 element and the plasmid linker, whereas another Tn2006 was inserted near orfBA. AB27 had two copies of ISAba1, one copy inserted at the tniE on the Tn6022 backbone and the second copy inserted at tniA of Tn6172. Two copies of Tn2006 were seen, one present on Tn6022 at orf4 with the second adjacent to orfBA on Tn6172. Insertion of sul2, rcr2, and hypothetical protein at the left inverted repeat of the Tn6172 element was also observed. Additionally, insertion of the arsenic resistance encoding gene, arsR, and the mobilization gene, mobL, was present on the Tn6172 element of all three genomes  and on an incomplete RepB family plasmid (blaOXA–23 in △Tn2006). Both AB14 and AB15 showed the presence of two prophage regions, namely, PHAGE_Mannhe_vB_MhM_3927AP2_NC_028766 and PHAGE_Acinet_YMC11/11/R3177_NC_041866. AbaR4 that were mapped to Tn6022 backbone and Tn2006 linked blaOXA–23 locus was present in both the genomes  and ST2441 Oxf/ST575Pas (AB11), whereas AB23 corresponded to ST447Oxf/ST10Pas and had an MDR phenotype. AB10 and AB11 were blaOXA–23 producers, while AB23 was found to be a non-carbapenemase producer. Two, one, and three plasmids were identified in AB10, AB11, and AB23, respectively. Three intact prophages such as PHAGE_Pelagi_HTVC010P_NC_020481, PHAGE_Acinet_Bphi_B1251_NC_019541, and PHAGE_Acinet_ vB_AbaS_TRS1_NC_031098 were seen in AB10. In AB11, PHAGE_Acinet_YMC11/11/R3177_NC_041866, PHAGE_ Acinet_Bphi_B1251_NC_019541, and PHAGE_Mannhe_ vB_MhM_3927AP2_NC_028766 were observed. A single prophage, PHAGE_Acinet_Bphi_B1251_NC_019541, was present in AB23. Similar to IC7, both XDR isolates carried AbaR4 on the chromosome but were absent in the MDR isolate, AB23 , an insertion of ISAba16, TnpB, and an IS66 transposase, there was no upstream presence or insertional inactivation  genes. When we analyzed and compared the p1AB28 plasmid sequence with the reference plasmid, pA85-3 (accession number–KJ493819), we found the presence of a complete blaOXA–23 gene with one complete and an incomplete copy of an ISAba1 locus. However, some transposon-related genes such as uspA and sulP were intact. IS66 family transposase with its accessory protein, tnpB, was also encoded within the p1AB28 plasmid but was absent in the pA85-3 reference plasmid. The plasmid, p1AB28, also carried putative tra genes that are required for mating pair formation and trwC and trwB genes that are needed for plasmid mobilization ::ISAba42]  phosphotransferase and confers resistance to amikacin. Previous studies from European and Asian countries have reported isolates of A. baumannii with large conjugative plasmid such as repAci6, carrying both the blaOXA–23 and aphA6 genes, which contribute to the dissemination of resistance to carbapenems and amikacin, respectively  that belonged to IC1 but lacked an AbaR3 type island. Instead, it carried an IC2-specific Tn6022-derived backbone, which indicates the possibility of independent acquisition. Tn6022-derived elements and AbaR4 and AbGRI variants are typically confined to IC2. In this study, we also found that none of the IC2 isolates carried AbaR4; instead, it was present among isolates belonging to other ICs such as IC7 and IC8. All the study isolates belonging to IC2 possessed either the AbGRI1 variant or the AbGRI variant with complex chimeric structures. Although the genetic events behind this process are unclear, such complex structural variation in the AbaR backbones might have resulted either due to the target sequences favorable for MGE insertion or due to the exposure of AbaRs with different MGEs in different clones. These findings indicate that AbaRs with diverse backbones might have evolved separately.Genomic analysis of AbaRs in this study unveiled novel genetic configurations specific to backbones, which involve either insertion of MGEs or structural modifications driven by known MGEs. For example, insertion of ISA. baumannii.Overall, to the best of our knowledge, this study is the first that provides comprehensive profiling of RIs together with the MGEs, acquired AMR genes, and the distribution of clonal lineages among CRAb from India. Although this study provides a clear picture of the Indian scenario, further comparative analysis with an extensive collection of global isolates is required to understand the structural diversity and the evolution of these MGEs that drive the genome plasticity of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/SV: laboratory methods, data analysis and interpretation, and manuscript writing. JJ: data analysis, interpretation, and manuscript writing. KV: hybrid genome assembly and other bioinformatics methods. PM, PR, SA, IB, and KW: manuscript correction. AN and AB: data analysis. BV: study design and supervising, manuscript writing, and manuscript correction. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Alphacoronavirus in the family Coronaviridae. It causes acute watery diarrhoea and vomiting in piglets with high a mortality rate. Currently, the GII genotype, PEDV, possesses a high separation rate in wild strains and is usually reported in immunity failure cases, which indicates a need for a portable and sensitive detection method. Here, reverse transcription–recombinase aided amplification (RT-RAA) was combined with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas12a system to establish a multiplexable, rapid and portable detection platform for PEDV. The CRISPR RNA (crRNA) against Spike (S) gene of GII PEDV specifically were added into the protocol. This system is suitable for different experimental conditions, including ultra-sensitive fluorescence, visual, UV light, or flow strip detection. Moreover, it exhibits high sensitivity and specificity and can detect at least 100 copies of the target gene in each reaction. The CRISPR/Cas12a detection platform requires less time and represents a rapid, reliable and practical tool for the rapid diagnosis of GII genotype PEDV.Porcine epidemic diarrhoea virus (PEDV) is a member of the genus  Alphacoronavirus of the family Coronaviridae, the porcine epidemic diarrhoea virus (PEDV), causes acute intestinal infectious diseases, which manifest as severe dehydration, diarrhoea, vomiting, and a high mortality rate in piglets (Coronaviruses (CoVs) are a type of enveloped virus consisting of a positive-strand RNA genome and includes numerous viruses of veterinary and human importance . SARS, M piglets . PEDV wa piglets . Seven y piglets .Porcine epidemic diarrhea virus may be divided into two different genotypes, GI and GII, according to multiple INDEL and point mutations on the S1 subunits of the spike (S) protein . The wid2 copies/μl for PEDV  platform, which enables rapid and specific detection of human papillomavirus in patient samples, was established by combining Cas12a ssDNase activation with isothermal amplification -Cas systems are adaptive immune systems found in bacteria and archaea. The CRISPR system is based on CRISPR RNAs (crRNAs) that bind to CRISPR-associated (Cas) proteins to form a complex, which directly cleavages complementary sequences . CRISPR-fication .The emergence and development of CRISPR enable new opportunities for the rapid and convenient diagnosis of viral diseases. Here, we report a molecular method for the detection of the dominant GII PEDV spike (S) gene using a CRISPR/Cas12a system, which is not limited by expensive instrumentation, skilled analysis, and complex processes. This platform relies on a fluorescent detection system (FDS) or two visual detection systems for signal sensing of the trans-cleavage activity of the Cas12a protein triggered by the spike (S) gene of PEDV. It can specifically distinguish GI and GII PEDV with high sensitivity and specificity, providing an effective detection tool for prevention and control of this virus.The FastPure Cell/Tissue Total RNA Isolation Kit V2 (RC112-01), the HiScript® III 1st Strand cDNA Synthesis Kit (R312-01), and T7 High Efficiency Transcription Kit (JT101-01) were purchased from Vazyme Biotech Co. Ltd. . The Recombinase Aided Amplification (RAA) kit (WLB8201KIT) was purchased from Warbio Biotech Co. Ltd. . The EnGen™ LbCas12a (#M0653T) and NEBuffer™ 2.1 (#B7202S) were purchased from New England Biolabs . RNase inhibitor and PerfectStart® II Probe qPCR SuperMix were purchased from TransGen Biotech Co. Ltd. . Synthesis of the target sequence for positive control, crRNA, 12 nt oligo reporter, primer, and