Whether immunotherapy improves the efficacy or worsens adverse events of subsequent chemotherapy remains unclear. We performed a Phase 2 study to evaluate the efficacy and safety of nanoparticle albumin‐bound paclitaxel (nab‐paclitaxel) as a treatment for advanced non‐small cell lung cancer (NSCLC) after treatment with programmed cell death 1 or programmed death ligand 1 [PD‐(L)1] inhibitor failure.2) was administered on Days 1, 8, and 15 of a 28‐day cycle to patients with advanced NSCLC within 12 weeks after the failure of PD‐(L)1 inhibitor treatment. The primary endpoint was objective response rate (ORR) in all patients; the secondary endpoints were disease control rate (DCR), progression‐free survival (PFS), overall survival (OS), and safety.Nab‐paclitaxel  and the DCR was 86.2% (95% CI: 65.9%–97.0%). The median PFS was 5.6 months (95% CI: 4.4–6.7 months), and PFS rates at 1‐ and 2‐year timepoints were 34.5% and 13.3%, respectively. The median OS was 11.9 months (95% CI: 0.8–23.0 months). Good performance status and responder of previous PD‐(L)1 inhibitor therapy were independent predictors of PFS. Grade 3 or higher toxicities included leukopenia (27.6%), neutropenia (31.0%), peripheral sensory neuropathy (6.9%), increased alanine aminotransferase and aspartate aminotransferase levels (3.4%), and interstitial lung disease (3.4%).Nab‐paclitaxel therapy improved ORR after PD‐(L)1 inhibitor treatment failure with a durable response of 13% and acceptable toxicities in patients with advanced NSCLC. However, nanoparticle albumin‐bound paclitaxel (nab‐paclitaxel) has recently demonstrated significantly longer progression‐free survival (PFS) than docetaxelSeveral chemotherapy and PD‐1 or PD‐L1 [PD‐(L)1] inhibitor combination regimens are currently available for patients with advanced NSCLC. Among these, the combination of nab‐paclitaxel and carboplatin with a PD‐(L)1 inhibitor has been reported to be both effective and well tolerated.22.13, neutrophil count ≥1500/mm3, hemoglobin content ≥9.0 g/dL, platelet count ≥100,000/mm3, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels ≤100 IU/L, total bilirubin concentration ≤1.5 mg/dL, creatinine concentration ≤1.5 mg/dL, urine protein level ≤1+, prothrombin time‐international normalized ratio ≤1.5, and resting arterial oxygen saturation in room air ≥90%. A life expectancy of more than 3 months was required. Previous treatment with molecular‐targeted drugs for driver oncogenes, radiotherapy, and chemotherapy without solvent‐based paclitaxel or nab‐paclitaxel was permitted.The criteria for patient eligibility included an age of 20 years or older, diagnosis of advanced or recurrent NSCLC confirmed histologically or cytologically, within 12 weeks after PD‐(L)1 inhibitor (single or combination therapy) treatment failure due to disease progression or unacceptable adverse events, an Eastern Cooperative Oncology Group performance status (ECOG‐PS) of 0–2, and measurable lesions according to Response Evaluation Criteria in Solid Tumor (RECIST) version 1.1.The key exclusion criteria included patients who had a history of severe drug allergies or hypersensitivity to nab‐paclitaxel or albumin; had been previously treated with solvent‐based paclitaxel or nab‐paclitaxel; were receiving more than 10 mg of systemic prednisolone or an equivalent amount of another corticosteroid at the time of registration; had Grade 2 or higher peripheral neuropathy; had interstitial pneumonia (apparent on chest X‐ray); had pleural effusions, ascites, and/or pericardial effusions requiring drainage; had symptomatic central nerve metastasis; received therapeutic or palliative radiation therapy within 2 weeks of study Day 1; underwent major surgery within 4 weeks of study Day 1; had a concurrent active malignancy; were lactating/breastfeeding; had a positive pregnancy test; and/or were positive for hepatitis B surface antigen(s).2.22 on Days 1, 8, and 15 (every 4 weeks). At the beginning of the second and subsequent cycles, patients had to meet the following criteria: a neutrophil count ≥1500/mm3, platelet count ≥10,000/mm3, hemoglobin ≥9.0 g/dL, AST and ALT ≤2.5 times the upper limit of normal, total bilirubin concentration ≤1.5 mg/dL, creatinine concentration ≤1.5 mg/dL, and peripheral neuropathy of ≤Grade 2. Prior to administration of nab‐paclitaxel on Days 8 and 15, patients had to meet the following criteria: a neutrophil count ≥500/mm3, platelet count ≥50,000/mm3, and peripheral neuropathy of ≤Grade 2. Dose reductions of 20 mg/m2 (by a minimum of 60 mg/m2) were allowed if the following criteria were met during treatment: Grade 4 neutropenia, Grade 3 or higher neutropenia that delayed the start of each cycle by at least 7 days, Grade 3 or higher thrombocytopenia, febrile neutropenia, Grade 2 or higher skin disorders, and/or Grade 3 or higher nonhematologic toxicities other than alopecia. Treatment continued until progressive disease (PD) was confirmed or unacceptable adverse events were identified.This trial was a three‐center, open‐label, single‐group, Phase 2 study. After confirming the baseline assessment, patients received a 30‐min infusion of nab‐paclitaxel  at a dose of 100 mg/m2.3https://www.umin.ac.jp/ctr/index.htm (UMIN000030994). This trial was self‐funded and designed by the principal investigators. All authors vouch for the accuracy and completeness of the data and for the adherence of the experiments performed for this research paper to the described protocol.This study was approved by the Research Ethics Committee of the University of Fukui (No. 20170185), the Institutional Review Board of the Japanese Red Cross Fukui Hospital, and the Institutional Review Board of the Municipal Tsuruga Hospital (No. 248). It was performed in accordance with the Declaration of Helsinki and the guidelines for Good Clinical Practice. All patients provided written informed consent. The clinical trial was registered at 2.417 were performed using computed tomography or magnetic resonance imaging (MRI) scans of the chest and abdomen at intervals of 8 weeks or less until PD. The response was confirmed at least 4 weeks after the initial response. Although brain MRI scanning was performed in all patients at the time of NSCLC diagnosis, it was not mandated at the time of inclusion except in patients with known or suspected brain metastases. In patients with brain metastases, a brain MRI was also performed at intervals of 8 weeks or less during nab‐paclitaxel treatment.Disease assessments using RECIST version 1.1The primary endpoint was ORR as determined via investigator assessments according to RECIST version 1.1.The secondary endpoints were disease control rate (DCR), PFS, which is the time from the date of enrollment to the date of disease progression, OS, which is the time from the date of enrollment to the date of death from any cause, and safety. Adverse events were evaluated in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events Version 4.0.In subset analyses, we examined ORR, PFS, and OS by age, sex, ECOG performance status, histological type (nonsquamous or squamous cell carcinoma), history of smoking , the degree of PD‐L1 expression, and number of previous regimens.2.5p < 0.05 indicated significant differences.Response was summarized with the use of frequency counts and percentages, with exact 95% confidence intervals (CIs) calculated. Fisher's exact test was used to examine the association between two categorical variables. PFS and OS were estimated using the Kaplan–Meier method, and 95% CIs were calculated. The Cox proportional hazards model using the stepwise method was used to identify prognostic factors. Statistical analysis was performed using SPSS Statistics V.26.0 (IBM). https://stattools.crab.org/index.html). At the time of study design, nab‐paclitaxel therapy was not established as a standard second‐line therapy; thus, docetaxel was used as the reference. Shepherd et al. reported an ORR of 7.1% for docetaxel second‐line therapy,α = 0.05 (one side) and β = 0.2, the required number of subjects was 24. Therefore, considering some ineligible cases, the planned number of cases was set to 30.To calculate the sample size, one arm binomial was used in SWOG's Statistical Tools (33.1EGFR) mutations were positive for the driver oncogene in only three patients. Twenty‐eight patients terminated ICI treatment due to confirmed PD and subsequently received nab‐paclitaxel therapy. In only one patient, nivolumab was discontinued due to Grade 3 liver enzyme increase; thus, nab‐paclitaxel was administered before PD was confirmed. In this case, the liver injury improved spontaneously and no steroids were administered.Between February 2018 and December 2020, 30 patients with advanced NSCLC were recruited, and 29 patients were included in the analysis  achieved complete response (CR), 15 (51.7%) had partial response (PR), 9 (31.0%) had stable disease (SD), and 4 (13.8%) experienced PD. The ORR was 55.2% (95% CI: 28.1%–79.6%) and the DCR was 86.2% (95% CI: 65.9%–97.0%), suggesting high efficacy (Table .4% achiep = 0.003) and responder of previous ICI treatment  were indicators of longer PFS (Table p = 0.013) and responder of previous ICI treatment  were indicators of longer OS, and multivariate analysis showed age (<70 years) and responder of previous ICI treatment as independent predictors of OS. In contrast, no significant associations were observed between PD‐L1 expression and both PFS and OS. Figure p = 0.0012) and responder of previous ICI treatment  had significant longer median PFS. Similarly, longer median OS was observed in patients with younger age , PS 0 , and responder of previous ICI treatment  . Three patients (10.3%) received taxane‐based anticancer agents as a prior therapy in combination with docetaxel and ramucirumab, and all three patients showed a response PR to nab‐paclitaxel therapy.Although no statistically significant differences were observed, younger patients (<70 years) and those with PS 0, squamous cell carcinoma, and 1 previous regimen tended to have a good response, while patient populations with apparently low ORR <40%) could not be identified Table . Interes0% could Figure 3.3The median number of treatment cycles was 6 (range: 1–38). One‐stage dose reduction occurred in four patients, and two‐stage dose reduction occurred in four. Two‐stage dose reductions were due to a decline in neutrophil count and peripheral sensory neuropathy. Four patients could not continue treatment because of adverse events, and two could not continue because of patient refusal.3.4Table 4This prospective Phase 2 study evaluated the efficacy and safety of nab‐paclitaxel therapy in patients with advanced NSCLC treated with ICI alone or in combination with chemotherapy and ICI as a pretreatment and demonstrated an ORR that was higher than expected, with acceptable safety. The ORR in this study (55.2%) was higher compared to the ORR (28.1%) that was previously reported for nab‐paclitaxel as a second‐line therapy for patients with NSCLC who had not received ICI as prior therapy.Several retrospective studies have reported improved efficacy of chemotherapy after immunotherapy for NSCLC.