Ketamine may become an important drug for multimodal analgesia regime again because of its strong analgesic effects and retaining the advantage of spontaneous breathing. The present study was designed to explore the influences of different dosages of S‐ketamine anesthesia induction regimes on psychiatric complications and postoperative prognosis in patients undergoing gynecological operations. In this prospective, triple‐blinded, randomized, controlled study, patients undergoing elective gynecological surgery were randomized to one of three treatment groups: low‐dose S‐ketamine (LDSK) group (a 0.3 mg/kg bolus for anesthesia induction), minimal‐dose S‐ketamine (MDSK) group (a 0.2 mg/kg bolus for anesthesia induction), and placebo (CON) group . The main outcome measures were as follows: intraoperative vital signs, extubation time, anesthesia recovery time and postanesthesia care unit (PACU) stay duration, incidence of psychiatric complications, Ramsay sedation scale (RSS) 1, 2, 24, and 48 h, postoperatively, and overall prognosis. One hundred and eighty female participants were finally included in this study from April 2021 to December 2021. Significant differences were not observed in age, height, weight, American Society of Anesthesiologists physical status classification, or history of mental illness between the groups. No statistically significant differences were discovered with regard to intraoperative vital signs, extubation time and PACU stay duration, incidence of psychiatric complications, and RSS scores at 1, 2, 24, and 48 h postoperatively in the three groups. However, the visual analog scale (VAS) scores of the CON group at 10 min after extubation and at the time point leaving PACU were much higher than that of the LDSK and MDSK groups. The VAS scores at 48 h after surgery in the MDSK group were also lower than that of the CON group and the CON group had received more analgesic drug treatment in the surgical wards consequently. Postoperative nausea and vomiting (PONV) occurrence at 24 and 48 h, postoperatively, increased sharply in the CON group than in the other two experimental groups, which led to an increase in the use of postoperative antiemetic drugs in this group. According to the postoperative satisfaction survey, patients in the CON group had lower medical satisfaction. Our data demonstrate that a small dosage of S‐ketamine anesthesia induction can reduce postoperative pain and the incidence of PONV without increasing hemodynamic fluctuations or psychiatric complications. This article evaluated the effect of the new‐type drug S‐ketamine on the cognitive function of gynecological patients through a randomized controlled clinical study, hoping to optimize clinical anesthesia management. The anesthetic and analgesic potency of opioids is beyond doubt. However, many studies have demonstrated that opioids are not conducive to the prognosis of surgical patients.22.12, American Society of Anesthesiologists (ASA) physical status class Ⅰ–Ⅱ, operative duration between 0.5 and 3 h. Exclusion criteria: severe liver or kidney dysfunction, severe cardiovascular diseases, intracranial hypertension and central nervous system diseases , hyperthyroidism, pregnancy and lactation, history of drug or alcohol abuse, present or past psychotic disorders. Elimination criteria: subjects withdraw informed consent at any time; subjects were found unqualified after random division; ingestion of medication (other than the study protocol) within 48 h of surgery, or subjects might benefit less than the risk due to any clinical adverse event.After receiving approval to carry out the study from the institutional ethics review board of Zunyi Medical University (KLL‐2020‐049), eligible subjects were contacted 1 week before the operation and signed informed consent after they had been fully informed about the study process. The trial process had been registered at chictr.org.cn (no.ChiCTR2100046703) before being launched. Patients were scheduled for surgical treatment from April 2021 to December 2021. Inclusion criteria: patients undergoing elective gynecological surgery, aged between 18 and 65 years old, body mass index between 18 and 30 kg/m2.2All qualified patients were randomly divided into one of the three treatment groups, as follows:Low‐dose S‐ketamine (LDSK) group: a 0.3 mg/kg intravenous (i.v.) bolus of S‐ketamine was given for anesthesia induction.Minimal‐dose S‐ketamine (MDSK) group: a 0.2 mg/kg intravenous (i.v.) bolus of S‐ketamine was given for anesthesia induction.Placebo (CON) group: the control group received 0.9% saline in the same volume as the above two groups for anesthesia induction.2.3The study medication was prepared preoperatively using a computer‐generated randomization list, by an anesthetist who was excluded from any further involvement in the treatment or evaluation of patients. The syringes for the bolus injection were labeled with “Study Medication” and the randomization number of the patient in this study. All involved stuffs were strictly kept unknown about group allocation beforehand.2.42 was maintained between 35 and 45 mmHg. Intravenous infusion of propofol  4–8 mg/kg/h and remifentanil  6–10 μg/kg/h was used for anesthesia depth maintenance. Rocuronium  was added intermittently according to the operation requirements to maintain muscle relaxation. It was forbidden to use inhaled anesthetics or local anesthetics during the anesthesia process in this study. Invasive hemodynamic monitoring was selected according to the surgical routine. The infusion volume was adjusted according to the monitoring index during operation. After the operation, all subjects were transferred to the Postanesthesia Care Unit (PACU) for resuscitation.There was no preoperative medication in this study. After anesthesia preparation, the researchers obtained the results of a randomized grouping of patients according to the random number method. Anesthesia was induced and maintained following our institutional standards: propofol  2.5 mg/kg, sufentanil  0.3 μg/kg, rocuronium  0.6 mg/kg. After injection of the above drugs, patients in the three groups received different doses of S‐ketamine  or a normal saline placebo injection according to the grouping scheme. Endotracheal intubation was performed after the depth of anesthesia was reached. After endotracheal intubation, mechanical ventilation was performed immediately: tidal volume (TV) 8–10 ml/kg, respiratory rate (RR) 12–20 times/min, and the PetCO2.5Immediately at the end of the operation, the PCIA pump  was attached to the patients' intravenous cannula in a unified way for all three groups: sufentanil  (2 μg/kg) and metoclopramide  (40 mg) were jointly configured into 100 ml elastomeric pumps. PCIA lasted until 48 h after the operation. When required, postoperative pain was treated with 40 mg paresibuna . If the patient complained of pain again, sufentanil  5 μg was used as a pain remedy. To ensure the quality of research, all analgesia schemes for perioperative patients were determined by anesthesiologists.2.6The primary outcome endpoints were the intraoperative hemodynamic signs, extubation time, PACU stay time, incidence of psychiatric complications, and Ramsay sedation scale (RSS) scores at 1, 2, 24, and 48 h postoperatively. Our secondary outcomes included perioperative opioids consumption, visual analog scale (VAS) scores 10 min after extubation, at the time point leaving PACU, and at 1, 2, 24, and 48 h postoperatively, complications and prognosis, duration of hospital stay, satisfaction survey results, and also medical expenses.2.7β to 20%, we estimated that at least a total sample size of 90 patients would be required (30 in each group). To further reduce the bias, a population of 180 patients was considered to be included in this study.When designing the study, no published literature was available to estimate the effects of different doses of S‐ketamine on psychiatric complications of patients. We used the PASS 20.0 software  for calculation, setting a significance level of 0.05 and 2.8χ2 test or Fisher's exact test. Data were analyzed using SPSS . All statistical tests were two‐tailed, and a p < 0.05 was defined as statistically significant.Data were tested for normality by using the Kolmogorov–Smirnov test. Normally distributed variables are expressed as the means ± standard deviations, and abnormally distributed variables are expressed using the median . Categorical variables are expressed as numbers (percentages). Differences between groups were calculated by analysis of variance or the Kruskal–Wallis test, as appropriate. Categorical variables were analyzed using the 3Two hundred and sixteen patients were assessed for eligibility from April 2021 to December 2021, and 36 patients were excluded from this study for failing to meet the inclusion criteria. Of the 180 patients included and randomly assigned in this study, no participants were excluded for violation of study protocols or any other reasons. Eventually, 60 patients entered the analysis stage in each group. The complete research process is summarized in Figure 3.1p > 0.05).Significant differences were not detected in age, height, weight, ASA physical status classification, or history of mental illness between the groups. Likewise, no significant differences were found in the preoperative diagnosis, the laboratory test results, duration of anesthesia, operation mode, length of surgery (skin incision to closure), and intraoperative volume between the groups. The baseline characteristics of the three groups were tested to be comparable (Table 3.22) between groups at different observational time points were similar to each other. Severe hypertension or hemodynamic fluctuations were not found postoperatively .In this study, heart rate (HR), mean arterial pressure (MAP), and pulse oxygen saturation SpO between 3.3p > 0.05). Significant statistical differences were not observed with regard to RSS scores at 1, 2, 24, and 48 h, postoperatively . The details of psychiatric complications over time are presented in Table p > 0.05).No obvious delay was found in the total extubation time or PACU discharge time between the groups (Table 3.4p < 0.05). The anesthesia drug consumption had no significant difference between groups at PACU (Table p > 0.05), but with a difference in the surgical wards (Table p < 0.05). The CON group was reported to have the highest analgesic pump pressing times within 24 h after the operation. The incidence of PONV after 24 and 48 h after operation in the CON group was much higher, as nearly half (48.3%) of the patients in this group developed PONV within 48 h after the operation; the patients also experienced more severe postoperative PONV and drug antiemetic therapy (Table p < 0.05).Compared with the CON group, the VAS scores at 10 min after extubation and at the time point leaving PACU in the LDSK and MDSK groups were significantly lower. A significant difference was also reported between the MDSK and CON group at 48 h during rest, postoperatively . With regard to the medical satisfaction survey, compared with the CON group, patients in the S‐ketamine group experienced higher medical satisfaction (96.7%) (Table p < 0.05).There was no significant difference in postoperative fluid drainage amount, drainage tube extraction time, wound healing, bed rest time, total hospital stay, postoperative length of stay, and medical expenses between the two groups (Table 4N‐methyl‐d‐aspartic acid (NMDA) receptors, inhibiting the activation of glutamine on this receptor. The block of NMDA is time‐dependent and stimulation‐frequency‐dependent, so as to weaken the activity of neurons and achieve anesthetic and analgesic effects. According to previous pharmacological research, The affinity of NMDA receptors and u receptors of the S‐enantiomer was reported to be four times more potent than that of the tranditional levoisome, the affinity of M acetylcholine receptors of S‐enantiomer has been suggested to be two times more potent than the previous tranditional levoisome.Ketamine, the only intravenous general anesthetic with analgesic effects, was widely used in clinical anesthesia many years ago, especially in short‐term surgical anesthesia for children. In the past, ketamine was reported to have a great impact on hemodynamics with increased secretion and potential postoperative psychiatric complications, which severely limited its clinical application.2, perioperatively. No severe hypertension or tachycardia occurred in the two S‐ketamine groups during the study. The above results suggest that a small dose of S‐ketamine induction is safe and reliable in maintaining hemodynamic stability in elderly women. Moreover, during the induction of general anesthesia, general anesthetics including propofol have strong myocardial inhibition and vasodilation effects, which is unfavorable to advanced aged gynecological patients. The sympathetic excitation property of S‐ketamine avoided the occurrence of cardiovascular and cerebrovascular accidents related to severe hypertension or hypotension after induction. Small dose S‐ketamine used for general anesthesia induction is recommended by this study.Ketamine drugs indirectly excite the cardiovascular system by exciting the sympathetic nerve center, which is mainly manifested in the increase of HR, blood pressure, cardiac index, and peripheral vascular resistance. At the same time, it can also cause the sympathetic ganglion to release norepinephrine.Ketamine has an excitatory effect on the limbic system of the brain, resulting in the separation of consciousness and feeling, so there are many adverse reactions. The main manifestations are mental and nervous system reactions, such as illusion, schizophrenia, irritability, disorientation, cognitive impairment, delirium, moderate muscle tension, and tremor.The postoperative psychiatric complication is a major concern in perioperative medicine, and ketamine has been associated with a prolonged time to resumption of mental orientation. Psychodysleptic symptoms are well‐described side effects of