probe, as well as gene sequencing, was accomplished by Tsingke Biotech Co. Ltd. . The Spark® multi-mode microplate reader was purchased from Tecan Trading Co. Ltd. . Applied Biosystems StepOne™ Real-Time PCR System was purchased from Thermo Fisher Scientific Inc. .The genomic RNA or DNA of Transmissible gastroenteritis virus (TGEV), Porcine deltacoronavirus (PDCoV), and Pseudorabies virus (PrV) were presented by Zhang Xiaorong, Associate Professor, College of Veterinary Medicine, Yangzhou University . The genomic DNA of ASFV was presented by the National Research Center for Exotic Animal Diseases, Animal Health and Epidemiology Center . The virus strain Porcine circovirus (PCV) and Seneca virus A (SVA) were isolated from clinical samples in our laboratory.The clinical samples were all collected from a pig farm with an outbreak of PEDV. Rectal swabs were collected according to the standards recommended by the OIE.S gene sequences of Genotype I and Genotype II PEDV were downloaded from the National Center for Biotechnology Information (NCBI) database. The target sequences were analyzed by SnapGene to design specific crRNAs and primers of Genotype II PEDV. These designed sequences were used to alignment by BLAST with the swine genome (taxid: 9823), PDCoV, transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), and the swine acute diarrhea syndrome coronavirus (SADS-CoV) for verifying the specificity. When CRISPR/Cas12a proteins were bound to double-stranded DNA (dsDNA) guided by specific crRNA, its trans-cleavage activity was nonspecifically activated to cleave ssDNA-FQ-reporter followed by signal sensor.n = 72) were collected from the clinical samples of swine breeding farms with an outbreak of diarrhoeal. After three times of freezing and thawing, the supernatant of 200 μl was obtained by centrifugation. Subsequently, 30 μl of RNA were extracted from 200 μl supernatant of the clinical sample by the FastPure Cell/Tissue Total RNA Isolation Kit V2, and 1 μg of RNA was reverse transcribed into cDNA using HiScript® III 1st Strand cDNA Synthesis Kit.The rectal swabs (The full-length S gene fragment of PEDV/JSX2014 (GenBank: MH056658.1) was amplified by the primers PEDV-S-F/R listed in In 100 μl of reaction system of CRISPR, the fluorescence signal activated by trans-cleavage in 96-well Tecan black flat was read by the Spark® multi-mode microplate reader, and the initial value which was favorable for observing the change of fluorescence signal was obtained. Firstly, the optimal quantity of ssDNA-FQ-reporter was confirmed with a molar gradient of 0.0625 pmolto 2 pmol. The name and sequence of reporter are listed in Recombinase aided amplification of the PEDV S gene was performed by the commercial RAA kit , primers were designed by Primer 5.0 according to the manufacturer’s instructions. The PEDV RAA primers named PEDV-RAA-F/R listed in rat resistance were sprayed on NC film as quality control line and detection line. The principle of LFSs detection is shown in Preparation of gold nanoparticles by sodium citrate reduction method. Heat 0.01% chlorauric acid hydrate to a boil, then quickly add 1 ml 1% sodium citrate while stirring. Let the solution turn wine red and continue to boil for 15 min, then cool to room temperature. Transmission electron microscope was used to scan the ultrastructure of gold nanoparticles, and their aggregation state and particle size distribution were then observed. FAM antibody was coupled with gold nanoparticles and attached to the binding pad of the strip. Digoxin antibody and In lateral flow detection, the reporter ssDNA probe sensor was labeled with FAM and digoxin at the 5′and 3′ termini, respectively. The sequence of ssDNA-FD-reporter is listed in The TaqMan real-time PCR detection of the PEDV S gene was carried out using a StepOnePlus™ Real-Time PCR System  according to the method previously reported. The TaqMan RT-PCR was performed in a final volume of 20 μl containing 10 μl of 2× AceQR qPCR Probe Master Mix , 0.4 μl of primer PEDV-q-F, 0.4 μl of primer PEDV-q-R, 0.2 μl of TaqMan probe PEDV-probe, 2 μl of cDNA, and 7 μl of ddH2O. The PCR program was as follows: initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation (95°C for 10 s), annealing, and extension (60°C for 30 s). The fluorescence signal collection occurred at the 60°C annealing extension per cycle. The cycle value (Ct) ≤ 35.0 was judged as PEDV positive. The sequence of the primers and probe used for qRT-PCR are listed in All experiments were performed in triplicate, and data were shown as the mean ± SD. Statistical analysis and graphing were carried out with GraphPad Prism 8.0.All data that support the findings of this study are available from the corresponding author upon reasonable request.To sensitively and specifically detect PEDV genomic cDNA, a CRISPR/Cas12a fluorescence detection system (CRISPR/Cas12a-FDS) was established consisting of the LbCas12a protein, PEDV-specific CRISPR RNAs, and an ssDNA-FQ-reporter . As CRIS8 copies  and PEDV/CV777 (GI) were used as templates for CRISPR/Cas12a-FDS detection. All of the crRNAs bound specifically to the genomic template of GII PEDV; whereas crRNA4 and crRNA5 bound non-specifically to the GI PEDV genome template ,B. The c8 copies .8 to 102 copies per reaction , Jiangsu Agricultural Science and Technology Innovation Fund [CX(21)2038], and the Guidance Foundation, the Sanya Institute of Nanjing Agricultural University Research (NAUSY-ZD08).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.


As a classic Chinese medicine, Wendan Decoction (WDD) encompasses a marvelous impact on the remedy of hyperlipidemia. It is known that hyperlipidemia leads to cardiovascular injury, therefore anti-vascular endothelial cell injury (AVECI) may be an underlying molecular mechanism of WDD in the cure of hyperlipidemia. However, there is no relevant research on the effect of WDD on vascular endothelial cells and its pharmacodynamic substances. Therefore, the purpose of this study was to investigate the protective effect of WDD on vascular endothelial cells. (2) Methods: The chemical constituents of WDD were determined by LC-MS/MS technology. The protective effects of 16 batches of WDD on samples from human umbilical vein endothelial cells (HUVECs) were evaluated. Finally, gray relation analysis (GRA) and partial least squares regression (PLSR) were used to analyze the potential correlation between chemical ingredients and AVECI. (3) Results: The results indicated that WDD had apparent protective effect on endothelial cells, and pharmacological properties in 16 batches of WDD tests were apparently discrepant. The GRA and PLSR showed that trigonelline, liquiritin, hesperidin, hesperetin, scopoletin, morin, quercetin, isoliquiritigenin, liquiritigenin and formononetin may be the active ingredients of AVECI in WDD. (4) Conclusions: WDD has a protective effect on endothelial cell injury induced by palmitic acid, which may be related to its component content. This method was suitable for the search of active components in classical TCM. In recent years, significant changes have taken place in diet and lifestyle. The incidence of metabolic diseases, including hyperlipidemia, has increased, and patients have high levels of triglyceride, phospholipid and total cholesterol . HyperliHUVECs are monolayer flat cells, lining the inner surface of the vascular system, and are important components of vascular intima. They can synthesize, secrete and express a variety of antithrombotic and thrombogenic substances. Endothelium injury is regarded as a reason of pathogenesis and clinical sign of hyperlipidemic vascular injury, and a large number of essential and auxiliary metabolic disarrangements have been connected to endothelial injury ,4,5,6. ETCM has played a basic role in treatment of various ailments since its long-standing history of clinical utilization . In ChinWDD dates from “Bei ji qian jin yao fang ” (written by Sun Simiao of the Tang dynasty), and was recorded in the Catalogue of Classical Prescriptions (first batch) issued by the state administration of traditional medicine of China in 2018 . This was one of the primary 100 classic Chinese pharmaceutical medicines discharged and consisted of Pineilia ternata (Thunb.) Breit. , Glycyrrhiza uralensis Fisch. , Citrus reticulata Blanco , Phyllostachys nigra (Lodd.) Munro var. henonis (Mitf.) Stapf ex Rendle , Citrus aurantium