+ T cells in tumorsIn the present study, the long‐term response of 2 years or longer was observed in 13.3% patients. Long‐term responses are generally not observed with single‐agent anticancer therapy and are often observed in the course of immunotherapy. Furthermore, long‐term responses may be a corollary of the continued efficacy of immunotherapy. In fact, a single dose of PD‐1 antibody, binding peripheral blood T cells, can persist for approximately 5 months and may continue to be effective during the next treatment.Pretreatment with ICI might exacerbate the adverse events of the subsequent chemotherapy, and it was vital to evaluate the safety of treatment. Moreover, previous reports on single‐agent chemotherapy with nab‐paclitaxel for patients with NSCLC have reported a median number of treatment cycles of 3–5,Although these results were clinically relevant, the study has several research limitations. First, the sample size was relatively small, and caution must be exercised regarding survival probability and frequency of adverse events. A Phase 3 randomized trial is needed to accurately validate the results of this study. However, since designing such a trial is difficult, it is necessary to validate the results in a Phase 2 trial such as the present study or in a retrospective trial with a large number of cases. Second, nab‐paclitaxel was used in this study, but further studies are needed to determine whether other agents can provide similar efficacy. Third, because only ICI monotherapy was approved in Japan during most of the study period, chemoimmunotherapy was administered as pretreatment in only a few cases in the current study. Therefore, whether nab‐paclitaxel monotherapy after chemoimmunotherapy is as effective as the present results indicate needs to be verified in the future.In conclusion, this is the first prospective study to evaluate the effect of nab‐paclitaxel monotherapy after ICI treatment failure in patients with advanced NSCLC. The ORR of nab‐paclitaxel was clearly better than those of standard single‐agent chemotherapies, and a durable response lasting more than 2 years was observed in 13% of all cases. These results may demonstrate that ICI treatment enhances the effect of subsequent chemotherapy. The adverse events of nab‐paclitaxel immediately after ICI treatment are similar to those reported for nab‐paclitaxel therapy; however, the possible increase in the incidence of drug‐induced pneumonia should be considered cautiously. Although further studies are required, we suggest that nab‐paclitaxel after ICI is an appropriate treatment for advanced NSCLC.Tomoaki Sonoda: Data curation (lead); formal analysis ; project administration (lead); resources ; writing—original draft (lead). Yukihiro Umeda: Conceptualization (lead); funding acquisition (lead); methodology (lead); resources ; writing— original draft (lead). Yoshiki Demura: Conceptualization ; methodology (lead); resources ; supervision ; writing—review and editing (lead). Toshihiko Tada: Formal analysis ; resources ; writing—review and editing . Koki Nakashima: Formal analysis ; resources ; writing—review and editing . Masaki Anzai: Resources ; validation (lead);writing —review and editing . Makiko Yamaguchi: Resources ; validation ; writing—review and editing . Akikazu Shimada: Investigation ; resources ; writing—review and editing . Masahiro Ohi: Investigation ; resources ; writing—review and editing . Chisato Honjo: Resources ; software ; writing—review and editing . Yuko Waseda: Resources ; software ; writing—review and editing . Masaya Akai: Conceptualization ; methodology (supporting); supervision ; writing—review and editing . Tamotsu Ishizuka: Conceptualization ; methodology ; supervision (lead); writing—review and editing (lead).The authors have no conflict of interest to declare.The study was approved by the medical ethics committees at each site. All patients provided written informed consent.


In conclusion, we observed extremely high plasma calprotectin concentration in patients with LPI. Mechanism of this phenomenon is unknown.Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder affecting the transport of cationic amino acids. Elevated plasma zinc concentrations have been described in patients with LPI. Calprotectin is a calcium‐ and zinc‐binding protein, produced by polymorphonuclear leukocytes and monocytes. Both zinc and calprotectin have an important role in immune system. In this study, we describe plasma zinc and plasma calprotectin concentrations in Finnish LPI patients. Plasma calprotectin concentration was measured from 10 LPI patients using an enzyme‐linked immunosorbent assay (ELISA) and it was remarkably high in all LPI patients (median: 622 338 μg/L) compared to that in healthy controls (608 μg/L). Plasma zinc concentration was measured by photometry and it was normal or only mildly elevated (median: 14.9 μmol/L). All the patients had decreased glomerular infiltration rate (median: 50 mL/min/1.73 m Plasma calprotectin is extremely high in patients with LPI.1SLC7A7 gene  encoding y + LAT‐1 protein, the catalytic light chain subunit of the heteromeric amino acid transporter located at the basolateral membrane of the epithelial cells of the renal proximal tubules and intestine. All Finnish patients share the same homozygous variant, c.895‐2A > T (NM_001126105.2).Lysinuric protein intolerance (LPI) is a rare inherited autosomal recessive disorder of amino acid metabolism, which effects the transport of dibasic cationic amino acids lysine, arginine, and ornithine. LPI has been described worldwide but it is more prevalent in Finland (1/60 000) than in any other country.Due to the protein‐restricted diet, patients with LPI are at risk for nutritional deficiencies. Because meat and seafood are considered major dietary sources of zinc,Zinc is an essential trace element, occurring as a component at least 300 enzymes and participating in numerous cellular functions.Plasma calprotectin levels in LPI patients have not been reported before. In this study, we describe in detail plasma calprotectin and plasma zinc concentrations of 10 Finnish LPI patients.210 Finnish LPI patients  followed‐up at the Turku University Hospital were included in this study. We included only adult patients with genetically confirmed LPI, otherwise there were no specific inclusion or exclusion criteria. The median age of the patients was 46.5 years (range 27–65 years). All patients followed a protein‐restricted diet instructed by a nutritionist. Daily use of a multivitamin preparation containing 10 mg of zinc was recommended for all patients. Otherwise, no zinc supplementation was included in their treatment protocol. All patients were clinically examined, and none of the patients had signs of acute infections. None of the patients were on anti‐inflammatory medication. All patients used oral L‐citrulline. Eight patients were treated with cholesterol‐lowering drugs (statins). Six patients were treated with antihypertensive drugs (ACE inhibitors or AT2‐blockers). Eight patients had lysine supplementation. Two patients needed oral supplementation of sodium bicarbonate and six patients used sodium benzoate.4), plasma iron, plasma zinc, plasma copper, blood manganese, plasma calcium, total plasma cholesterol, high‐density lipoprotein (LDL), triglycerides, prealbumin, plasma, and urinary amino acids. Glomerular filtration rate (GFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation (CKD‐EPI). All laboratory analyses were performed using standard clinical laboratory methods. Ferritin and LDH levels were not routinely measured as they are constantly elevated in LPI patients without clear correlation with the clinical symptoms. All the patients had had markedly elevated ferritin and LDH levels at the time of diagnosis.Routine follow‐up laboratory tests were analyzed, including total blood cell count, plasma creatinine, serum cystatin C, urine beta‐2 microglobulin, plasma alanine aminotransferase (ALT), plasma alkaline phosphatase (ALP), plasma ammonium ion .Blood samples for plasma calprotectin measurement were collected in EDTA tubes and they were centrifuged at 3000 rpm for 10 min. Plasma samples were stored in aliquots at −75°C until use. Control plasma was collected from five non‐matched adult volunteer donors with no known illnesses.Plasma calprotectin was measured with an enzyme‐linked immunosorbent assay  from the CALPROLAB™  according to the instructions of the manufacturer. Absorbance was measured at 405 nm using an ELISA plate reader (Hidex Sense). The standard curve was based on measuring six standards in the range 0–500 ng/mL.Plasma samples were diluted  with the Sample Dilution Buffer (from CALPROLAB) at final dilution 1:10 000. The control samples were analyzed with dilution 1:20.Samples, standards, and controls were incubated in the duplicate microtiter wells 45 min on a horizontal plate shaker (600 rpm) at room temperature. At the end of the incubation time, the liquid was removed, and the wells were washed three times with Washing Solution (from CALPROLAB), using an automatic plate washer. After washes, the Enzyme Conjugate was added to each well, and the plate was incubated for 45 min on a horizontal plate shaker (600 rpm) at room temperature. After the incubation, the plate was washed as described above. Then Enzyme Substrate Solution was added to each well and the plate was incubated for 25 min at room temperature, protected from light. After the incubation, 100 μL 1 M NaOH was added to each well to stop the reaction.p‐values < 0.05 were considered statistically significant. This study was approved by the joint Ethics Committee of the University of Turku and Turku University Hospital.The data were analyzed using IBM SPSS Statistics 27 software. Correlations were calculated with the Spearman correlation coefficient. 3Characteristics of 10 patients with LPI are presented in Table 4 median was 27 μmoL/L (range: 20–66 μmol/L). Plasma prealbumin levels were normal in all patients.ALT was elevated in two patients  and ALP was elevated in four patients . NH2, range: 17–88 mL/min/1.73 m2). Urine beta‐2 microglobulin was measured from eight patients and was elevated in seven of them . Six patients had hypertriglyceridemia and three patients had elevated LDL cholesterol.Seven patients had elevated plasma creatinine  and serum cystatin C  levels. GFR was decreased in all patients .Calprotectin was measured from 10 patients using an enzyme‐linked immunosorbent assay (ELISA). In all patients, plasma calprotectin was extremely high  compared to the health controls . There was a significant positive correlation between plasma zinc concentration and plasma calprotectin concentration  and with the Helsinki Declaration of 1975, as revised in 2000(5). Informed consent was obtained from all patients for being included in the study.Supplemental Table 1. Individual plasma creatinine, plasma cystatine C, plasma calcium, plasma zinc, urine zinc (24 h) and plasma calprotectin values.Click here for additional data file.


