Tuberculosis (TB)-associated mortality in South Africa remains high. This review aimed to systematically assess risk factors associated with death during TB treatment in South African patients.We conducted a systematic review of TB research articles published between 2010 and 2018. We searched BioMed Central (BMC), PubMed®, EBSCOhost, Cochrane, and SCOPUS for publications between January 2010 and December 2018. Searches were conducted between August 2019 and October 2019. We included randomised control trials (RCTs), case control, cross sectional, retrospective, and prospective cohort studies where TB mortality was a primary endpoint and effect measure estimates were provided for risk factors for TB mortality during TB treatment. Due to heterogeneity in effect measures and risk factors evaluated, a formal meta-analysis of risk factors for TB mortality was not appropriate. A random effects meta-analysis was used to estimate case fatality ratios (CFRs) for all studies and for specific subgroups so that these could be compared. Quality assessments were performed using the Newcastle-Ottawa scale or the Cochrane Risk of Bias Tool.n = 12561) and two were restricted to children < 15 years of age (n = 696). The CFR estimated for all studies was 26.4% ; 37.5%  for drug-resistant (DR) TB; 12.5%  for drug-susceptible (DS) TB; 15.6%  for studies in which drug susceptibility was mixed or not specified; 21.3%  for people living with HIV/AIDS (PLHIV); 19.2%  in HIV-negative TB patients; and 6.8%  in paediatric studies. The main risk factors associated with TB mortality were HIV infection, prior TB treatment, DR-TB, and lower body weight at TB diagnosis.We identified 1995 titles for screening, 24 publications met our inclusion criteria . Twenty-two studies reported on adults  under the registration number CRD42018108622. This study was funded by the Bill and Melinda Gates Foundation (Investment ID OPP1173131) via the South African TB Think Tank.The online version contains supplementary material available at 10.1186/s13643-023-02175-8. A tuberculosis (TB) death is defined as any death during an episode of TB, regardless of treatment or the underlying cause of death . This maThe End TB strategy aims to reduce the annual number of TB deaths (as reported in 2015) by 75% by 2025 and by 95% by 2035, for a reduction in TB CFR from 15% to 6.5% by 2025 , and a TIn a systematic review of risk factors for mortality in people on TB treatment globally between 1966 and 2010, two distinct epidemics dependent on TB/HIV burden were described –12; failWe aimed to identify risk factors for TB mortality during TB treatment in South Africa for patients with DS-TB, DR-TB, with and without HIV, and for adult and paediatric populations.This systematic review is reported according to PRISMA guidelines  . Data inData extracted were tabulated and subgroups and comparability of risk factors assessed. Due to heterogeneity in effect measures and risk factors evaluated, a formal meta-analysis of risk factors for TB mortality was not appropriate. We conducted a random effects meta-analysis using the restricted maximum likelihood estimation method to establish CFRs for all studies and for specific subgroups to contribute to our understanding of mortality risk factors; however, this was not an original aim of the study. One author assessed the quality of evidence for case control and cohort studies using the Newcastle-Ottawa scale (NOS)  and usedn = 13), prospective cohort studies (n = 8), two RCTs and one case control study.We identified 1995 titles for screening and 24 publications met our inclusion criteria  rated well overall, as well as for selection criteria, comparability, and outcomes. However, in relation to loss to follow-up (LTFU), seven studies either reported attrition greater than 10% without demonstrating a lack of systematic difference between those followed and those LTFU or did not report the number of those LTFU , incidence rate ratios (IRR), odds ratios (ORs), and risk ratios (RRs). Twelve studies reported exclusively on DR-TB, and this accounted for 39% of all patients 5149/13257). Of the three studies reporting exclusively on DS-TB, one was of miners and one of children. The remaining nine studies included four on patients with mixed DST patterns and five where the DST was not specified. Only two studies represented children, and these were restricted to very specific populations. The first was of children (defined as 0–13 years) with TB meningitis (TBM)  and the /13257. On = 13257); 37.5%  for DR-TB; 12.5%  for DS-TB; 15.6%  for studies in which drug susceptibility was mixed or not specified and 6.8%  in paediatric studies. There was little difference in CFRs estimated for PLHIV  and HIV-negative TB patients ; this is because five of six studies reporting on HIV-negative patients were restricted to DR-TB patients, elevating the CFR for this subgroup ; 14 studies described treatment regimens; 13 the duration of treatment; 9 time from treatment initiation to death; 4 time from diagnosis to death; and 2 reported place of death  of patients were included from a single study of adult miners  and 33% nated TB . In thatnated TB . In the nated TB  (Table 302/1935 on = 5149), the lowest CFRs were reported for a small sample of HIV-negative patients  [n = 9) the highest CFRs were reported for a small sample of patients who had a positive urinary lipoarabinomannan (u-LAM) test  [Twelve studies reported on individuals with DR-TB studies %, 3/56)  and thos%, 3/56) . The hig/56  and %, 3/56) . In stud, 13/53)  and pati, 13/53) . The fac, 13/53)   , and 6.3Nine studies described the time to death from treatment initiation , 45, 49.We broadly characterised risk factors as demographic, baseline clinical characteristics, TB disease-related, TB treatment-related and HIV-related. However, factors falling into these categories were varied, some being unique to studies focusing on a sub-population  or severely underweight (BMI < 16 kg/m2) compared to those with a normal BMI (BMI 18.5 kg/m2–24.9 kg/m2) increased the odds of mortality among adults with DR-TB [Z-score <− 3) was associated with greater hazard of mortality [Weight was evaluated in three studies. Lower weight at TB diagnosis decreased the odds of survival among TB patients with XDR-TB ; being uth DR-TB ; and in ortality   [p value = 0.009 [HIV status (independent of ART) was reported as a risk factor in six studies , 33, 46.5–210.6) . Two stu5–210.6) , 49 with5–210.6) , aHR 0.3 = 0.009 ). Not be = 0.009 . Timing  = 0.009   reported by the WHO for 2018 , because WHO numbers reflect estimates for all incident TB whereas our review is restricted to those on TB treatment, and because the WHO estimate includes deaths prior to TB notification and reflects deaths over a one-year period whereas our review includes some studies with greater periods of follow up and specific populations with higher risk of mortality .In our review, the risk of mortality was highest among patients with DR-TB, where death was observed in more than a third of all patients. In contrast, in those diagnosed with DS-TB, the risk was lower, with death observed in one in eight patients. Risk of mortality was equal for PLHIV and HIV-negative TB patients, with death observed in one in five patients in each group; however, this is because the pooled CFR for the HIV-negative group is based primarily on studies of DR-TB patients.3 from suspected bacterial sepsis and disseminated TB, underscores the impact of failure to timeously diagnose and initiate treatment in patients for both TB and HIV, and possibly delayed patient health seeking behavior. In South Africa, the response to treating HIV and TB has shifted considerably with all PLHIV eligible for ART as of 2016 [Individual studies included in this review indicate that HIV remains a major risk factor for TB mortality. This is in line with findings from previous studies as well as a national study evaluating treatment outcomes of all TB patients started on treatment, reporting that patients with HIV on ART had a greater hazard of death compared to patients without HIV, and those not on ART had two-threefold increased risk of mortality compared to patients who were HIV-negative , 24, 51. of 2016 . This inPrevious TB treatment was reported as a risk factor in two studies we reviewed , 32. StuMultiple studies reported advancing age increasing the risk of mortality in adults , 41, 44;A strength of this systematic review is that we included studies with moderate to large samples of TB patients that were reasonably representative of target study populations; that used medical records and other objective measures to establish exposure and outcomes; and sampled comparison groups from comparable sources. Multiple factors compromised the quality of the evidence, including the lack of prospective studies and RCTs, and the reliance on statistical procedures rather than sampling or design strategies to control for potential confounders. This increased the potential for unknown systematic bias. Inconsistency in risk factors and comparator variables made it difficult to assess the quality for each of the outcomes. Variances could not be pooled and estimated across studies, and we were unable to perform a meta-analysis of risk factors for TB mortality. A further limitation is that collectively the studies in this review do not accurately represent the population of people with TB in South Africa. In this review, the proportion of patients with DR-TB or living with HIV was much higher than estimated for South Africa and the proportion of children was under-represented. In our review 41% of TB patients included were miners in South Africa, but we had insufficient data to evaluate additional risk factors for mortality in this population such as silicosis. This review did not provide sufficient data to consider co-morbidities and final causes of death, nor was there sufficient data relating to sociostructural determinants of health such as those related to socioeconomic status, healthcare system access and quality and behavioural factors (e.g. cigarette smoking). Finally, this study evaluated studies published between 2010 and 2018 and included data on patients treated between 1995 and 2015. We acknowledge that this does not include earlier studies which may inform the results, nor does it include studies which address the impact of COVID-19 on TB mortality.Introducing standardised variables and minimum reporting requirements for TB cohorts would support future comparative work. We recommend at a minimum including sex; age with WHO age bands as a minimum; weight, including BMI; HIV status including HIV unknown, negative, and positive with an indication of ART regimen and timing; immune suppression with CD4 counts; diagnostic tests used with DST; and the place or level of care where the diagnosis was made. Published articles should include explicit statements about follow-up and the duration of follow-up and should include all WHO TB treatment outcome categories with descriptors of those who are LTFU. While sex was not identified as a definitive risk factor for TB mortality, specific interventions which target improvements in case finding and retention in care should focus on the differing needs of males and females. Based on existing evidence it is important to further examine the impact of factors such as pregnancy, diabetes mellitus, heart disease, chronic lung disease, and malignancy, which are associated with increased TB mortality , 57–60. Additional file 1: Supplementary material. Description of data: Complete list of outcomes and variables for which data were sought; PRISMA 2020 checklist. Supplementary Table 1. Newcastle-Ottawa quality assessment scale for cohort studies, South Africa, 2010-2018 (n=21). Supplementary Table 2. Cochrane risk of bias tool for randomised trials, South Africa, 2010-2018 (n=2). Supplementary Table 3. Newcastle-Ottawa quality assessment scale for case control studies, South Africa, 2010-2018 (n=1). Supplementary Table 4. Demographic risk factors for TB mortality. Supplementary Table 5. Clinical risk factors for TB mortality, South Africa, 2010-2018. Supplementary Table 6. Tuberculosis treatment-related risk factors for TB mortality, South Africa, 2010-2018. Supplementary Table 7. HIV and antiretroviral therapy related risk factors for TB mortality, South Africa, 2010-2018.