ketamine. In the previous literature, the most reported postoperative complication related to ketamine was postoperative delirium. However, in our population, the risk for psychiatric complications was comparable, whereas there were no significant differences between all groups, which is in accordance with the current literature indicating that psychomimetic effects of S‐ketamine are dose‐dependent.Our data showed that minimal‐dose 0.2 mg/kg S‐ketamine induction was comparably effective to conventional LDSK in reducing postoperative pain. Moreover, pain scores differed significantly between the three groups at postoperative short‐term time points, and this analgesic effect even lasted until 48 h after the operation. It is known that tissue damage may trigger altered processing of noxious stimuli so that perception of pain persists even after cessation of nociceptive input. This is referred to as central sensitization and represents a mechanism of amplification of pain perception.Female patients are associated with a high risk of PONV. The use of opioids may activate NMDA receptors, leading to acute opioid tolerance and postoperative hyperalgesia, resulting in increased postoperative pain.The dosage of rocuronium consumption in the CON group was also much higher than that in the other two groups. Ledowski et al.In this study, no obvious difference was detected between the two S‐ketamine subgroups in pain control and prevention of mental complications. The prognosis results are similar, suggesting that a 0.2 mg/kg injection is enough for balanced anesthesia induction.The major limitation of our study is that we did not include all possible dose study groups, which would represent an interesting topic for further research. In our population, however, no significant differences were found in intraoperative opioid consumption between groups. Therefore, we conclude that a single injection of S‐ketamine has limited effects on saving opioid consumption.In conclusion, a minimal‐dose S‐ketamine regimen is effective in reducing postoperative pain following gynecological surgery and can reduce the risk of postoperative psychiatric complications when compared with a low‐dose regimen. We therefore suggest that subanesthesia S‐ketamine 0.2 mg/kg be used as a low‐risk component of balanced perioperative analgesia.Fan Zhang contributed to study design, research implementation, and writing. Jun Ding contributed to the patients' inclusion process. Man Luo, Hao‐Hua Luo, and Xiao‐Lin Sun contributed to participants' follow‐up in PACU and wards. Xu Fang, Lei Chen, and Jun Tao contributed to the statistical analysis. Zhao‐Qiong Zhu contributed to picture making, adverse event reports, and study quality control. All authors revised and approved the final article version, and agreed to be accountable for all aspects of the work.Zhao‐Qiong Zhu is a reviewer for Ibrain but is not involved in the peer review process of this manuscript. Other authors declare no conflicts of interest.The author confirms that this manuscript is an honest, exact, and transparent description of the reported research. There is no omission or concealment of any important aspect in the study or any differences from the planned study before the start.Ethics Committee of Affiliated Hospital of Zunyi Medical University approved all experiments on the involved patients (no. KLL‐2020‐049). This trial had been registered at chictr.org.cn (no. ChiCTR2100046703) and was conducted strictly in accordance with the Declaration of Helsinki. Written consent to participate was signed for all the patients. Eligible patients were contacted and informed about the study.


The role of guanylate‐binding proteins (GBPs) in various cancers has been elucidated recently. However, our knowledge of the clinical relevance and biological characteristics of GBPs in hepatocellular carcinoma (HCC) remains limited.A total of 955 HCC patients were enrolled from five independent public HCC cohorts. The role of GBP molecules in HCC was preliminarily investigated, and a GBP family signature, termed GBPs‐score, was constructed by principal component analysis to combine the GBP molecule values. We revealed the effects of GBP genes and GBPs‐score in HCC via well‐established bioinformatics methods and validated GBP1‐5 experimentally in a tissue microarray (TMA) cohort.+ T cells in HCC.GBPs molecules were closely associated with the prognosis of patients with HCC, and a high GBPs‐score highly inferred a favorable survival outcome. We also revealed high GBPs‐score was related to anti‐tumor immunity, the immune‐hot tumor microenvironment (TME), and immunotherapy response. Among the GBPs members, GBP1‐5 rather than GBP6/7 may be dominant in these fields. The TMA analysis based on immunohistochemistry showed positive correlations between GBP1‐5 and the immune‐hot TME with abundant infiltration of CD8Our integrative study revealed the genetic and immunologic characterizations of GBPs in HCC and highlighted their potential values as promising biomarkers for prognosis and immunotherapy. The genetic characterizations and prognostic values of GBPs in HCC were preliminarily disclosed. To fully integrate the values of GBP molecules, GBPs‐score was constructed by the principal component analysis (PCA) algorithm. We demonstrated the favorable effects of GBPs‐score on prognosis, anti‐tumor immunity, inflamed immune infiltration, and predicting immunotherapy responses in HCC patients from five independent cohorts. We also revealed the effects of individual GBP molecules through public cohorts and a tissue microarray (TMA) cohort. Overall, our study provides a better understanding of the immunologic patterns and clinical relevance of GBPs in HCC, as well as the predictive value of the GBP family signature for immunotherapy responses across cancers.22.1https://xenabrowser.net/datapages/) and the ICGC database (https://icgc.org/), respectively. GSE76427 and GSE54236 were retrieved from Gene Expression Omnibus (GEO) database (http://wwwncbinlmnih.gov/geo/). Gene expression profile and clinical information of the CHCC cohort were obtained directly from the supplementary materials of the published article.The workflow of this study is displayed in Figure http://research‐pub.Gene.com/imvigor210corebiologies/.http://tide.dfci.harvard.edu/download/).Moreover, eight immunotherapy groups were recruited to test the predictive value of GBPs‐score responses to immunotherapy: The IMvigor210 cohort  was obtained from 2.2To combine the values of GBP family genes and evaluate them in individual patients with HCC, we constructed a scoring system that we termed GBPs‐score. First, PCA was conducted. Secondly, the first two principal components (PC1 and PC2) were selected to construct the GBPs‐score. This approach has the advantage of focusing the score on the most well‐related (or anti‐related) genes to integrate the gene contributions fully.2.3HCC TMA cohort (HLivH180Su11) consisting of 94 HCC tissues was supplied by Shanghai Outdo Biotech together with detailed clinical information Table . Written2.4t‐test and Wilcoxon rank‐sum test were selected as the standard tests. The relationships between GBPs‐score and other continuous variables were assessed by the Pearson method. The correlation between GBPs‐score subgroups and clinical features was analyzed by the χ2 test. A two‐tailed p value < 0.05 was considered as statistical significance.All analyses were conducted using GraphPad (version 8.0), R (version 4.1.1), and SPSS (version 25) software. The Student's Other bioinformatics methods are detailed in the 33.1As shown in Figure 3.2p = 0.12), were significantly associated with better survival outcomes  survival analysis was performed, using the “survminer” R package to determine the optimal cutoff. In the TCGA cohort, patients with high expression of GBP molecules, except for GBP2  genes so that it fully integrates the gene contributions.3.4Next, we explored the clinical characteristics and other HCC‐related molecular signatures based on GBPs‐score. Since the ICGC, TCGA, and CHCC cohorts had relatively complete clinical information, we used these three cohorts for further analysis. The results displayed the patients in the low GBPs‐score group were associated with worse TNM stage and grade, larger tumor size, and higher percentages of tumor invasion in the ICGC cohort Figure . In the In addition, we compared GBPs‐score with other HCC‐related favorable or risk molecular signatures according to previous research.3.5To explore the biological behaviors between GBPs‐score subgroups, GSEA was conducted in the five cohorts Figure . Low GBPAs an IFN‐γ stimulated family, GBP molecules are closely related to immune responses.3.6+ T cell infiltration had the strongest positive association with GBPs‐score across all five cohorts  which is correlated with immune checkpoint blockade (ICB) response was introduced into the analysis.We also explored the effect of GBPs‐score in the immunotherapy response of other cancers  from eight immunotherapy‐related cohorts . In the IMvigor210 cohort, a higher GBPs‐score was significantly associated with the inflamed immune phenotype Figure , in whic4Previous studies have identified the roles of GBP family molecules in host‐pathogen infection and some cancers,Integrated evaluation on multiple independent cohorts of HCC displayed that GBPs‐score could act as an independent prognostic signature in HCC, and high GBPs‐score inferred better survival, which was verified by various methods  to avoid calculation errors. We also found strong positive relationships between GBPs‐score and favorable clinicopathological features, and other HCC‐related favorable molecular signatures. These data collectively suggested GBPs were associated with a favorable prognosis in HCC patients.+ T cells.+ T cells. Besides, higher CYT was associated with more MHC‐I‐associated neoantigens and enhanced the immune response.As members of the IFN‐γ stimulated superfamily, GBPs are also induced by inflammatory cytokines such as TNF‐α, IL‐1α and IL‐1β in immune cells.+ T cells with anti‐tumor activity.+ T cells and tumor clearance require the support of Th1 cells, which are the most important anti‐tumor helper T cells.+ T and Th1 cells) were more significant than the others, suggesting the improvement of the GBPs‐score was more related to the activation of anti‐tumor immunity. In terms of individual GBP genes, GBP1‐5 had stronger correlations with the infiltration of the anti‐tumor cells  genes in the set, while down‐weights contributions from genes with weak correlations. Therefore, the GBPs‐score was not a simple addition of gene effects, but rather a full integration of gene values. Thirdly, the GBP family signature (GBPs‐score) showed excellent performances, which were validated in multiple independent HCC cohorts. Additionally, its predictive ability in immunotherapy can be extended to other cancers. These data indicated the GBPs‐score can be a promising prognostic and immunologic biomarker for HCC.Some limitations of this study need to be noted. Firstly, although a TMA cohort with 94 human specimens was included, our study was mainly carried out using bioinformatics methods through a public database. Further experiments and clinical studies are required to verify the role of GBPs in the future. Secondly, although the excellent performance of the GBPs‐score was validated in multiple cohorts, we should explore in‐depth mechanisms of the relationships between GBPs in future researches. Thirdly, despite the excellent performance of GBPs‐score was shown, adding some non‐GBP genes with stronger statistical significance into the signature may further improve the diagnostic performance. Fourthly, due to lacking of immunotherapy data on HCC, the enrolled immunotherapy groups for analysis are non‐HCC, which cannot directly reflect the predictive value of GBPs‐score in HCC immunotherapy. To compensate for this limitation, we conducted the Submap analyses in HCC cohorts and PD‐L1 IHC assays in the TMA cohort, which could confirmed the correlation between GBPs and the response to anti‐PD1 treatment. Fifthly, we focused on the role of GBPs in tumor immune response, while the exploration of other biological effects of GBPs was limited.5This integrated analysis revealed the genetic characterizations and immunologic roles of GBP family genes in HCC. The GBPs‐score was able to quantify integrative values of GBPs, predict the prognosis of patients with HCC, identify distinct immunologic patterns of HCC microenvironment, and help guide more effective immunotherapy strategies in cancers.Yumei Ning: Data curation ; formal analysis (lead); methodology ; visualization ; writing – original draft (lead). Shilin Fang: Data curation (lead); methodology ; writing – original draft . Jun Fang: Conceptualization ; methodology ; supervision (lead). Kun Lin: Data curation (supporting); formal analysis (supporting). Haihang Nie: Visualization (supporting); writing – original draft (supporting). Peiling Xiong: Writing – original draft (supporting). Peishan Qiu: Methodology (supporting); writing – review and editing (supporting). Qiu Zhao: Funding acquisition (supporting); project administration (lead); writing – review and editing . Haizhou Wang: Formal analysis ; methodology (lead); writing – review and editing (lead). Fan Wang: Conceptualization ; funding acquisition (lead); writing – review and editing .The authors declare no conflict of interest.The use of human specimens was approved by Ethics Committee of Zhongnan Hospital of Wuhan University (No. 2020011). All methods were carried out by the relevant guidelines under the ethical approval and consent to participate section.Figure S1.Figure S2.Figure S3.Figure S4.Figure S5.Figure S6.Figure S7.Figure S8.Figure S9.Figure S10.Figure S11.Figure S12.Figure S13.Figure S14.Figure S15.Click here for additional data file.Data S1.Click here for additional data file.Data S2.Click here for additional data file.Table S5.Table S6.Table S7.Table S8.Table S10.Table S11.Table S12.Click here for additional data file.