TTN expression significantly correlated with the clinical stage , node metastasis , histological type , race  and TP53 mutation , considering The Cancer Genome Atlas database. However, for patients who had higher TTN expression, the overall survival was remarkably shorter than patients who had low TTN expression. Furthermore, TTN was lowly expressed in four CRC cell lines. TTN overexpression facilitated CRC cells in terms of the proliferation, metastasis and invasion. Based on gene set enrichment analysis, the ERB pathway might be responsible for TTN‐related CRC. Besides, TTN was involved in the response to azacitidine. Overall, TTN might serve as a potential novel therapeutic target for treating and overcoming chemotherapy resistance in CRC.Colorectal cancer (CRC) is identified as a primary cause of death around the world. The current chemotherapies are not cost‐effective. Therefore, finding novel potential therapeutic target is urgent. Titin (TTN) is a muscle protein that is critical in hypertrophic cardiomyopathy. However, its role in CRC is not well understood. The study focused on exploring the possible role of TTN in CRC carcinogenesis. TTN mRNA and protein expression levels presented an obvious downregulation in CRC tissue samples, relative to normal control ( TTN contains an N‐terminal I‐band and a C‐terminal A‐band. The A‐band possesses kinase activity and participates in active contraction. The I‐band contains two regions of the tandem immunoglobulin domain, which can bind proteins implicated in protein quality control pathways.TTN gene‐related lncRNA TTN‐AS1 was found to regulate the transcriptional activity of relevant genes by targeting miR‐16‐5p and the tumour suppressor gene PTEN, resulting in decreased sensitivity of tumour cells to sorafenib. The resistance significantly diminished upon depletion of TTN‐AS1.Titin is a muscle protein that is encoded by the 22.1CRC tissues and normal controls came from two cohorts receiving surgery at the Affiliated Hospital of Qingdao University. Samples of 19 CRC patients and 19 adjacent normal controls collected between 2016 and 2018 were included in Cohort 1. Samples of 32 CRC patients and 32 normal controls collected between 2017 and 2020 were included in Cohort 2. Clinicopathological features, including gender, age, TNM stage, tumour size and degree of differentiation, were recorded for the study cohorts. Our study has obtained the approval of the Ethics Committee of the Affiliated Hospital of Qingdao University, and obtained all participants' informed consent.2.2HCT116, LoVo, HT‐29 and SW620 CRC cell lines were provided by the Cell Bank of the Chinese Academic of Sciences. HCT116, LoVo, HT‐29 and SW620 CRC cell lines were cultured in DMEM/F‐12 (Hyclone) with 10% FBS (Gibco) and 1% penicillin–streptomycin (Hyclone). Mycoplasma testing has been carried out for the cell lines used.2.32O2) at room temperature (RT). The sections underwent one night of incubation at 4°C using rabbit polyclonal antibody against TTN . Later, the sections underwent the treatment of horseradish peroxidase conjugated anti‐rabbit IgG antibody (1:500) for 30 min at RT. The non‐specific binding and antibody detection were blocked by the DAB Detection kit. Tissue sections then received haematoxylin counterstaining. A light microscope served for the detection of immunostained sections. Image‐Pro Plus (version 6.0) served for the semiquantitative analysis. In addition, The Cancer Genome Atlas (TCGA) database assisted in validating the association of TTN expression with CRC clinical features.Twenty percent formaldehyde‐fixed tissue samples underwent paraffin embedding, followed by being cut into thin sections (5 μm) and rehydrated. Then heat‐induced antigen retrieval was conducted in citrate buffer or Tris/EDTA buffer. Tissue sections received 10 min of blocking in 3% hydrogen peroxide  served for extracting the total RNA from cell lines and colon samples. PrimerScript TM RT reagent Kit with gDNA Eraser (TAKARA) served for the synthesis of first‐strand cDNA following the protocol of the manufacturer. The primers for TTN included, F: 5′‐GCGTAAGACTCAGGCATCCA‐3′ and R: 5′‐AGTAGAGGTTGTCAGCGTTGT‐3′. We conducted qPCR under the conditions of 30 min of predenaturation at 95°C, 5 s of 40 cycles of denaturation at 95°C, annealing and 34 s of elongation at 60°C. Target gene mRNA expression was amplified and analysed with ABI Prism 7300 SDS Software using GAPDH as an internal standard. The 22.5Guide RNA (gRNA) for site‐specific gene editing mediated by CRISPRa was used to trans‐activate TTN expression. The plasmids lenti dCAS‐VP64‐puro, lenti sgRNA(TTN)‐MPH‐Blast were used for CRISPR‐Cas9 activation. Subcloning PCR‐amplified Single‐guide RNA (sgRNA) of TTN was sub‐cloned into the H7284 plenti‐U6‐gRNA‐2x(wt+f6)MS2‐CMV‐MCP‐P65‐HSF1‐IRES‐Blast vector (OBiO Technology) using the forward primer: 5′‐CCGGGTATGTGACACTACCAAAT‐3′ and reverse primer: 5′‐AAACATTTGGTAGTGTCACATAC‐3′, so as to generate the TTN expression construct. The LoVo cell line was transduced by lenti dCAS‐VP64‐puro and lenti sgRNA(TTN)‐MPH‐Blast. Stable cell lines with overexpressed TTN were selected using puromycin (2 μg/mL) and blasticidin (10 μg/mL). qPCR served for the detection of the mRNA expression level of TTN after transfection.2.63 cells/well). 0, 24, 48, 72 and 96 h of treatment was followed by the addition of 10 μL CCK‐8 solution into each well. The plates received 2 h of incubation at 37°C. An automatic microplate reader  served for reading the absorbance at 450 nm.Cell counting kit‐8 (CCK‐8) served for detecting the cell viability. We plated LoVo, LoVo‐H7281+vector and LoVo‐H7281 cells in 96‐well plates  at 0, 24, 48, 72, 96 and 120 h. Image‐pro plus 6.0 software was used for the analysis of the images. Next, a transwell assay served for the evaluation of the cell invasion. Briefly, cells seeded in a matrigel invasion chamber (Corning) received 48 h of culturing at 37°C. Invaded cells received crystal violet staining (Beyotime). A microscope illustrated related images.2.8t‐test or two‐way anova tests served for comparing the means between two or three groups. The Kaplan–Meier method assisted in drawing survival curves. A p < 0.05 reported statistical significance.Data were in the form of mean ± standard deviation. SPSS 20 (IBM) served for all statistical analyses. Student's 33.1n = 19) compared to adjacent normal tissues (n = 19). Representative images of the IHC experiment are shown in Figure p = 0.008) . Similarly, TTN presented an obvious downregulation in CRC tissues, relative to normal controls   Figure . To veri) Figure . TTN exp) Figure . Accordi3.2p < 0.05; normal vs. Stage 4, p < 0.05), node metastasis , histological type , race  and TP53 mutation    Figure . In summ) Figure .3.3p < 0.001)   Figure . This re) Figure . A trans) Figure .3.4p value, and te value  served for analysing the underlying pathways of TTN involved in the development of CRC, finding that six pathways, namely, histone modifications, response to azacitidine, mRNA splicing, ERB pathway, VDR pathway, regulation of TP53 activity through acetylation, were significantly differentially enriched in low‐expressed TTN phenotype in terms of normalized enrichment score (NES), nominal e Figure ; Table 24Over the last decades, although various approaches have been employed in CRC treatment,Manipulating the expression of TTN in tumour cells revealed the positive impact of TTN overexpression on colon cancer cells in terms of the proliferation, invasion and metastasis, proving the involvement of TTN in colon cancer progression. Low TTN expression may indicate a poor prognosis. We further verified the impact of TTN expression on prognosis through survival analysis, finding the lower OS in group with high expression relative to group with low expression, which was consistent with cellular results.However, the result of CRC sample based on IHC seemed contrasted with the functional experiments of the LoVo cell. The results from IHC showed TTN was downregulated in CRC tissue, while over‐expressed TTN promoted CRC cell proliferation and migration in vitro. The possible explanation for the above results is that TTN was mainly observed to express in inflammatory cells based on IHC results. While the normal colon tissue has higher proportion of inflammatory cells than CRC tissues, higher TTN expression in the normal tissues was showed in IHC results compared to tumour tissues. In fact, TTN might be a potential oncogene in the development of CRC. Therefore, in tissues with high tumour heterogeneity, single‐cell sequencing can be conducted to avoid the interference caused by inflammatory cells in the tumour microenvironment. These results also indicate the potential impact of inflammation on CRC development, which is consistent with previous studies.In the GSEA analysis results, we identified six pathways that play important roles in the CRC carcinogenesis or chemoresistance. Histone modifications play a crucial role in regulating gene expression and chromatin structure. Aberrant histone modifications, such as histone methylation and acetylation, can lead to dysregulated gene expression patterns, including the activation of oncogenes and the silencing of tumour suppressor genes.Overall, the present study provides evidence for the role of TTN in CRC and confirms that inflammation remarkably impacts CRC development. Furthermore, GSEA was used to analyse the possible mechanisms of TTN‐induced colon cancer and found that the ERB pathway was associated with TTN in colon cancer based on bioinformatics analysis. Besides, TTN was also associated with response to azacitidine, indicating its potential role in improving chemotherapy resistance. To sum up, TTN critically impacts CRC occurrence and development through the ERB pathway and its high expression in cancer cells indicates a poor prognosis of colon cancer. Therefore, TTN might be a novel therapeutic target specific to CRC.5According to the results in the study, TTN may remarkably impact colon cancer process through the ERB pathway and high expression of TTN indicates a poor prognosis of colon cancer patients. Importantly, TTN can indicate the prognosis of CRC and serve as a new therapeutic target.Hongyun Wei: Formal analysis (lead); funding acquisition (lead); methodology (lead); project administration (lead); software (lead); writing – original draft (lead). Keyu Ren: Software ; writing – review and editing (supporting). Qian Zhang: Formal analysis ; methodology ; writing – original draft (supporting). Yanchun Jin: Writing – original draft (supporting). Bin Cao: Data curation (lead); validation (lead); writing – review and editing . Zibin Tian: Conceptualization ; data curation (supporting); writing – review and editing (supporting). Tao Mao: Validation ; writing – review and editing . Linlin Ren: Conceptualization (lead); data curation (supporting); funding acquisition ; project administration ; supervision (lead); validation (supporting); writing – review and editing (supporting).The work was completed with the support of the National Natural Science Foundation of China  and the Natural Science Foundation of Shandong Province  to Linlin Ren, Shandong medical and health science and technology development plan project (Grant number 202003030357) and Postdoctoral Science Foundation of China under Grant RZ2100002858 to Hongyun Wei.The authors declare no conflict of interest.