With a high incidence and limited treatments, gastric cancer (GC) seriously threatens human health worldwide. Weikang Keli (WK) is a compound prescription summed up from clinical experience. In our previous studies, WK has been proved to exert antitumor effects. However, there are no research studies to discuss and verify its mechanism as a compound.  The aim of the study is to explore the potential molecular mechanism of WK in the treatment of GC with the aid of network pharmacology and verify it through experiments.  Related databases were used to obtain genes and targets of WK and gastric cancer. A protein-protein interaction (PPI) network is constructed and visualized by Cytoscape 3.7.2. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to analyze core targets. The cell viability of MFC and BGC-823 cells was determined by CCK8. Immunofluorescence was used to determine autophagy of GC cells. Moreover, the effect of WK on the MAPK signaling pathway in GC cells and tumor tissues of ICR mice was detected by Western blot.  A total of 106 cross targets of WK and GC were obtained. According to the enrichment analysis of GO and KEGG, we target the MAPK signaling pathway to discuss the mechanism of WK on GC. Cell experiments proved that WK inhibited the viability of gastric cancer cells in a dose-dependent and time-dependent manner. Autophagosome aggregation and an increase in the expression of an autophagy marker protein LC3-II can also be observed in WK groups. Further animal experiments showed that the tumor inhibition rate of WK showed a dose-effect relationship. Moreover, the expressions of p-JNK, p-p38, and p-ERR1/2 proteins in the MAPK signaling pathway in WK Group were downregulated both in the cell and animal experiments, compared with the blank control group.  WK showed an explicit antitumor effect on gastric cancer through the MAPK signaling pathway, and the curative effect varies in different concentrations. Besides, in model mice, the antitumor effect of high-dose WK group is close to that of platinum. This study provided a theoretical basis for the application of WK in the clinical treatment of gastric cancer. With its incidence ranking fifth, gastric cancer is one of the most common cancers in the world. It is widely recognized that the top treatment pick for early gastric cancer is surgery. For advanced gastric cancer, chemotherapy containing platinum and fluorouracil is still the first-line treatment. Other therapies, such as HER2-targeted drugs (trastuzumab), anti-angiogenesis drugs , and PD1 monoclonal antibodies (nivolumab and pembrolizumab) also play a more and more important role in advanced gastric cancer. Nevertheless, the overall survival rates of treatments mentioned above are still limited, and it is difficult to achieve a balance between efficacy and adverse reactions, especially for patients in poor physical conditions  (http://www.organchem.csdb.cn.) and related papers to make a complement. PubChem (https://pubchem.ncbi.nlm.nih.gov/) was used to identify 2D structures of related compounds. The 2D structures were put into SwissADME (http://www.swissadme.ch/) for ADME  screening. ‘GI absorption' in pharmacokinetics should be ‘High' and more than 2 items should be ‘Yes' in drug-likeness . After ADME screening, active ingredients were finally selected. Then, the 2D structures of the selected compounds were imported into Swiss Target Prediction (http://www.swisstargetprediction.ch/) for target prediction. Duplicates in the target information were removed and the left ones were imported into Cytoscape 3.7.2 to construct the “active ingredient-target” network of WK.We searched the compounds of WK in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  and DisGeNET (https://www.disgenet.org/) were searched. All targets are standardized according to Uniprot (https://www.uniprot.org/).With the keyword “gastric cancer”, gastric cancer-related targets in GeneCards  to achieve the relationships between proteins and visualize them via Cytoscape 3.7.2.Cross targets of WK and gastric cancer were obtained and were imported into String (https://metascape.org/) was conducted to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. GO analysis includes biological process (BP), cellular component (CC), and molecular function (MF) analysis.Metascape (https://www.rcsb.org/) and mol2 formats of the active ingredients of the compound were downloaded from TCMSP. Autodock and Autodock Vina were used to calculate the docking scores between target proteins and molecules after they were dehydrated and hydrogenated. The interaction between proteins and bioactive compound should be stronger if its docking score is higher.PDB was downloaded from RCSB Protein Data Bank , anti-JNK1 + JNK2 + JNK3 antibody (ab179461), and other reagents were purchased from ABCam (United Kingdom), and fetal bovine serum was provided by GIBCO (United States).Anti-p38 antibody (ab31828), anti-ERK1 + ERK2 antibody (ab54230), anti-PERK antibody (ab65142), Panax ginseng C. A. Mey. (10 g), Atractylodes macrocephala Koidz. (10 g), Arum ternatum Thunb. (10 g), Curcumae rhizoma (10 g), Actinidia chinensis Planch (15 g), and Rhodiola rosea L. (10 g) was provided by the traditional Chinese medicine (TCM) pharmacy of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. The Chinese medicine was authenticated to be in compliance with the 2005 edition of “Chinese Pharmacopoeia”. WK crude drugs were crushed and soaked in the tenfold double distilled water for 24 h. Then, the mixture was heated, filtered, and refluxed for 2 cycles to get concentrated solution. The concentrate was evaporated at 60°C for the WK extract and the extract was stored at −20°C.WK consisting of 2.The mouse gastric cancer cell line MFC and human gastric cancer cell line BGC-823 are provided by Nanjing Enjing Biotechnology Co., Ltd. MFC and BGC-823 cells were cultured in RPMI-1640 with 10% fetal bovine serum and 1% penicillin-streptomycin, under a humidified atmosphere at 37°C with 5% COICR mice are provided by Nanjing Enjing Biotechnology Co., Ltd. For the experiment, mice were maintained in the SPF laboratory animal room of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. The experiment was approved by Ethics Committee of Jiangsu Province Hospital on Integration of Chinese and Western Medicine  and complied with the regulations of Experimental Animal Ethics Committee.5 cells/mL and placed in a 37°C, 5% CO2 incubator for 24 hours. Then, the gastric cancer cells were treated by 100 μL WK of different concentrations (0.39～200 mg/mL), and distilled water of equal dose was added as the control group. Each group had 5 replicate wells and was placed in a 37°C, 5% CO2 incubator for 72 hours. Then, the 10 μL CCK-8 reagent was added to each well, incubation was continued for 4 hours in the incubator, the OD value was measured at 490 nm with a microplate reader, and GraphPad Prism v8.0.2.263 was used to calculate the cell inhibitory rate and IC50 value.EnoGeneCell™ Counting Kit-8 (CCK-8) was used to test cell viability. Cells were inoculated into a 96-well plate at a concentration of 1 × 10Stable eGFP-LC3 MFC cells were selected to establish a blank control group, WK groups , and positive control group (50 nM Rapamycin). The distribution of green fluorescence dots was observed under fluorescence microscopy after incubation in the thermotank for 48 h. Cells were lysed by Trizol and RT-PCR was conducted to detect mRNA expression of autophagy marker protein LC3-II in MFC cells.With the same dose of distilled water as the control group, WK extracts of different concentrations  were incubated with gastric cancer cells for 48 hours. The cell lysis occurred on ice and related protein was obtained. Western blot was used to determine the expression of ERK, JNK, and p38 proteins in the MAPK signaling pathway of MFC and BGC-823 cells.A mouse gastric cancer cell line MFC was grafted into 40 ICR mice. The mice were randomly divided into 5 groups: the blank control group (control) where the model mice were injected with normal saline through the tail vein; the WK extracts (WK) group in which WK of different concentrations were given by gavage: 9.75 g/kg (low dose), 19.5 g/kg (medium-dose), and 39 g/kg (high-dose); and the positive control group (cisplatin) where the mice underwent an intraperitoneal injection of cisplatin 5 mg/kg.3) = πab2/6, and a graph of tumor growth was drawn.The longest diameter (a) and shortest meridian (b) of axillary tumors were measured with digital calipers on days 1, 3, 5, 7, 9, and 11. The volume of transplanted tumor was calculated according to the formula: V (mmThe mice were euthanized after a 10-day administration. The transplanted tumors were stripped and weighed. The tumor inhibition rate (TIR) was calculated according to the following formula: TIR (%) = (C − T/C) × 100%, where C means the average tumor weight of mice in model group and T represents the mean tumor weight of mice in the administration group.The tumor tissue was quickly stripped from euthanized mice and was stored at −80°C for later use. The tumor tissue was lysed to obtain protein samples. Western blot was used to detect the expression of ERK, JNK, p-p38, and p38 proteins in the MAPK signaling pathway.P < 0.05 was considered statistically significant.All data are processed by SPSS 19.0. The measurement data were compared with single factor analysis of variance. The homogeneity of variance was tested by the LSD method, and the heterogeneity of variance was tested by the DunnettsT3 method. After preliminary screening, we found a total of 52 active ingredients in TCMSP. In addition, we supplemented 8 ingredients from the Chemistry Database [DB/OL] and checked relevant papers , 13 for 2978 and 298 targets for gastric cancer were retrieved from GeneCards and DisGeNET, respectively. After screening to remove duplicates, 1646 disease targets were finally obtained. Finally, we got 106 cross targets between WK and gastric cancer.The cross targets were imported into string for prediction, medium confidence (0.0400) was chosen, the disconnected nodes were removed, and the results were imported into Cytoscape 3.7.2 for analysis. A PPI network was built finally . The larThe cross targets were imported into Metascape to make GO and KEGG enrichment analyses. The top 10 results of BP, CC, and MF in GO were selected, ranking by Log10 (P) . The resTo explicit the relationship between the MAPK signaling pathway and WK, structures of MAPK14 (PDB : 2GFS) and the top 5 active ingredients  were selected. The docking score ranked from −8.7 to −5.9, which shows a good binding activity  for 48 h, Western blot was used to determine the expression of ERK, JNK, p-p38, and p38 proteins in the MAPK signaling pathway of MFC cells. It was found that compared with the blank control group, the expressions of p-JNK, p-p38, and p-ERR1/2 protein were significantly reduced, in a dose-dependent manner. While, WK had no significant effects on the expression of the p38 protein .50 for 24 h is 59.85 mg/mL, IC50 for 48 h is 26.62 mg/mL, and IC50 for 72 h is 8.64 mg/mL. It can be seen that WK inhibits the proliferation of BGC-823 cells in a dose-dependent and time-dependent manner.After BGC-823 cells were treated with 0.39～200 mg/mL WK extracts for 24 h, 48 h, and 72 h, the cell viability was determined by CCK-8. The results of the experiment are shown in After the WK extracts  were incubated with BGC-823 cells for 48 h, we used Western blot to determine the expression of p-JNK, p-p38, p38, p-ERK1/2, and GAPDH in BGC-823 cells. It was found that compared with the blank control group, the expressions of p-JNK, p-p38, and p-ERR1/2 proteins in the MAPK signaling pathway were significantly reduced, in a dose-dependent manner. While, WK had no significant effects on the expression of the p38 protein .Under the fluorescence microscope, the autophagosome green punctate fluorescent particles were evenly scattered in the cytoplasm of the control group. While, the positive staining around the nucleus and the aggregation of fluorescent particles could be observed in the cytoplasm of WK groups, which is similar to the positive control group (Rapamycin). According to PCR, the expression of LC3-II mRNA increased significantly in WK groups compared to the control group, which is positively correlated to the concentration of WK. The abovementioned results suggest that WK can induce autophagy of MFC cells in a dose-dependent manner .As is shown in As is shown in The incidence of gastric cancer has been rising in recent years. However, conventional chemotherapy and radiotherapy limited in advanced gastric cancer. Although new drugs such as targeted therapy and immune checkpoint inhibitors show certain efficacy in advanced gastric cancer, the five-year survival rate remains low –16. BesiP value), we tend to choose the MAPK signaling pathway as the key pathway in cancer treatment of WK [As an emerging discipline, network pharmacology integrates system biology, bioinformatics, and high-throughput histology. On the basis of drug and disease targets, network pharmacology predicts multiple mechanisms of drugs through a series of algorithms, providing a preliminary basis for drug research. TCM and its compound prescriptions have complex components and targets, and they often participate in the treatment of diseases through various pathways . Accordint of WK . To confAs an important bridge from extracellular signals to intracellular responses, the MAPK pathway is considered as a key pathway related to tumor cell growth. Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) participate in the construction of the MAPK signaling pathway. When stimulated by hormones, the growth factors (GFs) of the MAPK signaling pathway bind to transmembrane glycoproteins of the receptor tyrosine kinase (RTK) family and activate its signal transduction cascade. It promotes the development of RAS/RAF/MEK and ERK1/2 pathway. The dual phosphorylation activation of MAPK can phosphorylate some transcription factors, initiate the transcription and translation of response genes, regulate protein kinase activity, and participate in various physiological processes such as cell growth, differentiation, apoptosis, and death . FurtherAlthough several key targets predicted by network pharmacology tend to work in both PI3K-Akt and MAPK signaling pathways, the module analysis based on MCODE about GO-BP indicated that the biological process involved more kinases connected with the MAPK pathway. The result shows more preference for the MAPK signaling pathway. Besides, many studies have proved that the herbs consisting WK could activate the PI3K-Akt signaling pathway , 24. As However, in this study, the data in vitro experiment show that the antitumor effect of the WK high-dose group is close to that of platinum. According to clinical experience, it is almost impossible that TCM has an equivalent effect with platinum-based chemotherapy. We use a mouse gastric cancer cell line MFC to make tumor transplantation in ICR mice, which may differ from a human gastric cancer cell line. Moreover, ICR mice have a high heterozygous rate, which may cause individual differences. Therefore, the result of animal experiment cannot be extrapolated to human beings directly. Moreover, the concentration of the high-dose group is higher than routine dosage in clinic; this may also cause the strong antitumor effect. WK is a prescription summarized from clinical experience, which is usually used in combination with chemotherapy to enhance the antitumor effect of chemotherapy, and its antineoplastic effects have also been observed in clinic. In vivo and vitro experiments, we also proved that WK has a good inhibitory effect on gastric cancer cells. The tumor inhibition rate (TIR) of clinical routine dosage (medium-dose group) in tumor bearing mice (close to 50%), even is lower than the high-dose group, is also obvious enough to prove the antitumor potential of WK.This study also has some limitations. We only explored one pathway that we think is more likely to work. As we did not verify other possible pathways, we cannot verify whether WK can participate in other pathways to make a synergistic antitumor effect. In addition, we only proved that autophagy existed when gastric cancer cells are treated with WK. Whether there is a connection with the MAPK signaling pathway is still underway to be verified in further experiments.In this study, we explored the potential mechanism of WK in gastric cancer with network pharmacology and predicted possible pathways with a higher probability. Then, we verified that WK displayed an antitumor effect in gastric cancer by participating in the MAPK signaling pathway, and the antitumor effect has a dose dependency, laying a certain theoretical foundation for further exploration to expand the application of WK.


