Asthma is a complex, polygenic, heterogenous inflammatory disease. Recently, we generated a list of 128 independent single nucleotide polymorphisms (SNPs) associated with asthma in genome‐wide association studies. However, it is unknown if asthma SNPs are associated with specific asthma‐associated traits such as high eosinophil counts, atopy, and airway obstruction, revealing molecular endotypes of this disease. Here, we aim to identify the association between asthma SNPs and asthma‐associated traits and assess expression quantitative trait locus (e‐QTLs) to reveal their downstream functional effects and find drug targets.N = 32,817/Vlagtwedde‐Vlaardingen N = 1554) and an asthma cohort (Dutch Asthma genome‐wide association study N = 917). Functional enrichment and pathway analysis were performed with genes linked to the significant SNPs by e‐QTL analysis. Genes were investigated to generate novel drug targets.Association analyses between 128 asthma SNPs and associated traits  were conducted using regression modelling in population‐based studies  targeted by drugs currently available for eosinophilic asthma. Several novel targets were identified such as IL‐18, CCR4, and calcineurin.Many asthma SNPs are associated with blood eosinophil counts and genetically driven molecular pathways of asthma‐associated traits were identified. It is characterised by respiratory symptoms, such as wheezing and chest tightness, and pathophysiological mechanisms, like reversible airway obstruction, and airway hyper‐responsiveness. Previously, asthma was regarded as a single disease entity. However, currently, distinct phenotypes of asthma are recognised due to an increased understanding of its underlying mechanisms. These asthma phenotypes are based on clinical presentation, inflammatory patterns, severity and triggers and/or pathophysiological mechanisms. Thus, studies into the pathogenic mechanisms of asthma should address this disease heterogeneity.Genome‐wide association study (GWAS) is an approach that can be used to reveal the underlying mechanisms by identifying single nucleotide polymorphisms (SNPs) that are associated with asthma. Recently, we described a list of 128 independent single nucleotide polymorphisms that were previously associated with asthma in European/white populations at a genome‐wide significant level.It has been previously observed that many asthma SNPs associate with different asthma related phenotypes, indicating potential pleiotropic effects.In this paper, we analysed the association of asthma SNPs with various associated traits in the general population as well as in asthma patients only; next, we performed expression quantitative trait loci (e‐QTL) and pathway analyses to reveal the downstream functional effects of SNPs associated with specific traits and test the hypothesis that these form distinct molecular pathways. Finally, we verify if the implicated genes were already targeted by known asthma drugs and propose new drug targets and drugs for asthma, by repositioning drugs for other indications based on genetic evidence.22.1SNP selection was based on our previous review, where we performed a systematic analysis of GWAS to identify SNPs associated with asthma at genome‐wide significant level.1), , total Immunoglobulin E (IgE), skin prick test positivity , severity of airway hyperresponsiveness (AHR), and childhood (<18 years) onset asthma using linear and logistic regression modelling, as appropriate.These asthma SNPs were tested for association with seven asthma‐associated traits (ATs); blood eosinophil counts, forced expiratory volume 1st second  which represents a general population cohort, comprising two and three‐generation families.1, FEV1/FVC) in both the general population and asthma patients. In addition, we tested the association between asthma SNPs and childhood‐onset asthma.n = 1554.1, FEV1/inspiratory vital capacity [IVC]), atopy  and AHR. Since no reliable asthma diagnosis was present in the Vlagtwedde‐Vlaardingen study, this cohort was not investigated in the asthma only analysis. The third cohort is the Dutch Asthma GWAS (DAG) cohort which consists of 917 asthma patients.1, FEV1/FVC), atopy , AHR and childhood‐onset asthma. The three studies were approved by the Medical Ethics Committee of the University Medical Center Groningen and a written informed consent was provided by all participants. Detailed descriptions of these populations, the genetic datasets and phenotypes are provided in the Online 1, FEV1/VC and childhood‐onset asthma for cohorts representing the general population (Lifelines/Vlw‐Vld) and cohorts with asthma patients only . Although the LifeLines study contributed the largest sample size to our study, other smaller cohorts provided a broader phenotypic assessment, such as BHR, skin tests and total IgE levels. We subsequently investigated the functional consequences of associated SNPs on gene expression by performing cis‐eQTL analysis using eQTLGenFor this study, we conducted the analyses in three independent Dutch general population and patient cohorts as shown in Table 2.2p value < 0.05. The expected direction of the association was taken into consideration: positive association between the asthma‐associated risk allele and eosinophil counts, IgE, skin test, AHR, and childhood‐onset asthma and negative association between the asthma‐associated risk allele and FEV1, FEV1/FVC.Linear or logistic regression analyses of SNPs with asthma‐associated traits were performed in SAIGEgds,p‐value below 2.3*10−9, as reported previously.The functional consequences of the risk SNPs on gene expression were investigated using published cis‐eQTL datasets  from whole blood for SNPs associated with eosinophils, IgE, and childhood‐onset asthma, whole blood and lung for SNPs associated with airway obstruction and AHR, and whole blood and skin for SNPs associated with skin test. The selection of eGenes was based on the significance of biit.cs.ut.ee/gprofiler) to detect statistically significant enriched terms. The data sources that were used in g:profiler are Gene Ontology , KEGG, Reactome, Wikipathways, and Human Protein Atlas. For multiple testing correction, we used Benjamini Hochberg FDR below 0.05 as a threshold. For the term size, it was set to 200 since large pathways are of limited interpretative value. Next, eosinophil counts gene list was submitted to STRING (string‐db.org), to investigate significant gene co‐expression clustering and protein‐protein interaction. The minimum required interaction score was set to 0.7 (high confidence). Pathways and eGenes associated with eosinophil counts were submitted to open targets platform (platform.opentargets.org), to acquire information about potential targets, their drugs, and diseases.Functional enrichment analysis was performed on eGenes associated with blood eosinophil counts, by mapping genes to g:GOST on g:Profiler  we used Lifelines and VLW‐VLD cohorts and for the asthma population analyses (N = 3840) we used Lifelines (only asthma) and DAG cohorts.For the general population analyses  and 4 SNPs were associated in the asthma population only . In contrast to our expectation, 6 asthma SNPs showed nominal association with lower eosinophil counts in the general population , and 1 SNP in the asthma population (rs2786098 [CRB1]).In the general population, 38 asthma SNPs were associated with eosinophil counts, 16 of which had a 1 and/or FEV1/FVC), with 1 SNP overlapping between FEV1 and FEV1/FVC with a padj < 0.05 in FEV1. In the asthma population, 8 SNPs were nominally associated with airway obstruction, with 1 SNP that overlapped.In the general population, 11 SNPs were associated with airway obstruction  that was related to three associated traits: blood eosinophil counts, airway obstruction and atopy. We observed that 9 asthma SNPs were associated with eosinophil counts and atopy  and five SNPs with eosinophil counts and airway obstruction .Whereas all asthma SNPs were selected based on prior evidence of their association with asthma, we also inspected further overlap in association with associated traits Figure . We obse3.2.2BCL6, ITGB8, TESPA1, ADAMTS4, NRROS, HLA‐DPA1, HRNR, IL7R, RUNX3, MYC, EFHC1, POLI, PYHIN1, PDE4D, ITPR3, SMAD3, GATA3, ZBTB38, ZNF217, and IL2RA. Of interest, one asthma‐specific SNP (rs12365699), near CXCR5 gene, showed a different direction of effect in the asthma population , whereas in the general population, this SNP was associated with lower AHR.We observed several asthma SNPs to be associated with one or more asthma‐associated traits in asthma patients, but not in the general population Figure . These S3.2.3IL4RA and TSLP. Some potential targets and their role in eosinophilia are mentioned in Supplementary Table After conducting the cis‐eQTL analysis, gene expression levels of 120 unique genes were significantly associated with SNPs that showed association with asthma traits. For SNPs associated with blood eosinophil counts, we identified 82 eGenes. Amongst these genes, we identified genes encoding drug targets for eosinophilic asthma, such as MICA, HLA‐DRB5, HLA‐DQA2, HLA‐DRB6, INPP4B, USP38, TNFSF4) of SNPs associated with lower blood eosinophil counts. SNPs associated with airway obstruction, atopy, AHR and age of onset had 38, 48, 3 and 1 eGenes, respectively , which is another interesting type 2 inflammatory biomarker, was not present in the cohorts under study. We observed pleiotropic effects of 20 asthma SNPs but also observed that 22 SNPs had an association with an associated trait in asthma patients only. Our results, therefore, provide evidence for pleiotropic, as well as trait‐specific effects. Based on the shared association of asthma SNPs with blood eosinophil counts, we identified genetically driven pathways that may regulate blood eosinophil counts in asthma, which could be targets of future interventions for eosinophilic asthma.p‐value together with direction of effect in the Supplementary Tables Blood eosinophil counts were associated with 38 asthma SNPs in the general population. Our findings are consistent with previous reports in studies on subjects of European ancestry, in which nearly 84% of the asthma SNPs showed association with eosinophil countsAlthough all 128 SNPs were previously reported to be associated with asthma at genome wide significance in other populations, we observed that some SNPs were not associated with asthma related traits in our included cohorts. This may be due to lack of power, or that specific related phenotypes for that SNP  were not investigated in our study. Alternatively, association with asthma related traits may be partly due to residual confounding, although the statistical model in the general population accounted for the presence of asthma. Moreover, since the publication of our review, additional asthma SNPs have been proposed; ; we propose that future approaches should include these genes.FCER1G, IL7R, USF1, APOA2, NDUFS2, ZNF217, PPOX, TSHZ2, TOMM40L) annotated from 4 SNPs that associate with the trait only in the asthma population. Some of these genes are known for their role in eosinophilic inflammation. FCER1G is part of the high‐affinity IgE tetramer receptor Fc Epsilon Receptor I (FcεRI). IgE‐activated mast cells are the main producers of prostaglandin D2 (PGD2), which stimulate eosinophils.We observed SNP associations with asthma‐associated traits in the asthma population only, but not in the general population. These genes annotated from the SNPs are referred to as asthma‐specific targets. In blood eosinophil counts, there were 8 genes  and TSLP (tezepelumab).IL18, CCR4 and calcineurin.We generated asthma trait‐associated lists of SNPs that were annotated to potential drug targets. It has been previously shown that the success of drug development doubles when drug targets have genetic evidence, compared to those without genetic evidence.IL‐18 receptors (IL18R1 and IL18RAP) share the same loci 2q12 with IL33 receptor (IL1RL1) but not given much consideration. Previous studies show IL‐18 critical role in eosinophil development and transformation from naïve to pathogenic eosinophils in patients with allergic diseases.TNFSF4 (OX40L) which plays a role in modulating airway inflammation in asthma. Oxelumab is an anti‐OX40L, which was tested in a small clinical trial for adults with mild allergic asthma without clear beneficial effects.Currently, there is an unmet need for drugs positioned for non‐eosinophilic asthma. In this study, we have identified SNPs associated with low eosinophil counts. One of the interesting genes is IL4R, TSLP) targeted by drugs currently available for eosinophilic asthma.In conclusion, many asthma‐associated GWAS SNPs have distinct associations with asthma traits, such as eosinophilia, airway obstruction and atopy. We found evidence for pleiotropic genes, but also some disease‐specific genes were identified. This study shows that there are distinct sets of asthma SNPs that constitute distinct molecular pathways, according to protein interaction networks analysis. We identified genetically driven pathways regulating eosinophilia in asthma, with two genes ; Formal analysis (lead); Investigation (lead); Methodology ; Software  Validation ; Visualization ; Writing – original draft (lead); Writing – review & editing . Judith M Vonk: Data curation (supporting); Formal analysis (supporting); Supervision (supporting); Visualization (supporting). Maarten van den Berge: Data curation (lead); Resources (lead); Visualization (supporting). Reinoud Gosens: Formal analysis (Supporting); Investigation (supporting); Methodology (supporting); Project administration (supporting); Supervision (supporting); Visualization ; Writing – review & editing (supporting). Gerard H. Koppelman: Conceptualization (lead); Data curation ; Investigation ; Methodology (lead); Project administration (lead); Resources (supporting); Supervision (lead); Visualization (lead); Writing – original draft (supporting); Writing – review & editing (lead).Mr. El‐Husseini has nothing to disclose. Dr. Gosens reports grants from Marie Sklodowska‐Curie, during the conduct of the study; grants from Aquilo, grants from Boehringer Ingelheim, grants from Sanofi‐Genzyme, outside the submitted work. Dr. Vonk has nothing to disclose. Dr. Van den Berge reports grants from Chiesi, grants from GlaxoSmithKline, grants from Novartis, grants from Genentech, grants from Roche, outside of the submitted work. Dr. Koppelman reports grants from Marie Sklodowska‐Curie during the conduct of the study; grants from TEVA the Netherlands, GSK, Lung Foundation of the Netherlands, VERTEX, UBBO EMMIUS foundation, TETRI Foundation, and ZON‐MW outside the submitted work; and GHK participated in advisory board meetings for GSK, Astra Zeneca and PURE‐IMS (money to institution).Supporting Information S1Click here for additional data file.Supporting Information S2Click here for additional data file.