Nonsupplemented samples were used as a control. Sperm's progressive motility, acrosome reaction, and extracellular oxidative stress biomarkers such as MDA contents and GPx, SOD, and CAT concentrations and intracellular transcriptomic levels of SOD and CAT genes were assessed. GSH and taurine supplementation improved the sperm's kinetics by reducing cooling-associated stress, which was ascertained by lowering MDA concentration and increasing SOD, CAT, and GPx concentrations (P < 0.05). Increasing the levels of antioxidant enzymes in the extender was due to the increasing mRNA copies of the SOD and CAT genes (P < 0.05). Furthermore, GSH and taurine maintained the fructose levels in the extender and lowered the GPT levels, which implies sperm membrane stability is maintained through GSH and taurine supplementation. GSH and taurine supplementation to the extender had protective influences on the in vitro rabbit semen quality during chilled storage for up to 72 h, which were remarkable with increasing supplementation dose and cooling time at 5°C.This study assessed the influence of supplementing the rabbit semen extender with various concentrations of glutathione (GSH) and taurine at 24, 48, and 72 h postchilling at 5°C. Semen samples were collected from 20 New Zealand bucks, and ejaculates with standard color, motility (>85%), about 0.5 mL volume, and ∼400 × 10 With the increase in human population and the decrease in resources, especially with the global climatic changes, ensuring food and nutritional security is a fundamental requirement –5. Thus,Rabbit is an indispensable animal species to meet the nutritional requirements of human beings and for biomedical research. Rabbits are generally used as valuable meat sources due to their high feed conversion rate and fur production, and they are also used as pets . BesidesAI programs in rabbits are currently performed with fresh diluted semen collected within 6–18 hours, and bucks are typically kept on the same farm . Improvi2O2 production plus its oxidation to glutathione disulfide (GSSG). Moreover, the thiol group content of the amino acid cysteine in GSH penetrates the spermatozoa's plasma membrane enhancing the intracellular biosynthesis of GSH, which protects sperm's DNA, proteins, and its membrane-lipids content via scavenging of ROS [Many approaches have been utilized to preserve and improve semen quality and sperm viability, including the supplementation of antioxidant substances, such as glutathione (GSH), taurine, lycopene, cysteine, and glutamine directly to the animals or in semen extenders to increase the antioxidant abilities and lower the impacts of oxidative stress , 37. GSHg of ROS . The sulg of ROS .In the previous study of Ahmad et al, the exogenous antioxidants supplementation of GSH at 0, 1, 2, 4, and 8 mM to rabbit semen extender stored at 5°C for 24 h improved the semen attributes, especially the lower doses . HoweverThe current study was conducted following the ethical and regulation procedures of the Department of Animal Wealth Development, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt .n = 20) of the New Zealand white rabbit breed (about 2-3 kg body weight and 1–1.5-year-old). Bucks were individually labeled and caged with free access to feed and water. Animals were housed in well-ventilated rooms with a natural daylight supply. Bucks were trained for two weeks for semen collection using an artificial vagina and a mature female as a mount stimulator. Then, ten ejaculates from each buck were collected (one ejaculate/week), during the ten-week trial, for a total of 200 ejaculates. In each collection, fresh semen samples were microscopically examined and evaluated after removing the gel clot. The spermatozoa activity was microscopically evaluated at 100x  and 400x  using a phase-contrast microscope  [6/mL concentration were used for the study. During each time of collection, ejaculates were pooled (n = 20 with an average volume of ejaculates ∼ 8.75 ± 1.165 mL) to avoid individual variations and get sufficient semen volume for different treatments [This study used 20 rabbit bucks (Austria) . A Neubaeatments .The pooled semen samples were diluted in a 1 : 2 basic extender  containiμg/mL semen), and viability was morphologically assessed based on sperm's membrane integrity differences: sperms with damaged membrane were penetrated by PI stain showing partial or complete pinkish fluorescence, which indicates dead sperm. At the same time, the live sperm exhibited no fluorescence over the sperm head. Slides were examined at 1,000x magnification using a phase‐contrast microscope , and 200 sperms/slide were counted and evaluated.Sperm viability was evaluated for control and treated samples according to the literature . Sperms μL of every semen sample was incubated at 37°C for 20 min, placed on a prewarmed glass slide, and covered by a glass cover slider. Slides were examined at 400x. Sperm motility was assessed in five microscopic fields for each semen sample (in the five examined fields within 1 min). Sperm motility was evaluated as the percentage of spermatozoa that moved their flagellum. The average of the five fields was used to calculate the final sperm motility value [Sperms' progressive motility was evaluated by visually measuring the percentage of spermatozoa showing any movement of the flagellum using a phase-contrast microscope , that was equipped with a warm stage. The motility was measured after 24, 48, and 72 h of chilling for control and different treatments. Briefly, 5 ty value , 44.μL from each semen sample was smeared on glass slides, air-dried, fixed with 10% neutral formal saline for 15 min, washed in running water for 20 min, and allowed to dry. After drying, slides were immersed in Giemsa stain solution overnight. After incubation, the slides were rinsed with distilled water and air-dried. The acrosome status of 100 spermatozoa for each sample was examined at 1000x in each stained smear, using a phase-contrast light microscope . Purple heads characterized spermatozoa with intact acrosomes, whereas the damaged-acrosome spermatozoa had a pale lavender head. The percentage of acrosome-intact spermatozoa was calculated.Acrosome reaction was also evaluated after 24, 48, and 72 h of chilling in differently treated semen samples using Giemsa stain according to Watson's methods with some modifications . BrieflySemen samples at 24, 48, and 72 postchilling were centrifuged at 1,000 × g for 20 min, and the supernatant, composed of semen extender and seminal fluid, was used to assess oxidative stress biomarkers, using MDA contents and assessing the concentration of glutathione peroxidase (GPx), SOD, and CAT. Pellets of spermatozoa were washed by suspending them in cold fresh phosphate buffer saline, centrifuged at 3,000 × g for 10 min, and then frozen in liquid nitrogen for RNA extraction. Lipid peroxidation was evaluated by quantifying the MDA content (nmol/mL) using a commercial kit  according to manufacturer instructions. MDA was measured with 2-thiobarbituric acid, monitoring the absorbance change at 532 nm with the spectrophotometer . Antioxidant concentrations of CAT, SOD, and GPx were determined with commercial kits: CAT , SOD , and GPx , according to manufacturer's instruction.μl as a total reaction volume contained 10 μl SYBER green master mix, 2 μl cDNA, primers 0.8 μl (50 nm), and 6.4 μl RNase/DNase-free H2O [E = 100 × (10−1/slope-1). Samples for each treatment were repeated twice. The relative mRNA expression of the genes was calculated using the comparative threshold cycle method 2−ΔΔCT [Total RNA was extracted using TRI Reagent  following the manufacturer's protocol. Diluted semen samples were centrifuged at 3,000 × g for 20 min to eliminate semen extender and seminal fluid. After that, a pellet of spermatozoa was washed many times using ice-cold PBS. Lysis buffer was added to the sperm pellet and kept at room temperature for 10 min. Then, 1 ml TRI reagent was added, and extraction was completed. Afterward, complementary DNA (cDNA) was synthesized by reverse transcription reactions by iNtRON Biotechnology cDNA kits  according to the manufacturer protocol. Specific primers for antioxidant-related genes such as CAT, SOD, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, as shown in free H2O . The cycd 2−ΔΔCT , and theP < 0.05.Before data analysis, normality testing was performed by testing the Gaussian normal distribution with the Shapiro–Wilk normality test. Results of the current study were statistically analyzed using two-way ANOVA to test the effect of GSH and taurine supplementation, chilling time, and their interactions on semen quality, followed by Tukey's multiple comparison test using GraphPad Prism  –57. ResuP < 0.05).P < 0.05). At 24 h postchilling, 1, 5, and 10 mM taurine and 0.5, 1, and 2 mM GSH-supplemented semen samples had the lowest MDA content (P < 0.05). After 48 h of chilling at 5°C, all samples had less MDA content than those after 24 h of chilling (P < 0.05). However, 1 and 10 mM taurine supplementation had the lowest level of MDA compared to the other treatments and control (P < 0.05). Increasing the chilling time to 72 h was significantly associated with reducing MDA content in all treatments compared to the control (except 5 and 10 mM taurine as well as 0.5 mM GSH). GSH and taurine supplementation lowers oxidative stress in chilled semen samples, up to 72 h, by reducing lipid peroxidation, confirmed by decreasing MDA contents.P < 0.05). After 24 h chilling, higher concentrations of GSH and taurine  significantly increased SOD activities in semen samples, compared to the lower concentration (0.5-mM GSH and 1 mM taurine) and nonsupplemented semen (P < 0.05). Increasing the chilling time to 48 and 72 h was associated with increasing SOD in all GSH and taurine-supplemented samples, compared to nonsupplemented semen samples (P < 0.05). GSH and taurine supplementation in