It is becoming increasingly evident that the accurate assessment of fluidstatus is critical to ensure optimal care in patients undergoinghemodialysis (HD). Various fluid parameters, includingoverhydration (OH) and overhydration/extracellular water(OH/ECW%), which can be obtained using abioimpedance spectroscopy device have been used to indicate the hydrationstatus in such patients. This study aimed to explore the effect of thesefluid parameters on cardiovascular events and determine which parameter wasa better predictor of cardiovascular events (CVEs).A total of 227 patients who underwent HD at the Hangzhou Hospital ofTraditional Chinese Medicine were enrolled in this prospective study betweenDecember 2017 and August 2018. Clinical data were collected, and the fluidstatus of patients was assessed using a body composition monitor. Thepatients were followed up until December 2020. The primary outcomes wereCVEs. The association between fluid parameters and CVEs was analyzed usingCox proportional hazards models. The areas under the curve(AUCs) of receiver operating characteristic analysis andimprovement in the global chi-squared value were used to compare thepredictive values of fluid parameters for CVEs.p = 0.016] and OH/ECW% wereindependently associated with CVEs. The predictive ability of the absolutehydration index was superior to the relative hydration index based on AUCcalculations for CVEs. Furthermore, a greater change in2χ in predicting CVEs was notedfor the absolute hydration index.During a median follow-up of 31 months, 66 CVEs were recorded. Thepatients with a higher absolute hydration index (OH) and arelative hydration index (OH/ECW%) exhibited anincreased risk of developing CVEs. After adjusting for confounding factors,both OH [hazard ratio (HR) 1.279 per L, 95%confidence interval (CI) 1.047–1.562;Both absolute hydration index and relative hydration index were found to beindependent predictors of CVEs in univariate and multivariate analyses.Furthermore, the absolute hydration index had a better additive predictivevalue than the relative hydration index in predicting CVEs. Fluid overload is highly prevalent among patients undergoing hemodialysis(HD) and is strongly associated with poor outcomes, includingsystemic hypertension, pulmonary hypertension, left ventricular hypertrophy, andmortality . BecauseAt most hemodialysis centers, the volume status is usually based on clinicalcriteria, including the patient’s signs and symptoms, blood pressuremeasurements, and intradialytic hemodynamic instability . AlthougTo the best of our knowledge, this study is the first to analyze and compare thepredictive value of fluid parameters, measured using a portable whole-bodybioimpedance spectroscopy device, for CVEs in a representative Asian cohort ofpatients undergoing HD.This prospective study included patients from the HD Center of HangzhouTraditional Chinese Medicine Hospital, People’s Republic of China,between December 2017 and August 2018. Patient inclusion criteria were asfollows: aged between 18 and 80 years old; undergoing regular HD(three times a week in 4–4.5 h sessions).Patient exclusion criteria were as follows: those with clinical CVE during the3-month period before enrolment; those with metallic joint prostheses, implanteddefibrillators, or cardiac pacemakers; pregnant women, breastfeeding patients,or amputees; those with malignant tumors, severe liver disease, acuteinfections, or severe malnutrition. Finally, a total of 227 patients undergoingHD were recruited in the present study and followed up until December 2020. Thestudy protocol was approved by the ethics committee of the Guangxing Hospitalaffiliated with the Zhejiang University of Traditional Chinese Medicine(No. 2018SQ119), and all the participants signed an informedconsent form.Demographic data, clinical history, etiology of renal failure, comorbidities, anddialysis data were collected from the patient's electronic medicalrecords. We defined residual renal function as a 24-h urine output>200 mL .LaboratThe fluid status was assessed at baseline using a whole-body bioimpedancespectroscopy device (body composition monitor (BCM),Fresenius Medical Care, Bad Homburg, Germany) before dialysis. Thistechnique involves attaching electrodes to the patient’s non-fistulaforearm and ipsilateral ankle, with the patient in the supine position. Allmeasurements were conducted by nursing staff trained in themanufacturer’s protocol. This device accurately measures bodycomposition by analyzing electrical responses at frequencies between 5 and1000 kHz. At low frequencies, the current cannot penetrate the cellmembrane and instead passes through the ECW space, whereas at high frequencies,the current can flow through both ECW and intracellular water . Based oAll patients were regularly followed up until 31 December 2020. In our study,the patients who followed up were treated with maintenance hemodialysis andcame to the hospital for hemodialysis three times every week. Ourinvestigation was therefore performed during this time. Outcome events wereroutinely registered in the system by experienced nephrologists. Theendpoint events were CVEs, which included cardiac death, acute coronarysyndrome, cerebrovascular accident, hospitalization for congestive heartfailure, and acute peripheral artery occlusion . In patiData were expressed as mean ± standard deviations fornormally distributed data and as medians (interquartilerange) for variables that did not follow a normal distribution andfrequencies for categorical variables.The Cox proportional hazards regression model was applied to performunivariate and multivariate analysis, which was presented as hazard ratios(HRs) and 95% confidence intervals(95% CIs). For multivariate Cox regression analysis,the enter method was applied. Baseline variables, include age, gender, bodymass index (BMI), smoking status, presence of diabetesmellitus, previous CVEs, hemoglobin, serum albumin, highly sensitiveC-reactive protein, serum calcium, serum phosphate, Kt/V, and totalcholesterol, which could interfere with the association between fluidparameters and the endpoint were entered into multivariate Cox proportionalhazards regression models. Given the number of events available, variablesfor inclusion were carefully chosen to ensure parsimony of the final models.Receiver operating characteristic (ROC) curves were plottedand areas under the ROC curves (AUCs) were calculated todetermine the discrimination threshold of each hydration value. Pairwise AUCcomparisons were performed between fluid parameters using the DeLong test.The cut-off values appropriate for the optimal combination of sensitivityand specificity were determined using the Youden index. The incrementalvalue of absolute hydration index and relative hydration index over thebasic model was assessed to predict CVEs by assessing improvement in theglobal chi-squared value.Kaplan–Meier curves were generated, showing cumulative probabilitiesof new CVEs, and differences were compared using the log-rank test.These analyses were conducted using SPSS software, version 22.0  and MedCalc Statistical Software version11.4.2.0 . All statisticaltests were performed at a two-sided 0.05 level of significance.n = 131;57.7%), followed by diabetic nephropathy. At baseline, the mean absolute hydration index(OH) was 2.1 ± 1.4 L and themean relative hydration index (OH/ECW%) was13.6 ± 7.4%. Additional patient baselinecharacteristics of the cohort are shown in n = 26), acutecoronary syndrome(n = 19),cerebrovascular accident(n = 18), cardiac death(n = 2), and acuteperipheral artery occlusion(n = 1).In total, 66 CVEs and 23 all-cause deaths were recorded during a median follow-upof 31 months. For these 66 CVEs, the breakdown of the first eventsduring follow-up were as follows: heart failure. According tothe cut-off values of OH (2.5 L) and OH/ECW(13%), the sensitivity and specificity of OH forpredicting CVEs in patients undergoing HD were 60.6 and 79.5%,respectively, and those of OH/ECW were 75.8 and 59.6%, respectively. We then addedOH > 2.5 and OH/ECW > 13% into the basic model, and bothbasic model + OH >2.5 and basicmodel + OH/ECW >13% were more beneficialfor the prediction of CVEs compared with the basic model. Adirect comparison between the basic model + OH>2.5 and the basic model + OH/ECW>13% showed that the former had a better predictive value forCVEs .The incremental values of fluid parameters used for predicting CVEs are shown invs. OH≤2.5 L (log-rankp < 0.001). (B)OH/ECW >13% vs. OH ≤13%(log-rank p < 0.001). Atotal of 109 (48%) patients had OH/ECW>13%, 64 (28%) patients had OH>2.5 L, and among the patients with OH >2.5 L,the OH/ECW ratio was all >13%. Patients with OH>2.5 L had a significantly higher risk of heart failure andacute coronary syndrome. Similar results were observed for OH/ECW>13% ;p < 0.001, respectively]. Afteradjusting for various confounders, the multivariate Cox analyses (p = 0.016), and the adjusted HRof CVEs with each 1% increase in the OH/ECW ratio was 1.061.ECW>13% . In the ECW>13% , both thanalyses  revealedThe present study evaluated the role of BCM fluid parameters in predicting theoccurrence of CVEs in Chinese patients undergoing HD. Several major findings werenoted in the present study. First, a high absolute hydration index and relativehydration index showed a significant correlation with an increased incidence ofCVEs, which remained significant even after multivariable adjustment. Second, theoptimal cut-off values of both absolute and relative hydration indices forpredicting CVEs were 2.5 and 13%, respectively. Third, a direct comparisonof three multivariate models revealed that the basicmodel + absolute hydration index > 2.5 had thehighest predictive value for CVEs. To the best of our knowledge, this is the firststudy that analyzed and compared the predictive values of fluid parameters in ahemodialysis cohort with CVE end points.BCM is a commercially available device that can provide information important interms of fluid control . The indvs. 60.6%) but lower specificity (59.6vs. 79.5%) for CVEs than those exhibited by theOH cut-off value. Therefore, the relative hydration index could be used as a betterscreening tool in terms of identifying high-risk patients. By contrast, the absolutehydration index expressed in liters could provide an estimate of how much thepatient is overhydrated and have higher specificity in the detection of CVEs.Overall, the OH values displayed greater accuracy levels in the identification ofCVEs than those displayed by OH/ECW. In our study, a direct comparison usingmultivariate models showed that the basic model + OH>2.5 L was superior to the basicmodel + OH/ECW >13% in predicting CVEs.Similar results were also obtained in the ROC comparison. Our study supports the useof the absolute hydration index to predict CVEs. This parameter is particularlyuseful because it can provide an estimate of how much the patient is overhydratedand allow the clinician to set a new target weight for the patient to achieve [In addition, we constructed ROC curves to determine the optimal cut-off value forfluid parameters to predict CVEs. Our data showed that the cut-off values for OH andOH/ECW ratio in detecting CVEs were 2.5 L and 13%, respectively. Inlight of most literature, the 15% cut-off for OH/ECW ratio and 2.5 Lfor OH value was often defined as the upper limit of normohydration status, beyondwhich have been shown to predict poor prognosis . Due to  achieve . More im achieve . By cont achieve , they al achieve . ItseemOur study had several limitations. First, the fluid status was defined using a singlebaseline measurement, and the relationship between the fluid status over time andoutcomes could therefore not be analyzed. Second, additional confounding variablesmay have existed, which were not included in the multivariate-adjusted analysis.Third, our study had a relatively small sample size and was performed at a singlecenter. Because of the small number of deaths, the influence of fluid parameters onmortality rates could not be further investigated.In conclusion, our study revealed that both the absolute hydration index and therelative hydration index were independent predictors for CVEs in univariate andmultivariate analyses. Besides, the absolute hydration index, which can be obtainednon-invasively and economically, and without requiring additional calculations,showed a better predictive value than the relative hydration index in predictingCVEs. Our findings provide further evidence for the selection of the fluid index inpredicting CVEs in patients undergoing HD. Future large, prospective trials arewarranted to establish the benefits of BIA-guided fluid management in patientsundergoing HD.This study was reviewed and approved by the ethics committee of the GuangxingHospital affiliated with the Zhejiang University of Traditional Chinese Medicine(No. 2018SQ119).Linghong Cheng analyzed the findings and drafted the manuscript. Liyang Chang andRongrong Tian analyzed the data and revised the manuscript. Jianfang Zhou and FenxiaLuo revised the manuscript. Hongmei Zhang designed the study, revised themanuscript, and interpreted the results.Click here for additional data file.


Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species , and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy.The mushroom genus IMPORTANCE The therapeutic use of psilocybin, the active ingredient in “magic mushrooms,” is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species  is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis. Psilocybe (Fr.) P. Kumm. have become a major scientific and public curiosity since 1957 when R. Gordon Wasson published a popular account of his first-hand experience with psychedelic mushrooms in Life magazine  have been known to the scientific community since the early 19th century , meaning “God’s flesh,” the sacred mushrooms of Mesoamerica. While being first recorded by Europeans and Western scientists in the Nahuatl population, ritual usage of psychoactive mushrooms has occurred in indigenous Mexican cultures including the Chatins, Chinantecs, Matlazincs, Mazatecs, Mixes, Purepechs, Totonacs, and Zapotecs, with names for these mushrooms specific to each culture  as part of the “war on drugs” promulgated by Richard Nixon in 1971  P. Karst., and none of them are known to synthesize psilocybin or psilocin   –35. Thistion 1) –, 36, 37 tion 1) –, 39. On Psilocybe specimens may contain controlled substances, no evidence exists on their consistency, and psilocybin has been empirically identified in only ~45 accepted Psilocybe spp., which are only 32% of the ~140 putative bluing species  Singer and Psilocybe mexicana R. Heim that may provide monoamine oxidase-inhibiting roles and contribute synergistically to the activity of psilocybin   and non-Psilocybe species under older names, as well as reconciling synonyms, the final database consisted of 3,707 records of physical vouchers across 72 institutions found on MycoPortal. Many institutions either could not or refused to provide loan material due to the legal ambiguity of Psilocybe specimens. Despite the large number of vouchers present across these institutions, approximately one-third were identified only to genus level . Most of the specimens identified to species level were dominated by a few well-known taxa, i.e., P. cubensis , Psilocybe cyanescens Wakef. , and Psilocybe semilanceata (Fr.) P. Kumm. . Of the ~3,700 specimens requested, ~1,200 were received from 35 institutions.We curated a database of al data) . After fA total of 18 species names across 94 specimens were used for DNA barcoding. Across all samples, DNA extractions yielded 5.5 to 166.1 ng/μL with 260:230 values between 0.22 and 6.52 and with 260:280 values ranging from 1.29 to 2.04 . Our best extraction results usually came from larger material inputs between 10 and 15 mg.Psilocybe spp. from the National Center for Biotechnology Information (NCBI), we found a large number of likely incorrect or inconsistent names and sequences that of were very poor quality or that are highly divergent from mushroom-forming fungi. Four hundred twenty-three internal transcribed spacer (ITS) sequences in NCBI were labeled as Psilocybe or Deconica and other labels (when Psilocybe was included as a name in the sequence record), including one sequence labeled Stropharia rugosoannulata Farl. ex Murrill and one sequence labeled “Uncultured fungus.” Thirty-two of 39 sequences (82%) labeled as Deconica had specific epithets (9 species names), and six did not. Three hundred sixteen of 382 sequences (83%) labeled as Psilocybe had specific epithets (69 species names), and 66 did not. The most abundant Psilocybe species labels were P. cubensis , P. cyanescens , and P. semilanceata . Fifty-seven sequences labeled as Psilocybe were attributable to Deconica .In our analysis of all publicly available sequences identified as MN510675 to MN510688 and MG969992) had many ambiguous bases and potential errors that introduced excessive numbers of autapomorphies, leading to exceedingly long branches in the phylogenetic tree. Two sequences labeled as Psilocybe cubensis (ON415277 and ON415278) are instead Fusarium spp. (pathogenic fungi distantly related to mushrooms) based on BLAST results. One sequence (DQ900972) from an environmental barcoding study  Kumm. (which should be updated to the current name Deconica montana (Pers.) P. D. Orton; AY129360) clustered with P. cubensis. Sequences labeled as P. cubensis were recovered in six clades. Some of these were other species of Psilocybe  whereas others belong to Deconica (AY129351) or Galerina (AY281023). A specimen from the Universidad de Guadalajara (IBUG) identified as Psilocybe cubensis that was newly sequenced clustered with sequences labeled as Stropharia rugosoannulata. Similar to sequences labeled as P. cubensis, sequences labeled as P. cyanescens were recovered in three clades, two of which correspond to Panaeolus (EU029946) and Deconica (MF467896). Fifty-five sequences labeled as Psilocybe  clustered with sequences labeled as Deconica.A number of erroneous sequences were encountered in the NCBI database that will require either removal due to poor sequence quality or artifacts, or updates to the sequence identity. Some of these , together sharing a most recent common ancestor (MRCA) with P. chuxiongensis (100% BS) . However, the sequence labeled as P. natalensis in GenBank (OK491080) is not derived from the type and is of uncertain provenance. The GenBank record refers to an observation on an online forum (https://mushroomobserver.org/472561) and lists the sequence isolation source as “Cultivated from a spore print” and the country of origin as “South Africa,” but no voucher specimen is indicated, and it is unclear if the data are reproducible or the source can be reexamined. Overall, the inclusion of NCBI sequences resulted in a large amount of noise in our analysis, which intensified confusion and contributed to a severe lack of clarity in sequence-based identification of Psilocybe spp.Some phylogenetic relationships between Psilocybe-only analysis, we performed a second phylogenetic analysis utilizing species hypothesis (SH) sequences from the family Strophariaceae and the closely related genera Gymnopilus P. Kumm. and Agrocybe Fayod from UNITE (Psilocybe coprophila (Bull.) P. Kumm. and Psilocybe subviscida (Peck) Kauffman, both of which belong to the nonpsychoactive genus Deconica.Due to the large number of misidentifications and the noise in our om UNITE . Of the om UNITE . Like thPsilocybe sensu stricto to be a monophyletic group (98% BS) sharing a most recent common ancestor with species of the genus Agrocybe  Quél., and Tubaria (W.G. Sm.) Gillet. Additionally, multiple groupings within Psilocybe had incongruent taxonomic names, which suggests either misidentification or possible synonyms  P. Kumm., Pleuroflammula Singer, Psilocybe, Mythicomyces Redhead & A.H. Sm., Strophariaceae, and Tubaria]. Additionally, the branch belonging to Deconica included two SH sequences attributed to Psilocybe, six of our vouchered specimens, and eight SH sequences labeled Strophariaceae species SH sequences  that also included two of our vouchered specimens labeled as Psilocybe and SH sequences labeled as Pholiota spp.Although taxonomic inconsistencies occurred in every clade, some clades contained more divergent taxonomic names than others. Groupings within the sister clade to equences . AgrocybP. cubensis, P. cyanescens, and P. semilanceata) for chemical analysis and taxonomically verified each using full-length ITS barcoding that corresponded to the SH for their respective voucher identification. A single specimen of P. cubensis was misidentified , and one specimen of P. cyanescens was misidentified   . Very fe storage .P. cubensis voucher specimens from 0.55 to 1.9 μg/mg (dry weight), with only a single sample having detectable amounts of psilocin at 0.44 μg/mg (dry weight). Psilocybe cyanescens specimens contained relatively more and consistently higher alkaloid concentrations, with psilocybin content ranging from 3.0 to 15.6 μg/mg (dry weight) and psilocin content ranging from 0.22 to 5.22 μg/mg (dry weight). Psilocybe semilanceata specimens contained psilocybin content ranging from 0.33 to 15.77 μg/mg (dry weight) and psilocin content ranging from 0.1 to 3.88 μg/mg (dry weight)  of P. semilanceata had barely detectable levels of the indole alkaloids.Concentrations of psilocybin and psilocin, when detectable at all, varied in  weight) . Specimet test, we found no significant difference in psilocybin or psilocin between whole individual sporocarps (Psi 5 and Psi 6) from the same reproductive event in a single growth bin (psilocybin percentage point [pp] = 0.1349 and psilocin pp = 0.4756). Unpaired t tests also showed no significant difference in concentrations between the same anatomical tissues (caps and stipes) or between the two sporocarps Psi 5 and Psi 6 (psilocybin pp = 0.5081 and psilocin pp = 0.4563) or measured across Psi 4, Psi 5, and Psi 6 (psilocybin pp = 0.8182 and psilocin pp = 0.6898). However, at the full sporocarp level, quantities of psilocybin and psilocin were statistically different between growth bins (psilocybin pp = 0.0560 and psilocin pp = 0.0389).Psilocybin was the major alkaloid recovered from mycelium (6.44 μg/mg), caps (10.5 to 20 μg/mg), and stipes (15.44 to 18.44 μg/mg) . Psilocim/z) ratio and retention time. Using hierarchical clustering to compare the intensity profiles of each of our P. cubensis samples across the two growing environments, we found that metabolic profiles of caps and stipes were most like one another, with the mycelium being most different from all other samples, regardless of which container they were cultivated in ; , which are likely to be found in high quantities because these profiles were derived from freshly preserved specimens. However, three annotated spectral features were of particular interest as they had structural similarity to serotonin (MQscore = 0.9292), cocamidopropyl betaine (CAPB) (MQscore = 0.9191), and trazadone hydrochloride (MQscore = 0.73).In addition to looking at broad-scale patterns of metabolomic profiles, we also wished to investigate if any of the identified spectral features could be annotated to known compounds. We collected tandem mass spectrometry (MS/MS) data for sample Psi-6-Cap for comparison to the Global Natural Products Social Molecular Networking database ; . Using tPsilocybe spp. are regularly misidentified, little has been done to investigate the stability of the known psychoactive compounds under standard storage conditions, and the chemical complexity of these organisms has been overlooked by an almost exclusive focus on the principal psychoactive agents. These deficiencies cast a shadow on what little information is available on Psilocybe and underscore the increasingly urgent need for comprehensive basic research on them.Our study shows that fungarium specimens of