The mechanisms for atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI) catheter ablation are unclear. Non-PV organized atrial arrhythmias  are possible contributors; however the prevalence and effect of their ablation on recurrent AF are unknown. We hypothesize that the identification and ablation of non-PV organized atrial arrhythmias were associated with less AF recurrence.Patients who underwent repeat ablation for recurrent AF after prior PVI were retrospectively enrolled. The prevalence and characteristics of PV reconnections and non-PV organized atrial arrhythmias were identified. The outcomes of time to clinical AF recurrence, heart failure (HF) hospitalization, and mortality were analyzed in patients using multivariable adjusted Cox regression.2, 59% persistent AF), PV reconnections were found in 46 patients (61%), macro-reentrant atrial flutter in 27 patients (36%), and focal tachycardia in 12 patients (16%). Mapping and ablation of non-PV organized atrial arrhythmias were associated with a reduced recurrence of late clinical AF  and the composite outcome of recurrence of late AF, HF hospitalization, and mortality , with median follow-up of 1.6 (IQR 0.7–6.3) years. The presence of PV reconnections or empiric linear ablation was not associated with reduction in clinical AF or composite endpoints.In 74 patients with recurrent AF (age 66 ± 9 years, left atrial volume index 38 ± 10 ml/mThe ablation of non-PV organized atrial arrhythmias resulted in a reduction of late clinical AF recurrence and composite outcome. In this challenging population, alternate mechanisms beyond PV reconnections need to be considered. Prospective studies are needed.The online version contains supplementary material available at 10.1007/s10840-022-01160-w. Although catheter ablation has been proven to be effective at eliminating AF, recurrences of AF after catheter ablation are common and estimated to be between 15 and 50% after 5 years.2 The mechanisms of recurrent atrial arrhythmias after catheter ablation of AF are not well defined. Prior studies have reported pulmonary vein (PV) reconnections as the predominant mechanism of AF recurrence in up to 97% of patients.3,4 Consequently, the current standard of care and only Class 1 guideline recommendation during repeat catheter ablation of AF is to assess for PV reconnections.2 As technology has improved through the years, including the use of contact force catheters, PV isolation (PVI) has become more durable and effective. Indeed, recent studies have indicated that the incidence of PV reconnections in recurrent AF is not as common as previously cited, occurring in as low as 38% of patients with recurrent AF.5 Still, despite durable PV isolation, AF recurrence is still common.6Atrial fibrillation (AF) is the most common arrhythmia in the world and a leading cause of hospitalization and morbidity2,4 It is unclear whether additional ablations beyond PVI have any benefit in outcomes, although guidelines state that operators should consider more extensive ablation in recurrent AF.7,8If no PV reconnections are found, there remains uncertainty as to the best strategy to guide repeat ablation. Different techniques have been proposed in this setting, such as empiric linear ablation with a roof line or mitral isthmus line, posterior box isolation, left atrial appendage isolation, targeting ablation of complex fractionated electrograms (CFAE), ablation of provoked non-PV organized atrial arrhythmias commonly associated with non-PVs organized atrial arrhythmias, such as the superior vena cava.The aim of the present study is to determine the prevalence of PV reconnections and non-PV organized atrial arrhythmias  in patients with recurrent AF undergoing repeat AF ablation and evaluate the impact of their ablation on clinical outcomes.A total of 74 patients who underwent repeat catheter ablation for recurrent AF after initial PVI were analyzed retrospectively at 2 medical centers . The inclusion criteria included consecutive patients over 18 years of age who had previously undergone PVI for AF and who developed clinically significant recurrent AF as documented on ECG, telemetry event monitor, and implantable device interrogation. Patients were excluded if they were found to only have atypical AFL without clinical AF. This study was performed in accordance with approved retrospective Institutional Review Board protocols at each institution.The prevalence of PV reconnections, atypical AFL, AT, and PACs were identified using standard mapping techniques. Informed consent was obtained prior to all ablation procedures. Intravenous heparin was used to target an activated clotting time of 350–400 s. Electroanatomic mapping systems were used in all cases . Esophageal position and temperature were monitored during all left atrial ablations using a multipolar temperature probe  positioned in the esophagus behind the left atrium at the level of the ablation catheter, in order to avoid any temperature rise above 38 °C. Left atrial access with an ablation catheter and multi-electrode catheter  was obtained with single or double trans-septal puncture, performed under direct visualization with intracardiac echocardiography guidance and fluoroscopy.9,10 Of note, non-PV organized atrial arrhythmia sources were defined as any focal ectopic beat or tachycardia induced in the atria, regardless whether they directly induced AF.If recovery of conduction from PVs was observed, repeat PVI was performed using a segmental, circumferential, or both approaches. Next, organized atrial arrhythmias such as AFL, AT, ectopic PAC, or supraventricular tachycardias were mapped and ablated using entrainment mapping techniques and standard activation mapping with a multipolar electrode catheter. High-dose isoproterenol infusion (20 mcg/min) was infused to induce non-PV organized atrial arrhythmias, as previously described.Additional lesion sets outside the previously ablated regions were performed at the discretion of the operator, including left atrial roof line, mitral valve isthmus line, coronary sinus ablation, and CFAE ablation. Closed and open irrigated and non-contact and contact force sensing catheters were also used at the discretion of the operator. The endpoint of PVI was elimination of all PV potentials and demonstration entrance and exit block by pacing after a 30-min waiting period and elimination of a trigger or a line of bidirectional conduction block if adjunctive ablations were performed.Data regarding long-term clinical follow-up were obtained through chart review. Recurrence of atrial tachyarrhythmia was defined as AF, AFL, or AT > 30 s occurring > 3 months after repeat procedure lasting for > 30 s on 14–30 day ambulatory monitors at least 3 months, 6 months, and every year after ablation. Anti-arrhythmic medications were continued during the blanking period for 3 months and then discontinued at the discretion of the treating electrophysiologist. Data on heart failure hospitalization and mortality after ablation was collected.t-tests were used for the between-group comparisons of the categorical and continuous variables, respectively.The baseline characteristics of those with any inducible atrial arrhythmia versus no inducible atrial arrhythmia were reported as means ± one standard deviation and frequency (n and %) for continuous and categorical variables, respectively. Chi-square and P values < 0.05 were considered statistically significant. All analyses were performed using IBM SPSS Statistics Version 28 .Time to recurrence and event-free survival curves were analyzed using the Kaplan–Meier method. Cox proportional hazards modeling was used to analyze arrhythmia-free survival with a 3-month blanking period with results presented as hazard ratios (HR) with 95% confidence intervals (CI), after verifying proportionality assumptions. Patients who were lost to follow-up were censored at the date of last known follow-up. Variables in the adjusted model were chosen a priori based on potential for confounding, including age, gender, body mass index (BMI), cardiomyopathy, coronary artery disease, stroke/transient ischemic attack, obstructive sleep apnea, persistent AF, alcohol use, presence of PV reconnections, anti-arrhythmic drug (AAD) use (during the blanking period), any empiric line ablation and any inducible non-PV organized atrial arrhythmia . Two-sided 2. AAD were used in 41 patients (55%) during the blanking period, and there were no significant differences in AAD use between patients with or without clinical AF recurrence . In patients with inducible non-PV organized atrial arrhythmias, 8% were on Class I AAD (flecainide/propafenone), 35% were on Class III AAD , and 24% were on amiodarone. In patients without inducible non-PV organized atrial arrhythmias, 8% were on Class I AAD (flecainide/propafenone), 19% were on Class III AAD , and 16% were on amiodarone. There were no significant differences in clinical and echocardiographic characteristics between the patients with and without any inducible organized atrial arrhythmia. The characteristics of prior ablation lesions are listed in Table S1 (online supplement).A total of 83 consecutive patients were enrolled into the study. A total of 9 patients were excluded due to only having clinical AFL but not AF. The study patient flow is illustrated in Fig. In 74 patients with recurrent AF undergoing repeat ablation, PV reconnections were found and ablated in 46 patients (62%). PV reconnections occurred 30% in the right upper PV, 30% in the right lower PV, 23% in the left upper PV, and 18% in the left lower PV. There were 28 patients (38%) who had persistently isolated PVs.In addition to recurrent AF, macro-reentrant AFLs were identified in 27 patients (36%). As shown in Table A total of 16 PAC or focal AT sources were identified during sinus rhythm in 12 (15.8%) patients. The locations and distribution of PACs and AT throughout the atria are shown in Fig. Abbreviations: SVC: superior vena cava, LAA: left atrial appendage, RA: right atrial, CS: coronary sinusP = 0.03). The association strengthened after multivariable adjustment  as shown in Fig. P = 0.02) 0.08–0.85, P value 0.03). Findings were comparable after multivariable adjustment .A total of 55 74%) patients remained clinical AF free during long-term follow-up of 19 months (interquartile range 9–75 months) as demonstrated by the Kaplan-Meir analysis in Fig. % patientP = 0.26 and adjusted HR 0.33, CI 0.09–1.18, P = 0.09) or the composite outcome , as shown in Tables S3-S4 and Figures S1-S2 (online supplement). Furthermore, in Cox regression analysis, AAD use in the blanking period was not associated with decreased clinical AF recurrence (P = 0.42). In patients with persistently isolated PVs and inducible non-PV organized atrial arrhythmias, 82% were free from AF.The presence of PV reconnections or empiric linear line ablation was not associated with reductions in clinical AF recurrence  were free from recurrent clinical AF. This data suggests that the presence of inducible targets for ablation contributes to the ability to achieve reasonable