semen extender were associated with increasing SOD and increased chilling time.P < 0.05). In this context, all supplementing taurine and GSH doses except 0.5 mM GSH prominently increased GPx levels at 24 h postchilling. The same effect was also reported at 48 h and 72 h postchilling for all taurine and GSH doses except 0.5 mM GSH, which only was able to induce higher GPx after 72 h of chilling (P < 0.05).GPx activity in semen samples was also measured at different chilling times, and GSH and taurine supplementation . NonsuppP < 0.05). Increasing chilling time to 48 h was associated with higher activities of CAT in the samples supplemented with 1 mM and 2 mM GSH along with the 1 mM taurine compared to the other treated samples (P < 0.05). Nevertheless, at 72 h postchilling, all treatments had nearly the same CAT activities. Additionally, the effect of GSH and taurine supplementation was significantly modified by chilling time. The highest activities of CAT were measured at 24 h and 48 h postchilling in the case of 1 mM GSH and taurine. At the same time, no differences were noticed for the other supplemented groups.CAT activities in supplemented semen samples and at different chilling times were evaluated . After 2P < 0.05). Conversely, semen extenders supplemented with different doses of GSH and taurine showed slight changes in fructose levels at different chilling times. At 48 and 72 h postchilling, the fructose levels in nonsupplemented semen extender (control) were significantly higher than those in supplemented ones, especially if compared with those treated with 5 and 10 mM taurine (P < 0.05).P < 0.05). In comparison with the nonsupplemented group (control), all supplemented semen extenders, either by GSH or taurine (at different doses), had significantly fewer GPT levels (P < 005). However, no significant differences in GPT levels were found among supplemented semen extenders. Besides, the GPT level in nonsupplemented extenders significantly increased with increasing chilling times (P < 0.05). However, it remained unchanged in the case of supplemented extenders with either GSH or taurine.GPT levels were also influenced by supplementations of semen extender with GSH and taurine and chilling time (P < 0.05). The highest expression levels of SOD were determined in the case of 2 mM GSH and 1 mM taurine, followed by 0.5 mM and 5 mM taurine. Increasing the cooling time to 72 h was associated with higher expression levels of SOD, especially in the case of 1 mM GSH and 10 mM taurine, compared with nonsupplemented or GSH- and taurine-supplemented samples (P < 0.05).P < 0.05), except for the integration with 1 mM taurine. The highest expression levels were measured at 24 and 48 h postchilling for both treated (except 1-mM taurine) or untreated samples. The expression level fluctuated with different GSH supplementation doses; the highest level was found in the case of 1 mM GSH compared to 2 mM and 0.5 mM GSH, while a different response was reported for taurine supplementation. Increasing the taurine dose was associated with lowering CAT mRNA levels at 48 and 72 h postchilling. At 24 h postchilling, minor increases were found with increasing taurine dose.The CAT gene's relative expression level was modulated with GSH/taurine supplementation and cooling times. Increasing chilling time to 72 h was linked with a significant reduction of CAT mRNA copies in samples either nonsupplemented or supplemented with GSH or taurine (2−), hydrogen peroxide (H2O2), and lipid hydroperoxides [Cooling and freezing/thawing procedures of semen markedly alter the quality of preserved sperm. In this regard, long-term cooling deteriorates sperm quality and, consequently, its fertilizing ability because of the excessive release of ROS such as superoxide anion radicals  semen diluted in tris-based extender supplemented with 25 and 50 mM taurine, respectively [Supplementing the extender with antioxidant substances such as GSH and taurine improved the semen quality, as confirmed by the significant increase in the percentages of live spermatozoa, motile sperms, and acrosome reactions. However, at 24 h postchilling, GSH and taurine did not alter these parameters compared to the control. This effect is probably related to the natural antioxidant potency of sperm to scavenge ROS production . The posectively , 64. Addectively . Moreoveectively . Our finectively , 65, whoectively . The varectively , pH, andectively . Besidesectively .The improving effects of both GSH and taurine may also be due to the prevention of lipid peroxidation, as confirmed by lowering the MDA concentrations in GSH and taurine-treated semen compared to nontreated semen. This effect ascertains the protective role of both taurine and GSH against lipid peroxidation and sperm membrane damage. The high content of polyunsaturated fatty acids (PUFA) in the sperm plasma membrane increases its chance to get deteriorated and damaged, and the consequent release of PUFA, which binds to oxygen, increases ROS production . This fiCAT and SOD genes. The upregulations of CAT and SOD following GSH and taurine supplementation to semen extender might be linked to the APS kinase (APK) and glutathione S-transferases (GST) family genes modulation by GSH [Another interesting finding is that increasing SOD, GPx, and CAT concentrations are linked with increasing the supplementation dose and the cooling time. This finding might explain the improving effect of the exogenous supplementation of GSH and taurine on sperm quality. Cooling and cryopreservation processes impair the sperm's functions and lower its fertilizing capacity by either overproduction of ROS or reducing the antioxidant defense system . Interesn by GSH  and to tn by GSH .Therefore, evaluating the transcriptomic levels of these pathways following taurine and GSH supplementation is recommended. Despite the reported improving effects of GSH and taurine on semen quality, this study has some limitations, including predicting the real male fertility. Also, this study did not highlight the problem of spermatozoa that occurs after entering the female tract. Therefore, other tests such as sperm penetration, sperm viability, and tests examining the sperm's attachment and fertilizing capacity (pregnancy test) are recommended in the future. Moreover, extending this study to cover as long as possible, up to 5-6 days, is recommended.Exogenous supplementation of the extender with GSH and taurine improved the quality of rabbit semen cooled at 5°C by increasing the percentage of live spermatozoa, motile sperms, and acrosomal reaction, as confirmed by maintaining fructose level and decreasing GPT in extracellular fluid. Moreover, both GSH and taurine reduced cooling-associated oxidative stress by lowering MDA concentration and increasing SOD, CAT, and GPx levels, along with upregulating the transcription levels of SOD and CAT genes. These improving effects increased with the supplementation dose and the cooling time to 72 h. Therefore, based on these results, the 2 mM GSH and 10 mM taurine can be effectively supplemented with a rabbit's semen extender to enhance the in-vitro sperm quality and antioxidant status of New Zealand rabbits' sperm during chilled storage for up to 72 hours.


Sphagnum‐dominated zones. Vegetation plot and water chemistry data were collected from string‐flark fens, transition zones with indications of Sphagnum increase, and bog zones; thus, in a chronosequence with a decadal time span. We ask, is there a common trajectory or many alternatives of FBT in progress, and what are the main characteristics (species and traits) of transitional plant communities? We found a pattern of fen‐bog transitions via an increase in Sphagnum sect. Cuspidata , indicating a consistently high water table. Indicators only of transitional communities were scarce (Sphagnum lindbergii), but FBT had apparently facilitated shallow‐rooted aerenchymatous vascular plants, especially Scheuchzeria palustris. Water pH consistently reflected the chronosequence with averages of 4.2, 3.9, and 3.8, from fen to transition and bog zones. Due to weak minerotrophy of string‐flark fens, species richness increased towards bogs, but succession led to reduced beta diversity and homogenization among bog sites. Decadal chronosequence suggested a future fen‐bog transition through a wet phase, instead of a drying trend. Transitional poor‐fen vegetation was characterized by the abundance of Sphagnum lindbergii, S. majus, and Scheuchzeria palustris. Sphagnum mosses likely benefit from longer growing seasons and consistently wet and acidic conditions of aapa mires.Climate change‐driven vegetation changes can alter the ecosystem functions of northern peatlands. Several case studies have documented fen‐to‐bog transition (FBT) over recent decades, which can have major implications, as increased bog growth would likely cause cooling feedback. However, studies beyond individual cases are missing to infer if a common trajectory or many alternatives of FBT are in progress. We explored plant community and hydrology patterns during FBT of 23 boreal aapa mire complexes in Finland. We focused on mires where comparisons of historical (1940–1970) and new (2017–2019) aerial photographs indicated an expansion of  Sphagnum lindbergii, S. majus, and S. balticum) and Scheuchzeria palustris, highlighting the fact that under climate change, boreal fens may experience a shift to Sphagnum‐dominated bogs through a wet phase.We studied recent fen‐bog transitions and the characteristics of transitional plant communities in 23 boreal aapa mires in a spatial chronosequence with a decadal time span. We found a pattern of fen‐bog transitions via an increase in wet Sphagna ( In the three spatial zones, plots were recorded as pairs located on two microhabitats: (1) a hummock string and (2) a flark area between strings (lawns in bogs). In a previous study, we used a multiproxy approach