Klebsiella pneumoniae have important role in infection persistence. The aim of the study was to evaluate the association between the prevalence of aminoglycoside resistance and virulence genes and biofilm formation capacity in K. pneumoniae strains isolated from hospitalized patients in South‐West of Iran.Resistance to antibiotics and the capability to develop biofilm as two main virulent determinants of K. pneumoniae collected from Ahvaz teaching hospitals. Identification of species was performed by biochemical tests and then confirmed by polymerase chain reaction (PCR) of rpoB gene. The susceptibility to antibiotics was determined by Kirby−Bauer disk diffusion method. Biofilm formation was assessed by microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants including fimbrial genes, aminoglycoside modifying enzymes‐ and 16S rRNA methylase (RMTase) genes.A total of 114 non‐duplicate clinical isolates of n = 81) of isolates were non‐susceptible to aminoglycosides. Among aminoglycoside antibiotics, K. pneumoniae isolates showed the highest and lowest resistance rates to tobramycin (71%) and the amikacin (25%), respectively. All biofilm producer strains were positive for the presence virulence determinants including ecpA, fimA, mrkD, and mrkA. Of 81 aminoglycosides non‐susceptible isolates 33% were positive for the presence ant (2″)‐Ia as the most prevalent gene followed by aac (3′)‐IIa and armA (27%), aac (6′)‐Ib (18%), and aph (3′)‐Ia (15%).Totally, all collected strains were carbapenem resistant and showed multidrug‐ and extensively drug‐resistance phenotype . Seventy‐one percent ‐Ia, aac (3′)‐IIa, and armA genes in aminoglycoside‐resistant isolates. This microorganism is especially dangerous in immunocompromised patients and those who have indwelling medical devices on which biofilm could developed.K. pneumoniae isolates poses a serious problem to public health, mainly with the rise of carbapenem‐resistant K. pneumoniae (CRKP) isolates. The irrational use of antibiotics in clinical practice has fostered the spread of resistance to “old class antibacterials.”Nosocomial K. pneumoniae simplified by chromosomal and plasmid‐mediated LPS changes have steadily increased with increased colistin usage for treating CRKP isolates.K. pneumoniae infections are increasing in intensive care units, particularly in patients who ultimately depend on vascular catheters for long‐term or short‐term use.Clonal complex 258 (CC258) is one of the CRKP clones disseminating all over the world.K. pneumoniae isolates grow as biofilms.K. pneumonia isolates to survive in the rough environmental conditions such as the antibiotic pressure and the host immune response. Biofilm formation in therapeutic devices, including urinary catheters, as their important stage in their disease pathogenesis can result in biofilm‐associated drug tolerance in these isolates.K. pneumoniae, biofilm development is an important virulence characteristic, and managing biofilms is very challenging as they confer significant tolerance to drug effects and host defense responses.CRKP isolates can cause serious infections in association with prolonged hospital stays, narrow therapeutic choices, and increased mortality. Several investigations indicated that MDR bacteria isolated from hosts with persistent infections are often biofilm formers. Also, it was noted that intrinsic resistance to antibacterial drugs increases when Biofilm can be considered as a reservoir of genetic diversity. In biofilm, the emergence and spread of antibiotic resistance genes increase through horizontal gene transfer. The horizontal transmission of antibiotic resistance genes increase between bacterial cells in an extracellular polymeric substance matrix.In addition to biofilm, the ability to acquire antibiotics resistance is considered as another important virulence determinant which plays a significant role in the successful pathogenicity. Aminoglycosides are frequently used to treat infections caused by Enterobacteriaceae.Enzymatic inactivation of aminoglycosides through three main known classes of aminoglycosides modifying enzymes (AMEs) including N‐acetyltransferases (AACs), O‐nucleotidyltransferases (ANTs), and O‐phosphotransferases (APHs) is the most considerable mechanism of bacterial resistance to aminoglycosides. AACs cause resistance to aminoglycosides by transferring acetyl groups from acetyl‐coenzyme A to amino group of these antibiotics, while ANTs by transferring adenyl group from ATP, and APHs by transferring γ‐phosphate to hydroxyl groups of these antibiotics confer the resistance, respectively.armA, rtmA, rtmB, rtmC and rtmD, and npmA, are located on transposons within transferable plasmids which render high level of resistance to all clinically relevant aminoglycosides.K. pneumoniae from Iran. The objectives of this study were to: (1) determination of antibiotic susceptibility pattern and capacity to form biofilm, (2) detection of AME and virulence genes, (3) identification of the relationship between the biofilm formation and aminoglycoside resistance among K. pneumoniae strains isolated from hospitalized patients in Iran.Another recently emerged mechanism of aminoglycosides resistance is methylation of ribosomal binding site of the antibiotic by 16S RMTase. The six important genes encoding 16S RMTase including 22.1K. pneumoniae collected from different clinical specimens in Ahvaz teaching hospitals affiliated to Ahvaz Jundishapur University of Medical Sciences during 6 months between October 2020 and March 2021. This project confirmed by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (No. IR.AJUMS.REC.1398.724). Patients signed informed consent forms before the start of this research project. The research was carried out in accordance with the Helsinki Declaration. Among all collected samples, 114 isolates were identified as K. pneumoniae using by phenotypic methods.This descriptive cross‐sectional study was performed on clinical isolates of 2.2Escherichia coli ATCC 25922 was used as a quality control strain.The antibiotic susceptibility test was done by the disk diffusion method on the Mueller−Hinton agar plates (Merck) according to CLSI guideline2.3Pseudomonas aeruginosa was also used as a positive control for the biofilm assay. All samples were tested three times.The capacity of isolates to develop biofilm was measured using the microtiter plate (MTP) method. The isolates were classified into four categories using the previously described criteria by Haney et al.2.4The MTP method was used to evaluate the capacity of amikacin and gentamicin to eradicate biofilms, as described previously.2.5fimA, fimH, mrkA, mrkD, and ecpA) in all isolates, AME genes  and 16S rRNA methylase genes  in aminoglycosides resistant isolates, in a final volume of 25 µL, including the following ingredients: 12.5 μL of 2 × Master Mix (Amplicon), 1 μL of forward primer, 1 μL of reverse primer, 2 µL of DNA template, and 8.5 μL sterile distilled water. The following thermal program was then used to amplify the fragments: Initial denaturation temperature of 95°C for 5 min, followed by denaturation temperature of 95°C for 45 s, annealing temperature of 56−63.5°C for 30 s, elongation temperature of 72°C for 45 s, and final elongation temperature of 72°C for 5 min. The number of cycles selected to perform the reaction was 35 cycles. The oligonucleotide primer sequences used in the present work are shown in Table http://blast.ncbi.nlm.nih.gov/Blast).The polymerase chain reaction (PCR) was performed for detection of genes involved in biofilm formation . The results were presented as descriptive statistics in terms of relative frequency . Fisher's exact test was employed to analyze the significant differences between variables. In addition, 33.1K. pneumoniae were obtained from several clinical specimens which distribution of them in terms of sample type shown in Table K. pneumonia mostly were isolated from urine , sputum , and tracheal tube . Remaining specimens were collected from blood , peritoneal aspirate , wound , tonsil , and surgical drain . The sputum and tracheal tube samples were obtained from patients with age mean ± SD: 61.07 ± 8.25 and 66 ± 7.88 years old, respectively.Between October 2020 and March 2021, a total of 114 clinical isolates of 3.2K. pneumoniae strains in present study were resistant to at least one of the carbapenem antibiotic. So, all of our examined strains were categorized as carbapenem resistant. However, according to the International Expert proposal for Interim Standards Guidelines,n = 86/114) and 24.6% (n = 28/114) of isolates revealed MDR and XDR phenotypes, respectively. In total, 74.6% (85/114) of isolates were resistant to aminoglycosides. Among aminoglycoside antibiotics, K. pneumoniae isolates showed the highest and the lowest resistance rates to tobramycin  and the amikacin , respectively. The K. pneumoniae isolates showed