freedom from clinical AF in this challenging population.In patients who did not have PV reconnections (39%), organized atrial arrhythmias were found in almost half (46%). This is consistent with results from a prior study in which 11 of 27 (41%) of patients with recurrent AF and durable PVI had a non-PV trigger identified.12,13,14. The findings suggest that identification and ablation of all organized atrial arrhythmias may be important in reducing AF recurrence, regardless of whether they directly induce AF. However prospective randomized studies are needed to establish whether non-PV arrhythmia mapping and ablation and use of high dose isoproterenol reduce AF recurrence, and this strategy is currently listed only as a Class IIb indication in the 2017 AF ablation guidelines.2In contrast, the presence and ablation of any organized atrial arrhythmias was associated with a reduction in AF recurrence and composite endpoint. This supports the hypothesis that any atrial arrhythmia may exacerbate atrial structural and cellular remodeling that further perpetuates the maintenance of AF7 which showed that empiric linear ablation or CFAE ablation was not associated with decreased AF recurrence. The findings from this study support the importance of identifying electrical mechanisms that may contribute to recurrent AF.In this study, performance of empiric linear ablation was not associated with reduced AF recurrence or composite endpoint. This is consistent with the results of the STAR-AF2 trial12 Focal and rotational driver mapping in patients who had prior PVI resulted in freedom from AF of around 70–80%.15,16 However, driver-based ablation is not yet incorporated as a standard protocol in repeat ablations, and further research is needed in this area.2 As expected, both inducible and non-inducible organized atrial arrhythmia groups had a high prevalence of the commonly recognized risk factors for AF, including advanced age, obesity, OSA, diabetes, and alcohol use.There were no identifiable ablation targets for the recurrence of AF in 55% of the patients who did not have PV reconnections. The mechanisms and optimal repeat ablation strategy for these patients are still unknown. Electrically active drivers has been suggested as a possible mechanism maintaining AF.17, these observations still inform the association between presence and absence of inducible organized atrial arrhythmias in a high-risk cohort. Furthermore, the model assumed fixed covariates and thus does not account for the possibility of time-dependent changes in the covariates for each patient. However, most of the covariates used in this model would not be expected to change over time . Fifth, all patients received at least a 14-day event monitor (or implantable device interrogation) at 3 months and at 1 year and with any clinical symptoms according to clinical practice at these two institutions. Unfortunately, due to the retrospective study design, the type of post-ablation arrhythmia monitoring after 1 year varied in the cohort and was left to provider discretion. As a result, the recurrence of AF may have been underestimated due to the varying monitoring strategies.First, as a retrospective observational study, causal inferences cannot be made. Second, racial and ethnic description of the population was omitted due to high rate of missingness and unreliability. Third, the modest sample size may be underpowered to detect the effect of different ablation strategies and characteristics on outcomes. Fourth, residual confounding cannot be excluded as multivariable models were adjusted for available risk factors and clinical characteristics. While other factors may play a role in predicting outcomes, such as AAD use following the blanking periodIn patients with recurrent AF after prior ablation, the ablation of inducible non-pulmonary vein organized atrial arrhythmias was associated with the reduction of recurrent late clinical AF in a median follow-up of 1.6 years. Prospective randomized studies are needed to determine the influence of non-PV mechanisms on long-term outcomes in patients with recurrent AF after failed prior AF ablation.Dr. Ho: grants from NIH (KL2TR001444), AHA (19CDA34760021), equity in Vektor Medical Inc.Dr. Han: research support from Abbott.Dr. Hsu: honoraria from Medtronic, Boston Scientific, Abbott, Biotronik, Biosense-Webster, Pfizer, Bristol-Myers Squibb, Janssen Pharmaceuticals, research grants with Biotronik and Biosense-Webster, and equity interest in Acutus Medical and Vektor Medical.Dr. Hoffmayer: grants from the NIH (F32 HL10472702 and LRP), consulting for Samsung Electronics, Inc. and Vektor Medical Inc.Dr. Feld: equity and consulting for Acutus Medical, Adagio, Medwaves, Varian, and co-founded and consulted for Perminova, received CCEP Fellowship Training program stipend support from Medtronic, Biotronik, Biosense Webster, Boston Scientific, and Abbott.Dr Krummen: grant from the UCSD Galvanizing Engineering in Medicine Foundation and equity in Vektor Medical Inc.Supplementary file1 (DOCX 57 KB)Below is the link to the electronic supplementary material.


Thus, the cytotoxicity of Ag/D-PAA composite against erythrocytes was more pronounced compared with D-PAA and hybrid Au/polymer composite. Eryptosis is a more sensitive tool for assessing the biocompatibility of nanomaterials compared with fibroblast viability assays.The aim of the present research was to assess the cytotoxicity of gold and silver nanoparticles synthesized into dextran-graft-polyacrylamide (D-PAA) polymer nanocarrier, which were used as a basis for further preparation of multicomponent nanocomposites revealed high efficacy for antitumor therapy. The evaluation of the influence of Me-polymer systems on the viability and metabolic activity of fibroblasts and eryptosis elucidating the mechanisms of the proeryptotic effects has been done in the current research. The nanocomposites investigated in this study did not reduce the survival of fibroblasts even at the highest used concentration. Our findings suggest that hybrid Ag/D-PAA composite activated eryptosis via ROS- and Ca Noble element-based nanoparticles are promising bioengineering materials that can be used for diagnostic and therapeutic purposes. In particular, there is accumulating evidence that gold and silver nanoparticles can find the potential application in cancer diagnostics and treatment, bioimaging, biosensing, drug delivery, and treatment of infectious diseases as antibacterial, antifungal, and antiviral agents , 2. UniqThe field of noble metal nanotechnology has been expanding for years to improve the stability of nanomaterials, their efficacy and biocompatibility, and to reduce the toxicity . AnotherIt has been demonstrated that the toxicity of metal/polymer composites is associated primarily with metal components, i.e., fillers. In their turn, metals can exert toxic effects directly or they can be promoted by ion release . It is wDuring the past few years, gold and silver nanoparticles (AuNPs and AgNPs) with their unique properties have led to new and exciting developments with enormous potential in biology and medicine , 14. It in situ synthesis of metal NPs preventing their aggregation and allowing regulating the nanoparticle size /[molDextran]) and was equal to 5.Dextran (Sigma-Aldrich) with Mw = 70,000 g/mol was dissolved in deionized water, and argon was passed for 20 min. Then, the calculated amount of initiator was injected. Cerium (IV) ammonium nitrate (Aldrich) was an initiator of radical polymerization. The acrylamide (AA) monomer was added 20 min after the initiator. The monomer was prerecrystallized twice from chloroform. After the initiator injection, the oxidation-reduction reaction took place, and macroradicals on the dextran macromolecule were formed , 23. TheThe polymerization was carried out for 24 hours. Upon completion of the polymerization reaction, the polymer was precipitated in acetone and freeze-dried.The molecular parameters of the polymer sample determined by size exclusion chromatography are shown in 3 and HAuCl4 precursors using sodium borohydride (NaBH4) as reductant. All chemicals were purchased from Sigma-Aldrich. The synthesis of metal sols was carried out in situ into aqueous solution of dextran-graft-polyacrylamide copolymer.The Ag and Au nanoparticles were synthesized in situ into polymer matrix D-PAA. The silver and gold nanoparticles were synthesized by reduction of the AgNOC = 1 g∙L−1) and stirred for 20 min. Then, 0.1 mL of 0.1 M aqueous solution of NaBH4 was added. The final solution was stirred for 30 min. The appearance of ruby-red color indicated the formation of AuNPs, and the reddish-brown color pointed to the formation of AgNPs.Initially, 0.05 mL of 0.1 M precursors dissolved in bidistilled water was added to 1 mL of water polymer solution . In addition, eight rat fetuses (gestation days 15-20) were used for obtaining skin fibroblasts .The MTT assay was used to estimate the metabolic activity of fibroblasts and their viability . After a4 cells per well). They were incubated with the concentrations of composites described above during 24 h in a СО2-incubator. The neutral red dye solution (0.01%) in culture medium was added and the incubation lasted for 3 h in a СО2-incubator (5% CO2). The medium found in the samples was collected. Then, the dye was extracted and dissolved using 0.1 ml 50% ethanol and 3% acetic acid. After the dissolution of neutral red, the absorbance was measured at a wavelength of 570 nm. To quantify the viability degree, calculations were performed in accordance with the following formula: (%) = [100 × (sample absorbance)/(control absorbance)]. Numerical data were expressed as a percentage compared with the control samples.In addition to MTT assay, the fibroblast viability was assessed by the neutral red uptake assay . Brieflyμl) were aliquoted from each sample to be incubated with various concentrations (0-0.1-0.2-0.5-1-2-5 mg/L) of pure D-PAA, Ag-D-PAA, and Au-D-PAA composites. Furthermore, the mixtures contained 5 ml RPMI-1640 medium with stable glutamine purchased from Biowest (France) and 5% fetal bovine serum purchased from BioWhittaker®, Lonza (Belgium) for 24 h. Thereafter, samples were used to obtain erythrocyte mass by double washing in phosphate buffer saline .To assess the impact of polymer and hybrid metal-polymer composites on eryptosis indices, blood portions . To load the cells, 5 μl fluorescein isothiocyanate- (FITC-) labeled Annexin V purchased from BD Pharmingen™, BD Biosciences  was dissolved in 100 μl of the suspension described above. Loading was performed for 30 minutes in the dark. Directly prior to analysis of fluorescence by BD FACSCanto™ II Cell Analyzer , 400 μl 1× Annexin-binding buffer was added to each tube. Annexin V-FITC fluorescence was analyzed. The 488 nm laser was used for excitation, while the emitted light was collected at 520 nm. Per sample, 200,000 events were acquired. Postacquisition analysis was carried out by BD FACSDiva™ and FlowJo™ v10 software.Annexin V staining was used to assess phosphatidylserine exposure , 36. Accμl of erythrocyte mass was resuspended in PBS. The erythrocytes were loaded with the dye (5 μM) and incubated for 30 min in the dark. Thereafter, the cells were washed twice and resuspended in 0.5 ml PBS. After a cascade of modifications in erythrocytes, H2DCFDA is converted to dichlorofluorescein (DCF) in cells under the influence of ROS. The DCF fluorescence was acquired at 520 nm after excitation with the 488 nm laser.ROS generation in erythrocytes treated with polymer and its hybrid composites with metals was analyzed by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining . The dye2+ concentrations were assessed by FLUO4 AM staining. This Ca2+-sensitive dye was purchased from Becton Dickinson (USA). Its 5 mM stock solution was in anhydrous dimethyl sulfoxide  that was prepared and used to stain 2 μl of erythrocytes dissolved in 98 μl PBS to obtain 2.5 μM dye working solutions. The same volumes (400 μl) of PBS were added after the incubation lasted for 30 min in the dark. The FLUO4 fluorescence was acquired at 520 nm after excitation with the 488 nm laser.Intraerythrocytic Ca2O2 (0.1 mM) were used as positive controls.Fluorescence of Annexin V-FITC, DCF, and FLUO4 was expressed in arbitrary units (a.u.). Erythrocyte suspensions without the corresponding dyes were considered negative controls. Erythrocytes incubated with Hpost hoc Dunn's tests were used. Data are available as the median and interquartile range . p values not over 0.05 were interpreted as statistically significant. GraphPad Prism5.0  was used to analyze data statistically.Both for the experiments with fibroblasts and erythrocytes, the Kruskal-Wallis and The nanosystem characterization that was performed by using dynamic light scattering, transmission electron microscopy, and SAXS was referred in details in , 27–29. All synthesized nanosystems were shown to be stable in time, and the size of Ag and Au nanoparticles was equal to 10-20 nm.The cytotoxic effects of metal/D-PAA composites on fibroblasts were evaluated by analyzing their impact on fibroblast morphology, monolayer confluence, and cell adhesion. Microscopically, all the metal/polymer composites used in this study did not influence the cell shape and viability. The confluence of fibroblast monolayers was affected. The cells showed normal fibroblast morphology, and no cell debris was observed in the samples treated with 0.1-5 mg/L of various metal/D-PAA hybrid composites. Thus, fibroblasts were visually unaffected. Visually, no detached cells or small cell clusters were detected in the experimental samples suggesting that cell adhesion was unaffected in response to nanocomposites . Thus, oIn addition, cytotoxicity profiles of nanocomposites were assessed by cell viability assays. Both cytotoxicity assays demonstrated that D-PAA, hybrid Ag/polymer, and hybrid Au/polymer composites did not reduce the survival of cells, evidenced by the absence of statistically significant changes in the optical density of formazan produced from MTT and neutral red culture media obtained from the fibroblast culture incubated with them for 24 h  when used at a concentration of 5 mg/L, while lower concentrations had no influence of ROS production  pointing to an increase in intraerythrocytic calcium ion levels (FLUO4-AM staining allowed comparing the effects of various composites on intracellular CaNanomaterials have drawn a lot of attention as novel diagnostic and therapeutic tools. However, despite recent advancements in biomedical research, the bench-to-bedside transition is still below the desired level. Only a few dozen nanoparticles are under the clinical trials at the moment . Implemein vitro [Viability assays performed on fibroblast cell lines remain a simple, physiologically relevant and widely used tool to assess the nanotoxicity in vitro . Our finin vitro .The search for novel, more sensitive and effective approaches to assess the biocompatibility of nanosized materials has revealed that eryptosis is a promising model in nanotoxicology , 43.2+ entry are the hallmarks of eryptosis [2+-mediated pathways in hybrid Ag-polymer composite-induced eryptosis. Silver nanoparticles are known to induce ROS generation and promote oxidative stress [2+ influx. In its turn, calcium ions activate calpains, a family of cysteine proteolytic enzymes, responsible for degradation of erythrocyte cytoskeleton [2+-mediated pathways [2+- and ROS-dependent manner. Of note, the authors attribute the proeryptotic effects to coating [Eryptosis is a suicidal cell death of erythrocytes associated with cell shrinkage, membrane blebbing, and phospholipid scrambling on the plasma membrane, triggered under unfavorable circumstance such as energy deficit, hyperosmolarity, exposure to xenobiotics, calcium ion entry, ceramide accumulation, activation of caspases and calpain, and imbalance of redox homeostasis . Eryptosryptosis , 47. In e stress , 49. It skeleton . Howeverpathways . In addi coating .It can be stated that Au/polymer composite is less cytotoxic than a composite with Ag. These findings are consistent with the experimental data on the comparison of Ag and Au nanoparticle toxicity , 53. Thu2 nanoparticles as well [Eryptosis is a more sensitive tool for assessing the biosafety profile of nanosized materials compared with fibroblast viability assays. Of note, its sensitivity is higher than that of viability assays on cultured fibroblasts not only in case of noble metal-polymer hybrid composites, but for different TiO as well .Our findings suggest that metal/D-PAA composites show no cytotoxicity at low concentrations. Ag/polymer composite is more toxic compared to D-PAA and hybrid Au/D-PAA composite. Thus, they are promising biocompatible agents that can be used for medical purposes, partially as a biocompatible therapeutic effect-enhancing component in the multicomponent nanosystems. The study provides experimental evidence that eryptosis is a sensitive approach to assess the biocompatibility of nanomaterials.


Multivariate analysis demonstrated that right sidedness, R1 resection, low body mass index (BMI) and adjuvant chemotherapy were independent factors for poor prognosis for stage II colon cancer. Low BMI, perineural invasion, higher T stage and N2 stage were independent factors for poor prognosis for stage III colon cancer. The results were confirmed by multivariate analysis after propensity score matching. Our study revealed that RCC was an independent risk factor for recurrence in stage II colon cancer.The right and left side of the colon derived from the midgut and hindgut, respectively. Previous studies have reported different characteristics of right-sided colon cancer (RCC) and left-sided colon cancer (LCC), but oncological outcomes remain unclear. This study compared the outcomes of RCC and LCC. This retrospective study included 1017 patients who received curative colectomy for stage I-III colon cancer at a single institute between August 2008 and December 2019. Overall survival (OS) and time to recurrence (TTR) were analyzed as outcome measurements. No significant difference in the OS or TTR of patients with RCC and LCC were observed. In subgroup analysis, RCC was associated with shorter TTR than LCC in stage II colon cancer (HR 2.36, 95% confidence interval 1.24–4.48,  Radical surgical resection is the standard treatment for American Joint Committee on Cancer (AJCC) stage I to III colon cancer; postoperative adjuvant chemotherapy is also administered to patients with high-risk stage II and stage III colon cancer3.Colorectal cancer is one of the most prevalent cancers, especially in developed countries5. Patients with RCC are more likely to be older and female and present with a more advanced tumor stage, larger tumor size, and more poorly differentiated tumor cells than those with LCC11. The differing characteristics of RCC and LCC are thought to be caused by differences in embryologic origin, fecal exposure, and detection time6. Most previous studies have indicated that RCC was associated with a higher recurrence and lower survival rate than LCC13, although several studies have concluded that early-stage RCC had a better prognosis than LCC15. In light of the lack of consensus on the prognosis for RCC and LCC, this study investigated the impact of cancer sidedness and stage on outcomes.Since 1990, right-sided colon cancer (RCC) and left-sided colon cancer (LCC) have been regarded as distinct cancers based on their different embryology, epidemiology, pathology, and prognosisAJCC Cancer Staging Manual, 8th Edition) was used for staging. Only patients with colon adenocarcinoma were included in this study. Patients who were diagnosed with rectal cancer, carcinoma in situ, synchronous colon cancer, or stage IV colon cancer were excluded, as were patients who received palliative surgery or R2 resection. The final sample comprised 1017 patients who underwent curative resection for stage I, II, or III colon cancer , carcinoembryonic antigen (CEA) value, tumor location, histological type, tumor grade, tumor stage, chemotherapy history, K-RAS status, mismatch repair (MMR) status and surgical margin status were collected from patient records. R1 resection was defined as section margin less than 2 mm. Overall survival (OS) was defined as the time from primary tumor resection to death from any cause. Time to recurrence (TTR) was defined as the time from primary tumor resection to the first recurrence confirmed by radiological or histological features.p value of < 0.05 was considered statistically significant.Both K–S and S–W tests were used to confirmed the normal distribution of continuous variables. Variables normally distributed were presented as mean ± standard deviation, otherwise presented as median (Q1–Q3). Comparisons were made using independent-t test or Mann–Whitney test for analysis of continuous variables. Chi-square test was used for comparisons of categorical variables. Kaplan–Meier curves were calculated for all patients and patients with stage I, II, and III colon cancer separately to compare OS and TTR between patients with RCC and LCC. Univariate and multivariate survival analyses were performed with a Cox proportional hazards function; HRs and 95% confidence intervals (CIs) were used to estimate the impact of primary tumor location on survival outcomes. Propensity score matching (PSM) was performed with greedy nearest neighbor matching method, using 14 variables  that could potentially influence outcomes. The number of lymph node harvested was not included due to native difference of mesocolon taken during operation between RCC and LCC. The caliper requirement was ignored. A This study was approved by the Ethics Committee of Taipei Medical University  and was performed in accordance with the Declaration of Helsinki and its subsequent amendments or comparable ethical standards.Informed consent was not applicable due to the retrospective nature of this study.p < 0.01) and less likely to be male . A lower BMI was also noted in patients with RCC than in patients with LCC . Minimally invasive surgeries were performed more frequently in patients with LCC than in patients with RCC . No significant differences in preoperative CEA level, the rate of emergency operations, the rate of adjuvant chemotherapy, T stage, or postoperative follow-up time were observed. More lymph nodes were harvested in patients with RCC than in patients with LCC (26 (19–35.5) vs. 21 (16–28), p < 0.01). Tumors in patients with RCC were larger (4.5 (3.15–6.4) vs. 4.0 (3.0–5.5), p < 0.01) and of a more advanced histological grade  than the tumors in patients with LCC. However, patients with LCC had a more advanced N stage  and AJCC cancer stage  than did patients with RCC. A higher percentage of K-RAS mutation  and deficient MMR (dMMR)  were noted in patients with RCC than in patients with LCC. No significant differences in the rate of perineural invasion or lymphovascular invasion were apparent between the two groups. R1 resection was found in 17 with RCCand 19 patients with LCC with RCC and LCC. There is no significant difference between the two groups .We reviewed the medical records of 1588 patients, and 1017 patients were eligible for inclusion in the study. 