including a detailed analysis of dated peat stratigraphies to verify recent FBTs at five out of the 23 sites studied here  and new (2017–2019) aerial photographs indicated recent lateral expansions of bog zones by an increase in Specifically, we ask (1) what is the succession trajectory of aapa mire complexes in the present climate, or are there multiple successional pathways, (2) can plant communities of modern FBT be recognized and what are their main characteristics , (3) how water chemistry and water‐table depth (WTD) reflect fen‐bog transition, and (4) how fen‐bog transition alters the diversity patterns of mire complexes.22.1n = 23) locate in the north and middle boreal zones of Finland (Table open data). During the same period, MAT increased by 0.4°C per decade and annual precipitation sum by 16 mm per decade. In the southernmost site , MAT was 2.5°C and the average annual precipitation sum was 671 mm. From 1961 to 2020, MAT increased by 0.3°C per decade and annual precipitation sum by 7 mm per decade.The study sites , with some exceptions  of each plant species was estimated in six locations , although some bog hummocks had too deep water table for obtaining a sufficient water sample.Water‐table depth (WTD) was measured in each plot as a distance from the water level to the top of the bryophyte layer. Water pH was measured in the field with a portable pH/conductivity tester (Consort). A water sample of 50 mL was obtained in each plot for chemical analyses using a 50‐cm long perforated plastic pipe well  of the relevé data, using function metaMDS in the package A permutation‐based nonparametric multivariate analysis of variance (PERMANOVA) was used to test the difference in bryophyte and vascular plant communities between the mire zones using the relevé data. PERMANOVA analyses were run in PC‐ORD 7.08  using the relevé data. This was done in R version 4.1.1  and micro‐topographic patterns (flarks and strings), and their interactions were tested using a general linear model with zone and pattern as fixed factors and mire as a random factor. Tukey HSD post hoc tests were separately performed for strings and flarks to study pairwise differences between zones. General linear models and post hoc tests were run in SPSS Version 27 and using data from subplots of 0.25 mn = 66) as gamma diversity/alpha diversity, where gamma is the total species number and alpha mean species number in the two subplots. Differences in beta diversity between mire zones were tested using Wilcoxon signed rank test and function compare_means in the package ggpubr .Plant species nomenclature follows the Finnish Biodiversity Info Facility  and 22 mineral elements, but several mineral elements were under or close to detection limits and were omitted from the statistical analyses. Before DOC and elemental analyses, samples were filtered through 0.45 μm sterilized membrane  or syringe filters . Subsamples for DOC analysis (10–15 mL) were analyzed with a multi N/C® 2100 TOC analyzer  within 2 months after sampling. Subsamples for elemental analysis (10–15 mL) were analyzed by Inductively Coupled Plasma—Mass Spectrometry (ICP‐MS) using a NeXION 350D ICP‐MS instrument (PerkinElmer Inc.). Multi‐element standard solution  was used for the calibration of ICP‐MS.n = 68) because two to four water samples were collected per zone in each site. Normality of the data was assessed from Q–Q plots and using the Shapiro–Wilk normality test  in R version 4.1.1  between the mire zones . Average per zone was used in the comparisons (33.1p < .05) associations to single microhabitats, ten species were associated with combinations of two microhabitats, seven species were associated with three microhabitats, four species with four microhabitats, and one species with five microhabitats.Among the 66 species, 37 (56%) showed significant habitat associations in the indicator species analysis , fen strings , bog lawns , and bog hummocks . Sphagnum lindbergii was the only species to show a significant association only with the transitional flarks, while the transitional strings had no distinct indicators. Six species were indicative of all hummock surfaces, irrespective of mire zones, while only one flark species  had a joint indication of all wet surfaces.Several strong indicators were found for the fen flarks , and the number of all vascular plant species (p = .002), nonaerenchymatous (p < .001), and aerenchymatous species , nonaerenchymatous species (p < .001), and shallow‐ and deep‐rooted aerenchymatous species . In the cases of species richness, bryophyte cover, and the number of bryophytes, all vascular plants, and aerenchymatous species, the response depended on the pattern (significant interaction with p < .01). On average, species richness and the covers of bryophytes, all vascular plants, and nonaerenchymatous species were highest in the bogs, while the cover and number of aerenchymatous species were highest in the fens.In total, 78 species were recorded in the study sites . When both flark and string plots were included in ANOVA, significant differences between mire zones were found in overall species richness  compared with bog hummocks (mean 2.8). The number and cover of nonaerenchymous species were significantly higher in transition zones and bogs than in fens, while transition zone did not differ significantly from bogs. The number of aerenchymatous species was significantly lower in bogs than in transition zones and fens, but the cover did not differ significantly between zones. The cover of shallow‐rooted species was significantly higher in fen strings than in bog hummocks.p < .0024; Figure p = .0554) or between transition zones and bogs.Beta diversity, as defined for each string‐flark pair, was significantly higher in fens than in bogs (3.4p < .001), and significantly higher than in the bogs (p < .01). In DOC, no significant difference was found between the fen and transition zones, while DOC was significantly higher in the bog than in the fen (p = .021) and the transition zones (p = .028). Concentrations of Ca, Mg, and Mn were significantly lower in the transition zones than in the fen zones . Ca and Mg concentrations did not differ significantly between the transition zones and the bog zones, while Mn concentrations were significantly higher (p < .01) in the transition zones. On average, concentrations of Al and Fe were highest in the fen zones, but no significant differences were found.Generally, pH and all mineral element concentrations were low in all study sites. The string‐flark fen zones had the highest average pH , Ca concentration , and Mg concentration . Between the mire zones, significant differences were found in pH, DOC, and concentrations of Ca, Mg, and Mn  and all hummock surfaces (p < .05).The average WTD was 1 cm (SD 4.3) in the fen flarks, 7 cm (SD 5.2) in the transition flarks, and 12 cm (SD 8.6) in the bog lawns Figure . On humm44.1Sphagnum section Cuspidata. The most abundant species in transitional flarks, in descending order, were Sphagnum majus, Scheuchzeria palustris, Sphagnum balticum, S. papillosum, S. jensenii, S. lindbergii, and Eriophorum vaginatum. S. lindbergii was indicative only of transitional flarks, but there were no species that only occurred in transitional communities. Indeed, Gałka et al.  and fewer species that were shared with bog lawns  and many generalist hummock species . Most remarkably, many fen species had significantly decreased or were absent from transitional vegetation, including aquatic flark species  and sedges in strings . Thus, the transitional character in vegetation resulted from an altered selection between species, instead of the dispersal of transitional habitat specialists.Our study of spatial chronosequences in 23 boreal aapa mire complexes showed a fairly uniform pattern of fen‐bog transition (FBT) through a quaking moss carpet phase with an increase in a et al.  found S.Sphagnum mosses. Instead, Sphagnum expansion had favored some shallow‐rooted aerenchymatous vascular plants. Most importantly, Scheuchzeria palustris was particularly abundant in transitional flarks, probably facilitated by the formation of quaking Sphagnum carpets  compared with seedbeds of Cladopodiella fluitans, a common liverwort of fen flarks in aapa mires. As they justified, germination may be hindered on compact C. fluitans seedbeds due to the fact that they are difficult for large seeds to penetrate, while S. Cuspidata carpets are more porous and still have high water level, facilitating germination and better root development  likely belongs to the alliance Sphagnion medii of classification of European bog vegetation , and indicators of intermediate fens  were often present already in the flark fens and they continued to occur also in the bog lawns, where S. balticum became the dominant moss species. This trajectory reappeared in most of our study sites. Elsewhere in a rich fen, we found an increase in hummock Sphagnum instead of wet Sphagna . This site had experienced one of the most significant expansions of a Sphagnum‐dominated zone with S. fallax as the main species of the transition zone, and bog lawns had S. medium coll. and S. papillosum , and both Ca and Mg concentrations were under 1 mg/L in the fen flarks. As expected, the pH and concentrations of Ca and Mg were higher in the fen flarks than in transitional communities and bogs. Groundwater pH and Ca concentration are relatively low in the whole Fennoscandia due to glacial history and calcium‐poor bedrock ; formal analysis (lead); funding acquisition ; investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead). Teemu Tahvanainen: Conceptualization ; formal analysis (supporting); funding acquisition ; investigation (supporting); methodology (supporting); project administration (lead); supervision (lead); writing – review and editing (lead).We declare no conflict of interest.Appendix S1.Click here for additional data file.