Celecoxib‐tramadol co‐crystal (CTC) is a first‐in‐class analgesic co‐crystal of celecoxib and racemic tramadol with an improved pharmacologic profile, conferred by the co‐crystal structure, compared with its active constituents administered alone/concomitantly.We evaluated CTC in moderate‐to‐severe acute postoperative pain.This randomized, double‐blind, factorial, active‐ and placebo‐controlled phase 3 trial (NCT03108482) was conducted at 6 US clinical research centers. Adults with moderate‐to‐severe acute pain following bunionectomy with osteotomy were randomized to oral CTC (200 mg [112 mg celecoxib/88 mg rac‐tramadol hydrochloride] every 12 h), tramadol (50 mg every 6 h), celecoxib (100 mg every 12 h), or placebo for 48 h. Patients, investigators, and personnel were blinded to assignment. The primary endpoint was the 0–48 h sum of pain intensity differences (SPID0–48) in all randomized patients. Pain intensity was assessed on a 0–10 numerical rating scale (NRS). Safety was analyzed in patients who received study medication. Funded by ESTEVE Pharmaceuticals.p < 0.001), celecoxib , or placebo . Total treatment‐emergent adverse events (TEAEs) were 358 for CTC and 394 for tramadol. Drug‐related TEAEs occurred in 37.7% patients in the CTC group, compared with 48.6% in the tramadol group. There were no serious TEAEs/deaths.In 2017 (March to November), 1323 patients were screened and 637 randomized to CTC (n = 184), tramadol (n = 183), celecoxib (n = 181), or placebo (n = 89). Mean baseline NRS was 6.7 in all active groups. CTC had a significantly greater effect on SPID0–48  than tramadol (−109.1 ; CTC provided greater analgesia than comparable daily doses of tramadol and celecoxib, with similar tolerability to tramadol. CTC is approved in the United States. There is an unmet need for new multimodal treatment options that are well tolerated, with equal or superior efficacy, and reduced risk of adverse outcomes such as abuse and dependence, compared with current conventional opioid analgesics.Today, provision of multimodal analgesia is standard care. For appropriate patients with moderate‐to‐severe acute pain, opioids are often a useful component of multimodal regimens.Celecoxib‐tramadol co‐crystal (CTC) has been developed as a multimodal treatment option for the management of acute pain severe enough to require an opioid. CTC contains tramadol, a μ‐opioid agonist and norepinephrine and serotonin reuptake inhibitor, plus the cyclooxygenase‐2 selective inhibitor celecoxib.The co‐crystal structure of CTC changes the physicochemical properties of celecoxib and tramadol in a way not achieved by their fixed‐dose or free combination.CTC received US Food and Drug Administration approval in October 2021.rac‐tramadol hydrochloride 176 mg) compared with tramadol 50 mg four times daily or celecoxib 100 mg twice daily.Using an established model of acute pain, and comparable doses of active ingredients, the present study demonstrates the superior analgesic efficacy of CTC twice daily , and a safety profile similar to tramadol.Celecoxib‐tramadol co‐crystal (CTC) received US Food and Drug Administration approval in October 2021.Combining tramadol (a μ‐opioid agonist and norepinephrine and serotonin reuptake inhibitor) with celecoxib (a cyclooxygenase‐2‐selective inhibitor) in CTC provides multimodal analgesia via 4 complementary mechanisms of action in central and peripheral pain pathways. Multimodal pharmacologic analgesia may improve efficacy beyond that provided by single agents while minimizing dose requirements and dose‐dependent adverse events (AEs) and is recognized as an essential tool in pain management. CTC, therefore, targets an unmet need for efficacious and well‐tolerated multimodal analgesics. CTC also addresses an unmet need for analgesics that are suitable for treating acute pain severe enough to require an opioid and that might also reduce the risk of adverse effects associated with conventional opioids ; tramadol has a lower risk of these effects than conventional opioids.rac‐tramadol hydrochloride) every 12 h as needed for pain relief,CTC is formulated as a 100‐mg immediate‐release tablet containing 56 mg of celecoxib and 44 mg of tramadol for oral administration. The recommended dosing regimen is 200 mg  requirements for the design of factorial studies assessing fixed‐dose combinations.https://clinicaltrials.gov/ct2/show/study/NCT03108482. Trial registration (submission) date was March 30, 2017. Patients were screened between March 14 and November 1, 2017; randomization occurred between March 21 and November 7, 2017.This randomized, double‐blind, factorial, active‐ and placebo‐controlled, parallel‐group, phase 3 clinical trial (NCT03108482) was performed at 6 US clinical research centers. The protocol was approved by an Institutional Review Board; Sterling IRB . The study was conducted in accordance with the ethical principles originating from the Declaration of Helsinki, the International Council for Harmonization guidelines for Good Clinical Practice, and local regulatory requirements. The protocol and statistical analysis plan are available at Eligible patients were ≥18 years old with moderate‐to‐severe pain  within 8 h of popliteal sciatic block cessation, following primary unilateral first metatarsal osteotomy with internal fixation and no additional collateral procedure. Full criteria are described in the supplemental digital content. All patients provided written informed consent.Eligible patients were randomized 2:2:2:1 to oral CTC 200 mg every 12 h, celecoxib 100 mg every 12 h, tramadol 50 mg every 6 h, or placebo every 6 h. Each CTC dose was given as two 100‐mg immediate‐release tablets; each tablet contained 56 mg of celecoxib and 44 mg of racemic tramadol. Randomization was stratified by center and baseline pain score (moderate [NRS 5–6] or severe [NRS 7–9]). At study start, a computer‐generated randomization schedule was prepared and randomization numbers were assigned sequentially via central interactive response technology. No one involved in study conduct had access to the schedule before official unblinding. Patients, investigators, and study personnel were blinded to assignment. To maintain blinding, all medications, including placebo, were provided as over‐encapsulated tablets. In the CTC and celecoxib groups, placebo was administered at the 6‐h midpoint between active treatments.Study duration was ≤6 weeks, including a screening period of ≤28 days. Patients were admitted to the center on the morning of surgery, remained there for 3 nights , and returned for a follow‐up visit 5 to 9 days post‐surgery. The treatment period was 48 h.Patients underwent bunionectomy with osteotomy and internal fixation under standardized regional anesthetic, involving popliteal sciatic nerve block and continuous sciatic infusion via a catheter placed in the proximity of the popliteal sciatic nerve. Patients received midazolam and/or propofol for initial sedation, lidocaine 1% for local anesthesia, and ropivacaine 0.5% for popliteal sciatic nerve block. If popliteal sciatic nerve block was insufficient to provide adequate anesthesia, a standard Mayo block with lidocaine 2% was permitted. Regional anesthesia (mepivacaine 0.5% or ropivacaine 0.2%) was continued after surgery, via continuous infusion, and discontinued between 3:00 and 4:00 am the day following surgery. After this time, patients meeting the inclusion criteria for baseline pain intensity were randomly assigned to treatment. Supplemental analgesia with intravenous ketorolac was permitted until 1:00 am to help control breakthrough pain if the regional anesthetic infusion was insufficient to provide adequate analgesia.Patients could receive rescue medication for pain after discontinuation of anesthetic and initiation of study medication . Patients were encouraged to wait ≥1 h after the first dose of study medication before receiving rescue medication.Pain intensity was assessed using the NRS  at: 0 h (before study medication); 15, 30, and 45 min; 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 10, 12, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 46, and 48 h; and immediately before rescue medication. Pain relief scores were assessed at the same time points, using a 5‐point categorical scale. Patients were asked, “How much relief have you had since your starting pain?”, with response choices of none (score of 0), a little (1), some (2), a lot (3), and complete (4). The pain intensity assessment was completed before the pain relief assessment; patients were not permitted to compare their responses with previous responses. Pain intensity and pain relief results were recorded in patient diaries. Pain assessments were performed during the night only if the patient was awake (patients were awakened to administer study medication). Time to perceptible and meaningful pain relief was measured using the double stopwatch technique starting at time 0 . Patients were instructed to stop the first stopwatch when they perceived any pain relief, and to stop the second stopwatch when they experienced pain relief that was meaningful to them. Patients provided an overall assessment of study medication by answering the following global evaluation question at the end of the 48‐h treatment period: “How effective do you think the study medication is as a treatment for pain?” . Safety and tolerability were evaluated until follow‐up based on the incidence, severity, and relationship of treatment‐emergent adverse events (TEAEs) to study medication, physical examination findings, clinical laboratory test results, and changes in vital sign and electrocardiogram measurements. TEAEs were defined as AEs with onset at the time of or following the start of study medication, or starting beforehand but increasing in severity or relationship at the time of or following the start of study medication, occurring up to 7 days after the final dose of study medication.0–48). SPID was calculated as a time‐weighted sum of pain intensity difference values at each follow‐up time point (difference between starting pain intensity and pain intensity at the given assessment time point) multiplied by time (h) since the last non‐missing assessment.The primary efficacy variable was pain intensity measured by NRS. The primary efficacy endpoint was the sum of pain intensity differences (SPID) from 0 to 48 h (SPID0–48) were performed, the latter to permit estimation of the absolute efficacy of CTC.Secondary efficacy endpoints included SPID over 4, 6, 12, and 24 h; total pain relief  over 4, 6, 12, 24, and 48 h; pain intensity and change from baseline in pain intensity at each time point; response rates; time to onset of analgesia ; proportion of patients using rescue medication; time to first use of rescue medication; and patients' overall assessment of study drug. Post hoc analyses on use of oxycodone rescue medication and on placebo‐adjusted efficacy  of 124, at a 2‐sided alpha level of 0.05 and 90% power, 142 patients per group were required (71 for placebo). Allowing for a 20% rate of nonevaluable patients, 180 individuals per group (90 for placebo) were to be randomized—630 in total. For sample size re‐estimation, a planned blinded interim analysis was performed after about half the patients had completed