75% of the patients were followed for more than 48 months. Ten patients were found metachronous cancers. Of the included patients, 385 (37.9%) had RCC, and 632 (62.1%) had LCC . Patients with stage III LCC had a higher BMI than patients with stage III RCC . More patients with stage II and III LCC underwent minimally invasive surgery than did patients with stage II and III RCC . Lymphovascular invasion was higher in patients with stage II RCC than in patients with stage II LCC . Tumors in stage I and II RCC had more advanced histological grading than did tumors in stage I and II LCC . A higher rate of K-RAS mutation and dMMR was noted in stage II RCC than in stage II LCC  and in stage III RCC than in stage III LCC . More lymph nodes were harvested in RCC than LCC in all three stages (stage I: 22 (18.75–31) vs. 17 (14–24), p < 0.01; stage II: 24 (18–35) vs. 22 (17–29), p = 0.02; and stage III: 29 (21–38.75) vs. 22 (17–30), p < 0.01). More R1 resection was found in stage III RCC than in stage III LCC .The study included 188, 322, and 507 patients with stage I, II, and III colon cancer, respectively. Table p = 0.12). Overall, Kaplan–Meier curves revealed no significant differences in OS or TTR for patients with RCC or LCC  months in RCC and 43.0 (25.3–67.5) months in LCC (3.5–63.5 p < 0.01). The risk factors for shorter TTR in stage II colon cancer were a higher BMI , lymphovascular invasion , perineurial invasion , advanced T stage , adjuvant chemotherapy , and R1 resection . The risk factors for shorter TTR in stage III colon cancer were higher BMI , older age , emergency surgery , open surgery , perineural invasion , advanced T stage , and N stage , R1 resection , and adjuvant chemotherapy  were risk factors for shorter TTR in stage II colon cancer. On the other hand, emergent operation , perineurial invasion , advanced T stage  and N2 stage  were risk factors for shorter TTR in stage III colon cancer. BMI was associated with lower risk of recurrence in both stage II  and stage III  colon cancer.In multivariate analysis, RCC . Figure p = 0.01). Table p = 0.34).After PSM, 271 pairs of patients were successfully matched and were listed in Table This study was a large-scale retrospective study. We followed more than 1000 patients who underwent curative resection for stage I, II, and III colon cancer, with the longest follow-up time more than 10 years. This study demonstrated that the sidedness of colon cancer did not influence OS but influenced TTR in stage II colon cancer. We also observed that RCC, lower BMI, R1 resection and chemotherapy were risk factors for shorter TTR in stage II colon cancer, whereas lower BMI, perineurial invasion, advanced T and N stage were risk factors for shorter TTR in stage III colon cancer. To compensate for the possible bias in this retrospective study, we performed propensity score matching. The results of multivariate analysis were mostly compatible with the results before PSM, which makes this study more reliable.14. In a study of 77,978 patients, Meguid et al. reported that OS in stage II and III RCC was worse than in stage II and III LCC6. However, in a study of 53,801 patients over 65 years of age, Weiss et al. reported that RCC had longer OS than LCC in stage II but worse OS in stage III16. The age at diagnosis of RCC is approximately 3.0 to 4.6 years older than that for LCC18, and only one-third of patients with stage I–III colon cancer die from the cancer19. Therefore, OS does not truly reflect the difference in prognosis between RCC and LCC20; hence, we used TTR to compare the risk factors of RCC and LCC.In previous population-based studies, the OS rate was highly different between stage I, II, and III RCC and LCC. A study of 91,416 patients demonstrated that OS in stage I and II RCC was longer than in stage I and II LCC16; this was also true in our study. However, in our results, women only composed a higher proportion of patients with stage I and II RCC, not stage III. Second, several studies have noted that RCC has a larger tumor size and presents as a later stage than LCC21. In our study, tumor size was significantly larger in stage III RCC than in stage III LCC but not in stage II RCC. It is generally believed that LCC tends to manifest in symptoms such as changes in bowel habits and bleeding earlier than RCC, and consequently, LCC is diagnosed earlier than RCC22. However, the results of this study indicate that the consensus that LCC is often detected at an earlier stage than RCC may need to be reevaluated. A relatively small tumor size with lymph node metastasis may be the typical representation of LCC at the time of diagnosis. Third, lymphovascular invasion and tumor grade are powerful prognostic factors in colon cancer23, and many studies have reported that RCC has a higher rate of lymphovascular invasion and a higher tumor grade than LCC24. However, we observed that tumor grade and the incidence of lymphovascular invasion are significantly higher in stage II RCC than in stage II LCC but not in stage III RCC. The high rate of lymphovascular invasion and the presence of grade 3–4 tumor cells shortened TTR in stage II RCC compared with that for stage II LCC. In our study, although stage III RCC had a larger tumor size and more advanced T stage than stage III LCC, cancer recurrence did not differ if patients underwent appropriate radical resection and adjuvant chemotherapy; moreover, we observed no difference in tumor size between stage II RCC and LCC. However, RCC had a shorter TTR because of a higher proportion of lymphovascular invasion and tumor grade. Therefore, we believe that stage II RCC can be regarded as a unique category of colorectal cancer, and postoperative treatment and follow-up strategies need to be accordingly tailored.This study also noted differences between RCC and LCC and reinforced the consensus that RCC and LCC can be regarded as distinct cancers. However, our results contradicted previous studies regarding their specific differences. First, many previous population-based studies have reported a higher proportion of women with RCC than LCC26. A recent study reported that in patients with dMMR stage II colon cancer, 5-FU based chemotherapy did not improve survival and could even worsen prognosis27. The National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (2017) recommended that MMR status be checked in stage II colon cancer3. Our institute follows this treatment guideline and has tested MMR status for colon cancer since 2017. According to the limited MMR test data, nearly half of patients with RCC had dMMR (45.5%), which is 10–20% higher than in previous studies28. This also explains why chemotherapy was a risk factor for stage II RCC because many patients underwent adjuvant chemotherapy without an MMR status test before 2017.Several studies have also suggested that colon cancer heterogeneity is different between RCC and LCC. Microsatellite instability (MSI) and dMMR are more common in RCC. Patients with stage II colon cancer and MSI or dMMR tumors had a better prognosis in RCC than LCC, but in patients with stage III colon cancer, MSI or dMMR provided no benefit to prognosis29. In this study, however, patients with RCC and stage III RCC had significantly lower BMI than did patients with LCC and stage III LCC, respectively. This may be because patients with RCC are predominantly female and older, which are two groups that generally have a lower BMI. We observed increased BMI to be a positive prognostic factor for patients with both stage II and stage III cancer. By contrast, previous studies have reported obesity or a BMI of > 30 kg/m2 to be risk factors for cancer recurrence and cancer-related mortality in colorectal cancer33. In agreement with our study, Sinicrope et al. reported that overweight patients (BMI 25–29.9 kg/m2) had better OS in stage II and III colon cancer34; the authors speculated that relatively healthy patients in this BMI range can tolerate operations and adjuvant chemotherapy34. We speculate that patients with a low BMI have too little visceral fat to cover the tumor, and thus, the tumor can easily invade the surrounding organs or spread. Patients with obesity usually have comorbid diseases, and complications often occur during surgery or chemotherapy. Most patients (91%) in this study had a BMI below 30, and we observed that increased BMI was associated with longer TTR.In previous studies, no differences in BMI were reported between patients with RCC and LCC37. CME is not routinely performed for RCC at our institute, and CME status is not always recorded in medical records, thus affecting the analysis of prognosis.This study had several limitations. First, this was a retrospective study at a single institution, which makes our study susceptible to bias and confounding factors despite the large study population. Second, only 33 patients (0.3%), 110 patients (11%) and 488 patients (48%) had an MSI status, MMR status or K-RAS mutation test, respectively. Because of the low number of patients with an MSI status, MMR status or K-RAS mutation test, we did not analyze the effect of these variables on prognosis in this study. Third, detailed chemotherapy data were not available in this study. Patients who received oral chemotherapy, intravenous chemotherapy, and incomplete chemotherapy courses were all classified as having received adjuvant chemotherapy. This may underestimate the effects of chemotherapy. Finally, complete mesocolic excision (CME) in RCC increases OS and DFS, and cancer stage may be underdiagnosed in patients without CMEIn conclusion, sidedness is an independent risk factor for cancer recurrence in stage II colon cancer; patients with stage II RCC had shorter TTR than did those with stage II LCC. Furthermore, stage III RCC had a more advanced T stage, but this did not influence TTR in stage III colon cancer. Further research is needed to evaluate the differences of sidedness by using clinicopathological and genetic factors.