This study aimed to investigate the presentations and survival outcomes of the distant metastatic non‐small cell lung cancer (NSCLC) without lymph node involvement to obtain a clearer picture of this special subgroup of metastatic NSCLC.A least absolute shrinkage and selection operator (LASSO) penalized Cox regression analysis was used to select the prognostic variables. A nomogram and corresponding risk‐classifying systems were constructed. The C‐index and calibration curves were used to evaluate the performance of the model. Overall survival (OS) curves were plotted using the Kaplan–Meier method, and the log‐rank test was used to compare OS differences between groups. Propensity score matching (PSM) was performed to reduce bias.A total of 12 610 NSCLC patients with M1 category  were included. Regarding the N0 group, multivariate analysis demonstrated that age, sex, race, surgery, grade, tumor size, and M category were independent prognostic factors. A nomogram and corresponding risk‐classifying systems were formulated. Favorable validation results were obtained from the C‐index, calibration curves, and survival comparisons. Survival curves demonstrated that N0 NSCLC patients had better survival than N1‐3 NSCLC patients both before and after PSM. Furthermore, the survival of resected N0M1 patients was superior to that of those without surgery.In this study, a prognostic nomogram and risk‐classifying systems designed for the T1‐4N0M1 NSCLC patients showed acceptable performance. Primary lung tumor resection might be a feasible treatment for this population subset. Additionally, we proposed that lymph node stage might have a place in the forthcoming tumor‐node‐metastasis (TNM) staging proposal for NSCLC patients with M1 category. The central messages of this study are presented in this graphical abstract. NSCLC is non‐small cell lung cancer. Permission was obtained to retrieve SEER data files with the reference number: 12962‐Nov2019. Due to the fact that patient data in the SEER database are de‐identified, this study was dispensed with signing informed consent forms and acquiring ethical approval.All included cases fit the following criteria: [1] confirmed as NSCLC; [2] diagnosed as M1 category (8th TNM staging system); and [3] received treatments. The exclusion criteria were as follows: [1] clinicopathological characteristics unknown; [2] age < 18 years old; [3] previous or concurrent other cancers; and [4] survival time ≤ 1 month.The entire cohort was categorized into two subgroups: the N0 group and the N1‐3 group. The N0 group was further divided into the training group and the validation group with a ratio of 3:1, and the development of the prognostic nomogram was based on data in the training set. The study flow for patient selection is depicted in Figure 2.2The patients' demographic and clinicopathological features included sex , age , race , marital status (married and other), tumor location , histology , tumor size , tumor grade , surgery (no and yes), radiotherapy (no and yes), chemotherapy (no and yes), M category , bone metastasis (no and yes), brain metastasis (no and yes), liver metastasis (no and yes), patient status, and survival time. A complete data analysis was performed in this study. The 6th and 7th editions of the TNM staging systems were converted to the current 8th edition of the TNM staging system. Because several patients have not received surgical resection, so both the pathological and clinical TNM stage was used in this study.2.3Information on survival time and patient status was available in the SEER database. Patients who had definitive status and exact survival time were included in this study. Patients whose survival time was less than or equal to 1 month were excluded. Overall survival (OS), defined as the interval between the date of diagnosis and the date of death from any cause or the last follow‐up, was the primary endpoint of this study. The median follow‐up time was 10 months (range from 2 to 83 months) in the entire cohort, 13 months (range from 2 to 83 months) in the N0 group, and 10 months (range from 2 to 83 months) in the N1‐3 group.2.4http://www.r-project.org) and IBM SPSS Statistics . Least absolute shrinkage and selection operator (LASSO) regression analysis was performed to select and minimize the prognostic variables of the N0 group.χ2 test between groups. Continuous variables, provided as the mean and standard deviation (SD), and median (range), were compared using the Mann–Whitney U test between groups. Two‐sided P values < 0.05 was considered statistically significant.Statistical analyses were carried out by R version 4.1.1  and the N1‐3 group (N = 9565). The N0 group patients were randomly divided further into a training cohort (N = 2284) and a validation cohort (N = 761).From 2010 to 2016, a series of 360 702 NSCLC cases from the SEER dataset were retrospectively evaluated. A total of 12 610 eligible M1 cases were retained following the application of our inclusion and exclusion criteria and were separated into the N0 group (P < 0.001) and had smaller tumor sizes (P < 0.001). More poorly differentiated tumors were diagnosed in the N1‐3 NSCLC group than in the N0 group (P < 0.001). Patients in the N1‐3 NSCLC group were less likely to receive surgery (P < 0.001) and radiotherapy (P < 0.001), and almost all of the patients had received chemotherapy . The clinicopathological features of the patients with N0 and N1‐3 category after PSM were summarized in Table The general demographic, clinical, and pathological characteristics of the N0 and N1‐3 NSCLC cases are described in Table 3.2P < 0.001), sex , race , surgery , grade , tumor size , and M stage  were independent prognostic factors .LASSO regression model analysis was carried out to select the prognostic factors of N0 NSCLC with the best predictive performance using 10‐fold cross‐validation in the training set3.3Based on the results of multivariate Cox analysis, a prognostic nomogram was constructed Figure . The nom3.4P < 0.001, Figure P < 0.001, Figure P < 0.001, Figure Each category of the prognostic factors was assigned a score on the points scale in the nomogram. After summing the total score, the continuous scores were divided into two subgroups  based on the cutoff value (33) and into three subgroups  based on the cutoff values 26 and 33) , endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA), and mediastinoscopy are needed. However, the related data are unavailable in the SEER database, and we hoped other data including the distant metastatic NSCLC patients with definite N category could validate our results in the future. Second, the SEER database also lacks data on novel therapies, the treatment schedule and the reason why these metastatic patients received surgery, and all these variables are important factors in survival analysis. Third, the patient population used to formulate this nomogram was predominantly of White ethnicity, and external validation from the Asian population is warranted. At last, although large sample data from the SEER database might be less prone to the bias toward the null hypothesis, selection bias may still be present due to the retrospective design of the study. Efforts to include prospective study designs and broader clinicopathological variables are encouraged for future studies.5In conclusion, in this study, an efficient nomogram and risk‐classifying systems were designed for T1‐4N0M1 NSCLC that showed acceptable performance. Primary lung tumor resection may be a feasible treatment for this population subset. In addition, we proposed that lymph node stage might have a place in the TNM staging proposal for the M1 NSCLC patients.(I) Conception and design: Su‐Wen Zhang and Jing‐Sheng Cai. (II) Administrative support: Su‐Wen Zhang. (III) Provision of study materials or patients: Bao‐Wen Huang and Wen‐Qin Wang. (IV) Collection and assembly of data: Bao‐Wen Huang. (V) Data analysis and interpretation: Bao‐Wen Huang. (VI) Manuscript writing: all authors. (VII) Final approval of manuscript: all authors.The authors declare that there is no conflict of interest regarding the publication of this article.Permission was obtained to retrieve SEER data files with the reference number: 12962‐Nov2019. Due to the fact that patient data in the SEER database is de‐identified, this study was dispensed with signing informed consent forms and acquiring ethical approval.Figure S1. The calibration curves for predicting OS in the training cohort  and the validation cohort . Nomogram‐predicted survival probability is plotted on the x‐axis; Actual observed survival probability is plotted on the y‐axis. A curve along the 45‐degree line indicates perfect calibration models. OS, overall survival.Click here for additional data file.Figure S2. Validation of the risk‐classifying systems. (A) ROC curves comparison: Risk‐classifying system A vs. TNM staging system, (B) DCA comparison: Risk‐classifying system A vs. TNM staging system, (C) ROC curves comparison: Risk‐classifying system B vs. M staging system and (D) DCA comparison: Risk‐classifying system B vs. M staging system. ROC: receiver operating characteristic, DCA: decision curve analyses; TNM, tumor‐node‐metastasisClick here for additional data file.Table S1. The Clinical characteristics of the N0M1 and N1–3M1 patients after PSMTable S2. The Clinical characteristics of the N0M1 patients without surgery and with surgery after PSMClick here for additional data file.


To build a nomogram prediction model, assess its predictive ability, and perform a survival decision analysis on patients with muscle‐invasive bladder cancer (MIBC) to study risk factors affecting overall survival (OS).A retrospective analysis was performed on the clinical information of 262 patients with MIBC who underwent radical cystectomy (RC) at the Urology Department of the Second Affiliated Hospital of Kunming Medical University between July 2015 and August 2021. The final model variables that were included were chosen using single‐factor stepwise Cox regression, optimal subset regression, and LASSO regression + cross‐validation with the minimum AIC value. The next step was to do a multivariate Cox regression analysis. The establishment of a nomogram model by fitting and the screening out of independent risk factors impacting the survival of patients with MIBC having radical resection. Receiver Activity Characteristic curves, C‐index, and a calibration plot evaluated the prediction accuracy, validity, and clinical benefit of the model. The 1‐, 3‐, and 5‐year survival rates were then computed for each risk factor using a Kaplan–Meier survival analysis.p = 0.001), preoperative hydronephrosis , T stage , lymphovascular invasion , prognostic nutritional index , and neutrophil‐to‐lymphocyte ratio ], p = 0.026) were independent risk factor for the survival of bladder cancer patients. Create a nomogram based on the aforementioned findings, and then draw the 1‐year, 3‐year, and 5‐year OS receiver operating characteristic curves by the nomogram. The AUC values were 0.811 , 0.814 , and 0.787 , respectively, and the calibration plot matched the predicted value well. The 1‐year, 3‐year, and 5‐year decision curve analyses were higher than the ALL line and None line at threshold values of >5%, 5%–70%, and 20%–70% indicating that the model has good clinical applicability. The calibration plot for the Bootstrap 1000‐time resampled validation model was similar to the actual value. Patients with preoperative combination hydronephrosis, higher T‐stage, combined LVI, low PNI, and high NLR had worse survival, according to Kaplan–Meier survival analysis for each variable.262 eligible patients in total were enrolled. With a median follow‐up of 32 months, the follow‐up period ranged from 2 to 83 months. 