the study. In this analysis, estimated pooled variance of the primary outcome measure was substantially lower than that used for sample size calculation; thus, it was concluded that the study was adequately powered and there was no need to increase sample size.The study aimed to demonstrate superiority of CTC 200 mg over tramadol and celecoxib. To determine sample size, power for both comparisons of CTC (vs tramadol and vs celecoxib) was set at 90%; therefore, overall study power was 81%. To detect a difference in SPIDThe full analysis set comprised all randomized patients. The per‐protocol analysis included all patients in the full analysis set with no major protocol deviations. The safety analysis included all patients who received study medication. Efficacy analyses were performed on the full analysis set, sensitivity analyses on the per‐protocol analysis set, and safety analyses on the safety analysis set.0–48 across treatment groups using an analysis of covariance (ANCOVA) model, adjusting for center and baseline pain intensity. To account for use of rescue medication, a pain intensity assessment was performed upon each request for rescue medication, with the score used to replace all scores for timed assessments taken in the subsequent 4 h (considered to be the period over which rescue medication was active). ANCOVA analyses were performed for all quantitative efficacy outcomes; p‐values and 95% confidence intervals (CIs) for the least‐squares (LS) means for pairwise treatment differences were derived.The primary efficacy analysis compared SPIDAn additional analysis of pain intensity difference from baseline by time point was conducted using a mixed model for repeated measures analysis on the full analysis set using type 3 tests of fixed effects.The proportion of patients requiring rescue medication and of responders, and patients' overall assessment of study drug, were analyzed by logistic regression, adjusted by baseline pain and center. Time to onset of analgesia and first use of rescue medication were analyzed using time‐to‐event statistical methods. Safety data were analyzed and reported using descriptive statistics.Additional sensitivity analyses were conducted to assess the impact of rescue medication use and missing data imputation due to study discontinuation. Efficacy analyses were also performed by subgroups defined by baseline pain intensity, sex, age, body mass index (BMI), and race.All statistical analyses were conducted using SAS 9.4 .n = 184), tramadol (n = 183), celecoxib (n = 181), or placebo (n = 89)  discontinued treatment or withdrew from the study.Of 1323 patients screened, 637 were randomized to CTC  and white (74%). Mean age was 46 years, mean body weight was 76 kg, and mean BMI was 28 kg/m2 Table . Mean  provided a significantly greater analgesic effect than tramadol 50 mg administered 4 times daily , celecoxib 100 mg twice daily , and placebo  versus placebo .For SPID0–48 was approximately 1.9 and 2.2 times larger than that of tramadol and celecoxib, respectively , celecoxib , and placebo . In patients with moderate pain, CTC provided a significantly greater effect versus celecoxib  and placebo ; the comparison versus tramadol was not statistically significant . Results of subgroup analyses by sex, age, and BMI generally mirrored those of the main analyses, although treatment differences did not always reach statistical significance, particularly in smaller subgroups for which statistical power was low , over celecoxib at 1.25 h , and over tramadol at 3.5 h . Analyses of SPID and TOTPAR at 4, 6, 12, and 24 h and TOTPAR at 48 h were consistent with primary endpoint findings  reported that analgesia was good, very good, or excellent, versus 70.0% for tramadol, 59.7% for celecoxib, and 48.3% for placebo  and 3 in the tramadol group ; all events resolved. The most common TEAEs were nausea, dizziness, vomiting, and headache. No safety concerns were observed in laboratory tests, electrocardiograms, or vital sign assessments.Table 0–48 than comparable daily doses of tramadol or celecoxib, and placebo, thereby meeting the study's primary endpoint. SPID0–48 was selected as the study's primary efficacy endpoint as it provides a summary of treatment effects on pain intensity over a clinically meaningful time period. Results of sensitivity analyses using different imputation procedures for missing data and rescue medication use, and of a placebo‐adjusted analysis of treatment effects on SPID0–48, were consistent with those of the primary analysis. Subgroup analyses for the primary endpoint  were also generally consistent, although treatment differences did not always reach statistical significance, particularly in smaller subgroups.We evaluated the efficacy of CTC, a novel API–API co‐crystal containing celecoxib and tramadol, for the management of moderate‐to‐severe postoperative pain following bunionectomy with osteotomy. CTC was approved by the FDA in October 2021 and, to our knowledge, is the first API–API co‐crystal for analgesia to be specifically acknowledged as such within the prescribing information.Secondary efficacy endpoints supported primary findings, with most reaching statistical significance in favor of CTC. Reductions in mean pain intensity difference from baseline were statistically significantly greater for CTC over placebo, celecoxib, and tramadol at 1, 1.25, and 3.5 h post‐dose, respectively. Due to study medication, rescue medication, and/or natural post‐surgery resolution of pain, most study patients were classified as responders at some point during the 48‐h dosing period; response rates were not significantly different between groups but were highest in the CTC group. Most patients used first‐line rescue pain medication (intravenous acetaminophen), and about half used second‐line (oxycodone). Patients in the CTC group used less rescue medication, and used it later, than patients in other groups. Most patients in the CTC group (58%) did not require oxycodone, compared with 30% to 45%  in other groups, suggesting that CTC may have an opioid‐sparing effect while improving overall analgesia versus other treatments.The overall rate of TEAEs was similar in the CTC and tramadol groups and lower in the celecoxib and placebo groups. The most common TEAEs were consistent with known safety profiles of tramadol and were as expected in the acute postoperative setting. TEAEs included all events occurring from enrollment to follow‐up and, therefore, captured AEs arising from confounding factors . In phase 1 studies designed to avoid such factors, AE rates were lower with CTC than with tramadol alone, or with tramadol and celecoxib in free combination.This study used an established model of acute surgical pain (bunionectomy with osteotomy) associated with pain of sufficient intensity to warrant opioid use, and of sufficient duration to measure efficacy over multiple days; the model provides good sensitivity for approximately 72 h post‐surgery.Given ongoing problems with opioid misuse and abuse,The recommended CTC dose translates into a maximum daily dose of 224 mg of celecoxib and 176 mg oftramadol—about half the maximum daily doses of its individual active components based on the full prescribing information of celecoxib and tramadol for acute pain.Tmax [h] of 1.5, 3.0, and 2.5 from CTC, celecoxib alone, and free combination with tramadol, respectivelyThe study represents a well‐designed and fair comparison between CTC and its individual APIs. However, possible limitations should be acknowledged. First, doses of tramadol and celecoxib used in comparator groups may have been lower than full therapeutic doses. Although differences in tramadol and celecoxib dosage between CTC and comparator groups mean that the study might not be a true factorial study, the FDA considered that the study met requirements for a well‐controlled, full‐factorial, phase 3 study and proposed the dosing used . Second, most patients enrolled were female, although this is typical for the clinical population seeking treatment for bunionectomy. Subgroup analysis of the primary endpoint by sex showed no significant interactions, albeit this analysis was not well powered due to the small number of males. Third, the study did not include a head‐to‐head comparison versus the individual reference products taken together. However, earlier data show that the pharmacokinetics of the APIs in CTC are different to those observed when commercially available celecoxib and tramadol are administered in free combination.In conclusion, the API–API co‐crystal CTC provided greater analgesic efficacy than did comparable daily doses of tramadol and celecoxib alone. This was accompanied by lower utilization of rescue pain medication, including significantly lower use of opioid rescue medication. The safety profile of CTC 200 mg (112 mg celecoxib/88 mg tramadol) administered every 12 h was similar to that of tramadol 50 mg administered every 6 h. Our findings suggest that CTC could be an efficacious and well‐tolerated alternative for appropriately selected adult patients with acute pain severe enough to require an opioid, by providing multimodal analgesia with an anti‐inflammatory component, via a means not replicated by free or fixed‐dose combination.E.R. Viscusi, O. de Leon‐Casasola, J. Cebrecos, A. Jacobs, A. Morte, M. Sust, A. Vaqué, M.E. Kuss, S. Videla, N. Gascón, and C. Plata‐Salamán conceived or designed the study. J. Cebrecos, E. Ortiz, I. Gottlieb, S. Daniels, J.S. Gimbel, D. Muse, P. Winkle, and S. Videla contributed to data collection. J. Cebrecos, A. Jacobs, E. Ortiz, and M. Sust analyzed the data. E.R. Viscusi, O. de Leon‐Casasola, J. Cebrecos, A. Jacobs, A. Morte, M. Sust, A. Vaqué, I. Gottlieb, M.E. Kuss, and C. Plata‐Salamán interpreted the data. All authors drafted, edited, or reviewed drafts of the manuscript and approved the final version. All authors had access to the underlying data. E.R. Viscusi, J. Cebrecos, A. Jacobs, A. Morte, E. Ortiz, M. Sust, A. Vaqué, I. Gottlieb, S. Videla, N. Gascón, and C. Plata‐Salamán verified the underlying data.This study was supported by ESTEVE Pharmaceuticals S.A. E.R. Viscusi reports consulting fees for ESTEVE Pharmaceuticals, Fresenius, Heron Therapeutics, Innocoll Pharmaceuticals, Merck, and Salix Pharmaceuticals. O. de Leon‐Casasola reports personal fees for advisory board membership for ESTEVE Pharmaceuticals during the conduct of the study, and for ESTEVE Pharmaceuticals, Stimgenix, and Medtronic outside the submitted work. J. Cebrecos, A. Morte, E. Ortiz, M. Sust, A. Vaqué, and N. Gascón are employees of ESTEVE Pharmaceuticals. A. Jacobs is an employee of Premier Research . I. Gottlieb reports grants for ESTEVE Pharmaceuticals for participation as a principal investigator in the current study and personal fees for ESTEVE Pharmaceuticals for consulting projects. S. Daniels is an employee of Optimal Research . M.E. Kuss was an employee of Premier Research during the conduct of the study. S. Videla was an employee of ESTEVE Pharmaceuticals during the conduct of the study. C. Plata‐Salamán was an employee of ESTEVE Pharmaceuticals and has pending or issued patents relevant to CTC. The remaining authors have no conflicts of interest to declare.Appendix S1Click here for additional data file.