Pohlia nutans subsp. LIU) to drought stress. We found that drought stress made the gametophytes turn yellow and curled, and enhanced the contents of malondialdehyde and proline, and the activities of antioxidant enzymes. Totally, 2,451 differentially expressed genes (DEGs) were uncovered under drought treatment. The representative DEGs are mainly involved in ROS-scavenging and detoxification, flavonoid metabolism pathway, plant hormone signaling pathway, lipids metabolism pathway, transcription factors and signal-related genes. Meanwhile, a total of 354 differentially changed metabolites (DCMs) were detected in the metabolome analysis. Flavonoids and lipids were the most abundant metabolites and they accounted for 41.53% of the significantly changed metabolites. In addition, integrated transcriptome and metabolome analyses revealed co-expression patterns of flavonoid and long-chain fatty acid biosynthesis genes and their metabolites. Finally, qPCR analysis demonstrated that the expression levels of stress-related genes were significantly increased. These genes included those involved in ABA signaling pathway , jasmonate signaling pathway , flavonoid pathway , antioxidant and detoxifying functions , and transcription factors (ERF and DREB). In summary, we speculated that P. nutans were highly dependent on ABA and jasmonate signaling pathways, ROS scavenging, flavonoids and fatty acid metabolism in response to drought stress. These findings present an important knowledge for assessing the impact of coastal climate change on Antarctic basal plants.Most regions of the Antarctic continent are experiencing increased dryness due to global climate change. Mosses and lichens are the dominant vegetation of the ice-free areas of Antarctica. However, the molecular mechanisms of these Antarctic plants adapting to drought stress are less documented. Here, transcriptome and metabolome analyses were employed to reveal the responses of an Antarctic moss ( The Southern Ocean and Antarctic coastal seas are undergoing large-scale changes in environmental conditions as a result of warming, ozone depletion, and a positive Southern Annular Mode . In receDeschampsia antarctica and Colobanthus quitensis dominate the vegetations of Antarctica Peninsula  results in inactivation of type 2C protein phosphatases . Meanwhile, we employed the UPLC–MS/MS platform to make a high-throughput analysis of the metabolites of P. nutans and detected 354 differentially changed metabolites (DCMs). Among them, flavonoids and fatty acids were the most significantly changed metabolites. An integrated analysis demonstrated that the accumulation of flavonoids was accompanied by the upregulation of flavonoid synthase genes, indicating flavonoid biosynthesis pathway might play an important role in the adaptation of P. nutans to drought stress.Bryophytes and lichens are the dominant organisms on the Antarctic continent . BryophyP. nutans subsp. LIU was collected near the Great Wall Station on the Fildes Peninsula  in Antarctica in March 2014. Plants were grown on a mixed soil  and cultured at 16°C, 50 μmol photons·m−2·s−1 light with a 16-h-light/8-h-dark photoperiod . The plants without PEG6000 treatment were collected as the control group (CG). The samples were quickly frozen in liquid nitrogen and stored at −80°C.The toperiod . To retatoperiod . Plants g/min for 10 min. POD catalyzed the reaction of hydrogen peroxide (H2O2) and the enzyme activity was calculated by measuring the absorbance values at 420 nm (Product No. A084-3-1). CAT decomposing H2O2 could be quickly terminated by adding ammonium molybdate, and the remaining H2O2 reacted with ammonium molybdate producing a pale-yellow complex. The absorbance values were measured at 405 nm, and the activity of CAT was calculated (Product No. A007-1-1). APX was mainly present in chloroplasts, and its activity was determined by catalyzing the reaction of ascorbic acid with H2O2 (Product No. A123-1-1). GSH-PX could promote the reaction of H2O2 and reduce glutathione to generate water and oxidized glutathione. The activity of GSH-PX could be expressed by the speed of its enzymatic reaction (Product No. A005-1). SOD activity was based on a plate assay using xanthine oxidase and the water-soluble tetrazolium  to detect biochemical characteristics. Briefly, 1 g of moss gametophytes with various treatment times were cut and ground into powders in liquid nitrogen. Then, 0.1 g of the powder was added to the appropriate extracting buffer to generate a 10% tissue homogenate. The supernatant was obtained after centrifugation at 4000 r/min for 10 min. The supernatant was used to detect the contents of malondialdehyde (MDA) and proline (PRO) using the commercial kits (Product No. A003-2-3 and A107-1-1), respectively. Meanwhile, to measure the enzyme activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), the samples were homogenized in physiological saline to make 10% tissue solution and the supernatant were collected after centrifugation at 4000 × Trizol reagent  was used to extract total RNA from the samples, and the RNA quality was analyzed by 1% agarose gel electrophoresis. The integrity of RNA was tested using Agilent RNA Nano6000 kit and Agilent 2,100 bioanalyzer . The concentration of total RNA was measured using Qubit® RNA Assay Kit (Thermo Fisher Scientific). Magnetic beads were used to purify mRNA and then to break it down into smaller fragments. Following the manufacturer’s instructions, sequencing libraries were generated using the NEBNext®UltraTM RNA Library Prep Kit for Illumina® . Finally, the libraries were sequenced on Illumina Hiseq 2500 platform by Novogene Biotech Co., Ltd. .P. nutans (p < 0.05 and |log2(Fold Change)| > 1. DEGs were annotated using Swiss-Prot databases. GO enrichment analysis of the DEGs was implemented by the TBtools software  were also removed. The clean reads were generated and used for the following analysis. Firstly, we performed the alignment program for mapping sequencing reads to the reference genome of . nutans . This pr. nutans . We then. nutans . We emplsoftware . KEGG pasoftware .TransZol Up Plus RNA Kit . 500 ng of total RNA were employed for cDNAs synthesis using TransScript® All-in-One First-Strand cDNA Synthesis Super Mix Kit (TransGen). We tested four house-keeping genes  and finally chose GAPDH as reference gene, because it was stable expression under control condition and drought stress (Perfect Start® Green qPCR Super Mix Kit (TransGen). The cycling regime was 95°C for 2 min, followed by 45 amplification cycles . The reaction was repeated three times for each template. The experiment was repeated three times. Relative gene expression levels were calculated by using the Ct(2−ΔΔCt) method  method .g for 10 min, and filtered using a 0.22 μm membrane . The supernatant was subjected for LC–MS/MS analysis and conducted by Wuhan Metware Biotechnology Co., Ltd. following the standard procedure . The samples were then ground with a hybrid grinder . To dissolve lyophilized powder, 1.2 ml of 70% aqueous methanol was added to the plant samples at 4°C overnight. The supernatant was obtained after centrifugation at 10,000 × rocedure . We usedt-test .Data are expressed as mean ± standard error of the mean (SEM). Significant differences between the drought group and CG were calculated using Student’s P. nutans were shown in P. nutans after PEG6000 treatment for 12 h  was greater than 1.0, and the value of p was less than 0.05. A total of 2,451 DEG were identified between drought group and CG , allene oxide cyclase (AOC), allene oxide synthase (AOS), jasmonate ZIM domain protein  and jasmonate signaling pathway  were markedly upregulated under drought stress , ABA receptor  and type 2C protein phosphatases (PP2C), as well as SnRK1-activating protein kinase 2 SnAK2; . Similarein JAZ; . We perft stress ,B. Ethylt stress . In addit stress .P. nutans under drought stress, a widely targeted metabolome analysis was performed based on the UPLC–MS/MS platform. A total of 559 metabolites were obtained  and VIP, and heatmaps were drawn to show the overall changes of metabolites (2(Foldchange)| ≥ 1 and VIP ≥ 1 | in the comparison of drought stress with CG (2(Fold change) of 13.83. Flavonoids and phenolic acids were very abundant in DCMs. Our results showed that there was a substantial accumulation of flavonoids and phenolic acids in P. nutans under drought stress.To reveal the function of the differential metabolites, KEGG pathway enrichment analysis was performed by rich factor, value of ed genes . value oynthesis . In planynthesis . The pheynthesis . The top with CG . The oveP. nutans under drought treatment, we performed a transcriptome and metabolome co-expression network analysis. We selected DEGs and DCMs with Pearson correlation coefficient greater than 0.8 to draw a histogram. Genes and metabolites with negative correlation in quadrants 1, 2, 4, 6, 8, and 9. Metabolites were overproduced in quadrants 1, 2, and 4, where genes were unaltered or downregulated. However, in quadrants 6, 8, and 9, genes were upregulated, and metabolites were unchanged or under-produced. Only in the 3rd and 7th quadrants, genes and metabolites had positive correlation, and the changes of metabolites might be positively regulated by genes (p < 0.05), while those in flavonoid biosynthesis and alpha-linolenic acid metabolism were close to being consistent , flavone , flavonol (quercetin-3-O-(2″-p-coumaroyl) galactoside) and two flavanols  were significantly accumulated under drought stress . MeanwhiPnUFGT-2 . Taken tt stress .P. nutans under the treatment of 20% PEG6000  but not JA or its amino acid conjugates, which indicates that OPDA regulates a signaling pathway distinct from JA  might promote the synthesis of VLCFAs (KCS can enhance resistance to drought stress by changing the composition of VLCFAs in cuticular wax (MpFAE2 and MpFAE3) of Marchantia polymorpha are known to synthesize beta-ketoacyl-CoA synthase  under the BioProject number PRJCA008231. The genome assembly and annotation data were deposited in Genome Warehouse (GWH) under the accession number GWHBHNB00000000. The transcriptome sequencing data of Pohlia nutans were also deposited in the Genome Sequence Archive  under the BioProject number PRJCA008962 and the sequence accession number CRA006553.The original data presented in the study are publicly available. The whole genome sequence data were available in the National Genomics Data Center  and Central Public-Interest Scientific Institution Basal Research Foundation of China (GY2019Q05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Conventional second window indocyanine green (SWIG) technique has been widely attempted in near-infrared fluorescence (NIRF) imaging for intraoperative navigation of tumor radical resection. Nevertheless, the overuse of indocyanine green (ICG) led to an increased risk of drug lethal allergy and high medical cost. This prospective study was to explore clinical application of modified low-dose SWIG technique in guiding dermatofibrosarcoma protuberans (DFSPs) radical resection.Patients with DFSPs were randomly assigned to control and experimental group. The ICG was injected intravenously 24 h before surgery, at a dose of 3.5 mg/kg in the control group and 25 mg/patient in the experiment group, respectively. Intraoperative NIRF imaging included serial views of gross tumor, tumor bed and cross-sectional specimen.P = 0.000). Data in both groups displayed that there was a positive correlation of tumor size in cross-sections between integrated histopathologic photomicrographs and NIRF imaging of specimen views (P = 0.000). NIRF imaging of cross-sectional specimens had a significant decrease in time cost, and an increase in the ability of examining more surgical margins (P = 0.000).Although NIRF imaging of gross tumor and tumor bed in the experimental group demonstrated similar sensitivity and negative predictive value, the specificity and positive predictive value were obviously higher compared to control group. The tumor-to-background ratios of cross-sectional specimens in the experimental group was significantly higher than in the control group (This is the first study to demonstrate that a low-dose SWIG technique could improve the accuracy of near-infrared