different levels of resistance to antibiotics as follows: gentamycin , tigecycline , piperacillin‐tazobactam , imipenem , meropenem , ertapenem , cefazolin , cefoxitin , cefotaxime , ceftazidime , ceftriaxone , cefoperazone , ciprofloxacin , azithromycin , and ampicillin‐sulbactam .3.3n = 88/114) of isolates were biofilm producer. Of these, 65.9% (n = 58/88) of isolates were strongly able to produce biofilm and remaining isolates were considered as moderate  and weak  biofilm formers. There was significant relationship between resistance phenotype and different degrees of biofilm production, so that the prevalence of strong biofilm producers among extensively drug resistant (XDR) phenotype was higher than MDR group . Majority of non‐biofilm producer isolates  were exhibited MDR phenotype. Biofilm producer strains were significantly placed in aminoglycosides non‐susceptible isolates  of non‐biofilm producer isolates were resistant to aminoglycosides.According to our results, 77.2%  and for amikacin between 0.25 and 128 μg/mL (MBEC50: 16 and MBEC90: 64). Data indicated that antibiotics‐resistant isolates in the planktonic mode did not show increase in resistance to antibiotics when grow in biofilm state. However, stronger biofilm former showed a dramatic increase in resistance to antibiotics when grow in biofilms mode compared with moderate and weak biofilm former strains. The gentamicin and amikacin MBECs against stronger biofilm former strains were ≥16 and ≥32 μg/mL, respectively, while, against moderate and weak biofilm former, the gentamicin MBEC was ≤8 μg/mL and the amikacin MBEC was ≤16 μg/mL.To investigate the ability of gentamicin and amikacin in eradication of preformed biofilm, we determined MBEC. So those, after treatment with antibiotics at the different concentrations of amikacin and gentamicin, living bacteria were counted in all tested isolates. Table 3.5n = 70/85) had AME genes. Some isolates had only one gene and some others harbored a combination of two or more genes  by PCR and sequencing. Of the 70 positive isolates for at least one of the AMEs or RMTase genes, only 2 isolates carried one gene encoding AMEs and 15 isolates harbored armA alone, 38 isolates carried two genes, and 15 isolates carried three genes. Overall, 10 different combination patterns were determined in mentioned isolates ‐Ia , aac(3′)‐IIa , armA , aac(6′)‐Ib , and aph(3′)‐Ia . None of the mentioned isolates were positive for rmtA, rmtB, rmtC, rmtD, and npmA genes. Our findings revealed that the most frequent AME related genes and RMTase gene were ant(2”)‐Ia, aac(3′)‐IIa, and armA genes followed by aac(6′)‐Ib. As shown in Table ant(2”)‐Ia was found to coexist with aac(3′)‐IIa , aph(3′)‐Ia , and with aac(6′)‐Ib . The mentioned coexistences were the most common combinations in tobramycin and gentamicin non‐susceptible isolates. All amikacin resistant isolates harbored armA either alone or in combination with ant(2”)‐Ia and aac(6′)‐Ib. It is noteworthy that, isolates positive for ArmA in combination with mentioned AMEs genes were simultaneous resistance to tobramycin and amikacin.The prevalence of genes encoding AMEs among 85 aminoglycoside non‐susceptible  n = 38; 4.7%, aacecpA, fimA, mrkD, and mrkA) were detected in all biofilm producer K. pneumoniae strains, whereas fimH was determined in 93.2% (n = 82/88) of biofilm forming isolates. FimA and ecpA were detected in 42.3% (n = 11/26) and 38.5% (n = 10/26) of non‐biofilm producer strains, respectively. Among these strains mrkA, fimH, and mrkD genes were not identified. Most isolates harboring AMEs genes were also positive for presence biofilm related genes including ecpA, mrkA, and fimbrial genes. Table In the current study, the prevalence of genes involved in biofilm formation and virulence ‐IIa and aac(6′)‐Ib in our study was higher than that reported from previous studies from Iran and China in which 20.75%−30.2% and 19.8%−21.7% of clinical isolates of K. pneumonia were positive for the presence of aac(3)‐IIa and aac(6′)‐Ib genes, respectively.ant(2″)‐Ia was the most frequent AME gene. In line with this study, Miller et al.ant(2″)‐Ia gene as the dominant determinant in the United States, in which gentamicin was the most commonly used aminoglycoside. Furthermore, 75.7% of our isolates harbored combination of two or three AMEs and/or RMTase genes among which armA‐ ant(2″)‐Ia, armA‐aac(6′)‐Ib, and aac(6′)‐Ib‐aac(3′)‐IIa were more prevalent. The prevalence of combination of armA and AMEs genes reported by two studies from Greeceaac(6′)‐Ib and aac(3′)‐IIa was reported as the most common genotype.Indiscriminate and uncontrolled use of antibiotics, especially in third world countries like Iran, is one of the reasons for increasing antibiotic resistance. Two of the most principal mechanisms of resistance to aminoglycosides are AME and RMTase genes, which are known to be present in gram‐positive and gram‐negative bacteria. The existences of these coding genes has been reported in several studies from Iran.K. pneumoniae isolates. Moreover, the majority of isolates were biofilm producers. These findings were in line with the previous studies that revealed significant correlation between biofilm formation capacity and antibiotic resistance among K. pneumonia isolates.K. pneumoniae strains. MDR K. pneumoniae is a significant source of concern because it not only causes severe and fatal infections, but it also prolongs hospitalization, resulting in higher treatment costs.One of the notable findings in this study was the 100% rate of carbapenems resistance of K. pneumoniae isolates were more resistant to gentamicin and amikacin than those of non‐biofilm producers. In this study, the majority of XDR isolates were strong biofilm producers. In line with our results, Vuotto et al.K. pneumoniae strains. However, the presence of association between antibiotic resistance and biofilm formation capacity is still debated by some researchers.Our findings revealed that biofilm producer strains were significantly aminoglycosides non‐susceptible isolates that was in agreement with a previous study by Karimi et al.ecpA, fimA, mrkD, and mrkA genes were harbored by all biofilm producing K. pneumoniae strains, whereas fimH was determined in 93% (82/88) of isolates. The prevalence of mrkA, ecpA, and fimH (100%) in our study were almost similar to that reported by Alcántar‐Curiel et al.K. pneumoniae isolates were able to produce biofilm and all isolates carried the mrkA gene. Moreover, our results showed that all MDR/XDR carbapenem‐resistant biofilm producing K. pneumoniae isolates carried the ecpA, fimA, fimH, mrkD, and mrkA genes. These results may indicate the importance of these virulence determinants in association with carbapenem resistance in K. pneumoniae. So far, no study has investigated the association of aforementioned genes with carbapenem resistance in K. pneumoniae isolates.This study showed that This study was performed during the pandemic of COVID‐19 and due to the crisis raised from this situation and the traffic restrictions, there were several limitations. In this study we did not evaluate the clonality of the isolates by genotyping methods. Also, we did not evaluate the other possible aminoglycoside resistance mechanisms including efflux pumps.K. pneumoniae isolates were carbapenem‐resistant. Moreover, K. pneumoniae isolates showed the highest and the lowest aminoglycoside resistance rates to tobramycin and amikacin, respectively. Majority (more than 70%) of isolates were biofilm producers and there was significant association between antibiotic resistance pattern and the strength of biofilm production. Also, ant(2”)‐Ia and aph(3′)‐Ia were the most and the least frequent AME genes, respectively. Finally, most AMEs harboring isolates were also positive for presence of the biofilm related genes including ecpA, mrkA, and fimbrial genes.In conclusion, this study showed that all Saeed Khoshnood: Resources. Sousan Akrami: Conceptualization. Morteza Saki: Data curation. Moloudsadat Motahar: Formal analysis. Sara Masihzadeh: Resources. Sara Daneshfar: Resources. Hossein Meghdadi: Validation. Effat Abbasi Montazeri: Visualization. Marjan Abdi: Writing—original draft. Zahra Farshadzadeh: Conceptualization; investigation; supervision.The authors declare no conflict of interest.The lead author Zahra Farshadzadeh affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.Protocols for collection of samples as well as the experiment plan and all methods were performed in accordance with the guidelines and regulations of Ahvaz Jundishapur University of Medical Sciences and approved by the institutional ethical committees. Written informed consent was obtained from participants or parents before their involvement. The present research was supported by the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (OG‐9838). All authors have provided consent for this publication.