Halomonas ventosae JPT10 promoted the salt tolerance of both dicots and monocots. Physiological analysis revealed that JPT10 reduced reactive oxygen species accumulation by improving the antioxidant capability of foxtail millet seedlings. The metabolomic analysis of JPT10‐inoculated foxtail millet seedlings led to the identification of 438 diversely accumulated metabolites, including flavonoids, phenolic acids, lignans, coumarins, sugar, alkaloids, organic acids, and lipids, under salt stress. Exogenous apigenin and chlorogenic acid increased the salt tolerance of foxtail millet seedlings. Simultaneously, JPT10 led to greater amounts of abscisic acid (ABA), indole‐3‐acetic acid (IAA), salicylic acid (SA), and their derivatives but lower levels of 12‐oxo‐phytodienoic acid (OPDA), jasmonate (JA), and JA‐isoleucine (JA‐Ile) under salt stress. Exogenous JA, methyl‐JA, and OPDA intensified, whereas ibuprofen or phenitone, two inhibitors of JA and OPDA biosynthesis, partially reversed, the growth inhibition of foxtail millet seedlings caused by salt stress. Our results shed light on the response of foxtail millet seedlings to H. ventosae under salt stress and provide potential compounds to increase salt tolerance in foxtail millet and other crops.Plant growth‐promoting bacterias (PGPBs) can increase crop output under normal and abiotic conditions. However, the mechanisms underlying the plant salt tolerance‐promoting role of PGPBs still remain largely unknown. In this study, we demonstrated that  Halomonas ventosae JPT10 promoted the salt tolerance of both dicots and monocots. JPT10 could improve the antioxidant capability of foxtail millet and the levels of abscisic acid, indole‐3‐acetic acid, salicylic acid, and their derivatives but reduce the levels of 12‐oxo‐phytodienoic acid (OPDA), jasmonate (JA), and JA‐isoleucine (JA‐Ile).We demonstrated that  Researchers have been drawn to creating biotechnological methods that use PGPBs to enhance the growth of stressed plants. PGPBs from naturally salty environments could provide real candidates that more efficiently lessen the impacts of salt stress , tomato, and Arabidopsis  secretions under salinity , chlorogenic acid (CGA), and other antiocidants, increasing the activity of important antioxidant enzymes, as well as ABA, IAA, and SA content but decreasing the levels of JA, OPDA, and JA‐Ile. Furthermore, exogenous AP, CGA, and inhibitors of JA and OPDA biosynthesis partially reversed the growth inhibition caused by salt stress. As a result, our findings provide potential active substances for improving salt tolerance in crops, and offer physiological mechanisms for JPT10‐mediated salt tolerance of foxtail millet and other crops.In this study, we performed a series of physiological experiments to explain whether and how halophilic 22.1To investigate the impact of JPT10 on the performance of foxtail millet seedlings under salt stress, foxtail millet seedlings were watered with various concentrations of NaCl solution with or without JPT10. After 21 days of salt exposure, foxtail millet seedlings showed slow growth and senescence Figure . Both th3  homeostasis was measured to determine how it boosts the salt tolerance of foxtail millet. The ROS levels were determined by aminophenyl fluorescein (APF), a fluorescence probe for hydroxyl radical and peroxynitrite . We evaluated metabolite changes in whole foxtail millet seedlings treated with 200 mM NaCl in the presence or absence of JPT10 for 6 and 24 h. A total of 438 metabolites with significant changes were identified Table . To find2.3We examined the impact of AP and CGA, representative antioxidants belonging to flavonoids and phenolic acids, respectively, on the salt tolerance of foxtail millet seedlings. Three‐day‐old foxtail millet seedlings were treated with the indicated amounts of AP or CGA for 7 days. Under salt stress, foxtail millet seedlings grew better when exposed to certain amounts of AP than when exposed to salt stress alone Figure . AP‐treaThen, we detected that the effect of JPT10 on exogenous AP and CGA increased seed germination of foxtail millet. The results showed that salt stress inhibited seed germination was partially recovered by JPT10 and exogenous AP and CGA Figure . The com2.46‐isopentenyladenine (IP) and dihydrozeatin (DZ), remained unaltered under all three situations  accumulated more ROS, including HA Figure , indicatA Figure , and YugA Figure  experiens Figure , indicatare mutant, which have a flavonol 3‐hydroxydase (F3H) deficiency and exhibit reduced flavonol content and increased ROS formation in pollen grains  is more sensitive to salt treatment in terms of the root meristem   ‘Yugu1’ was used in this investigation. The plants were grown in a greenhouse at 25°C and 60%–70% relative humidity, with a 16 h/8 h (day/night) photoperiod. In addition, Glycine max, Solanum lycopersicum, Triticum aestivum L., and Zea mays were cultured under the same conditions.Foxtail millet . Luria Bertani (LB) medium with 850 mM NaCl was used for isolation. The strain has been stored in China Center for Type Culture Collection (CCTCC), the accession number is CCTCC M 2018548. JPT10 was cultured in 15 mL LB medium containing 2 M NaCl at 28°C for 24–48 h. The cultured cells were harvested and re‐suspended with water containing the indicated NaCl concentrations to 1 × 1010 CFU/mL.Endophytic 10 CFU/mL strain JPT10 was applied to the different plants. All plants were treated every 3 days and treated three times. When the distinct phenotype manifested, pictures were taken, and data were gathered. Three biological replicates of each experiment were performed.Uniformly sized seeds of foxtail millet, soybean, tomato, wheat, and maize were selected for planting and then thinned at the two‐leaf stage. The evenly developed seedlings were placed in each designated pot (10 × 10 cm), which contained a 1:3 volume mixture of matrix and vermiculite, and every big pot contained 12 designated pots. A 2 L volume of 200 mM NaCl concentrations was irrigated in the NaCl groups, and the MOCK groups were irrigated with an equal amount of sterile water. For the +JPT10 groups, a volume of 200 mL solution containing 104.32O2 and O2−, seedlings were stained using DAB  or NBT  according to previously described methods , O2− , SOD , and POD  contents were detected using assay kits provided by Beijing Solabio Life Sciences & Technology Co., Ltd. The kits for APX (G0203F), CAT (G0303F), GR (G0209F), MDA (G0109F), and proine (G0111F) were bought from Suzhou Grace Biotechnology Co., Ltd.The collected seedlings were washed in distilled water to remove the soil and then dried off. The fresh weight was calculated. Acetone (80%) was used to extract chlorophyll in the dark for 24 h at 25°C. The absorbance at wavelengths of 663 nm and 645 nm was assessed using a photometer . The chlorophyll amount per gram of fresh weight was calculated according to a previous study Arnon, . To dete4.4http://www.anpel.com.cn/) before UPLC‐MS/MS analysis. Utilizing a planned protocol, the metabolites were quantified  based on the AB Sciex QTRAP 6500 LC‐MS/MS platform. Three replicates of each assay were performed.Fresh plant materials were harvested, weighted, immediately frozen in liquid nitrogen, and stored at −80°C until needed. Plant materials (50 mg fresh weight) were frozen in liquid nitrogen, ground into powder, and extracted with methanol/water/formic acid . The combined extracts were evaporated to dryness under nitrogen gas stream, reconstituted in 80% methanol (V/V), and filtrated  before LC–MS/MS analysis. JA , CK , auxin , SA, and ABA contents were detected by MetWare  in SPSS software (version 24). All experiments had at least three biological replicates and the data were presented as means ± SEM.Statistical analyses were performed using one‐way analysis of variance , the Natural Science Foundation of China (32241039), the National Key R&D Program of China (2022YFD1201704/2022YFD1201700).The authors declare they have no conflict of interest.Figure S1.Figure S2.Figure S3.Figure S4.Figure S5.Figure S6.Figure S7.Figure S8.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.