L. , Zingiber officinale Rose. . This prescription is the basic prescription of “expectorant”. Clinically, it is generally utilized within the treatment of diabetes, chronic kidney disease and hyperlipidemi ,15,16. TThe identification method of chemical constituents in WDD refer to a previous study . In all,The single mass spectra of 20 compounds were studied by pouring the relevant standard solutions into the mass spectrometer in both positive and negative ion modes. The optimization results are shown in The contents of 20 compounds in 16 batches of WDD were determined by UPLC-QQQ-MS/MS within 10 min. The degree speed had major upgrades over the HPLC method, so ensuring that we may checkout still more cases in one day. v/v) methanol-water. The slopes, intercepts and correlation coefficients (r2) were obtained by weighted (1/x) linear regression. The estimation ranges (S/N ≥ 10), lower limit of quantitation (LLOQ) and straight twists of diverse chemical fixings are tabulated in The 20 primitive courses of action were precisely debilitated with 50%  is the standard to evaluate the quality of traditional Chinese medicine compound, which should not be based on the ingredients of a few traditional Chinese medicines but should reflect the situation of the whole compound . These iThe research still has some shortcomings, such as small selection of the components. It still cannot fully represent the efficacy of WDD.The 16 batches, comprising of WDD counting of BX, GC, ZS, CP, SJ, ZR were included in this research. These 16 batches of Chinese homegrown drugs come from different pharmaceutical markets of China. All homegrown solutions were distinguished by Teacher Jin Guangqian, TCM recognizable proof master of Shandong Institute of TCM. All the herbs met the necessities of Ch. P (2020 Version) as shown in Hesperidin (wkq20030407), tangeretin (wkq20041611), naringin (wkq21020606), nobiletin (wkq18020111), isoliquiritigenin (wkq18033006), formononetin (wkq18022712), naringenin (wkq21022409), glycyrrhetinic acid (wkq16070701) and glycyrrhizic acid (wkq16032502) were purchased from Sichuan Weikeqi Biotechnology Co., LTD., China. Daidzein (PS000251), isoquercitrin (PS001042), hesperetin (PS000219), trigonelline (PS000427), morin (PS020344), scopoletin (PS010525), liquiritigenin (PS010083) and liquiritin (PS012028) were provided by Chengdu Pusi Biotechnology Co., LTD., Sichuan Province, China. Obacunone (111923-201102) and furosemide  (100544-201503) were obtained from National Institutes for Food and Drug Control. Quercetin (100081-200406) and synephrine (0727-200105) were obtained by China National Institute for the Control of Pharmaceutical and Biological Products. All the reference substances were HPLC ≥ 98%. Acetonitrile for UPLC-QQQ-MS/MS was acquired from Merck KGaA .HUVEC cell line was purchased from Wuxi Bohe Biomedical Technology Co., LTD. . RPMI-1640 basal medium was obtained from Hyclone Laboratory Media . Pancreatin was obtained from Shanghai Beyotime Biotechnology Co., LTD, China. The detection kit of MTT was bought from Sigma Company, Saint Louis, MO, USA. Penicillin-streptomycin was purchased from biological sharp, from Shanghai, China. Dimethyl sulfoxide (DMSO) was obtained from Invitrogen .The preparation of medicinal materials was based on previous studies . It was The freeze-dried powder of WDD extricate (0.5000 g) was precisely weighed and ultrasonically broken up in methanol . The arrangements were blended and balanced to a volume of 5 mL and diluted to the proper consistence. The test arrangements were sifted through a 0.22 µm channel film and the filtrate was stored at 4 °C for later UPLC-QQQ-MS/MS examination.All standard solutions were weighed accurately and diluted in methanol to form a reserve solution, which was stored at 4 °C. All reserve fluids were diluted to the desired concentration and then mixed immediately prior to analysis.LC–MS/MS investigations were carried out employing a Thermo Scientific Vanquish LC system connected to a TSQ Quantum Ultra triple quadrupole mass spectrometer  equipped with a heated electrospray ionisation source (H-ESI). Xcalibur 4.4 Software (Thermo Scientific) was used for method setup, data processing