Colorectal cancer (CRC) is a common gastrointestinal malignancy with high morbidity and fatality. Chemotherapy, as traditional therapy for CRC, has exerted well antitumor effect and greatly improved the survival of CRC patients. Nevertheless, chemoresistance is one of the major problems during chemotherapy for CRC and significantly limits the efficacy of the treatment and influences the prognosis of patients. To overcome chemoresistance in CRC, many strategies are being investigated. Here, we review the common and novel measures to combat the resistance, including drug repurposing , gene therapy , protein inhibitor , natural herbal compounds , new drug delivery system , and combination therapy. These common or novel strategies for the reversal of chemoresistance promise to improve the treatment of CRC. Chemoresistance is the major cause of treatment failure and disease relapse in colorectal cancer (CRC). In addition to some common reversal measures, some novel strategies have sprouted in recent years, including drug repurposing, gene therapy, protein inhibitor, natural herbal compounds, new drug delivery system, and combination therapy, which promise to improve the treatment of CRC. It can also be divided into primary drug resistance and multidrug resistance (MDR) in terms of the drug resistance spectrum. Multidrug resistance is seem to be the major determinant of cancer chemotherapy failure. Nowadays, drug resistance not only undermines the therapeutic effects of anticancer chemical drugs but also causes CRC refractory. Therefore, drug resistance has become a thorny issue and urgently needed to be solved. Various strategies have been applied or investigated to the drug resistance reversal in CRC. The most common treatment among them is the combined use of chemical drugs and marketed medicines with already known effects such as drug repurposing  and natural herbal compounds . Gene therapy, as an emerging technology, has also been studied in chemoresistance reversal, including ribozymes, RNA interference, CRISPR/Cas9, epigenetic therapy, antisense oligonucleotides, and noncoding RNAs, which opens the door to a new genetical world in this field. Protein inhibitors are countermeasures at protein levels, which exserts their reversal effects directly by reacting with specific proteins such as EGFR inhibitor, S1PR2 inhibitor, and DNA methyltransferase inhibitor. It has been known that conventional drug delivery systems have many shortcomings comprising low bioavailability and cytotoxicity, which contribute to drug resistance. New drug delivery systems, including nanocarriers, liposomes, exosomes, and hydrogels, can overcome the drawbacks of traditional delivery systems and transport the therapeutic molecules directly to the specific tumor site, which plays an important role in reversing chemoresistance , metformin, dichloroacetate, enalapril, ivermectin, bazedoxifene, melatonin, and S‐adenosylmethionine. Due to their low expenses, low toxicity, and high safety, drug repurposing is a promising strategy for combating chemoresistance in CRC Table .2.1Nonsteroidal anti‐inflammatory drugs (NSAIDs), as a kind of widely used medicine worldwide, have potent anti‐inflammatory, analgesic, and antipyretic activity. The main mechanism of the action is the inhibition of cyclo‐oxygenase (COX), which is responsible for the biosynthesis of prostaglandins and thromboxane.2.2Metformin (MET), which suppresses hepatic glucose production, is the first‐line therapy for type 2 diabetes mellitus.2.3Dichloroacetate (DCA), originally utilized to treat metabolic diseases as a metabolic regulator, was discovered to overcome chemoresistance in CRC through p53/miR‐149‐3p/pyruvate dehydrogenase kinase 2 (PDK2) glucose metabolic pathway.2.4Enalapril, an antihypertensive and antiheart failure drug, was found to inhibit cell migration and invasion and overcome chemoresistance in the 5‐FU‐resistant CRC cells at clinically acceptable dosages without extra toxicity. And the effect may be primarily achieved by the suppression of cell proliferation, angiogenesis, and NF‐κB/signal transducer and activator of transcription 3 (STAT3)‐regulated proteins, including Cyclin D1, c‐Myc, matrix metalloproteinase‐2 (MMP‐2), matrix metalloproteinase‐9 (MMP‐9), vascular endothelial growth factor (VEGF), Bcl‐2, and X‐linked inhibitor of apoptosis protein (XIAP).2.5Ivermectin, an antiparasitic drug, could reverse the chemoresistance in VCR‐resistant HCT‐8/VCR cells by diminishing P‐gp expression via reducing the activation of epidermal growth factor receptor (EGFR) and its downstream extracellular signal‐regulated kinases (ERK)/Akt/NF‐κB signaling pathway.2.6Bazedoxifene, the third‐generation of selective estrogen receptor modulator, also a novel IL‐6/glycoprotein 130 (GP130) target inhibitor, improved the anticancer effect of 5‐FU in CRC via impeding the IL‐6/GP130 signaling pathway and the phosphorylation of the downstream effectors such as AKT, ERK, and STAT3, suggesting that the block of IL‐6/GP130 might reverse chemoresistance in CRC.2.7Melatonin, used for insomnia in usual, was found to reduce cell viability, promote apoptosis, and reverse 5‐FU resistance in SW480/5‐FU and HCT116/5‐FU cells by downregulating thymidylate synthase (TYMS) via upregulating miR‐215‐5p.2.8S‐adenosylmethionine (AdoMet), a natural chemical and generally utilized as a dietary supplement, was capable of conquering 5‐FU chemoresistance in CRC cells through reverting the P‐gp upregulation induced by 5‐FU and suppressing the activation of the key anti‐apoptotic factor NF‐κB implicated in P‐gp‐related chemoresistance.2.9Vitamins are common dietary supplement used in daily life and their potential effects on chemoresistance reversal in CRC have been found, including vitamin C and vitamin D. Vitamin C, also called ascorbic acid, could sensitize the human CRC cells to L‐OHP by inducing cell apoptosis.3Gene therapy has become a novel strategy to conquer chemoresistance in CRC by gene correction, immunogene therapy, prodrug activation, oncolytic viruses, and other technologies manipulated on genes.3.1Ribozymes are small RNA molecules with catalytic functions, which can degrade specific mRNA sequences. Transfection of the anti‐MDR1 ribozyme, bound to the carcino‐embryonic‐antigen (CEA) promoter, could reverse DOX resistance by reducing P‐gp expression in SW1116R MDR CRC cells.3.2Antisense oligonucleotides (ASOs) are synthesized nucleic acids, containing 12–25 nucleotides in general, which exert their inhibitive effect by binding to the target RNA through Watson–Crick hybridization.3.3RNA interference (RNAi) regulates gene expression and contributes to the targeted therapy through 20–24 bp RNA including small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs).3.3.1Knockdown of the ubiquitin fusion‐degradation 1‐like protein (UFD1) could restore the sensitivity of hydroxycamptothecin (HCPT)‐resistant SW1116/HCPT colon cancer cell line to HCPT probably via increasing caspase‐3‐mediated apoptosis and inducing endoplasmic reticulum (ER) stress.3.3.2A disintegrin and metalloproteinase 17 (ADAM17) shRNA could inhibit cell proliferation, increase apoptosis, and reverse L‐OHP resistance in L‐OHP resistant HCT‐8 cell line by suppressing the EGFR/PI3K/AKT signaling pathway.3.4CRISPR/Cas9 technology, a powerful, efficient, easy, and specific gene editing tool, is extensively administrated in tumor pathogenesis, treatment, and drug resistance by inserting or knocking out the related genes.3.5Micro‐RNAs (MiRNAs), defined as small ncRNAs containing 21–23 nucleotides, regulate gene expression at the level of posttranscription by repressing mRNA translation and inducing mRNA degradation.3.6Long noncoding RNAs (LncRNAs), which are transcripts that exceed 200 nucleotides, can regulate CRC progression by acting as competitive endogenous RNAs (ceRNAs) by interacting with miRNAs or protein‐coding mRNAs and encoding short peptides.3.7CircRNAs, a special subclass of ncRNAs, have a covalently closed circular structure with no 5′ cap structure and 3′ polyA tail, which is associated with tumor recurrence and chemotherapy resistance in CRC.3.8CCNEI, CCNDBP1, CHL1, DDX43, and PON3. Treatment of 5‐aza‐2′‐deoxycytidine  could restore the sensitivity to 5‐FU in the SW48 cell line.Epigenetic therapy is a coping strategy to modify the aberrant epigenetic alterations in cancers, including CRC. It has been found that epigenetic alterations like higher DNA methylation and mRNA N6‐methyladenosine (m6A) are related to the occurrence of chemoresistance and tumor relapse in CRC.4In the treatment of CRC chemoresistance, except for gene‐level therapy, direct protein inhibitors also play an important role. Various inhibitors, including inhibitors of classical resistance‐related proteins, EGFR inhibitors, and sphingosine 1‐phosphate receptor (S1PR) modulators have been found to reverse chemoresistance in CRC.4.1An increment in drug efflux could reduce drug concentration and accumulation in tumor cells, which leads to drug resistance. Targeting ATP‐binding cassette (ABC) transporters like breast cancer resistance protein (BCRP) and P‐gp provides a potential approach to eliminating drug resistance during the treatment of CRC. GF120918, a potent P‐gp and BCRP inhibitor, markedly increased the oral bioavailability of topotecan from 40% to 97% in cancer patients.4.2EGFR is a transmembrane receptor belonging to a family of receptor tyrosine kinases that mediates a series of intracellular pathways that promote tumor proliferation, invasion, metastasis, and neovascularization.4.3Sphingosine‐1‐phosphate (S1P), one of the sphingolipids, could regulate cancer survival by binding sphingosine 1‐phosphate receptors (S1PRs), which activate multiple cell growth‐related pathways.4.4Notch and Wnt signaling‐associated proteins were upregulated in HCT116/5‐FU and HCT116/L‐OHP cells, including Notch1 receptor intracellular domain NICD1 and nonphosphorylated β‐catenin and Notch target gene HES1. Wnt inhibitor XAV939 and Notch inhibitor RO4929097 could restore cell viability in L‐OHP‐treated HCT116 cells.5Natural herbal compounds have become putative adjuncts of conventional chemotherapy in CRC and are able to reverse acquired drug resistance by different mechanisms.5.1Hovenia dulcis), could inhibit both MRP2 expression and its promoter activity by inhibiting NF‐κB‐Nrf2 signaling in HCT116/L‐OHP and HCT‐8/VCR cell lines, contributing to the reversal of L‐OHP/VCR‐resistant CRC cells.Dihydromyricetin, a flavonoid compound (belonging to polyphenols) extracted from the Japanese raisin tree , and glutathione S‐transferase (GST), which reverses chomoresistance in HCT‐8/5‐FU and LoVo/5‐FU cell lines.5.6Salvia miltiorrhiza Bge. (Labiatae), could reverse MDR in HCT‐8/VCR cells through elevating ROS levels, which promoted apoptosis and lowered the expression of P‐gp, which increased the chemosensitivity of drug‐resistant cancer cells.Salvianolic acid B, a phenolic acid isolated from the dried root and rhizome of 6The low solubility and bioavailability, off‐target, and cytotoxicity to normal tissues of chemotherapy decreases the therapeutic effect and promotes drug resistance in CRC, which causes relapse and poor prognosis of CRC patients. To deal with it, a new drug delivery system including nanocarriers, liposomes, exosomes, and hydrogels has been used to overcome chemoresistance in CRC.6.1Lower toxicity and fewer side effects in blood and noncancerous tissues, increased aqueous solubility and bioavailability, specific target feature and multifunctional drug combination make nanoparticle‐based formulations become an effective strategy to overcome drug resistance in CRC combined with conventional chemotherapy.6.2Liposomes, artificial colloidal vesicles with unique lipid bilayered membranes, can target tumor cells after chemical modification or physical treatment and have been used in the targeted therapy of cancer.6.3Exosomes are endocytic extracellular vesicles (EVs) ranging from about 40–160 nm in diameter, which is associated with cancer proliferation, migration, invasion, and drug resistance.6.4Hydrogels, an