The aim was to develop and validate a nomogram for the prediction of brain metastases (BM) in small cell lung cancer (SCLC), to explore the risk factors and assist clinical decision‐making.We reviewed the clinical data of SCLC patients between 2015 and 2021. Patients between 2015 and 2019 were included to develop, whereas patients between 2020 and 2021 were used for external validation. Clinical indices were analysed by using the least absolute shrinkage and selection operator (LASSO) logistic regression analyses. The final nomogram was constructed and validated by bootstrap resampling.A total of 631 SCLC patients between 2015 and 2019 were included to construct model. Gender, T stage, N stage, Eastern Cooperative Oncology Group (ECOG), haemoglobin (HGB), the absolute value of lymphocyte (LYMPH #), platelet (PLT), retinol‐binding protein (RBP), carcinoembryonic antigen (CEA) and neuron‐specific enolase (NSE) were identified as risk factors and included into the model. The C‐indices were 0.830 and 0.788 in the internal validation by 1000 bootstrap resamples. The calibration plot revealed excellent agreement between the predicted and the actual probability. Decision curve analysis (DCA) showed better net benefits with a wider range of threshold probability . The model was further externally validated in patients between 2020 and 2021 with a C‐index of 0.818.We developed and validated a nomogram to predict the risk of BM in SCLC patients, which could help clinicians to rationally schedule follow‐ups and promptly implement interventions. In this study, we constructed a nomogram to predict the probability of brain metastasis in SCLC patients. We eventually identified 10 factors, including gender, T stage, N stage, ECOG, HGB, LYMPH, PLT, RBP, CEA and NSE, which are readily available in clinical practice. The inclusion criteria were as follows: (1) SCLC was the primary tumour, which was confirmed by histological or cytological evidence; (2) There was a continuous record of diagnosis and treatment; (3) Imaging data such as computed tomography (CT), magnetic resonance imaging (MRI) or positron emission tomography‐CT (PET‐CT) were used to confirm the occurrence of BM. We excluded patients with incomplete clinical data , serological indices , clinicopathological characteristics  and sodium concentration (Na+). Finally, tumour markers included carcinoembryonic antigen (CEA), non‐small cell lung cancer associated antigen (CYFRA211) and neuron‐specific enolase (NSE).Blood routine included the counts of red blood cells (RBCs), haemoglobin (HGB), platelet (PLT), as well as white blood cells (WBCs) including neutrophils (NEUT), lymphocytes (LYMPHs) and monocytes (MONOs). Liver function was indicated by the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), adenosine deaminase (ADA), sialic acid (SA), glutamate dehydrogenase (GLDH), albumin (ALB), globulin (GLO), ALB/GLO (A/G) and superoxide dismutase (SOD). Furthermore, renal function indices included β2‐microglobulin (BMG), retinol‐binding protein (RBP) and complement C1q (C1q). Blood biochemistry indices included glucose (GLU), carbon dioxide . ECOG was divided into <2 and ≥2 to assess performance status. mGPS was calculated using the levels of serum albumin and CRP to assess systemic inflammation. For CRP >10 mg/L, 1 point was given if ALB value was normal, and 2 was given when ALB <35 g/L. Whereas, 0 point was awarded regardless of ALB values, as long as CRP was normal. In the study BM population, all indices were extracted from the records of first diagnosis. In the study population with BM, all indices were extracted from the records within 24 h of admission of the first diagnosis. In addition, SCLC‐related factors within 24 h after admission in patients without BM were collected from the first diagnostic records of SCLC.2.3p‐value of <0.05 was considered as the significance threshold for all statistical analyses.Baseline characteristics of the enrolled population were presented as median  for continuous variables, or as numbers and percentages for categorical variables. The Wilcoxon–Mann–Whitney test was used to compare statistical differences of non‐normally distributed variables between the SCLC patients with and without BM. Categorical variables were analysed using chi‐square test. A two‐sided p‐value were calculated and recorded. The performance of the nomogram was assessed by discrimination, calibration and clinical usefulness in succession. The predictive discriminative ability of the model was displayed by the C‐index and was equivalent to the area under the receiver operating characteristic curve (AUC). Similar to the AUC, the C‐index ranging from 0.5 (no relationship) to 1.0 (perfect concordance) was also used. The calibration plot and Hosmer–Lemeshow test were applied for evaluating calibration. The decision curve analysis (DCA) was displayed to determine the clinic usefulness of the model by quantifying the net benefit at disparate threshold probabilities. The training cohort underwent 1000 bootstrap resamples for internal validation, and external validation was performed on the nomogram by the validation cohort. Finally, we showed the predictive risk points of each predictive risk factor in the nomogram. In addition, the predictive potentials of different cut‐off values for BM in SCLC patients' probability in the nomogram were evaluated by calculating the sensitivity and specificity. The study adhered to the TRIPOD  statement for reportingThe selection of significant variables relied on the results of univariate logistics regression analysis, clinical importance and predictors identified in previously published articles. We then extracted the following risk factors for the prediction model: gender, HGB, the absolute value of lymphocyte (LYMPH #), PLT, RBP, CEA, NSE, tumour (T) stage, node (N) stage and ECOG. Next, we used the least absolute shrinkage and selection operator (LASSO) method to select the optimal variables with non‐zero coefficients as potential predictors and avoid overfitting of this model. Above factors were selected to develop the final nomogram. β (the regression coefficient), odds ratios (ORs) with 95% confidence intervals (CIs) and http://www.r-project.org/).All statistical analyses were conducted using SPSS, version 26  and R language, version 4.0.3  between the two groups (p > 0.05). The partly baseline characteristics of SCLC patients with BM or without BM subgroups are summarized in Table A total of 737 SCLC patients were included in our study Figure , and BM 3.2p < 0.05) (Table p < 0.001), HGB , NSE , PLT , LYMPH # , ECOG , T stage , N stage , CEA  and RBP   Table . After w5) Table . These p3) Table .3.3The above 10 predictive factors were used to construct a visualized nomogram Figure . The pre9/L, PLT <125 × 109/L, RBP >70 mg/L, CEA >10 ng/mL and NSE >16.3 ng/mL, was defined to be 0.839 (95% CI: 0.250–0.988), which was higher than the risk score, and the patients were classified to the high‐risk group. In addition, a SCLC male with T1N2, ECOG <2, HGB <115g/L, LYMPH # between 1.1 and 3.2 × 109/L, PLT between 125 and 350 × 109/L, RBP <25 mg/L, CEA between 0 and 10 ng/mL and NSE >16.3 ng/mL was defined to be 0.024 (95% CI: 0.005–0.105), which was lower than 0.194, the patients were classified to the low‐risk group. As identifying higher than the cut‐off value, this SCLC patient was the high‐risk group that could provide more direct information for clinicians to take early intervention.A SCLC individual with a risk score >0.194 was regarded as a high‐risk patient for BM. For example, if a SCLC patient that was a male, combined with T2N2, ECOG ≥2, HGB <115 g/L, LYMPH # <1.1 × 103.4p‐value of 0.562 for the Hosmer–Lemeshow test further indicated ideal results. In addition, the results of DCA showed a better net benefit with a broader range of threshold probability with a net clinical benefit of 1%–58% , indicating that the model possessed a good discriminative ability. Moreover, the optimal cut‐off value of the nomogram was 0.194 according to the Youden's method. The specificity and sensitivity of this model were 78.9% and 71.7%, respectively , as well as with a lower incidence of metachronous brain failure.Although the above studies revealed independent risk factors for BM in SCLC patients, no study has conducted a systematic model construction. Other studies analysed risk factors for BM, and the results were not exactly the same as our study. We supposed that the different inclusion and exclusion criteria adopted by the studies contributed to the heterogeneity. Even after the previous multivariate analysis, a shift towards visualization and assisting clinical decision‐making has not been achieved. This study innovatively used a clinical prediction model to perform LASSO regression analysis, which was used to minimize the risk of overfitting and contributed to the development of the optimal model. Furthermore, a visualized nomogram and a free‐accessed web calculator were constructed. We found some indicators innovatively to participate the development of BM in SCLC patients: HGB, PLT, LYMPH # and RBP. Low HGB levels and PLT counts were identified as an adverse prognostic factor in BM from solid extracranial cancers.The prediction model was validated to have good performance in the clinic. The C‐index was 0.830 with a specificity of 78.9% and a sensitivity of 71.7%, indicating good discriminative ability. And DCA showed a better net benefit with a wider threshold probability. PCI is considered to be the standard treatment of SCLC and could extend the limited stage small cell lung cancer OS. However, it is not clear in patients with stage I‐II SCLC with low risk of brain metastasis and patients ≥70 years old or in poor health. Therefore, risk assessment should be individualized, and treatment decisions should be discussed with patients. This predictive model could help doctors predict that SCLC patients have high risk for BM and correspondingly develop appropriate therapeutic strategies.This study has the following limitations. First, this study was a retrospective study and cannot guarantee the integrity of the data, resulting in a lack of some valuable indicators, such as metastatic sites, pro‐gastrin‐releasing peptide (ProGRP) and treatment . Second, our study had no further survival data to investigate about the differential prognosis between high‐ and low‐risk of BM subgroups. Therefore, rigorous and prospective cohorts with larger sample size are needed. Meanwhile, basic experiments should also be used to explore the key steps in SCLC BM.5In conclusion, we constructed a visualized nomogram to predict the risk of BM in patients with SCLC, which covered common clinical indicators and showed good discriminative performance. We believe that the nomogram established in this study will assist clinicians in clinical decision‐making regarding BM and ultimately provide more benefit to the high‐risk population.Weiwei Li: Conceptualization; investigation; writing—original draft; writing—review and editing; data collection and interpretation. Can Ding: Conceptualization; methodology; resources; and statistical analysis. Wei Sheng, Qiang Wan, Zhengguo Cui and Guiye Qi: Provide expert advice and project supervision. Yi Liu: Supervised the study planning and design; and statistical analysis.The authors declare no conflicts of interest.The studies involving human participants were reviewed and approved by the Shandong Provincial Hospital Medical Ethics Committee . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Table S1. Baseline features of SCLC patients with BM and without BM subgroups.Table S2. Univariate logistic regression analysis for brain metastases in patients with SCLC.Table S3. Identification of predicted variables for nomogram.Table S4. Predictive risk points of each variable in the nomogram.Click here for additional data file.Data S1. TRIPOD Checklist. Prediction Model Development and ValidationClick here for additional data file.