171 cases (65.27%) survived while 91 cases (34.73%) perished. Age (HR = 1.06 , This study might conclude that PNI and NLR were separate risk factors that affect a patient's OS after RC for MIBC. The prognosis of bladder cancer may be predicted by PNI and NLR, but additional confirmation in randomized controlled trials is required. It occurs more frequently and in younger people.Reduced post‐operative survival rates are a result of radical cystectomy's high level of invasiveness, higher T‐stage bladder cancer, and patients' suboptimal nutritional and immunological conditions before surgery.PNI can be used to assess a patient's health status, surgical risk, and postoperative complications in the early stages of their illness, based on the serum albumin level and the total number of lymphocytes. It reflects the nutritional and immune status of patients with malignant tumors. Numerous studies have demonstrated that PNI is a reliable prognostic indicator for a variety of tumor types and is an independent risk factor for predicting malignant tumors.The Nomogram, a straightforward graphical representation of a statistical prediction model, is frequently used to forecast cancer prognosis by estimating the likelihood of a clinical event. The Nomogram is recommended as an alternative or perhaps as a new standard because it outperforms the conventional TNM staging system for many malignancies. Make customized predictions using the Nomograms to classify and identify patients for clinical trial participation. Combined with the above research background, for patients with MIBC who underwent RC, a survival prediction model and decision analysis were created in this study using PNI and NLR.22.1The clinical data of 262 patients with MIBC who underwent RC from July 2015 to August 2021 in the Department of Urology of Second Affiliated Hospital of Kunming Medical University were retrospectively analyzed. Inclusion criteria: (I) pathologically confirmed MIBC; (II) RC performed; (III) complete routine blood and blood biochemistry data 1 week before surgery; (IV) immunohistochemical indices such as Ki 67, P53 and P63 available; (V) regular post‐operative follow‐up. Exclusion criteria: (I) concurrent other tumors; (II) concurrent other systemic significant diseases such as inflammatory diseases, liver diseases, auto‐immune diseases, hematological diseases, and cardiovascular diseases; (III) pre‐ and post‐operative co‐infectious diseases; (IV) severe complications in the perioperative period; (V) patients receiving pre‐operative neoadjuvant therapy; (VI) patients with no follow‐up records or missed follow‐ups. All patients had no preoperative neoadjuvant therapy, no other major systemic diseases in combination, and no severe complications in the perioperative period. The Ethics Committee of the Second Affiliated Hospital of Kunming Medical University gave its clearance to this investigation . The study was done by the Declaration of Helsinki, and each participant provided their informed permission.2.29/L); PNI = serum albumin (g/L) +5 × peripheral blood lymphocytes cells (×109/L). Kaplan–Meier survival analysis based on a log‐rank test determined the optimal cut‐off values of PNI, NLR, and Ki‐67 and divided them into high and low groups.The blood routine and blood biochemical data of all patients within 1 week before the operation was calculated according to the following formula: NLR = neutrophils/lymphocytes , and orthotopic neobladder are all examples of urinary diversions.2.5x2 test. The data were expressed as x¯±s¯ and the independent sample t‐test was run if the measurement data fit the normal distribution and homogeneity of variance. The data were reported as median [IQR] and the nonparametric rank‐sum test (Wilcoxon test) was run if the data did not fit a normal distribution and/or the variance was not uniform. Univariate Cox regression analysis, optimal subset regression, LASSO regression, and cross‐validation were used to find the best set of variables. The multivariate COX analysis included the factors that were screened by the three techniques. The final filtered variables of the three methods were determined using the backward stepwise regression method, and the model with the smallest Akaike Information Criterion (AIC) value was constructed. Among the three comparison methods, the model with the smallest AIC value was used to construct the Nomogram. Nomograms were created to predict 1‐, 3‐, and 5‐year OS based on multivariate models. The Nomogram used a graphical depiction to connect several prognostic risk variables of patients to the likelihood of post‐operative OS in MIBC patients. The area under the receiver operating characteristic (ROC) curve, also known as the Concordance index (C‐index), which measured the Nomogram's discrimination, was almost identical to the area under the ROC curve. Using a calibrated plot, the Nomogram's calibration was intuitively assessed. When the model's anticipated value and the patient's risk match, the 45° line denoted a flawless calibration. Under‐prediction or over‐prediction, respectively, was indicated by deviations above or below the 45° line. The clinical value of the Nomogram was confirmed using the decision curve analysis (DCA). A curve that was above the None line and the All line denoted a net advantage and suggested that the model was more clinically applicable. The cutoff for this study's statistical significance was p < 0.05. (Download version R4.2.1 from Tsinghua University and the TUNA Team in China at mirrors.tuna.tsinghua.edu.cn/CRAN/).For statistical analysis and the creation of statistical graphics, R4.2.1 software was utilized. Count data were presented as percentages and tested using either Fisher's exact probability test or the 33.1p > 0.05). Urothelial carcinomas (UC) with 63 (24%) squamous differentiation, 11 (4.2%) glandular differentiation, 3 (1.15%) micropapillary, 1 (0.38%) lymphoepithelioma‐like, 1 (0.38%) microcystic, 1 (0.38%) clear‐cell, 1 (0.38%) neuroendocrine variant, 1 (0.38%) small‐cell cases. 1 case (0.38%) each of simple intestinal adenocarcinoma, plasmacytoid (0.38%), and 2 cases (0.76%) each of simple lymphoepithelioma .Based on the inclusion and exclusion criteria, 262 cases were finally included Figure . In this3.2p > 0.05), while age history of hypertension, presence of pre‐operative Hydronephrosis, T‐stage, Presence of LVI, PNI and NLR and their subgroups were statistically significantly different from OS (p < 0.05).Among the 262 patients, 91 (34.73%) died, and 171 (65.27%) survived. Each potential risk factor was analyzed separately to see whether it affected the OS of the patients. Statistical results showed (Table p < 0.05). The groups for PNI and NLR were high and low, respectively. The two variables were then reorganized into four groups: high PNI low NLR, high PNI high NLR, low PNI low NLR, and low PNI high NLR. According to Kaplan‐log‐rank Meier's test for survival analysis .The aforementioned statistically significant risk factors were subjected to a univariate Cox regression analysis Table . Age, his Figure , the grop < 0.05). Among them, the risk factors discovered by the aforementioned univariate Cox regression analysis were submitted to multivariate Cox regression analysis (Table p < 0.05). The presence of pre‐operative hydronephrosis was p > 0.05 among them. Even yet, using techniques like cross‐validation and optimal subset regression, the presence of preoperative hydronephrosis was identified as one of the most effective sets of factors. Preoperative hydronephrosis was proven to be a separate risk factor for survival following RC for bladder cancer in earlier research.The risk factors obtained from the above single‐factor Cox regression analysis were subjected to multi‐factor Cox regression analysis Table . Stepwisis Table , and the3.33.3.1According to the results of multivariate Cox analysis, independent risk factors such as age, preoperative hydronephrosis, T stage, lymphatic vessel invasion, PNI, and NLR were fitted to construct the Nomogram of the 1‐year, 3‐year, and 5‐year OS columns after RC in MIBC patients Figure .3.3.2This study used internal validation to assess and validate each of the three aspects of the model: discrimination, calibration, and clinical benefit.The ability to predict 1‐year OS, 3‐year OS, and 5‐year OS was evaluated using the C‐index and the area under the ROC curve Figure , but it Utilize calibration plots and Bootstrap techniques to validate prediction models. For this study's internal validation procedure, the predictive model's precision was examined. The estimation of 1‐year, 3‐year, and 5‐year OS following RC was confirmed using 1000‐time resampling of raw data using the R soft. Calibration plots were also created Figure . The calDecision curve analysis (DCA) was used to assess the extent of patient clinical benefit. 1‐year, 3‐year, and 5‐year decision curve analyses showed that the decision curves were always higher than the “None” line and “All” line, and the model has clinical applicability Figure . The DCA4The most prevalent malignant tumor of the urinary tract, bladder cancer has a high fatality rate, an increasing prevalence, and an aging population. MIBC is becoming more prevalent. The current gold standard still includes PLND and RC and neoadjuvant cisplatin‐based chemotherapy (NAC). The clinical questions, which reflect a variety of survival issues, include, however, OS and 1–5 year survival after radical cystectomy in patients with bladder cancer. This study developed and validated a nomogram model to predict OS following radical cystectomy using two prognostic variables, PNI and NLR. The Nomogram had a fantastic net clinical benefit, according to the DCA. As a result, the nomogram model from this study aids doctors in predicting OS and clinical choice following radical cystectomy.PNI is a prognostic indicator first proposed by Buzby et al.In many solid tumors, NLR has been used to predict survival and postoperative problems.p = 0.012), and multivariate analysis revealed that LVI status was an independent prognostic factor for MIBC patients following RC.LVI is crucial for spreading and engulfing bladder cancer cells. Positive LVI implied a substantial relationship between lymph node metastases, histological grade, and pathological stage.p > 0.05). Based on earlier investigations, it was still nevertheless incorporated into the model. The accuracy and validity of the model were not affected by including preoperative hydronephrosis, and Kaplan–Meier survival analysis  but not an independent risk factor (p = 0.8618) for OS after RC. Both Claps et al.A multi‐center, multi‐laboratory analysis in 1058 RC patientsThe prediction model using age, presence of pre‐operative hydronephrosis, T‐stage, LVI, PNI, and NLR had a good C‐index and correlated well with the actual occurrence. The calibration plot also showed that the Nomogram could effectively predict OS after RC in patients with MIBC, which had good clinical application. The DCA results showed that the model had good clinical applicability.The Nomogram can guide clinicians in making personalized clinical decisions and improve post‐operative follow‐up and timely identification of problems for appropriate management. In addition, the PNI and NLR used to construct the Nomogram were easy to obtain and low cost, which made the nomogram model clinically applicable. At the same time, there are some shortcomings in this study. First, this study was a retrospective study with a small sample, which prevented any bias in the follow‐up of patients. Second, the values of the Nomogram in this study were validated internally but not externally to validate the general applicability of the model further. Third, the endpoint of this study was death, and for various reasons, patients were not followed up for the time to recurrence. Tumor‐free survival and tumor‐specific survival were not described in this study. For another, this study did not include lymph node metastasis, which was different from previous studies. It may be related to the individual differences of the included patients and whether they underwent postoperative adjuvant chemotherapy. In addition, short follow‐up and low single‐center sample size were also limitations of this study. Finally, this study only analyzed factors other than surgery and did not investigate the prognostic impact of different urinary diversion methods.5In conclusion, this study can conclude that age, history of diabetes mellitus, history of hypertension, pre‐operative hydronephrosis, high T‐stage, presence of lymph node metastasis or regional extra‐lymph node metastasis, combined LVI, high Ki‐67 expression, low PNI, and high NLR were prognostic influencing factors affecting post‐operative RC in patients with MIBC; among them, age, pre‐operative hydronephrosis, high T‐stage, combined LVI, low PNI, and high NLR were independent risk factors affecting OS after RC in patients with MIBC. PNI and NLR may be predictive factors for predicting bladder cancer prognosis, but further validation in multicentre randomized controlled trials is needed.Jincheng Wang: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; resources ; software ; visualization ; writing – original draft ; writing – review and editing . Hongjin Shi: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; resources ; software ; visualization ; writing – review and editing . Zhinan Fan: Conceptualization ; data curation ; formal analysis ; methodology ; resources ; software ; visualization ; writing – review and editing . Jiaxin Yang: Data curation ; investigation ; software ; visualization . Yanghuang Zheng: Data curation ; investigation ; software ; visualization . Dan Zeng: Data curation ; investigation ; software ; visualization . Jinsong Zhang: Conceptualization ; data curation ; formal analysis ; funding acquisition ; methodology ; project administration ; software ; supervision ; writing – review and editing . Bing Hai: Conceptualization ; data curation ; formal analysis ; methodology ; project administration ; resources ; supervision ; writing – review and editing .This work was supported by the National Natural Science Foundation of China (Grant Nos. 82060464 and 81860452).