emerging drug carrier, are three‐dimensional (3D) frameworks comprised of cross‐linked hydrophilic polymers. Due to their excellent biocompatibility and biodegradability, hydrogels have been extensively utilized for the treatment of cancer chemoresistance and relapse in recent years.5‐FU loaded in a thermal sensitive hydrogel system (a biodegradable PEG‐polycaprolactone (PCL)‐PEG (PECE) triblock copolymer), prolonged drug release time and markedly inhibited the growth and dissemination of CT26 cells in colorectal peritoneal carcinomatosis (CRPC) murine model.7Combination therapy is the common treatment for CRC. FOLFOX, capecitabine and oxaliplatin (CapeOX), and leucovorin, fluorouracil, and irinotecan hydrochloride (FOLFIRI) are the first‐line regimen for chemotherapy in CRC. Combination therapy could enhance anticancer efficacy, prolong survival, prevent drug resistance, and tumor relapse, which always makes an effect of “1 + 1 > 2.” Various combination therapies combating chemoresistance have been clarified in the upper five sections, so the combined therapies not belonging to these sections are illustrated below.7.1Lactobacillus Plantarum Supernatant (LP SN) combined with 5‐FU could reverse the resistance to 5‐FU and enhance the therapeutic effect of 5‐FU in 5‐FU‐resistant HT29 cells and HCT116 cells by inactivating the Wnt/β‐catenin signaling, reducing CSCs, and increasing apoptosis.Auranofin, a forkhead box 3 (FoxO3) agonist and thioredoxin reductase 1 (TR1) inhibitor, could enhance the sensitivity of HCT‐8/5‐FU and SW620/5‐FU cells to 5‐FU in vitro and the sensitivity of SW620/5‐FU cells to 5‐FU treatment in vivo by inhibiting the nuclear factor erythroid 2‐related factor 2 (Nrf2)/TR1 signaling pathway. Auranofin in combination with 5‐FU induced the most cell death compared to treatment with auranofin or 5‐FU alone and suppressed tumor cell invasion obviously. In SW620/5‐FU cells xenograft nude mice, fewer lung metastatic nodules were observed in auranofin plus 5‐FU group than in the 5‐FU or auranofin groups.7.2Administration of Toll‐like receptor agonists R848 combined with L‐OHP reversed the functional orientation of myeloid‐derived suppressor cells (MDSCs) toward tumoricidal M1‐like macrophages and strengthened the antitumor effect of L‐OHP.7.3Tamoxifen, a selective estrogen receptor modulator, combined with DOX can reverse MDR in CRC nude mice, reduced weight and volume of transplanted tumors, which is independent of the expression of estrogen receptors and has no influence on MDR1 expression.8CRC is one of the most prevalent cancers across the world with increasing incidence in recent years, and the chemoresistance caused by long‐term use has evaded the therapeutic process of CRC. Chemical drugs, combined with clinical common drugs, herbal medicine monomers, gene therapy, and protein inhibitors, could restore the chemosensitivity of tumors to chemodrugs. Nanocarriers, liposomes, exosomes, and hydrogels, as drug delivery systems, could combine multiple drugs together well, transport these drugs to the tumor site directly, and control the drug release, which dramatically enhances the drug effect and reverse the drug resistance occurred in mono‐drug therapy. Reversal drugs involved in drug repurposing and herbal medicinal compound are already marketed drugs, and are the safest and fastest mean able to apply in the clinic, thus more efforts should be contributed in these fields. Gene therapy and protein inhibitors, belonging to gene and protein levels, respectively, become a hot spot in cancer treatment and chemoresistance. Moreover, gene therapy in chemoresistance reversal still stays in the early stage but own huge developmental potential. So far, although various potential strategies have been found, there is still a long way to go from laboratory to clinic. Therefore, further and consistent investigation is required to the realization of the victory in the chemoresistance combat of CRC.Shuchang Ma: Visualization (lead); writing – original draft (lead); writing – review and editing (lead). Jiaqi Zhang: Visualization (lead); writing – review and editing (lead). Tianhua Yan: Writing – review and editing . Mingxing Miao: Writing – review and editing . Yemin Cao: Writing – review and editing . Yongbing Cao: Writing – review and editing . Lichao Zhang: Writing – review and editing . Ling Li: Supervision (lead); validation (lead); visualization (lead); writing – review and editing (lead).This study was supported by grants from the National Natural Science Foundation of China (No. 81973551), the Science and Technology Commission of Shanghai Municipality , the Innovative projects of Shanghai University of Traditional Chinese Medicine (Y2021030), the Future plan for Traditional Chinese Medicine development ofScience, the Health Commission of Shanghai Municipality (ZY(2021‐2023)‐0203‐04) and the Technology of Shanghai Municipal Hospital of Traditional Chinese Medicine (WL‐HBBD‐2021001 K).The authors declare no potential conflict of interest.


C, comprising the ligand‐binding domains of both EGFR and ERBB4 and able to trap all ERBB ligands. Next, we generated a transgenic mouse model (CBATRAP/0) expressing TRAP‐FC ubiquitously under the control of the chicken‐beta‐actin promoter and crossed these mice with KRASG12D/+ mice (Kras) to generate Trap/Kras mice. The resulting mice displayed decreased emergence of spontaneous pancreatic lesion areas and exhibited reduced RAS activity and decreased activities of ERBBs, with the exception of ERBB4, which showed increased activity. To identify the involved receptor(s), we employed CRISPR/Cas9 DNA editing to singly delete each ERBB receptor in the human pancreatic carcinoma cell line Panc‐1. Ablation of each ERBB family member, especially the loss of EGFR or ERBB2/HER2, altered signaling downstream of the other three ERBB receptors and decreased cell proliferation, migration, and tumor growth. We conclude that simultaneously blocking the entire ERBB receptor family is therapeutically more effective than individually inhibiting only one receptor or ligand in terms of reducing pancreatic tumor burden. In summary, trapping all ERBB ligands can reduce pancreatic lesion area and RAS activity in a murine model of pancreatic adenocarcinoma; hence, it might represent a promising approach to treat PDAC in patients.Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of cancers. Attempts to develop targeted therapies still need to be established. Some oncogenic mechanisms in PDAC carcinogenesis harness the EGFR/ERBB receptor family. To explore the effects on pancreatic lesions, we attempted simultaneous blockade of all ERBB ligands in a PDAC mouse model. To this end, we engineered a molecular decoy, TRAP‐F C, was overexpressed in a mouse model for pancreatic ductal adenocarcinoma. The TRAP‐FC molecule, which comprises ligand‐binding domains of both EGFR and ERBB4, is able to trap all ERBB ligands. Mice overexpressing the decoy show significantly reduced pancreatic lesions and decreased EGFR and ERBB2 activation compared with control animals.An engineered decoy molecule, TRAP‐F ADMacinar‐to‐ductal metaplasiaAREGamphiregulinBrdU5‐bromo‐2′‐deoxyuridineBTCbetacellulinCBAchicken‐beta‐actinCRISPR/Cas9clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9DMEMDulbecco's Modified Eagle MediumECDextracellular domainEDTAethylenediaminetetraacetic acidEGFepidermal growth factorEGFRepidermal growth factor receptorEPGNepigenEREGepiregulinFBSfetal bovine serumGAPDHglycerinaldehyd‐3‐phosphat‐dehydrogenasegRNAguide RNAHBEGFheparin‐binding EGF‐like growth factorICDintracellular domainKOknockoutKrasKRASG12D/+ miceMABmonoclonal antibodyMAPKmitogen‐activated protein kinaseMEKmitogen‐activated protein kinase kinasePanINpancreatic intraepithelial neoplasiaPBSphosphate‐buffered salinePDACpancreatic ductal adenocarcinomaPFApara‐formaldehydePI3Kphosphoinositide 3‐kinasePKBprotein kinase BRAFrapidly accelerated fibrosarcomaRASrat sarcomaRTroom temperatureRTKreceptor tyrosine kinaseTtransgenic TRAP miceTGFAtransforming growth factorTrap/KrasPtf1aCre/0; KRASG12D/+; CBATRAP/0 miceTKItyrosine kinase inhibitor1C, by recombining the extracellular domains (ECDs) of EGFR and ERBB4, fused by a linker domain. Secretion of the decoy is ensured by the presence of a synthetic leader sequence. TRAP‐FC is able to sequester all ERBB ligands, thereby inhibiting a broad range of ERBB signaling. We previously reported that after TRAP‐FC‐treatment the proliferation rate in the human pancreatic cancer cell lines BxPC3, Panc‐1, and MiaPaCa2 were significantly reduced and in BxPC3 cells [C even reduced proliferation after the exposure to growth factors, like EGF, TGFA, and HBEGF. Furthermore, TRAP‐FC in combination with gemcitabine prolonged the survival of subcutaneously BxPC3‐injected tumors in mice [C under the control of the ubiquitous chicken‐beta‐actin promoter (CBATRAP/0) to evaluate the potential of this therapeutic approach in a genetic pancreatic cancer model. In addition, we studied the consequences of the deletion of each ERBB receptor in a human PDAC cell line. Independently, we deleted EGFR, ERBB2, ERBB3, and ERBB4 in Panc‐1 cells by using CRISPR/Cas9 gene editing, in order to analyze the dependency of Panc‐1 cells on the individual ERBB receptors in terms of ERBB and intracellular signaling, cell migration, proliferation, and RAS activity.Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of all cancers in Europe and the US , 2. AlthC3 cells , TRAP‐FC in mice . In our 22.1EcoRI side downstream of the chicken‐beta‐actin (CBA) promoter and rabbit‐beta‐globin splice acceptor into the expression vector pUC‐CAGGS , and upstream to the rabbit‐beta‐globin 3′‐flanking region and polyadenylation signal. The plasmid was linearized by restriction enzyme digest using SalI and HindIII to generate the microinjection construct, which was injected into zygotes of C57BL/6N mice, which were purchased from Janvier . Thus, we obtained the CBATRAP0 mouse line ubiquitously expressing the TRAP‐decoy molecule. The genotypes of CBATRAP0 mice were verified by PCR , employing genomic DNA from tail tips by using the oligonucleotides: pTORU‐seq: 5′‐CTACAGCTCCTGGGCAACGTG‐3′, and TRAP‐Rev: 5′‐ATCTTGCCAGTGTATAGTGTCAGC‐3′. Mice carrying floxed KRASG12D+ (B6.129S4‐Krastm4Tyj/J) [Ptf1a‐Cre; Ptf1atm1creHnak) [Ptf1aCre0; KRASG12D+ (herein referred to as Kras) mice with CBATRAP0 mice (herein referred to as Trap), herein referred to as Trap/Kras mice. In our mouse facility Ptf1aCre0; KRASG12D+ mice were crossed over 20 generations in the C57BL/6N background. Mice were maintained in the C57BL/6N background and housed under specific pathogen‐free conditions in the closed barrier facility of the Gene Center Munich at 23 °C, 50% humidity, and with a 12‐h light/dark cycle (lights on at 7 AM). They had free access to water and a standard rodent diet .All animal experiments were approved by the Committee of Animal Health and Care of the local governmental body of the state of Upper Bavaria , Germany, and were performed in strict compliance with the European Communities Council Directive (86/609/EEC) recommendations for the care and use of laboratory animals. Pronuclear microinjection was used to generate a mouse line that expresses the TRAP molecule consisting of the ECDs of murine EGFR and ERBB4, which are connected by a linker and a synthetic leader sequence to ensure that the molecule will be secreted. The TRAP sequence was ligated into the Tyj/J)  alleles 2.22. At a confluence of 90%, cells were starved for 3 h (1% FBS) and subsequently stimulated with 25 ng·mL−1 of recombinant human BTC  for 10 min. Cells were then lysed in a TRIS‐based buffer  freshly supplemented with protease and phosphatase inhibitors (Roche) and the lysates were subjected to western blot analysis.Panc‐1 cells (Research Resource Identifier: RRID:CVCL_0480) were purchased from CLS  4 months before the experiments were performed. All human permanent cell lines in the CLS cell bank were authenticated by using the STR DNA profiling analysis. Mycoplasma testing was done every 6 months using a mycoplasma detection kit . The cells were cultured in Dulbecco's Modified Eagle Medium , supplemented with 10% fetal bovine serum  and 1% Penicillin/Streptomycin  and maintained at 37 °C and 5% CO2.3EGFR, ERBB2, ERBB3, and ERBB4 genes were designed using benchling  were performed as described previously [Ensembl genome database; indels were verified for each ERBB gene locus; and the ERBB knockouts (KOs) were verified by western blot analysis. sgRNA sequences, KO‐genotyping primer sequences, miSeq primer sequences can be found in Tables Guide RNA (gRNA) sequences targeting the desired loci of rs Table  for MiSe2.4Male and female mice were sacrificed by cervical dislocation at the age of 4 months. The pancreas was isolated, blotted dry, and weighed to the nearest milligram. Parts of the head, tail, and central part of the pancreas were dissected, pooled, frozen on dry ice, and stored at −80 °C. The remaining tissue was fixed in 4% paraformaldehyde  overnight and subsequently embedded in paraffin for histopathological examination.2.5t‐test and plotted in graphpad prism .We performed a histomorphological analysis as described previously . In brie2.6−1) or unstimulated. Three hundred microgram of total lysate was incubated with the GSTRaf1Ras binding domain for 1 h at 4 °C, washed, eluted under denaturing conditions, and applied to western blot analysis detecting RAS. Total protein was analyzed by western blot analysis of the total lysates detecting total RAS and GAPDH. The amount of active RAS was measured by densitometry using imagej , referenced to total RAS and GAPDH, and plotted in graphpad prism. Data were analyzed by the Student's t‐test.To evaluate pancreatic RAS activity, the Active Ras Detection Kit  was used according to the manufacturer's instructions. In brief, dissected, the frozen pancreas was homogenized or Panc‐1 cells were lysed in LBW buffer, freshly supplemented with phenylmethanesulfonyl fluoride. Panc‐1 cells were either lysed after BTC  stimulation ). Equal amounts of protein were electrophoresed on 10% SDS gels and blotted to PVDF membranes . The membranes were blocked using 5% milk and incubated in primary antibodies overnight at 4 °C. After washing, the membranes were incubated in the appropriate horseradish peroxidase‐conjugated secondary antibody at room temperature for 1 h. Immuno‐reactive bands were visualized using an ECL kit . Antibodies and dilutions are provided in Table imagej 1.52a and plotted in graphpad prism (GraphPad Prism version 5.0). Western blots were repeated three times to confirm the shown results.Murine pancreas was homogenized in LBW buffer , freshly supplemented with protease and phosphatase inhibitors (Roche), chilled on ice for 10 min, and centrifuged at maximum speed, and cell debris was discarded. Human cells were lysed in a TRIS‐based buffer  using standard protocols.2.93 cells per well into a 2‐well culture insert  and allowed to adhere overnight. The insert was removed carefully, the cells were washed twice with PBS to remove debris and the media was changed. The 500 μm gap was photographed under a light microscope at the indicated time points of n = 3 replicates per cell clone and control, respectively. The area of the gap was measured using imagej 1.52a and plotted in graphpad prism .For the migration assay, the ERBB‐KO clones and the control cells were seeded at 21 × 102.104 cells per well were seeded in a 96‐well plate and cultured for 48 h. We performed the cell proliferation ELISA, BrdU (colorimetric) Kit  according to the manufacturer's instructions. In brief, the cells were labeled with BrdU for 3 h at RT and subsequently fixed by the incubation of FixDenat solution for 30 min. The cells were then incubated with a monoclonal antibody detecting BrdU, conjugated with peroxidase for 90 min at RT. Following washing, the cells were further incubated with the substrate tetramethyl‐benzidine for 20 min and the substrate reaction was stopped by adding 1 m H2SO4. The absorbance of the wells was measured at 450 nm (reference wavelength: 690 nm) in an ELISA reader. Per cell clone, four replicates were analyzed and the measurements were plotted relative to the control Panc‐1 cell line as fold change in GraphPad Prism. To evaluate the viability of the cell clones, we measured the relative number of living cells to all cells using fluidlab R‐300  and compared them to Panc‐1 control cells by plotting the fold change using graphpad prism.For the proliferation assay, 1 × 102.116 cells/mouse in 20 μL of Matrigel; 1 : 1 with DMEM). Tumors were harvested 4 weeks postinjection and weight was recorded.All animal experiments and methods were approved by the Weizmann Institutional Animal Care and Use Committee. Genetically modified Panc‐1 cells  were injected orthotopically into the pancreata of female 14‐week‐old CD1‐Foxn1nu mice  and Tukey's multiple comparison test. All data were analyzed with graphpad prism . P‐values < 0.05 were considered statistically significant.Data are presented as mean ± SEM and compared by the two‐tailed unpaired Student's 33.1C under the control of a ubiquitous chicken‐beta‐actin (CBA) promoter. TRAP‐FC transgenic mice (Trap) at the age of 4 months were phenotypically not distinguishable to their age‐matched control littermates, since we could not detect a difference in body and organ weight , and the IgG1‐Fc sequence of the decoy. Antibodies targeting the intracellular domain (ICD) of the ERBB receptors were used to investigate receptor expression and phosphorylation stage in pancreas, lung, and liver ), herein referred to as Trap/Kras mice. To compare the pancreatic lesions of Trap/Kras and Kras mice, the pancreatic tissue of 4‐month‐old mice was analyzed. The absolute pancreas weight of Trap/Kras mice was significantly decreased in female and male Trap/Kras animals compared with Kras control littermates  cell lines, compared with Panc‐1 control cells. Firstly, we performed a migration assay and revealed alterations in all ERBB‐KO cell lines compared with the MOC Panc‐1 cell line. The monolayer wound of ERBB3‐ and ERBB4‐KO clones closed significantly earlier, after 24 (ERBB4‐KO) or 30 (ERBB3‐KO) hours Fig.  comparedlls Fig. . Moreovells Fig. .4in vivo [Staphylococcus aureus superinfection, and hematopoietic side effects such as papulopustular rush, nail inflammation, dry skin, alopecia, increased growth of eyelashes and facial hair, and strong pruritus [C mice in the KRASG12D/+ PDAC mouse model and revealed that Trap/Kras mice acquired reduced pancreatic lesions, possibly due to a decreased RAS activity compared with age‐matched Kras controls. Thus, along with efficacy tests, our animal model would permit in‐depth, long‐term exploration of side effects of the decoy molecule. It is noteworthy that so far we have followed our Trap animals for 6 months but have observed no overt toxicity. Our results indicate that TRAP‐FC is able to reduce the pancreatic lesion area without showing any detectable toxicity. All ERBB receptors were significantly less expressed in the pancreas of Trap/Kras mice, except for ERBB3. As expected, the phosphorylation state of EGFR, ERBB2, and ERBB3 was significantly reduced. These data, altogether, point to a reduction in pan‐ERBB signaling. In our recent publication, we have shown that the genetic deletion of a single ligand of the ERBB family, BTC, also led to a reduced tumor burden in Kras mice, which was due to decreased EGFR‐signaling [Trap mice. As we have shown by western blot, in trap mice are the levels of ERBB ligands TGFA and EGF only reduced and not deleted, but this seems to be sufficient enough to reduce tumor growth and does not cause side effects. However, ERBB4 phosphorylation was increased, indicating a reciprocal regulation in Trap/Kras compared with Kras mice, which does not fit the remaining results. The ERBB4 receptor has many faces and it turned out that its function has to be looked at in a context‐dependent manner. First, the affinity of ERBB4 ligands to the ERBB4‐TRAP domain might be weaker than to the physiologic receptor so that some ligands might bind and activate the wild‐type ERBB4 receptor in the presence of TRAP‐Fc. Second, ERBB4 might act autonomously in response to the down‐regulation of the remaining receptors, as we have observed previously in Kras mice lacking EGFR [Kras mice from developing PDAC. Indeed, ERBB4 might have tumor‐suppressive functions in different cancers and its role in cancer remains controversial [Trap/Kras mice after signaling by the three other ERBB members was silenced through the inhibition of their ligands.Pancreatic ductal adenocarcinoma is a lethal disease with a high mortality rate , 25 and in vivo . Antibodin vivo , 9, 10. pruritus , 33. Thepruritus , 35 and pruritus . Patientpruritus , 37. A npruritus , colorecpruritus , bladderpruritus  or head pruritus . Since tignaling . Both stignaling , EGF [43ignaling  shows noignaling , 46, HBEignaling , EREG [4ignaling , and AREignaling  leads toing EGFR . Howeveroversial . On the oversial . Hence, in vivo PDAC animal model, all four ERBB receptors should be equally affected by the TRAP decoy. To investigate, which receptor affects the tumor cells the most in PDAC, we chose a human in vitro model, as it is easier to analyze the signaling pathways of each receptor in cell culture. To evaluate ERBB receptor dependencies of PDAC in human cells, we deleted each single ERBB receptor using CRISPR/Cas9 technology in Panc‐1 cells and investigated the activation of the ERBB signaling network. We found that ERBB2 activation was highly dependent on the presence of EGFR. On the contrary, EGFR activation was partially dependent on ERBB2 and also, but to a lesser extent, dependent on ERBB4. ERBB4 expression was increased in ERBB2‐KOs, which indicates that these three receptors share a co‐dependency upon BTC stimulation, which is in line with what has previously been observed in a murine PDAC model [In our AC model . HoweverAC model . By contAC model  and is iAC model , it mighAC model .GTP molecules [Loss of EGFR or ERBB2 in human Panc‐1 cells results in delayed migration, reduced proliferation rate, and increased tumor growth behavior. Just an increased proliferation rate and also an increased migration are important hallmarks of cancer . The wouolecules . Possibl5in vivo model and demonstrating that an ERBB ligand trap reduced pancreatic lesion area in mice. This study and our recently published work emphasize the urgency of using pan‐ERBB strategies instead of blocking single receptors of the family. Unfortunately, a recent study showed that afatinib, a TKI that inhibits EGFR and ERBB2, in combination with the cytostatic drug gemcitabine, did not improve gemcitabine treatment alone [Since the inhibition of a single ERBB receptor seems insufficiently effective in treating pancreatic adenocarcinoma, our study provides evidence that inhibiting pan‐ERBB‐signaling by trapping all ERBB ligands is a more promising approach. In line with this concept, anti‐tumorigenic effects of pan‐ERBB have been demonstrated already in pancreatic cancer cell lines . Our stunt alone . The newThe authors declare no conflict of interest.MD, KH, and YY involved in the study concept and design. MD and KH involved in the acquisition of data. MD, KH, ML, AB, TH, and NBN involved in the analysis and interpretation of data. MD, KH, and YY involved in drafting of the manuscript. MD, KH, ML, and YY involved in the critical revision of the manuscript for important intellectual content. MD, KH, ML, and KEA involved in statistical analysis. Obtained funding was not applicable. MD, KH, YY, ML, AB, TH, and NBN involved in administrative, technical, or material support. MD and YY involved in study supervision.https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/1878-0261.13473.The peer review history for this article is available at Fig. S1. Expression of TRAP‐FC in transgenic mice.Click here for additional data file.Table S1. Guide RNA‐design for ERBB‐KOs.Table S2. Primers used for ERBB‐KO‐genotyping.Table S3. Primers used for miSeq.Table S4. Antibodies used for western blot analysis.Click here for additional data file.