The LV end‐systolic elastance (Ees) did not change immediately after TAVI . The Ea/Ees improved after TAVI , demonstrating an immediate improvement of VAC. The stroke work (SW) did not change , but the pressure‐volume area (PVA) decreased  after TAVI. The SW/PVA increased after TAVI  representing an improvement of LV efficiency. We proposed a novel minimally invasive method for PV analysis in patients with severe AS receiving TAVI. The VAC and LV efficiency improved immediately after TAVI.The ventriculo‐arterial coupling (VAC) and left ventricle (LV) mechanics are crucial and play an important role in the pathophysiology of aortic stenosis (AS). The pressure‐volume (PV) analysis is a powerful tool to study VAC and LV mechanics. We proposed a novel minimally‐invasive method for PV analysis in patients with severe AS receiving transcatheter aortic valve implantation (TAVI). Patients with severe AS were prospectively enrolled in a single center. LV pressure and cardiac output were recorded before and after TAVI. We constructed the PV loop for analysis by analyzing LV pressure and the assumed flow. 26 patients were included for final analysis. The effective arterial elastance (Ea) decreased after TAVI (3.7 ± 1.3 vs. 2.9 ± 1.1 mmHg/mL,  Reconstructed pressure volume loop by LV pressure and cardiac output. Right heart pressure and cardiac output were recorded by Swan‐Ganz catheter at the beginning and the end of TAVI, simultaneously LV and aortic‐root pressure recording were also measured by a fluid‐filled system as well. The signal of pressure and surface electrocardiogram were recorded in a hemodynamic system , and the hemodynamic data was subsequently sampled and analyzed by MATLAB (MathWorks Inc.).2.3tri) as an assumed flow of LV before and after TAVI  from a non‐linear least‐squares approximation technique with curve‐fitting method . Veed is effective end‐diastolic volume = LV end‐diastolic volume—V0. The slope from the original point to the end‐systolic equilibrium point was Ea, the results are shown in Figure By analyzing the constructed pressure‐ejected volume curve, the Ees is the slope from the point indicating estimated peak LV isovolumic pressure to the end systolic equilibrium point. The intercept of the Ees at the 2.5Ea/Ees is widely used to represent VAC  is determined from the SW to the theoretically maximal value . The results were presented as mean ± standard deviation (SD) for continuous variables, and percentage for categorical variables. The comparison of pre and post TAVI data were performed by paired 32. The Zva was 2.4 ± 0.9 mmHg/mL.From September 2019 to August 2021, total 26 patients were included for analysis. The baseline and procedural characteristics are presented in Table 96.2% received consciousness sedation for TAVI. 61.5% of patients received Sapien S3 (Edwards Lifesciences), and 38.5% received Evolut R (Medtronic)3.1p = 0.0197; cardiac output, 3.7 ± 0.8 vs. 4.3 ± 1.1 L/min, p = 0.0004). The LV end‐systolic pressure (LVPes)  and LV peak systolic pressure (LVPmax)  decreased after TAVI. The isovolumic peak LV pressure (LVPisomax) did not change after TAVI . The stroke volume increased after TAVI . Ea decreased after TAVI , while Ees did not change . The value of Ea/Ees and Q load both improved after TAVI , demonstrating immediate improvement of VAC. The SW did not change , while PV area (PVA) decreased after TAVI , SW/PVA increased after TAVI , demonstrating the improvement of LV efficiency.The acute change of hemodynamic parameters after TAVI are summarized in Table 4In patients with AS, left ventricle is contracting against the stenotic valve and the arterial load. Previous studies had discussed the load condition, but VAC was neglected , demonstrating the improvement of energy transferal immediately after TAVI. Our study also showed the immediately improvement of LV efficiency . The SW equals to the LV systolic pressure × stroke volume. In our study, the LV systolic pressure decreased and stroke volume increased immediately after TAVI, thus the SW did not change significantly. There might also be partial effects of short‐acting vasopressor that could lead to incomplete resolution of LV pressure, thus the SW did not change significantly immediately after TAVI. Although the SW did not change, the improvement of LV efficiency was seen immediately. which was contributed to the decrease of PVA after TAVI. The improvement of energy transfer and LV efficiency were consistent with previous studies (Bastos et al., The comprehensive PV analysis can provide detailed information of pathophysiology. Currently the diagnosis and risk stratifications of AS are based on non‐invasive echocardiographic measurements and clinical symptoms. However, the decisions could be inappropriate in missing the timing of intervention in patients with marginal aortic valve area and pressure gradient, or at their later stage of AS due to LV dysfunction. Fundamental physiologic characteristics such as Ea, Ees, and VAC, may actually provide more detailed information for the pathophysiology of AS. Analysis of PV loop offered more in‐depth information of myocardial damage and contractility than echocardiography (Ishikawa et al., Conventional method of PV analysis has major limitations such as the complexity for pressure/flow acquisition and its invasiveness. Our method is less invasive and simpler compared with conventional invasive method for PV analysis. However, our method developed from the mice study, using the disease model of diastolic heart failure. The accuracy of our model in AS required further study to validate. Our study is a proof of concept study. Immediately after TAVI, the Ea decreased while the Ees did not change. The post‐TAVI VAC and LV contractile efficiency improved immediately which is compatible with previous studies (Marino et al., 5The major limitation of the present study was the lack of good validation method. We did not perform conventional PV analysis due to its invasiveness and complexity. Also, our sample size was small and lack of long‐term follow‐up data. The clinical impacts and the long‐term results of the hemodynamic data were uncertain. Further study for clinical outcome comparison was warranted.6We demonstrated a minimally invasive method for PV analysis in patients with severe AS receiving TAVI. The Ea and VAC improved immediately after TAVI. The LV contractile efficiency improved which contributed to the decrease of PVA.Tsung‐Yu Ko contributed to planning/collecting data/writing. Chia‐Chuan Chuang/Kuo‐Chu Chang contributed to data analysis and collection. Mao‐Shin Lin/Yi‐Chang Chen/Ying‐Hsien Chen/Ching‐Chang Huang/Chih‐Fan Yeh/Ming‐Jiuh Wang contributed to collecting data. Hsien‐Li Kao/Yi‐Lwun Ho contributed to planning/writing.No funding information provided.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vascular remodeling is a basic pathological process in various diseases characterized by abnormal changes in the morphology, structure, and function of vascular cells, such as migration, proliferation, hypertrophy, and apoptosis. Various growth factors and pathways are involved in the process of vascular remodeling. The transforming growth factor‐β (TGF‐β) signaling pathway, which is mainly mediated by TGF‐β1, is an important factor in vascular wall enhancement during vascular development and regulates the vascular response to injury by promoting the accumulation of intimal tissue. Vascular endothelial growth factor (VEGF) has an important effect on initiating the formation of blood vessels. The Hippo‐YAP/TAZ signaling pathway also plays an important role in angiogenesis. In addition, studies have shown that there is a certain interaction between the TGF‐β/Smads signaling pathway, Hippo‐YAP/TAZ signaling pathway, and VEGF. Many studies have shown that in the development of atherosclerosis, hypertension, aneurysm, vertebrobasilar dolichoectasia, pulmonary hypertension, restenosis after percutaneous transluminal angioplasty, and other diseases, various inflammatory reactions lead to changes in vascular structure and vascular microenvironment, which leads to vascular remodeling. The occurrence of vascular remodeling changes the morphology of blood vessels and thus changes the hemodynamics, which is the cause of further development of the disease process. Vascular remodeling can cause vascular smooth muscle cell dysfunction and vascular homeostasis regulation. This review aims to explore the mechanisms of the TGF‐β/Smads signaling pathway, Hippo‐YAP/TAZ signaling pathway, and vascular endothelial growth factor in vascular remodeling and related diseases. This paper is expected to provide new ideas for research on the occurrence and development of related diseases and provide a new direction for research on the treatment of related diseases. TGF‐β/Smads signaling pathway, Hippo‐YAP/TAZ signaling pathway, and vascular endothelial growth factor: their mechanisms and roles in vascular remodeling related diseases. The pathway was originally studied in the fields of inflammation, tissue repair, and embryonic development. In recent years, TGF‐β has been shown to play an important role in the regulation of cell growth, differentiation, and immune function.1.21.2.1The TGF‐β superfamily has more than 40 members, including TGF‐βs, bone morphogenetic protein (BMP), activin, and related proteins.1.2.2Smad transcription factors are the core of the TGF‐β signaling pathway. The TGF family binds to TβRI and TβRII and forms a complex. To date, a total of eight different Smad proteins have been identified in mammals and are divided into three subfamilies according to their functions: receptor‐regulated Smads (R‐Smads), universal chaperone Smads (Co‐Smads), and inhibitory Smads (I‐Smads). Under general conditions, R‐Smads are mainly located in the cytoplasm, I‐Smads are mostly located in the nucleus, and Co‐Smads are distributed in the nucleus and cytoplasm.1.3TGF‐β1, TGF‐β3, and activin have high affinity for the type II receptor. Therefore, a unique interface can be generated by first forming a complex with TβR II and then recruiting TβR I to the complex. In contrast, TGF‐β2 has a very low affinity for TβR II. Therefore, TβR I and TβR II are required to first form a complex or coreceptor, such as betaglycan, which facilitates TGF‐β2 assembly within the complex.waf1 promoter and suppresses cell growth. On the other hand, pro‐inflammatory cytokines (CKs), such as tumor necrosis factor‐α (TNF‐α) activate c‐Jun N‐terminal kinase (JNK), which phosphorylated the linker regions of Smad2 and Smad3. pSmad3L translocate with Smad4 to nucleus and binds plasminogen activator inhibitor type 1(PAI‐1) promoter. PSmad2L is localized in the cytoplasm, and Smad2 translocates to the nucleus only after COOH‐tail phosphorylated by TβRI. PSmad2L/C in cooperation with pSmad3L and Smad4 stimulated PAI‐1 transcription and ECM deposition. In addition, pSmad3L upregulates c‐Myc and stimulates cell growth.In the Smads‐mediated signaling pathway, phosphorylated Smad2 and Smad3 also have positive and negative regulating effects  is a homodimeric glycoprotein with a molecular weight of 45 kDa.3.2The VEGF family consists of multiple members: VEGF‐A (with multiple subtypes), VEGF‐B, VEGF‐C, VEGF‐D, VEGF‐E, VEGF‐F, placental growth factor, and the newly discovered endocrine‐derived vascular endothelial growth factor (EG‐VEGF).44.1YAP/TAZ plays a role in vascular development, and studies have shown that TAZ promotes SMC differentiation through a nonclassical TGF‐β/Smads signaling pathway.4.2The Hippo‐YAP/TAZ signaling pathway plays an important role in VEGF‐induced angiogenesis and development. VEGF stimulation leads to cytoskeletal changes and promotes YAP/TAZ activation and nuclear entry, which in turn regulates the expression of related proteins and affects cytoskeletal movement Figure . Studies5Vascular remodeling is a change in vessel wall structure, which often occurs under pressure, inflammation, trauma, and other stimulation conditions. Oxidative stress and inflammation are often the pivotal players in endothelial dysfunction during abnormal vascular remodeling.5.1TGF‐β/Smads signaling pathway has different biological effects on angiogenesis in different pathological environments and concentrations and can inhibit or promote angiogenesis.5.2YAP/TAZ in endothelial cells is an important regulator of angiogenesis in the growth and development of the embryo and postnatal life. This important role of YAP/TAZ was first expressed in the central nervous system after birth. As the body grows and develops, this effect will also be observed in the liver and other organs.5.3Under various stimulation conditions, VEGF, angiotensin II (Ang II), and FGF induce the activation of matrix proteolytic enzymes (ECM), resulting in the degradation of ECM and the shedding of pericytes on the vascular wall. VEGF contributes to the remodeling of the ECM by increasing the permeability of the vascular endothelium, leading to the extravasation of plasma proteins and deposition in the ECM.6At present, the occurrence and development of many diseases are related to vascular remodeling, such as atherosclerosis, hypertension, aneurysm and arterial dissection, and vertebrobasilar dolichoectasia (VBD).6.1Vascular