fluorescence image-guided dermatofibrosarcoma protuberans resection.Clinical Trial Registration: ChiCTR2100050174; date of registration: August 18, 2021 followed by “retrospectively registered” To improve local control, it is preferably recommended that tumor athology –3. Theorathology , 5. GiveWith the development of intraoperative navigation, a variety of technologies, such as BrainLab intraoperative 3-dimensional magnetic resonance imaging (MRI) , 7, RamaNevertheless, the dose of ICG was overused on patients and violated package insert in previous clinical trials of SWIG approach, which led to an increased risk of drug lethal allergy and extremely high medical cost. In contrast to excessive dose of ICG, we designated 25 mg ICG for each patient as low-dose approach. Our current research was to explore clinical application of modified low-dose SWIG technique in guiding DFSP radical resection.. The main exclusion included seafood/iodine allergy, hyperthyroidism, pregnancy, myasthenia gravis, or acute severe hypertension. The study, registered under chictr.org.cn (ChiCTR2100050174), was approved by the Medical Ethics Committee of the Chinese PLA General Hospital and rigidly obeyed the principles of the Declaration of Helsinki. All informed consent was obtained before ICG administration.Patients with DFSPs in heads, extremities or trunk were recruited between October 2019 and May 2022via median cubital vein. After 20 min, if the test result was negative, the antiallergic preconditions consisting of 10 mg dexamethasone sodium phosphate and 20 mg diphenhydramine were administrated. Finally, the surplus ICG was infused intravenously. The adverse effects of ICG were recorded.The patients enrolled in current research were randomly divided into control and experimental groups with a 1:1 allocation ratio based on block randomization by IBM SPSS software version 20.0  . The progross tumor view”. Patients underwent radical tumor resections at a safe margin distance of 2 cm from tumor fluorescent signals. After tumor removal, NIRF imaging was captured by visualizing surgical cavity again to detect residual cancerous tissues, which was named as “tumor bed view”. The suspicious lesions discerned by high fluorescent signals were further removed and diagnosed by histopathologic examinations. If there was no fluorescent signal in tumor beds, surgeons were designated to collect tissue as negative tumor base for final histopathologic examination. Sequentially, based on pathologic protocols, representative areas with close margins and the greatest horizontal and perpendicular dimensions of the gross specimens were cut transversely into cross-sections with the thickness of 10 mm in the vertical direction to the skin. Each cross-section was inspected using NIRF probe. The final ICG fluorescence imaging was defined as “specimen view”, which offered the information of TBRs and tumor size in cross-sectional specimens. The fluorescent imaging of “specimen view” could not affect the range of tumor resection in accordance to panel's recommendation. However, all cross-sections were evaluated by the final pathologic diagnoses. During this phase, the scanning time and number of surgical margins assessed by multiple cross-sections were also compared with conventional rapid frozen pathology. Fluorescent camera system  was applied to scan the tumor, with an 785-nm laser excitation source and 830 to 900-nm emission filter. The computer screen with a 3,820 × 2,160 pixel video resolution could offer white light view and pseudocolor view. The probe was positioned 20 cm above the operative area. Initially, the preoperative fluorescence imaging was referred to as “.After completing “specimen view”, the dissected specimen was retrieved to an intact one by suturing. In addition to routine specimen collection, each cross-section was subsequently cut into multiple small portions, the purpose of which was to measure tumor size by assembling into a complete photomicrograph by Adobe Photoshop . Pathological assessment included histologic type, tumor grade and size, and resection margin status. The surgical margins was reported based on the American Joint Committee on Cancer (AJCC) classification/R systems , which dhttps://imagej.nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA) was used to analyze the TBRs and tumor size in fresh resected cross-sections from NIRF images of specimen views. Region of interest (ROI) size was defined as 100 mm2, given that the diameter of tumor in our study was more than 20 mm and the distance between tumor margin and surgical margin was more than 20 mm. The TBRs were obtained by manually drawing ROI areas of tumor and normal tissue to measure mean grey values on black-and-white images of cross-sectional specimens.ImageJ  and negative predictive value (NPV) with 95% confidence intervals. TBRs of cross-sectional specimens between two groups were compared by Mann-Whitney U test. Spearman correlation analyses were performed to compare the maximum tumor dimension in cross-sections between integrated H&E slides and NIRF imaging of specimen views among respective group. An independent t-test was used to analyze the examining time between rapid frozen pathology and NIRF imaging of specimen view. Mann-Whitney U test was used to compare number of surgical margins between rapid frozen pathology and NIRF imaging of specimen view. All statistical analyses were performed using IBM SPSS software version 23.0 .PASS software was used to calculate sample size. Fisher's exact test, Mann-Whitney P = 0.351), gender (P = 1.000), histologic grade (P = 1.000), tumor location (P = 0.943), tumor size (P = 0.76) and body mass index (BMI) (P = 0.91). Comparison of patient demographics between two groups are summarized in The needed sample size in this prospective two-arm randomized study was estimated by an alfa-value of 5%, a power of 85% and a difference in TBR of 1 between the two groups. The estimated sample size was 12 patients in each group. Hence, a total of 36 patients were enrolled in our study. 1 patient in the control group developed a skin rash during ICG administration. One operation was cancelled duo to nonmedical reason in the control group. At last, there were 16 patients in the control group and 18 patients in the experimental group . There w16 DFSPs in 16 patients (100%) in the control group displayed significant NIRF imaging on gross tumor views. Four positive fluorescent signals in 4 of 16 patients were detected on tumor bed views, whereas these 4 lesions were confirmed as normal tissue or fibrous tissue based on histopathologic diagnoses. Based on the final pathology, the sensitivity, specificity, PPV, and NPV of NIRF imaging were 100%, 75.0%, 80.0%, 100%, respectively . All tumP < 0.001). It was noted that DFSPs in the experimental group showed significantly more intensive fluorescent signals within tumor sites. On the other side, tumor size calculated by the largest diameter did not differ between these two groups . The data from both groups displayed that there was a positive correlation of maximum tumor size in cross-sections between integrated H&E photomicrographs and NIRF imaging . Representative cases in each group are shown in To identify the optimal SWIG technique, we compared the fluorescent intensity of cross-sections in “specimen views” between these two groups. The difference in TBRs of cross-sectional specimens between these two groups was statistically significant (3.90 (3.45–4.45) in the control group vs. 4.90 (4.60–5.50) in the experimental group; P < 0.001). In addition, the number of surgical margins evaluated by NIRF imaging of specimen view was considerably increased compared with that by rapid frozen-section diagnosis (5.00 (5.00–5.00) vs. 13.00 (10.00–16.00), P < 0.001).To compare with rapid frozen pathology, some corresponding parameters of intraoperative “specimen view” were recorded. Firstly, there was a statistically significant decrease in examining time between rapid frozen-section diagnoses and NIR fluorescence imaging of specimen views . Hence, we conclude that the low-dose SWIG technique (25 mg/patient) displayed an improved performance of identifying DFSP lesions by intraoperative NIRF visualization system.DFSP is a common type of soft tissue sarcomas (STSs). In theory, ICG as macromolecule is liable to be accumulated within the DFSP, characterized by capsule or pseudocapsule neoplasm surrounded by avascular subcutaneous fat tissue. At present, radical resection with at least 2 cm surgical margins is primary treatment to minimize the risk of local recurrence. To our knowledge, this is the first clinical trial to display intraoperative NIRF fluorescence imaging of DFSP. We designed traditional SWIG technique as control group. Firstly, we compared the low-dose SWIG technique with conventional high dose group in terms of the accuracy of identifying malignant tissue by NIRF views of gross tumor and tumor bed. NIRF imaging in the experimental group demonstrated similar sensitivity (100% vs. 100% in the control group), higher specificity (93.8% vs. 75.0% in the control group), increased PPV (95.2% vs. 80.0% in the control group) and similar NPV (100% vs. 100% in the control group). In both groups, all gross tumors were detected by NIRF imaging. 3 positive fluorescent signals were observed from tumor bed views in experimental group, two of which were confirmed as residual DFSP tissue. In contrast, 4 positive fluorescent signals in tumor beds were validated as non-malignant tissue in the control group. Therefore, we assumed that low-dose SWIG technique appeared to be more sensitive in visualizing tumor bed. Secondly, we analyzed fluorescent signals in cross-sectional specimens. The TBRs of dissected cross-sections in the experimental group was significantly higher than in the control group (4.90 (4.60–5.50) vs. 3.90 (3.45–4.45), Our NIRF visualization system has more sensitive femtomolar probe detecting fluorescent dye at a concentration of 300 pm/L, the light source output of three homologous wavelength and the camera with three channels. The highly sensitive NIRF optical system decreases the dosage of contrast agent from 5 mg/kg to 0.2 mg/kg, which is one of the reasons why we chose 25 mg ICG in one ampoule as the dose in the experimental group. Besides, the images are processed to reduce background signal by a convolutional neural network approach. Therefore, compared to previous reports, our NIRF imaging system is capable of producing more favorable outcome than conventional SWIG technique.Although the incidence of lethal complication is less than 0.1%, the iodine, a main component of ICG, is liable to induce a severe allergic reaction, even shock and death. Since the number of patients in our study was small, we didn't analyze the incidence of drug allergy. However, 1 patient had a mild skin rush during ICG administration in the control group. Take a patient with a weight of 65 kg as an example. The ICG in the control group costs 1,458 RMB (162 × 9), and that in the experimental group spends 162 RMB, which is obviously a very different cost. Given that drug safety, convenience of drug administration, and inpatient cost, 25 mg ICG as a single bolus has also a distinct advantage over high-dose infusion (2.5–5.0 mg/kg ICG).Even so, there are still some issues to address. Firstly, 25 mg ICG are suitable for patients with normal body mass index (BMI). The individualized dosage in modified SWIG technique should be verified in the following study. Secondly, the inclusion criteria stipulated that only patients with DFSPs were recruited in our study. In fact, intraoperative NIRF imaging using this low-dose SWIG technique might play a positive role in visualizing other malignant soft tissue tumors, such as other types of sarcomas and cutaneous squamous carcinomas. Besides a great deal of confidence in the prospect of low-dose SWIG technique for NIRF imaging, we also discovered that intraoperative NIRF imaging with SWIG technique could evaluate resected specimen in a more efficient, thorough, and quantitative manner.At present, some problems in traditional histopathologic diagnosis remain to be solved. Firstly, there are several classification schemes of surgical margins in soft tissue sarcomas, such