Temporary cardiac pacemaker implantation (PM) via the femoral and subclavian veins is widely used in clinics to treat patients with severe bradycardia or tachycardia, but it is technically challenging and potentially associated with various complications.This study investigated the feasibility and safety of a novel method of PM implantation via the median cubital vein.n = 107), via the femoral vein; S‐control (n = 67), via the subclavian vein, and N‐group (n = 105), via the median cubital vein. The sheath placement time (SPT), electrode placement time (EPT), electrode arrival rate (EAR), rate of sensing and pacing (RSP), radiation quantity (RD), electrode dislocation rate (EDR) and average electrode retention time (AERT) were recorded and evaluated. In addition, the Hamilton Anxiety Scale (HAMA) and Self‐Rating Depression Scale (SDS) were used to evaluate the comfort levels of patients in the three groups.A total of 279 patients of the First Affiliated Hospital of Xiamen University between March 2020 and December 2021 who required no‐emergency PM implantation were enrolled. The patients were divided into three groups based on the temporary PM implantation routes: F‐control (p > 0.05). However, the N‐group had significantly lower SPT than the F‐control and S‐control groups . Additionally, the F‐control had significantly higher EDR than the S‐control group and the N‐group (11 (10.3%) vs. 2 (3.0%) and 3 (2.9%), p = 0.036). Besides, comparison of the HAMA and SDS scores before and after PM implantation showed significant differences in the S‐control group (p = 0.010) and the N‐group (p = 0.000).There were no significant differences between the groups with regard to age, EAR, RSP, EPT, RD, and AERT  implantation were included in this study and were assigned to different groups according to the route of PM implantation. Two commonly used venous access routes are the femoral and subclavian veins.Here, we report a novel method for the implantation of temporary PMs via the median cubital vein, a superficial vein. Compared with the other two venous access routes, puncturing through the median cubital vein is quicker, simpler, and more effective, with fewer complications. Our findings suggest that this new venous access route should be widely used for temporary PM implantation in clinical practice.22.1n = 107) and the median cubital vein route  . All patients provided written informed consent for their anonymized medical data to be analyzed and published for research purposes.Exclusion criteria ① Pregnant women and lactating patients; ② Patients who received emergency temporary cardiac pacing treatment; ③ Patients with obvious bleeding tendencies and blood system diseases; ④ Patients with end‐stage diseases; ⑤ Patients who were unable to cooperate or declined to undergo temporary PM placement surgery.2.2The operating area was disinfected and locally anesthetized with 1 mL 1% lidocaine. A guide wire was inserted after puncturing the femoral or subclavicular vein Embedding indwelling needle in the median cubital veinAn indwelling needle was routinely embedded in the median elbow vein in the ward by specialist nurses  Figure .(2)Disinfection of the operating area, placement of sterile towels, and local anesthesia administration in the Cardiac Catheterization Room.The indwelling needle consisted of a stainless steel core, a soft outer sleeve, and a plastic base. After disinfection and placing sterile drapes, local anesthesia was administered with 1% 1 mL lidocaine for the surrounding skin. The soft outer sleeve was then cut off, leaving about 2 cm Figure .③ PM electrode placementThe guide wire was inserted along the outer sleeve into the vein for about 10 cm Figure . A sheat2.4Y = in + (1.25×) was used to obtain the standard total score (Y). The cut‐off value for depression with SDS was 41 points for the total score and 53 points for the standard score.We conducted questionnaire surveys for the enrolled patients using the Hamilton Anxiety Rating Scale (HAMA) and Self‐Rating Depression Scale (SDS) to evaluate anxiety and depression. A HAMA score over 29 indicates high anxiety, over 21 indicates significant anxiety, over 14 indicates moderate anxiety, over seven indicates low anxiety, and below seven indicates no anxiety. For SDS, the 20 items were summed to obtain a total score and the formula conversion 2.52.5.1The time from the administration of local anesthesia to successful placement of the sheath tube.2.5.2The time from when the electrode entered the sheath tube in the median cubital vein to the time when the operating electrode was in the right position and the ECG indicated that the temporary PM pace was well‐driven.2.5.3Accurate positioning for the electrode was defined as the fixation of the right ventricular apex by the electrode tip under X‐ray fluoroscopy, confirmed when the ECG indicated that the temporary PM pace was well‐driven. EAR (%) = the number of cases with the electrode in the right position/the total number of cases in the group.2.5.4The temporary PM sensed the frequency and drive according to a set frequency. A typical ECG is shown in Figure 2.5.5The frame frequency was set at 7.5 p/s. The fluoroscopy mode was used during the surgical procedure and images obtained under this mode were saved. For each patient, images of the anteroposterior and left anterior oblique 30° were retained to confirm the PM electrode position. The digital subtraction angiography system automatically calculated the total amount of radiation for each patient upon completion of the operation.2.5.6During the period of temporary PM implantation, the occurrence of transient, intermittent, continuous PM perception, or poor drive was defined as electrode dislocation, excluding factors such as battery depletion and the PM itself.2.6t‐test. Categorical variables were expressed as percentage and analyzed using the χ2 test. All analyses were conducted using SPSS version 17.0 (SPSS Inc.). Statistical significance was set at p < 0.05.Continuous variables with normal distribution were expressed as mean ± standard deviation (SD) and analyzed with the Student 33.1n = 107), S‐control (n = 67), and N‐group (n = 105). None of these patients required emergency PM implantation. The details for the basic characteristics of all participants in these three groups are listed in Supporting Information: Table p > .5), indicating that the three methods evaluated were all safe.A total of 279 patients were included in this study and were divided into three groups: F‐control .The operation parameters in the three groups are outlined in Table p = 0.123). However, the F‐control group had a significantly higher electrode dislocation rate (EDR) than the N‐group and the S‐control group (11 (10.3%) vs. 2 (3.0%), and 3 (2.9), p = 0.036). Hence, the N‐group and S‐control group were safer than the F‐control group with regard to the dislocation rate.The details of the safety analysis of three groups of patients are shown in Table 3.4p = 0.913) and 22.0 ± 11.0, 19.2 ± 8.2, and 17.0 ± 9.5 after implantation (p = 0.002), respectively. The corresponding SDS scores were 48.3 ± 19.5, 48.1 ± 22.1, and 48.5 ± 19.4 before PM implantation (p = 0.990) and 42.8 ± 21.2, 38.4 ± 21.4, and 36.6 ± 20.5 after implantation (p = 0.086), respectively. The values before and after PM implantation were significantly different in the S‐control group (p = 0.010) and the N‐group (p = 0.000).As shown in Table These two psychological scale scores were further analyzed by gender , the subclavian vein (S‐control group), and the median cubital vein (N‐group). We found that there were no significant differences between these groups in laboratory test results before and after PM implantation, indicating that PM implantation through these routes was safe and did not negatively affect kidney function, cardiomyocyte viability, cardiac function, and hematology.We used a number of operational parameters, including EPT, EAR, RSP, and RD, to evaluate the effectiveness of the three routes. While we did not observe any significant differences in EPT, EAR, RSP, and RD between the groups, we observed that SPT, an indicator that is frequently used to assess the effectiveness of the PM implantation procedure,We further analyzed the safety of the three methods. In the F‐control group, femoral artery puncture occurred in 13 patients during the procedure and local ecchymosis occurred in six patients. In the S‐control group, subclavian artery puncture occurred in three patients and local ecchymosis occurred in one patient. However, in the N‐group, local petechiae occurred in two patients. And no hematoma, pneumothorax, pseudoaneurysm, arteriovenous fistula, or thrombosis happened in our study. Taken together, these observations indicated that the novel vein puncture method had fewer and milder side effects than the conventional approaches and was a safe option.The deep veins used for puncture in the F‐control and S‐control groups are potentially associated with a variety of complications, including artery puncture, hemopneumothorax, infection, hemorrhage, hematoma, pseudoaneurysm, arteriovenous fistula, thrombosis, vasculitis, nerve injury, and ecchymosis.In the present study, we analyzed two psychological scales, HAMA and SDS, for the patients in these three groups. HAMA is widely used to assess the responses of patients, mainly anxiety, to medical interventions,The limitations of our study should be noted. For example, our study was performed in a single center with a limited number of cases. Therefore, the findings from this study should be further corroborated in multicenter large‐cohort studies in the future.5In conclusion, although the current technology for temporary PM implantation is mature, deep‐vein puncture still has a number of disadvantages, including the need for skilled operators and potential complications. Temporary cardiac PM implantation via the median cubital vein has several significant advantages over the conventional subclavian and femoral vein approaches. First, the puncture is easier because the median cubital vein is relatively superficial. Second, puncture is safer because the procedure is associated with much fewer and milder complications. Third, puncture is more efficient because it is easier and the period from the puncture time to the EPT is greatly shortened, resulting in more time for rescue if necessary. Hence, we concluded that puncture through the median cubital vein for temporary cardiac PM implantation is simpler, safer, and more effective and should be popularized.Dehua He, Ziguan Zhang: Conceptualization; data curation; formal analysis; investigation; methodology; validation; writing—review and editing. Huiqing Huang: Conceptualization; data curation; formal analysis; resources; software; visualization; writing—review and editing. Kaimin Lin, Yan Ge, Xiongbiao Lin, Qiang Xie: Conceptualization; data curation; formal analysis; writing—original draft; writing—review and editing. Weihua Li, Zhengrong Huang: Conceptualization; data curation; formal analysis; funding acquisition; project administration; supervision; writing—original draft; writing—review and editing.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.