remodeling can be classified as positive or negative remodeling according to the remodeling index (RI). The RI is the ratio of the cross‐sectional area (CSA) of the vascular external elastic membrane (EEM) at the lesion to the CSA of the EEM of the proximal or distal vessel. RI > 1.05 indicates positive remodeling, RI < 0.95 indicates negative remodeling, and 0.95 > RI > 1.05 indicates intermediate remodeling. Initially, it was showed that positive vascular remodeling was more beneficial to atherosclerosis than negative vascular remodeling. However, with further research, it has been found that although positive vascular remodeling can compensate for luminal stenosis, the plaques of patients with positive vascular remodeling are more prone to thrombosis than those with negative vascular remodeling.6.2In hypertension and hypertensive diseases, arterial remodeling is stimulated by hemodynamic changes, resulting in reduced vascular compliance. The remodeling process is mainly changed by endothelial dysfunction, the proliferation and migration of medial VSMCs, the apoptosis and transformation of extravascular fibroblasts, and the synthesis, degradation, and rearrangement of the ECM.Endothelium‐dependent vasodilation is impaired in animal models of balloon injury‐induced vascular remodeling, so vascular remodeling is thought to lead to endothelial dysfunction.6.3Aortic aneurysm formation is mainly due to the weakening of the aortic wall, resulting in aortic enlargement.6.4VBD is a rare cerebrovascular disease, and the main manifestations are vertebrobasilar artery prolongation, tortuosity, and dilation.6.5According to the pathological and hemodynamic characteristics, PH can be divided into five categories, including arterial pulmonary arterial hypertension (PAH), pulmonary hypertension (PH) caused by left heart disease, PH caused by respiratory diseases and hypoxia, PH caused by pulmonary obstructive diseases, PH caused by unknown causes and multifactorial PH. PH caused by respiratory diseases includes interstitial pulmonary diseases, chronic obstructive pulmonary disease (COPD), and sleep apnea. Pulmonary vascular remodeling refers to vascular structural changes caused by injury factors such as hypoxia, high blood shear stress, and inflammation, which are the main pathological features of pulmonary arterial hypertension (PAH). Vascular remodeling in PH is characterized by the accumulation of different vascular cells in the pulmonary artery and the infiltration of inflammatory cells around the vessel.Although current advances in the treatment of PAH have improved patient outcomes, 5‐year and 10‐year survival rates of 52%–75% and 45%–66%, respectively.6.6Restenosis after percutaneous transluminal angioplasty (PTA) is a major factor affecting the long‐term outcome of this technique. It was previously thought that the occurrence of restenosis after PTA was similar to wound healing. That is, after PTA balloon expansion, vascular wall injury causes vascular elastic retraction, thrombosis, and the migration and proliferation of VSMCs.7Although exploration of the pathological mechanism of vascular remodeling has made progress in recent decades, the specific and comprehensive mechanism is still not fully elucidated. This article reviews the possible mechanism of vascular remodeling and its role in the occurrence and development of many diseases according to current research progress. In conclusion, vascular remodeling can be triggered by various stimuli, including high blood pressure, trauma, oxidative stress, and inflammation, to make up for the aberrant changes in hemodynamics and vascular microenvironment brought on by vascular injury. Proper vascular remodeling can compensate for vascular dysfunction and even restore vascular function. On the contrary, excessive vascular remodeling can aggravate the disease progression of vascular dysfunction and vascular remodeling‐related diseases. Treatments for these disorders that reduce inflammation are currently making significant progress. Despite this, there are currently no effective treatments for vascular remodeling. More research is required to determine how to prevent excessive vascular remodeling and slow the progression of disease. Numerous signaling pathways are active during vascular remodeling, according to recent studies. By inhibiting the excessive conduction of these signaling pathways, the occurrence of aberrant vascular remodeling can be prevented and the disease progression of vascular remodeling‐related diseases can be delayed.The Hippo‐YAP/TAZ signaling pathway, TGF‐β/Smads signaling pathway, and VEGF play important roles in the process of angiogenesis and the repair of pathological damaged blood vessels, and there are certain interactions among them. According to recent research, inhibiting the Hippo‐YAP/TAZ signaling pathway, TGF‐β/Smads signaling pathway, and VEGF can prevent aberrant vascular remodeling and hence slow the progression of illness. On the other hand, we discovered that there is a relationship between the TGF‐β/Smads signaling pathway, the Hippo‐YAP/TAZ signaling pathway, and VEGF. Therefore, inhibiting one of the three may also indirectly inhibit the effect of the other. By studying the mechanism of the three factors in vascular remodeling and related diseases, we hope to provide new therapeutic ideas for vascular remodeling‐related diseases.Hui Liu: Writing—original draft; writing—review and editing. Mingyue Sun: Writing—review and editing. Nan Wu: Writing—review and editing. Bin Liu: Writing—original draft; writing—review and editing. Qingxin Liu: Writing—original draft; writing—review and editing. Xueli Fan: Writing—original draft; writing—review and editing.The authors declare no conflict of interest.


Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase‐aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology.In this study, we developed and validated the multiplex reverse transcription recombinase‐aided PCR (multiplex RT‐RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process.Using recombinant plasmids, the sensitivity of multiplex RT‐RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT‐RAP showed no cross‐reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT‐RAP and the results were found to be consistent with those of corresponding RT‐qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT‐RAP was two to eightfold higher than that of corresponding RT‐qPCR.We conclude the multiplex RT‐RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load. A novel nested nucleic acid amplification assay that integrates isothermal amplification technique RAA and traditional qPCR technique. The technique starts with a first step of rapid enrichment by RAA technology, followed by a second step of PCR amplification and TaqMan probe detection. This method is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load. The human‐derived Human RNaseP protein p38 gene (HRPP) was added in this method to improve the authenticity and credibility of the assay results. A paraffin (docosane) was added as a barrier to separate the RAA mix from the quantitative PCR (qPCR) mix. The multiplex RT‐RAP was further validated with clinical samples.22.1The clinical specimens of the respiratory tract were obtained from the Hunan Provincial Center for Disease Control and the Capital Institute of Pediatrics. The types of clinical specimens contained pharyngeal swabs and alveolar lavage fluid. The total DNA and RNA from 200 μL of these clinical specimens were extracted by Viral RNA/DNA Isolation Kit (Tianlong) following the manufacturer's instructions. The nucleic acids were eluted in 50 μL of elution buffer into 1.5 mL EP tube and stored at −80°C refrigerator.2.2https://www.ncbi.nlm.nih.gov/) and aligned using Vector NTI 11.5.1 to locate the highly conserved regions. RAA primers (outer primers of RAP) for HADV3, HADV7, HRSV, and HRPP were cited from previous studies in our laboratory.Complete sequences of HRSV were downloaded from the Nation Center for Biotechnology Information database , B (HADV14 and 55), C , and E (HADV4). The procedures were operated according to the optimized working conditions.The sensitivities of multiplex RT‐RAP were measured with the serial dilutions of the HADV3, HADV7, and HRSV plasmids ranging from 102.6The clinical samples were simultaneously tested by previously reported RT‐qPCR, RT‐RAA, and multiplex RT‐RAP. Subsequently, three positive clinical samples for each of HADV3, HADV7, and HRSV were selected, fourfold serially diluted and detected by RT‐qPCR, RT‐RAA, and multiplex RT‐RAP. The results were analyzed by SPSS 26 software.33.1The volume ratio of RT‐RAA mix to PCR mix was previously determined to be 1:4.2 . The mix B (40 μL) contained 200 nM of each qPCR primers and 100 nM of each qPCR probe, 0.5 mM of each dNTP, qPCR buffer, and nuclease‐free water. The RT‐RAP mix consisted of mix A, the barrier (30 μL docosane), and mix B, the mix A was above the barrier and mix B was below the barrier. The procedure of RT‐RAP was as follows: 39°C for 20 min, 95°C for 5 min, followed by 20 cycles at 95°C for 10 s, 55°C for 15 s, and 72°C for 15 s. The whole detection time was within 1 h, which is faster than traditional RT‐qPCR.Accordingly, we finalized the optimal reaction procedure for multiplex RT‐RAP. The RT‐RAA reaction mix was prepared according to the manufacturer . The RT‐RAA pellet was dissolved with rehydration buffer, nuclease‐free water, 140 nM of each RAA primers, 14 mM of magnesium acetate, and then the mix was divided into four RT‐RAP volume and added 1–2 μL template that were called mix A (10 μL). The pre‐qPCR mix was called as mix B that was prepared following manufacturer's protocol but reduced the volume of MgCl3.2The sensitivity of multiplex RT‐RAP for simultaneous detection of HADV3, HADV7, and HRSV was determined by detecting serial dilutions of the plasmids. The sensitivity of the assay was determined by Probit regression analysis for HADV3, HADV7, and HRSV to be 18, 3, and 18 copies per reaction, respectively Table . During 3.3Totally, 252 clinical specimens were tested simultaneously by RT‐qPCR, RT‐RAA, and multiplex RT‐RAP methods to evaluate the clinical performance of multiplex RT‐RAP assays. Multiplex RT‐RAP identified 28 HADV3 positive specimens , 39 HADV7 positive specimens , and 41 HRSV positive specimens , which was consistent with those of RT‐qPCR/qPCR and RT‐RAA/RAA. Compared with RT‐qPCR and RT‐RAA assay, both the specificity and sensitivity of the multiplex RT‐RAP assay using 252 clinical samples were 100%  serially diluted clinical samples also indicated that multiplex RT‐RAP possessed higher sensitivity than that of correspond qPCR or RT‐qPCR assay. The rapidity of multiplex RT‐RAP  saves running time compared to qPCR/RT‐qPCR. Multiplex RT‐RAP can be performed in a common fluorescence qPCR instrument, and does not require the purchase of a special supporting instrument for versatility.So far, multiplex RT‐RAP has been applied in our laboratory in the detection of various pathogens including HIV, Norovirus GII, Rotavirus, and SNP detection of Mycobacterium tuberculosis. Our preliminary results indicate multiplex RT‐RAP is a universal sensitive and rapid nucleic acid detection technique. Although, we have made various improvements over the previous single‐plex RAP technique, which has greatly improved the operation and stability of the method, the components of the RAA system are not yet compatible with those of PCR, for example, the RAA mixes still need to physically block the two mixes, which cannot really achieve a single mix throughout the detection process. In addition, there is no obvious correlation between RAA enrichment and the amount of initial viral nucleic acid, the quantitative detection of RAP method still needs further research. This method is not sufficiently automated or mechanized. Our ongoing attempt is to use microfluidic chip technology to improve the automation and the throughput of the assay.Despite the current multiplex RAP technology still has some shortcomings, it remains a robust, rapid, and sensitive nucleic acid detection technique that can be applied to viral, bacterial, and mutant loci detection. In addition, due to its high sensitivity, it might be useful for rapid screening of asymptomatic patients with infections, sensitive detection of cerebrospinal fluid samples with low viral load, and low frequency monitoring of drug‐resistant pathogens.In conclusion, the proposed closed‐tube multiplex RT‐RAP is a rapid, highly specific, and highly sensitive nucleic acid detection platform, which has great potential to be used in the detection of various pathogens.XM and XS designed the study. GF, RZ, FT, XS, MZ, and FT performed the experiments. GF, RZ, and XH analyzed and interpreted the data. XM and GF wrote the study. All authors provided a critical review and approved the final article.This work was supported by grants from the National Key R&D Program of China (2021YFC2301102), National Natural Science Foundation of China (82202593), Natural Science Foundation of Shandong Province, China (ZR2022MH115), and the key R&D projects in Zibo city (2020kj100011).The authors declare that there is no conflict of interest in this work.