as the Musculoskeletal Tumor Society (MSTS) classification , the AmeP = 0.000). The average examining time of rapid frozen-section diagnoses was 47 min, which was evidently longer than intraoperative NIRF imaging of specimen views with a mean time of 10 min. Technically, since the NIRF imaging has the advantage of rapid scanning, it is capable of inspecting the whole resected specimen within a few minutes. This efficient approach of evaluating gross surgical specimen could increase the number of positive margins along with a great amount of cross-section scanning. Additionally, NIRF imaging can offer more detailed information about tumor size and margin distance in every cross-section, which is impossible in rapid frozen diagnosis. In our study, the data in both groups displayed that there was a positive correlation of largest diameters of malignant tissue in cross-sections between integrated H&E photomicrographs and NIRF imaging of specimen views . Although margin distances were not analyzed in our research, the measurement of margin distance is feasible and beneficial for surgeons, especially when resected margins are suspicious.In the present study, we dissected surgical specimen into multiple cross-sections and scanned cross-sectional specimens in sequence using NIRF imaging with SWIG technique before rapid frozen-section histopathology. There was a significant increase in the number of surgical margins assessed by NIRF imaging of specimen views compared to that by rapid frozen-section diagnoses (13.00 (10.00–16.00) vs. 5.00 (5.00–5.00), We limited the quantity of dissected cross-sections at the current stage, so that the pathologists could assess the intact tumors before sampling. We plan to dissect surgical specimens from comprehensive and multidimensional viewpoints in the future study. Furthermore, we will proceed with investigating whether intraoperative NIRF imaging has potential in assisting pathologists with improving the performance of histopathologic diagnosis. To our knowledge, this was the first time that low-dose SWIG technique was investigated in NIRF imaging. The low-dose SWIG technique demonstrated improved accuracy of detecting tumor fluorescent signal in the intraoperative navigation of DFSP radical resection compared with conventional SWIG technique. Taking into consideration the benefit of drug safety, convenience of drug administration, and inpatient cost, we assume that this modified approach has a good prospect for extensive clinical application. Additionally, our research was the first to explore the potential application of intraoperative NIRF imaging for evaluating resected specimens. Further investigations of NIRF imaging using low-dose SWIG technique will focus on the following issues: a refinement in ICG dosage, broadening clinical application in a variety of malignant tumors, as well as optimizing NIRF imaging of specimen view for assisting pathologic diagnosis.


Studies have suggested that the postoperative length of stay (PLOS) of esophagectomy patients under the enhanced recovery after surgery (ERAS) pathway should be >10 days as against the previously recommended 7 days. We investigated the distribution and influencing factors of PLOS in the ERAS pathway in order to recommend an optimal planned discharge time.This was a single-center retrospective study of 449 patients with thoracic esophageal carcinoma who underwent esophagectomy and perioperative ERAS between January 2013 and April 2021. We established a database to prospectively document the causes of delayed discharge.The mean and median PLOS were 10.2 days and 8.0 days (range: 5–97), respectively. Patients were divided into four groups: group A (PLOS ≤ 7 days), 179 patients (39.9%); group B (8 ≤ PLOS ≤ 10 days), 152 (33.9%); group C (11 ≤ PLOS ≤ 14 days), 68 (15.1%); group D (PLOS > 14 days), 50 patients (11.1%). The main cause of prolonged PLOS in group B was minor complications . Severely prolonged PLOS in groups C and D were due to major complications and comorbidities. On multivariable logistic regression analysis, open surgery, surgical duration >240 min, age >64 years, surgical complication grade >2, and critical comorbidities were identified as risk factors for delayed discharge.The optimal planned discharge time for patients undergoing esophagectomy with ERAS should be 7–10 days with a 4-day discharge observation window. Patients at risk of delayed discharge should be managed adopting PLOS prediction. Esophageal cancer is the seventh most commonly diagnosed cancer in the world and the sixth most common cause of cancer-associated mortality . More thThis was a single-center retrospective cohort study of 449 patients with thoracic esophageal cancer who were treated at the Department of Thoracic Surgery between January 01, 2013 and April 30, 2021. All patients underwent surgery, and perioperative ERAS measurements were performed by the same team. The study was conducted according to the principles enshrined in the Declaration of Helsinki and the study protocol was approved by the Ethics Committee (EC) for Medical Research and New Medical Technology of Sichuan Cancer Hospital (SCCHEC-02-2022-042). Written informed consent of patients was waived by the EC. Data pertaining to clinical status, tumor characteristics, neoadjuvant therapy, surgical approach, surgical duration, intraoperative blood loss, anastomotic approach, anesthesia, PLOS, and perioperative complications  were recorded. Causes of delayed discharge were monitored prospectively.Clinical staging was based on preoperative contrast-enhanced cervicothoracic and abdominal CT, endoscopic ultrasound, fibreoptic bron choscopy, neck and abdominal ultrasound, and bone scan, with additional contrast-enhanced head MRI, and PET-CT scans to rule out distant metastases, if necessary. Tumor pathology was staged according to the 8th edition of the International Union Against Cancer (UICC). Neoadjuvant chemoradiotherapy, chemotherapy, or immunochemotherapy was administered according to the National Comprehensive Cancer Network guidelines, the Chinese Society of Clinical Oncology guidelines, registered clinical trials, and the patients’ wishes. Patients were treated with McKeown esophagectomy and three-field lymph node dissection if the tumor was located in the thoracic segment; the Ivor-Lewis or transhiatal surgical approach was used for tumors located at the esophagogastric junction. Clinical protocols of patients undergoing esophagectomy are shown in t test. Non-normally distributed continuous variables were expressed as median (range) and between-group differences assessed using the Mann-Whitney U test. Categorical variables were expressed as frequency (percentage) and between-group differences assessed using the Chi-squared test or Fisher's exact probability method. All statistically significant perioperative variables were included in a multifactorial analysis model using binary logistic stepwise regression analysis. Variables that were not statistically different were phased out until the model was explained by the lowest number of variables. P values < 0.05 were considered indicative of statistical significance.Data analyses were performed using SPSS 24.0 software. Normally distributed continuous variables were expressed as mean ± standard deviation and between-group differences assessed using independent samples A total of 449 patients  who were selected for the perioperative ERAS pathway were included in this study. The baseline characteristics are summarized in n = 179, 39.9%); group B ; group C ; and group D . The PLOS data had a skewed distribution. The PLOS of 73.8% patients was within the range of 3 days from the standard PLOS plan .The distribution of the PLOS in our cohort is shown in The most important reason for delayed discharge was postoperative complications . PatientSix patients were readmitted within one month of discharge (readmission rate: 1.3%). Two patients were in group A, three in group B, and one in group C. All six patients were readmitted because of complications . Clinical manifestations and blood indices of these patients were within the normal range, and all readmitted patients were eventually discharged .P < 0.001), access modality (P = 0.04), American Society of Anesthesiologists (ASA) score (P = 0.01), intraoperative bleeding (P = 0.01), surgical duration (P = 0.04), classification of surgery-related complications (P < 0.001), and severe comorbidities (P < 0.001) (P = 0.88). PLOS score, operative time, intraoperative bleeding, and age were transformed into categorical variables.In this study, there were no significant differences between patients with PLOS ≤10 days and those with PLOS >10 days with respect to sex, tumor location, T stage, N stage, surgical approach, or neoadjuvant therapy. However, there were significant differences between the two groups with respect to age (< 0.001) . There wP = 0.515, OR = 1.180, 95% CI: 0.717–1.942) and ASA score >2 . The Hosmer-Lemeshow test revealed that the model explained 23.9% (Nagelkerke R2) of delayed discharges and correctly predicted 78.2% of discharges, with a sensitivity of 95.5%, specificity of 29.7%, positive predictive value of 79.2%, and a negative predictive value of 70.0%. The results of the multivariable binary logistic regression model are shown in Variables that showed significant differences in the univariable analysis were included in the multivariable binary logistic regression models. Multivariable binary logistic regression was performed after excluding patients with intraoperative blood loss of >150 ml  . Five ofThe database on which this study is based was primarily developed to address causes of impact-planned discharges. The database actively records impact events and causes. This is critical for an accurate analysis of the reasons for delayed discharge. Continuous analysis of these reasons can improve the quality of the ERAS protocol and provide a basis for developing the optimal PLOS plan for each patient. In this study, 33.9% of patients were discharged within 8–10 days; whether this group of patients could achieve the standard PLOS pathway (PLOS ≤ 7 days) warrants further analysis. In group B, the reasons for delayed discharge included prolonged chest drainage, pulmonary infection, and minor complications, such as recurrent laryngeal nerve injury, electrolyte disturbance, or pneumothorax. Esophagectomy and lymph node dissection result in large intrathoracic wounds that impede lymphatic return and produce more exudate , 19. PosIn group C, more than 10% of patients had delayed discharge due to severely prolonged drainage, poor pulmonary function, recurrent laryngeal nerve injury, gastrointestinal disturbances, or poor wound healing. More than 5% had delayed discharge due to pulmonary infection. Patients in groups B and C had similar complications; however, complications in group C were more severe, and the patient's underlying condition tended to be worse. In patients with risk factors undergoing esophagectomy, it is particularly important to selectively dissect the lymph nodes surrounding the recurrent laryngeal nerve in order to avoid damage to it . This isWe compared various factors in the perioperative period of the two subgroups (PLOS ≤10 days and PLOS >10 days), and factors with significant differences in univariable analysis were included in a multivariable logistic regression model. We found that a surgical duration >240 min, age >64 years, surgical complication grade >2, and severe comorbidities were risk factors for increased PLOS. This result is consistent with previous studies , 22–25. Some limitations of our study should be considered while interpreting the results. This was a retrospective single-center study, and the conclusion was based on the single-center ERAS mode. Larger multicentre studies are required to investigate the applicability of our findings to other ERAS pathways.The optimal planned discharge time for the ERAS pathway for esophagectomy should be between 7 and 10 days postoperatively, with a 4-day discharge observation window. For patients with risk factors for delayed discharge, prediction methods should be implemented to determine the planned PLOS. Individualized perioperative management protocols should also be provided.