Psilocybe and other closely related groups further exacerbates the problem. Without DNA barcodes from type or other authentic material, names can be applied inconsistently, leading to different applications of species names to modern collections. This also makes it difficult to know when a specimen represents a new species, resulting in further misapplication of named species to novel taxa. We found that close to 13% of the specimens we studied that were cataloged as Psilocybe belonged to other genera, none of which are known to contain controlled substances. This result challenges the use of names in collections for meta-analysis or evidence-based decisions on their custody and care. Many publicly available sequences are incorrectly labeled or are of very poor quality, further aggravating the problem for identifying specimens or other samples using a DNA barcoding approach. Even some of the most recognizable and frequently collected species (P. cubensis and P. cyanescens) had sequences that belonged to other related genera and in two cases to fungi from a different phylum (Ascomycota). Some of these are likely misidentification of the source material, including cultures, highlighting the need for an authoritative, expert-curated reference database of DNA sequences derived from types or other authentic specimens.Fungi often exhibit cryptic morphology and can be difficult to identify, even for highly trained experts. Cryptic traits, lack of extensive training, and even clerical errors in databasing can all contribute to misidentification , 51. In Psilocybe specimens makes misidentifications detrimental not only to scientific research but also to properly applying regulatory policies. Misidentification of putative psilocybin-producing mushroom specimens may lead to an unnecessary effort and poor use of resources, or even miscarriage of justice in legal cases.While misidentifications in museum collections and public DNA repositories are not uncommon , 39, thePsilocybe suggests that 165 belong to Psilocybe sensu stricto while 98 belong to Deconica or other genera . Fifty-two species that likely belong to Deconica are currently still classified in Psilocybe. However, these likely include synonyms and cryptic species that are presently unknown and do not account for undocumented species that likely exist. Only five species of Psilocybe sensu stricto are represented by ITS barcodes derived from types. Critically, sequences from types of the best-known species, such as Psilocybe cubensis, do not exist. This fundamental information is critical for accurate application of names and should be considered an urgent priority to aid in the identification of this medically and societally important group.A review of the currently known species of Psilocybe, new phylogenetic patterns were uncovered. While Ramírez-Cruz et al. (P. cubensis shared a most recent common ancestor (MRCA) with the southeast Asian species Psilocybe thaiaerugineomaculans Guzmán, Karun. & Ram.-Guill., our inclusion of the South African P. natalensis and the more recently described Asian species P. chuxiongensis revealed that P. cubensis likely shares an MRCA with P. natalensis and not the Asian species. This is consistent with the African-origin hypothesis for P. cubensis and/or its progenitor proposed by Guzmán et al. , and so while the geographic implications remain, the taxonomic ones are uncertain. To gain a better understanding of the geographic origins of Psilocybe spp., especially the enigmatic P. cubensis, new fieldwork to expand documentation of Psilocybe in Africa and Asia should be a priority.While our data set had limited representation of the known species of z et al.  reportedn et al.  and Froen et al.  rather tn et al.  and Ma en et al. . HoweverPsilocybe and methods for measuring its metabolites render most previous studies unreliable and unreproducible. In our review of the published literature on alkaloid chemistry in Psilocybe, there was an almost complete lack of thorough sample identification, few studies designated vouchers, and literature citations frequently contained incorrect information or opposing statements . In a few instances, reports for some species, such as Psilocybe ovoideocystidiata Guzmán & Gaines and Psilocybe kumaenorum R. Heim, lacked peer review or were not accessible. Although our focus was specifically on literature pertaining to Psilocybe, psilocybin has been reported from other species such as Agrocybe praecox (Pers.) Fayod (Psilocybe subcubensis Guzmán (Decades of inconsistency in the identification of .) Fayod , 56, alts Guzmán . UnfortuPsilocybe to determine their presence and stability over time. One recent study showed that the method of drying and extent of light exposure can affect psilocybin/psilocin stability in P. cubensis (P. cubensis (WTU-F-054869) deposited around the same time as specimens used for previous chemical analysis (P. cubensis contained over 5.0 μg/mg (dry weight) of psilocybin. However, when we measured the WTU-F-054869 voucher, now 42 years old, we detected less than half of their reported psilocybin content (1.9 μg/mg) and no psilocin  specimen having 6.11 μg/mg psilocybin and 3.66 μg/mg psilocin. Similarly, in P. semilanceata the youngest sample contained 11.2 μg/mg of psilocybin and 3.88 μg/mg psilocin, while the oldest contained 0.2 μg/mg psilocybin and no detectable psilocin. One possible explanation for the generally better chemical preservation in P. cyanescens and P. semilanceata is that these species have sporocarps that are comparatively lower in biomass than those of P. cubensis. This may result in a more rapid desiccation during heat-assisted drying, minimizing the opportunity for spontaneous and/or enzymatic degradation of psilocybin and psilocin. Another possible explanation may be from differences in the expression or catalytic activity of the enzymes involved in conversion of psilocybin and psilocin  from multiple sporocarps within the same flush (Psi 5 and Psi 6) and from sporocarps between two growth containers (Psi 5/Psi 6 versus Psi 4). Mycelium showed one-third the content of psilocybin and five to 20 times less psilocin than did the stipe and cap, respectively  Lloyd, have been shown to produce different profiles based on the presence of other fungi  can have as many as 9,000 spectral features that differ depending on the life cycle stage in which they are investigated  Quél., some species of which also produce psilocybin , a chemical component of coconut . CAPB is72–e toxins , 76. Thie toxins .Psilocybe due to accumulating evidence of the therapeutic potential of psilocybin, the current state of knowledge of Psilocybe and the psychoactive compounds it produces is still in its infancy. Our study shows that much more research is needed to achieve a more thorough, accurate, and reliable understanding of this enigmatic and socially important group to develop meaningful policies around its use and care, and improve its application for human wellbeing.Despite a surge in interest in (i) Fungarium specimens. We created a curated database of voucher information for all collections identified as the genus Psilocybe using MycoPortal  DNA extraction and barcode sequencing. Total DNA was extracted from the hymenophore in an amount ranging from 5 to 15 mg. These fragments were ground to a fine powder by placing them in 2.0-mL screw-cap tubes containing a single 3.0-mm bead and 8- by 1.5-mm stainless steel beads and shaking them in a BeadBug microtube homogenizer  for 120 s at speed setting 350. The ground sample was used as input to the Monarch genomic DNA purification kit  following the manufacturer’s instructions except using twice the volume of lysis buffer and increasing the amount of wash buffer to 550 μL during both of the washing steps. These samples were amplified using the PCR primers ITS-8F (5′-AGTCGTAACAAGGTTTCCGTAGGTG-3′) and ITS-6R (5′-TTCCCGCTTCACTCGCAGT-3′), which were specifically designed for Agaricomycetes  and trimming the 5′ and 3′ ends to conserved motifs 5′-CATTA- and -GACCT-3′ following the method in reference Complementary sequencing chromatograms were then processed by creating a consensus sequence using Sequencher 5.4 ((iii) Phylogenetic analysis. To validate the identities of the voucher specimens, we subjected the ITS sequences to phylogenetic analysis using two data sets. The first consisted of all currently available ITS sequences from the National Center for Biotechnology Information (NCBI) identified as Psilocybe spp. using the query ‘Psilocybe [ORGN] AND “internal transcribed spacer”’ (n = 384). The second included all sequences belonging to the Species Hypothesis (SH) (generated at the 1.5% similarity threshold) of family Strophariaceae Taxon Hypothesis (Psilocybe) found in the UNITE database  (Agrocybe (TH012107) and Gymnopilus (TH012136) were included based on phylogenetic relationships provided by JGI MycoCosm . The motivation for using the UNITE SH sequences independent of NCBI sequences is that the UNITE database is curated to minimize noise by reducing minor sequence variation using representative sequences chosen manually by expert curators or automatically using a standard dynamic similarity threshold. All SHs are assigned a unique digital object identifier (DOI) to allow stable, unambiguous reference across studies, even in the complete absence of meaningful taxonomic names (pothesis  TH00680305.2021) . Outgrouic names .Psilocybe, expert verification has been completed for 9/48 SHs, for which a representative sequence has been manually chosen, whereas the remaining SHs are represented by a sequence automatically chosen by the SH algorithm.For ITS barcode sequences generated in this study were combined with data set 1 and data set 2 for phylogenetic analysis.http://tree.bio.ed.ac.uk/software/figtree/).For all data sets, sequences were automatically aligned using the multiple sequence alignment software MAFFT v7.475  with the(i) Specimen preparation and chemical extraction. Eight Psilocybe cubensis vouchers , nine P. cyanescens samples, and eight P. semilanceata samples were used for chemical analysis across multiyear time points . Samples were ground to a fine powder, as described above, prior to solvent extraction as described in the work of Fricke et al.  UHPLC-MS method to identify and quantify psilocybin and tryptamine alkaloid intermediates. Hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry was performed using an Acquity Arc UHPLC-MS  in both positive and negative mode with a Waters Acquity UPLC ethylene-bridged hybrid (BEH) amide column  set to 40°C. The λ range was set at 200 to 500 nm with a resolution of 1.2 nm and a sampling rate of 10 points/s. The mass range in positive mode was set at 100 to 800 Da with a gain of 1, probe at 600°C, a capillary voltage of 0.8 kV, and collection at 10 points/s. A flow rate of 0.5 mL/min and a linear solvent gradient of 99.9% solvent A  to 30% solvent B  over 7 min were used. All samples were resuspended to a concentration of 100 μg/mL in methanol-water (50%:50%).