and reporting. The mass spectrometer was operated with a H-ESI interface handled in both positive and negative ionization modes and was utilized for the multiple reaction monitoring. Instrument settings were: positive ion: 3600 v, negative ion: 3400 v, sheath gas 0.3 mL/min, aux gas 0.3 mL/min, ion transfer tube temp 345 °C, vaporizer temp 350 °C.Chromatographic fractionation was accomplished on a Thermo Hypersil GOLD C18 column  at 30 °C, and versatile stage comprised 0.1% formic corrosive and water for dissolvable A and acetonitrile for dissolvable B at a stream rate of 0.3 mL/min. The gradient elution conditions of mobile phase B are as follows: 0–0.5 min: 5%, 0.5–2 min: 5–8%, 2–2.1 min: 40%, 2.1–4 min: 40–50%, 4–6 min: 50–60%, 6–6.1 min: 60–70%, 6.1–8 min: 70–80%, 8–8.1 min: 80–5%, 8.1–10 min: 5%. The sample size for analysis was 3 μL.The standard curve was established in the method validation part with internal standard method. The precision, stability, repeatability and recovery were investigated. The precision was calculated by 6 consecutive injections of the same sample solution, and repeatability was evaluated by six samples from the same source. The sample solution was put into the sample bottle at room temperature and injected for analysis at different times  after preparation to observe the sample stability at room temperature. The measured WDD content was precisely weighed and added equal to 100% of the measured reference substance in WDD to determine the recovery of the sample.2 at 37 °C. The PA was broken down in DMSO (200 mM) as a stock solution and after that diluted with a medium containing RPMI-1640 basal medium with 10% FBS for later use . The control treatment: cells were cultured with RPMI-1640 basal medium with 10% FBS. The experimental group added different concentrations of PA with the climate containing 5% CO2 at 37 °C. All the treatments were performed in triplicate. According to the above grouping, 10 μL of MTT was included in each well with the air brake containing 5% CO2 at 37 °C for 3–4 h in a dark incubator. After the liquid absorption, 150 μL DMSO was added and shaken at the shaking table at room temperature for 10 min. Finally, the optical density (OD) value at the same time point was calibrated at 492 nm wavelength by enzyme labeling instrument, and the measured OD value was used for cell viability analysis.The HUVECs were stored in a RPMI-1640 basal medium containing 10% FBS and other essential development variables in a humidified incubator with the climate containing 5% COThe 16 batches of WDD powder were absolutely weighed. The volume was settled to 1 mL by PBS, and the microscopic organisms were sifted by 0.22 μm channel layer. Later the cells were included, and each batch of concentrated arrangement was weakened to 5 concentrations of 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL by RPMI-1640 basal medium with 10% FBS. They were isolated into control group and the WDD treatment group. The cells treatment strategy was rehashed. All groups were in triplicate.GRA’s significant concept is to select whether the filiation is close by characterising the geometric resemblance between the reference data course of action and a couple of comparison data columns. It mirrors the relationship degree between bends . The datPLSR could be a measurement strategy which brings the calculated variable amount and watched variable into an unused room to look out for a direct relapse demonstrate . To encoA fast, sensitive and dependable LC-/MSMS strategy, combined with MTT and scientific factual examination strategy, was set up for the screening of numerous dynamic fixings of WDD with AVECI action. The results show that the extracts of WDD significantly increased the proliferation of endothelial cells and protected endothelial cells. However, the interaction between them can be additive, synergistic and antagonistic, and the mechanism remains unclear. More appropriate models should be included in the follow-up to conduct a comprehensive pharmacodynamics evaluation of WDD, laying a foundation for accurately and comprehensively revealing the active component group of WDD, so as to provide the basis for the rational use of WDD.