LAMA5, which has been recently discovered and encodes the laminin α5 chain. The laminin α5β2γ1 heterotrimer is an essential component of the glomerular basement membrane and is necessary for embryogenesis and immune modulation. Biallelic LAMA5 variants have been identified in one adult and ten pediatric nephrotic syndromes (NS) patients with variable phenotypes. Biallelic truncating mutations in this gene have recently been proven to cause SRNS. Here, we present another case of infantile SRNS related to novel compound heterozygous variations of LAMA5 , the first reported case with one missense and one nonsense allele. A 10-month-old female patient presented with eyelid edema and massive proteinuria without any extrarenal symptoms or family history. The patient was diagnosed with SRNS. Renal biopsy revealed focal segmental glomerulosclerosis with widely effaced epithelial foot processes and a “moth-eaten” appearance. She progressed to end stage kidney disease (ESKD), requiring dialysis at 31 months of age, and underwent a deceased-donor kidney transplant at 6 years of age. Four months after transplantation, she developed Ebstein-Barr Virus (EBV) infection related to post-transplantation lymphoproliferative disorder (PTLD). After chemotherapy, the patient remained healthy with adequate renal function without disease recurrence for the past 7 years. We also identified previous cases of biallelic LAMA5 variants associated with the nephrotic phenotype and analyzed the available clinical and genetic information. All reported patients had an onset of NS ranging from 3 months to 8 years, with no other syndromic features. Response to therapy and renal outcomes varied greatly; most patients exhibited steroid resistance, five progressed to ESKD, and two received kidney transplantation (KT). There was one report of PTLD. Our patient’s phenotype was markedly more severe than those with biallelic missense variants and somewhat less severe than those with two truncating variants. LAMA5 defects may also play a role in PTLD, though no conclusions can be made with such limited cases. LAMA5 should be considered a candidate gene for SRNS and should be actively tested in cases with no other genetic diagnosis.Single gene pathogenic mutations have been implicated in up to 30% of pediatric steroid-resistant nephrotic syndrome (SRNS) cases, mostly in infantile patients. Among them is  LAMA5, which encodes laminin α5, is the predominant alpha chain of laminin in the mature GBM (Steroid-resistant nephrotic syndrome (SRNS) is a subtype of nephrotic syndrome (NS) characterized by proteinuria, hypoalbuminemia, and edema that does not respond to standard steroid therapy. Fifty percent of patients with SRNS are reported to develop end-stage kidney disease (ESKD) within 15 years, indicating a poor prognosis. Over 70 genes, often encoding proteins of the podocytes or the glomerular basement membrane (GBM), have been reported as causative factors of SRNS . Of thosture GBM .Laminin-α5 is composed of three globular domains  and three rod-like elements , followed by a long coiled-coil domain (LCC), ending with five laminin G -like domains . TogetheLAMA5 have been associated with kidney disease in murine models since 2000, in addition to abnormalities in embryogenesis, extracellular matrix formation, and immune modulation  showed reduced secretion of the laminin α5β2γ1 heterotrimer and, in turn, altered GBM composition  in 2013 , and her weight was 7.7 kg (10–25 p). She showed mild periorbital swelling and abdominal distension. The initial laboratory findings were as follows. Serum albumin 1.6 g/dl, serum BUN/creatinine 4/0.4 mg/dl, serum cholesterol 712 mg/dl, IgG/A/M respectively 236/33/131 mg/dl, and C3/C4 103/16 mg/dl. Serological testing was negative for HBV, HCV, HIV, and RPR/VDRL. Urine dipstick examination showed albuminuria (3+) and a random urine protein/creatinine ratio of 27.47 mg/mg. Kidney ultrasonography findings were unremarkable, with no signs of increased echogenicity or renal cysts. A kidney biopsy was performed, and its pathology was consistent with focal segmental glomerulosclerosis (FSGS) of the perihilar type. Four of 101 glomeruli (3.9%) showed global sclerosis, three (2.9%) showed segmental sclerosis, and five (5.0%) showed small crescentic lesions. Focal GBM abnormalities were noted ultra-structurally, with widely effaced epithelial foot processes and a moth-eaten appearance. The patient did not show any remarkable extrarenal abnormalities. Upon admission in November 2007, at the age of 12 months, she was tested for known mutations in the She was treated with oral steroids and angiotensin-converting enzyme (ACE) inhibitors, but her proteinuria and renal function worsened, along with occasional viral infections. The patient progressed to ESKD within 31 months and was started on peritoneal dialysis. She underwent a deceased-donor kidney transplant at 6 years of age. She then developed Ebstein-Barr Virus (EBV)-associated post-transplant lymphoproliferative disorder (PTLD) of the intestine 4 months after transplantation, with EBV titers rising to 166,571 copies/ml in the blood. The patient was seronegative for EBV before transplantation, and the kidney donor was seropositive for EBV. Ileo-colectomy was performed, followed by intensive chemotherapy for 3 years, and remission was achieved in 2015. At her most recent follow-up in July 2022, the patient had been off therapy for 7 years and had maintained adequate renal function without recurrence. The clinical course of the patient is shown in in silico prediction; c.6883C > T (p.Gln2295*) is a nonsense variant classified as likely pathogenic (LP) by the American College of Medical Genetics, inherited from the father, and c.3434G > A (p.Cys1145Tyr) is a de novo missense variant classified as a variant of unknown significance (VUS).In June 2021, new whole exome sequencing technology became available for use in still undiagnosed patients. Only then was the patient was found to have compound heterozygous variations of LAMA5 through whole exome sequencing. Both variants were predicted to be deleterious by LAMA5-related nephropathy in Korea, the only reported case with one missense allele and one nonsense allele, and the second case of PTLD. This case fits well with previous reports, with the onset of SRNS at 10 months and progression to ESKD at 31 months with no other syndromic features. LAMA5 variants for whom clinical information was available. Reported pediatric patients with LAMA5 mutations had an onset of NS ranging from 3 months to 8 years. Except for the two Japanese siblings with bilateral cataracts and one syndromic case with Arg286Leu variation, all patients exhibited no extra-renal manifestations or syndromic features. Most patients were steroid-resistant and progressed to ESKD by ages 1–6. Renal histology revealed by renal biopsy was consistent with previous reports of both mouse models and clinical data: FSGS, foot process effacement, and thick and thin “moth-eaten” GBM.This is the first case report of de novo variant to be harmful (The variants c.6883C > T (p.Gln2295*) and missense variant c.3434G > A (p.Cys1145Tyr) have not been previously reported. This missense variant is in a highly evolutionarily conserved sequence in zebrafish. The variants were not found in control databases such as ClinVar, gnomAD, ExAC, and 1000G . Twelve  harmful . StaininWhile our patient had only one truncating allele, her phenotype was more severe than those with biallelic missense variants and somewhat less severe than those with two truncating variants. This missense mutation is in the L4a subunit, where other pathogenic missense variants have been reported in humans and mice. Analysis of a murine model generated by Falcone et al. with an L4a domain variant (Gly3685Arg) showed depletion of laminin α5 and different protein composition and organization of the GBM compared to wild mice . FalconeLAMA5, with some steroid-sensitive forms. Still, among the steroid-resistant, the few cases reported to use immunosuppressants showed no response, especially those with infantile-onset (According to the International Pediatric Nephrology Association SRNS guidelines, comprehensive genetic panel testing should be performed in all children with primary SRNS if available . After ile-onset , 19. OurLAMA5 is implicated in transplant tolerance and immunity by regulation of T cells in mouse studies (LAMA5-related NS has been described in four cases, including this report. As expected from genetic NS, none recurred after KT ( studies . The posafter KT ; howeverafter KT ; in our after KT , 22, theLAMA5 variants and the first published case with one missense and one nonsense variant. Because identifying a precise, monogenic diagnosis helps guide therapy and predict recurrence risk after renal transplantation, LAMA5 should be recognized as a candidate gene and actively tested in cases of nephrotic patients with no other genetic diagnosis.In summary, we report a Korean case of infantile SRNS associated with