(iii) Chemical standard curve creation and calculation. First, we calculated control curves by measuring the content of six chemical standards for baeocystin, norbaeocystin, norpsilocin, psilocin, psilocybin, and tryptophan at three different concentrations in triplicate . This allowed us to identify our compounds in spectral data based on retention times and calculate the concentration of each compound in each sample based on extracted m/z intensities (Table S4). Standard curves were measured by running standards at 1.0-, 1.2-, and 1.5-μg injections in triplicate and in three replicates. The baseline integrations were recorded for each, and the standard curves were generated using the line of best fit from the standard measurements.P. cubensis were cultivated from spore syringes acquired from Spore Works  inoculated on 2% malt agar plates with 50 μg/mL ampicillin antibiotic. Isolated sections of contiguous mycelium from initial spore plates were subcultured by removing ~0.2 cm of tissue from the edge of the growing mycelium with a flame-sterilized scalpel to another plate of 2% malt agar and ampicillin with a thin sheet of sterilized cellophane plastic placed on the surface to facilitate the removal of mycelium for chemical processing. A single culture was used for chemical analysis as well as cultivation of sporocarps. Agar plugs from this culture were placed into spawn growth medium and incubated at room temperature without a consistent light cycle for ~2 to 3 weeks until medium was entirely colonized by mycelium based on visual inspection. Spawn growth was achieved using 1-L wide-mouth Mason jars  containing one bag of Ben’s Original Ready Rice whole-grain brown rice , topped with custom lids that included a 0.22-μm filter for sterile airflow , with autoclaving for 40 min on a liquid cycle. To induce sporocarps, fully colonized spawn was mixed aseptically with one brick of coconut coir , 300 g of vermiculite , and enough tap water  to fill half a container with a moist but not muddy consistency within an 18-qt plastic bin .Specimen cultivation and processing were carried out as follows. Specimens of After 2 to 3 weeks, the lid was left ajar for increased airflow to promote sporocarp production (~1 week). Mycelium and sporocarps underwent lyophilization prior to chemical analysis. Viable culture plugs were retained for long-term storage at room temperature in 50 mL of distilled water , and lyoP. cubensis sporocarps and vegetative mycelium. Tissue samples were lyophilized, ground, and extracted in 80:20 (vol/vol) methanol-water, producing 2 mL of retained supernatant from 100 mg (±2.5 mg) of sample for spectroscopic analysis. Small molecules  from the extraction were analyzed using UHPLC  and mass spectrometry (Waters Xevo G2 quadrupole time of flight [QToF]) (UPLC-MS) in positive and negative ionization mode. Additionally, MS/MS spectra were acquired for sample Psi-6-Cap by running data-dependent acquisition mode (DDA), whereby MS/MS data were collected for all metabolites that were ionized above a set threshold .We applied a UPLC-MS-based untargeted metabolomics pipeline to identify shared and unique metabolites across three cultivated centWave’ ; peak grouping method ‘density’ (bw = 3); and retention time correction method “peakGroups,” in which retention time standards containing eight known compounds as a reference sample and integrate-areas-of-missing-peaks method “FillChromPeaksParam” were used. We then used the R package Camera (m/z-retention time combinations) derived from one compound into pc groups. The parameters used were peak grouping after retention time ‘groupFWHM’ , verify grouping ‘groupCorr’, annotate isotopes ‘findIsotopes’, and annotate adducts ‘findAdducts’ (polarity = ‘positive’).Raw data from the UPLC-MS analysis were processed using the R package XCMS  for peake Camera  to assigChemical profile comparison was performed by using the intensity values for each identified factor across all cultivated samples. Intensity values were processed and normalized by the total using the decostand command from the R package Vegan . NormaliThe identification of compounds from metabolic studies is often one of the most difficult parts of chemical profiling, as thousands of compounds and their derivatives are often produced but most lack standard references. In an attempt to make annotation and exploration of metabolomic data sets more accessible, GNPS was created as an initiative to make a standardized and accessible platform to compare spectral data across numerous organisms and chemical databases, as well as providing a platform that would allow older data to be reanalyzed as databases become better over time , 93. GNPhttps://ccms-ucsd.github.io/GNPSDocumentation/) on the GNPS website (http://gnps.ucsd.edu). The data were filtered by removing all MS/MS fragment ions within ±17 Da of the precursor m/z. MS/MS spectra were window filtered by choosing only the top 6 fragment ions in the ±50-Da window throughout the spectrum. The precursor ion mass tolerance was set to 2.0 Da, and the MS/MS fragment ion tolerance was set to 0.5 Da. A network was then created where edges were filtered to have a cosine score above 0.7 and more than 6 matched peaks. Further, edges between two nodes were kept in the network only if each of the nodes appeared in the other’s respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set to 100, and the lowest-scoring edges were removed from them until the molecular family size was below this threshold. The spectra in the network were then searched against the GNPS spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks.A molecular network was created using the online workflow (https://massive.ucsd.edu/ProteoSAFe/private-dataset.jsp?task=1b798991ec6b40d48b49b74e971e2ecf.All edited ITS sequence data for all specimens were submitted to the National Center for Biotechnology Information with NCBI accession numbers reported in Table S2. The collection database, multiple sequence alignments, and trees are available in Figshare project number 129332. The whole metabolomic spectra have been submitted as a MASSIVE data set available at


P < 0.05). There was a higher concentration of serum ANXA3 in the KD-CAL group than in the KD-NCAL group (P < 0.05). Neutrophil cell counts and serum ANXA3 levels were higher in the KD group than in the HC group (P < 0.05) and quickly decreased when the patients were treated with IVIG after 7 days of illness. Platelet (PLT) counts and ANXA3 levels concurrently exhibited significant increases 7 days after onset. Furthermore, ANXA3 levels were positively correlated with lymphocyte and PLT counts in the KD and KD-CAL groups. ANXA3 may be involved in the pathogenesis of KD and CALs.Kawasaki disease (KD) is an acute, self-limited vasculitis, and the etiology is still unclear. Coronary arterial lesions (CALs) are a major complication of KD. Excessive inflammation and immunologic abnormities are involved in the pathogenesis of KD and CALs. Annexin A3 (ANXA3) plays crucial roles in cell migration and differentiation, inflammation, cardiovascular and membrane metabolic diseases. The purpose of this study was to investigate the effect of ANXA3 on the pathogenesis of KD and CALs. There were 109 children with KD in the KD group  and 58 healthy children in the control (HC) group. Clinical and laboratory data were retrospectively collected from all patients with KD. The serum concentration of ANXA3 was measured by enzyme-linked immunosorbent assays (ELISAs). Serum ANXA3 levels were higher in the KD group than in the HC group ( Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is classified as acute systemic vasculitis, and approximately 80% of affected patients are children under 5 years old , 2. Thro2+-dependent soluble and hydrophilic proteins that reversibly bind to negatively charged phospholipid head groups  and the KD without CALs group .The diameter and All blood samples were obtained from KD patients during the first week of onset before treatment with anticoagulants and intravenous immunoglobulin (IVIG). Then, the samples were centrifuged at 3000 rpm for 10 min, and the serum was stored at −80°C.All experiments complied with the ethics committee from Chongqing Medical University, and written informed consent for all participants was provided before the study by their parents or legal guardians.Serum concentrations of ANXA3 were quantified with a human ANXA3 ELISA kit  according to the manufacturer's instructions. The intra-assay and interassay precision were CV% < 8% and CV% < 10%, respectively.The serum concentrations of ANXA3 were tested in KD and HC groups. The important clinical data of all the subjects with KD, including the duration days with fever and the time point of IVIG administration, were recorded. General laboratory data of all the subjects in KD were examined, including white blood cell counts (WBC), platelet counts (PLT), neutrophil counts, lymphocyte counts, alanine transaminase (ALT) levels, aspartate transaminase (AST) levels, albumin (ALB) levels, procalcitonin (PCT) levels, C-reactive protein (CRP) levels, the erythrocyte sedimentation rate (ESR) levels, and blood coagulation parameters such as thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT) and plasma fibrinogen (FIB) levels. Eighty-eight KD patients had creatine kinase isoenzyme (CK-MB) results, 70 KD patients had brain natriuretic peptide (BNP) results, and 48 KD patients had cytokine results, including the levels of IL-6, IL-10, TNF-α, and INF-γ. Moreover, ANXA3 were tested in 15 paired KD-CAL patients both before and after day 7 disease onset.P value (2-tailed) < 0.05. Student's t test was used to compare data from two cohorts that followed a normal distribution, while the Mann–Whitney U test was used to compare nonnormally distributed data. The Wilcoxon signed ranks test was used to test paired samples for which data failed to follow a normal distribution. Correlations between serum ANXA3 levels and various laboratory parameters were assessed via Spearman rank correlation analysis.All data in this study are described as the mean ± standard deviation (SED) or median and interquartile range (IQR). Statistical significance was defined as a SPSS 23.0 was used to analyze all statistical results. GraphPad Prism 8.0 and Adobe Illustrator 2021 were used to create the figures.P > 0.05). The levels of serum ANXA3 in the KD group [6.617 pg/ml  n = 109] were significantly higher than those in the HC group  (P < 0.001). In addition, there were significant