To investigate the effects of brain-derived neurotrophic factor (BDNF) gene polymorphism on cognitive function, neuroimaging and blood biological markers in patients with subcortical ischaemic vascular dementia (SIVD).n = 26), Val/Met (n = 35), and Met/Met (n = 20) groups. A comprehensive neuropsychological evaluation and multimodal brain MRI scan were performed. MRI markers for small vessel disease were visually rated or quantitatively analysed. Moreover, 52 patients were further evaluated with blood marker assays, including amyloid beta (Aβ), phosphorylated tau at threonine-181 (P-tau181), glial fibrillary acidic protein (GFAP), total tau (T-tau) and neurofilament light chain (NfL).A total of 81 patients with SIVD were included. According to their BDNF gene polymorphism, the participants were divided into the Val/Val (There were no significant differences in demographics, disease duration or MRI markers of small vessel disease between the three groups. Compared with the Val/Val and Val/Met groups, the Met/Met group showed worse performance in the verbal fluency test and higher levels of plasma NfL.The rs6265 polymorphism of the BDNF gene is associated with semantic language fluency in patients with SIVD. The Met genotype may be a risk factor for cognitive impairment and neuronal injury. Subcortical ischaemic vascular dementia (SIVD), which often has an insidious onset and manifests as gradual cognitive decline, gait instability, urinary incontinence and abnormal mood and behaviour, is the most common subtype of vascular dementia VaD; . PatientBrain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family, is mainly produced by neurons in the hippocampus and cerebral cortex, and is widely expressed in the central nervous system. Previous studies have shown that BDNF plays a key role in supporting neuronal survival and differentiation, enhancing synaptic transmission and plasticity, and consolidating memory . BDNF plThe cognitive correlation of the BDNF gene has been demonstrated in several central nervous system diseases. It has been shown that Met allele carriers have a higher incidence rate of AD; in contrast, the age of disease onset in AD patients with Val carriers is delayed . CompareWhilst numerous studies have investigated the association between BDNF polymorphism and neurocognitive changes in ageing and various pathological backgrounds, the effect of the Val66Met polymorphism on cognition and neurodegeneration is still not fully understood, particularly in patients with VaD, although a previous study found that Val carriers were more quickly diagnosed with dementia after ischaemic stroke . This stEighty-one patients with SIVD were enrolled in a longitudinal MRI study of Alzheimer’s disease and subcortical ischaemic vascular dementia (ChiCTR1900027943) at Tianjin Medical University General Hospital. All participants were aged 50~85 years and received a systematic evaluation, including medical history collection, physical and neurological examinations, neuropsychological evaluation, laboratory tests, and brain MRI. This study was approved by the Ethics Committee of Tianjin Medical University General Hospital. All participants and their legal guardians signed written consent forms.All SIVD patients were diagnosed according to the criteria for major neurocognitive disorder in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders  and the The exclusion criteria were as follows: (1) cognitive impairment caused by other central nervous system diseases, such as AD, dementia with Lewy bodies, PD, frontotemporal lobar degeneration (FTLD), hydrocephalus, and multiple sclerosis; (2) cognitive impairment caused by systemic diseases, such as vitamin B12 deficiency, thyroid dysfunction, syphilis or HIV infection; (3) cognitive impairment caused by mental disorders, such as schizophrenia and severe depression; (4) alcohol or drug abuse affecting cognitive assessment; (5) inability to cooperate with MRI scan or cognitive assessment.All participants were evaluated with the clinical dementia rating (CDR) scale and received a comprehensive neuropsychological battery as previously described . In addiMultimodal brain MRI scans were performed on all participants using a 3.0 T superconducting magnetic resonance scanner , including T1 weight imaging (T1WI), T2 weight imaging (T2WI), diffusion-weighted imaging (DWI), fluid-attenuated inversion recovery (FLAIR), and gradient echo (GRE) sequences. The T1WI parameters were as follows: repetition time (TR) = 8.2 ms; echo time (TE) = 3.2 ms; time of inversion (TI) = 450 ms; flip angle (FA) = 12°; field of view (FOV) = 256 mm × 256 mm; layer thickness = 1 mm; and number of layers = 188. The following T2WI parameters were used: TR = 2,500 ms; TE = 80 ms; FA = 90°; FOV = 230 mm × 230 mm; layer thickness = 1 mm; and number of layers = 376; The FLAIR parameters were as follows: TR = 8,400 ms; TE = 150 ms; TI = 2,100 ms; FA = 111°; FOV = 240 mm × 240 mm; layer thickness = 6 mm; and number of layers = 18; The following DWI parameters were used: TR = 2,100 ms; TE = 65.4 ms; FA = 90°; FOV = 256 mm × 256 mm; layer thickness = 6 mm; and number of layers = 36. Finally, the GRE parameters were as follows: TR = 200 ms; TE = 3.9 ms; FA = 30°; FOV = 256 mm × 256 mm; layer thickness = 6 mm; and number of layers = 18.All MRI scans were reviewed by two investigators who were trained to be consistent and blinded to the clinical information and neuropsychological testing results. Small vessel disease markers were defined according to the standards for reporting vascular changes on neuroimaging . Primer-BLAST software was used for primer design. After purifying the polymerase chain reaction (PCR) product, BDNF genotyping was performed using the ABI 3730XL analyser .Plasma was obtained from blood samples within 2 h of collection by being centrifuged at 2,500 × g for 15 min at 4°C and then was stored at −80°C until biochemical analysis. The levels of plasma amyloid beta (Aβ)42, Aβ40 and total tau (T-tau) were quantitatively detected using the Neurology 3-Plex A Assay Kit , plasma phosphorylated tau at threonine-181 (P-tau181) was quantitatively detected using the P-tau 181 Assay Kit V2 , and plasma glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) were quantitatively detected using the Neurology 2-Plex B Assay Kit . All measurements were performed on the single molecule array (Simoa) HD-X analyser platform  according to the procedure previously described , 2023. Tp < 0.05 was considered the threshold for statistical significance.All analyses were performed using SPSS version 22.0. Quantitative data are presented as the mean ± SD, and categorical data are presented as n (%). Participants were divided into three groups  according to their BDNF genotype. The differences in demographic and clinical data, neuropsychological scores, MRI markers for small vessel disease, and plasma biomarkers were compared between groups using one-way ANOVAs for continuous variables or chi-square tests for categorical variables. Fisher’s least significant difference was used for multiple comparisons. All hypothesis tests were two-tailed, and The participants, 50 males and 31 females, had an average age of 71.40 ± 7.24 years, average course of disease of 3.98 ± 2.13 years, and average education level of 11.06 ± 3.38 years. There were 26 patients with Val/Val, 35 with Val/Met, and 20 with Met/Met BDNF genotypes. There were no significant differences in age, sex, years of education, or proportions of hypertension and diabetes between patients with different BDNF genotypes .Post hoc analysis showed that the Met/Met group had lower scores on the VFT than both the Val/Val group  and the Val/Met group . No significant differences were observed in global cognition measured with the MMSE and the MoCA and other scores for various cognitive domains between groups (p > 0.05).There was a significant difference in VFT scores amongst the three groups . Post hop > 0.05; There was no statistically significant difference in any small vessel disease markers between groups (p = 0.002) and the Val/Met group , multiple sclerosis, and some mental disorders , the eviConsistent with the observed tendency of the Met carriers to show worse cognitive performance in our study, the influence of Met genotype on impairment in various cognitive domains has been reported in several previous studies. In persons with preclinical AD, Met carriers have significantly decreased cognitive functions, such as episodic memory, executive function and language function, compared with noncarriers , 2017. MVerbal fluency was the main cognitive domain affected by the Val66Met polymorphism in the present study. A previous study also found this effect in epilepsy patients . The verSince no specific biomarkers for SIVD have been identified yet, we tested plasma biomarkers for AD pathology, neuroinflammation, and neurodegeneration in this study. No differences in Aβ and P-tau or GFAP, which is a special intermediate filament component of mature astrocytes, were observed between SIVD patients with different Val66Met polymorphisms, indicating that the effect of Met may not be attributable to increasing AD pathology or activating astrocyte-related inflammation.Interestingly, NfL levels were significantly increased in SIVD patients with Met homozygotes compared with those with Val homozygotes and heterozygotes. Although it has been demonstrated that plasma NfL, an important protein component of the neuronal axon cytoskeleton, could be a sensitive biomarker for neurodegeneration and predict cognitive decline in many central nervous system diseases, such as AD, FTLD and VaD , there iIn this study, there was no difference in MRI markers for small vessel disease, including WHM, lacunes, microbleeds and PVS, between SIVD patients with different BDNF gene polymorphisms. The effect of the Val66Met polymorphism on vascular markers was contradictory in previous studies. For instance, Met66 allele carriers show a larger WMH volume amongst elderly individuals with depression . HoweverThere are some limitations of the study to address. First, although strict MRI criteria, including the medial temporal lobe atrophy score, were included in participant recruitment, we did not detect Aβ and tau markers using CSF or PET to exclude patients with mixed AD pathology, which could aggravate cognitive impairment and neurodegeneration in patients with SIVD. Second, apart from BDNF gene polymorphism, other genetic factors , which might also play a role in pathogenesis and disease progression, were not analysed in this study. Third, although the Met/Met group showed worse performance on most tests, such as the AVLT, the BVMT-R, the Stroop, the TMT-A, the DST, the SDMT, and the JLO, the difference between the 3 groups was only statistically significant on the VFT. Therefore, the cognitive correlation of Val66Met polymorphism needs validation in more studies. Fourth, biomarker results were only obtained from individuals who were enrolled during the latter half of the study, which might not be representative of all participants. Finally, since this was a cross-sectional study, we could not completely determine the causal relationship between BDNF polymorphism and clinical and neurobiological correlations in SIVD patients. It is worth further investigating these correlations in persons with cerebral small vessel injury but not dementia with a long-term follow-up.The Met/Met genotype in the rs6265 polymorphism of the BDNF gene may accelerate cognitive impairment in patients with SIVD, and this effect was correlated with neurodegeneration measured with plasma NfL but independent of vascular lesions.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Ethics Committee of Tianjin Medical University General Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.XY and GY drafted the manuscript. XY performed the statistical analysis. TF, ZT, YL, and FC performed the neuropsychological assessment. TF and ZT processed, rated, and analysed the images. PC and JC conducted the plasma biomarker measurement. NZ designed the study and revised the manuscript. All authors contributed to the article and approved the submitted version.