differences in the serum levels of ANXA3 among the KD-CAL group, KD-NCAL group and HC group (P < 0.001). The serum levels of ANXA3 in the KD-NCAL group  were higher than those in the HC group  (P < 0.001) but lower than those in the KD-CAL group  (P = 0.033) (There were no differences in age or sex between the KD and HC groups (= 0.033) .9/L , n = 109] in comparison with the HC group  (P < 0.001). The neutrophil cell counts were markedly decreased when the patients were treated with IVIG after 7 days of illness   in the KD group and significant positive correlations with the time point of IVIG (day)  in the KD-CAL group. In addition, lymphocyte and PLT counts showed significant positive correlations with ANXA3 in the KD and KD-CAL groups  (There were distinctly positive correlations between ANXA3 and WBC (ctively) .Compared with the initial data obtained, the serum levels of ANXA3 and PLT counts showed a concurrent increase at day 7 after onset of illness in KD-CAL patients .P < 0.05)  .P < 0.001 and P = 0.002, respectively). The cutoff values were 3.033 pg/ml and 9.846 pg/ml, respectively (The AUC values of the KD group and KD-CAL group were 0.930 and 0.679, respectively (95% confidence intervals: 0.892 to 0.969 and 0.578 to 0.779, respectively; ectively .KD as the main cause of acquired cardiovascular disease in children. It is presumably triggered by an infection-related abnormal inflammatory reaction in genetically susceptible children. The abnormal response of immune cells and vascular endothelial dysfunction are important causes of CALs in patients with KD . FindingFirst, studies showed that ANXA3 is expressed by neutrophils and monocytes, particularly in neutrophils , 29. ANXIn this study, the higher level of serum ANXA3 was accompanied by an increase in the neutrophil cell count, and when KD patients were treated with IVIG, the neutrophil cell count decreased. However, serum ANXA3 remained at a higher level, so the higher levels of serum ANXA3 may come from PLTs. In addition, Meng H et al. demonstrated that the silencing of ANXA3 promotes the repair and healing of myocardial tissue through PI3K/Akt signaling pathway activation in rats with acute myocardial infarction . The ROCIn conclusion, this study first verified that the serum concentration of ANXA3 was markedly higher in the KD group than in the HC group and higher in the KD-CAL group than in the KD-NCAL group. There was a positive relationship between ANXA3 and WBCs, lymphocytes and PLTs. The timing of the ANXA3 concentration increase coincides with neutrophil and PLT counts. Accordingly, we postulate that ANXA3 may be an intermediary agent between neutrophils and PLTs in the pathogenesis of KD and further in the development of CALs in different stages of KD. Therefore, ANXA3 may be a potential target in the prevention of KD and KD-CALs.This study had limitations. One is due to the small number of KD samples, further studies are need to demonstrated the signaling pathway of ANXA3, neutrophil and PLTs that is involved in the pathogenesis of KD and CALs. The other is all the patients were enrolled from in a single center, multicenter experimental evidence were needed in the further.


Part-solid nodules (PSNs) have gradually shifted to defining special clinical subtypes. Commonly, the solid portions of PSNs show various radiological morphologies, of which the corresponding pathological basis and prognosis are unclear. We conducted a radiological–pathological evaluation to determine the histopathologic basis of different consolidation radiographic morphologies related to prognosis.A cohort of 275 patients with a surgical pathological diagnosis of lung adenocarcinoma were enrolled. Preoperative computed tomography (CT) images of the PSNs were recorded and assessed. A panel of 103 patients with complete pathological specimens was selected to examine the radiological–pathological associations, and follow-up was performed to identify the prognosis.P < 0.001). Visual punctate solid components on CT correlated with tertiary lymphoid structures, stripe solid components on CT correlated with fibrotic scar, and irregular solid components on CT correlated with invasion. PSNs with regular consolidation had a better prognosis than those with irregular consolidation.Of the 275 patients, punctate consolidation was observed radiologically in 43/275 (15.7%), stripe consolidation in 68/275 (24.7%), and irregular consolidation in 164/275 (59.6%) patients. The radiological morphology of the solid components was significantly associated with the histopathological subtypes (Radiological morphology of solid components in PSNs can indicate the pathological basis and is valuable for prognosis. In particular, irregular solid components in PSNs usually indicate serious invasive growth, which should be taken with caution during assessment. The universal application of low-dose computed tomography (CT) in lung cancer screening has increased the detection rate of subsolid nodules. Persistent subsolid nodules are always considered malignant and have drawn considerable attention –3. BasedGenerally, the solid component size indicates the aggressive behavior of the disease and is correlated with patient prognosis –10. PartThis retrospective study was approved by our institutional review board. We enrolled patients diagnosed with lung adenocarcinoma pathologically from January 2016 to January 2020 at the following hospitals: the General Hospital of the People’s Liberation Army, Affiliated Beijing Shijitan Hospital of Capital Medical University, and Affiliated Hospital of Qingdao University. The inclusion criteria were as follows: (a) patients with primary lung adenocarcinoma confirmed by postoperative pathology, (b) patients undergoing at least one CT examination within 1 month before surgery and with available complete thin-slice images, and (c) patients with a maximal diameter of subsolid nodule ≤ 3 cm. The exclusion criteria were as follows: (a) patients with incomplete clinical and CT data or without raw thin-slice images, (b) patients with a history of radiotherapy or chemotherapy before surgery, and (c) patients with pneumonia or atelectasis. Finally, 275 patients were included in this study  or Siemens SOMATOM Sensation 128-Slice CT scanner . The parameters for CT screenings were as follows: routine section thickness, 1.0–1.5 mm; section thickness after reconstruction, 0.625–1.25 mm; 80–120 kV; and 200–300 mAs.These CT images were reviewed and assessed by setting the lung  and mediastinal  windows. A cardiopulmonary radiologist  and a pulmonary radiologist  conducted the evaluation work in consensus. The evaluation results were assessed and confirmed by a senior radiologist . The following CT characteristics were recorded for each lesion: (a) lesion location, (b) lesion size , (c) solid component morphology findings , and (d) size (> 5 mm or < 5 mm). Figure All postoperative histopathological slides after staining with hematoxylin and eosin were analyzed in consensus by a senior pulmonary pathologist  and a pulmonary pathologist . The histopathological subtype was determined according to the international multidisciplinary classification of lung adenocarcinoma. Other recorded features included the presence of tertiary lymphoid structures, fibrotic scars within the tumors, and the presence of invasion.P-values < 0.05 were considered statistically significant.The Statistical Package for the Social Sciences version 26.0  was used for statistical data analyses. Continuous variables, such as age, tumor size, and smoking index, are expressed as means ± standard deviations with ranges. Categorical variables, including sex, smoking history, symptoms, and CT characteristics, are expressed as counts and percentages and compared using the Pearson chi-squared or Fisher exact test, when appropriate. The log-rank test and Kaplan–Meier analyses were used to estimate the survival curves of recurrence-free probability. As demonstrated in Table P < 0.001)  of the 275 patients had complete and available pathological specimens. The slides were then re-reviewed and evaluated. Punctate, stripe, and irregular consolidations were observed radiologically in 9/103 (8.7%), 28/103 (27.2%), and 66/103 (64.1%) patients, respectively. Pathological specimens included minimally invasive adenocarcinoma, invasive adenocarcinoma (lepidic predominant), invasive adenocarcinoma (acinar predominant), invasive adenocarcinoma (micropapillary predominant), invasive adenocarcinoma (papillary predominant), and invasive adenocarcinoma (solid predominant) in 8/103 (7.8%), 32/103 (31.0%), 52/103 (50.5%), 4/103 (3.9%), 4/103 (3.9%), and 3/103 (2.9%) patients, respectively. The visual radiological morphology of the solid components was significantly associated with histopathologic subtypes (P < 0.001)  patients Fig. A, stripeP = 0.132), possibly because of the sample size, as shown in Fig. In our study, follow-up was performed on 103 patients with complete pathological specimens. Recurrence was identified in 6 5.8%) of the 103 patients. Of the six patients with recurrence, one had PSN with stripe consolidation and five had PSNs with irregular consolidation. As in the examples provided in Fig. .8% of thLung adenocarcinoma is the leading fatal subtype of non-small cell lung cancer and is more prevalent among young female patients –17. A reThe punctate consolidation in PSN on CT scans was previously considered to be a tiny invasion focus . HoweverFollow-up results showed that no recurrence occurred in PSNs with punctate solid components. In contrast, PSN with irregular solid components experienced recurrence or metastasis. Previous studies have explored the correlation between the size or proportion of solid components of PSNs and prognosis in patients with lung adenocarcinoma. The conclusion was that the solid component size was not prognostic in part-solid lung cancer due to the presence of ground-glass opacity –33. GrouOur study has some limitations. First, this was a retrospective study with a small sample size, and further studies from multiple centers are required to confirm the results. Second, deviation in the accurate judgment of CT signs might exist because of the small nodules. Third, there was no precise definition of the three different solid component morphologies in PSN, and further refinement is required in future studies. Finally, due to the indolent nature of subsolid nodules, the follow-up time should be sufficiently long and require further confirmation.In conclusion, the radiological morphology of the solid components in PSN can reflect a pathological basis and is valuable for prognosis. In particular, irregular solid components in PSNs usually indicate serious aggressive behavior, which should be taken with caution during assessment.