In an articleThe correct Figures The correct GEO dataset ID number are shown below:In Section 2.1, row 3. “The GSE135918 dataset was captured from the GEO Database”.GSE135918 should be GSE126093.In Section 2.2, row 2. “Screening for miRNAs linked with colon cancer phenotype, WGCNA was performed on TCGA‐miRNA and GSE195918 datasets”.GSE195918 should be GSE126093.In Section 2.4, row 2. “The “limma” package was employed to select differentially expressed miRNAs,17 with |LogFC>1| and FDR <0.05”.|LogFC>1| should be |LogFC| > 1.In Section 3.1, row 2. “Weighted gene co‐expression network analysis was performed on the TCGA‐miRNA dataset and GSE135918”.GSE135918 should be GSE126093.In Section 3.1, row 8. “TCGA‐miRNA data and GSE135918 were also evaluated by differential analysis”.GSE135918 should be GSE126093.In Section 3.1, row 11. “30 differentially expressed miRNAs were screened from GSE135918”: GSE135918 should be GSE126093.The authors apologize for the errors.


Two‐step changes in paced QRS morphology during the left bundle branch area pacing threshold test. It suggests that capturing occurs at multiple sites of the left bundle branch‐Purkinje system. Left bundle branch (LBB) area pacing (LBBAP) is widely performed as conduction system pacing because it has a lower threshold and is technically easier than His bundle pacing. Although LBBAP can capture the LBB–Purkinje conduction system and create a narrow‐paced QRS duration (pQRSd), several different capture types and paced QRS morphologies are observed. Recently, left bundle fascicular (LBF) capture has been reported to be the predominant type of LBBAP in clinical practice.A 69‐year‐old woman presented with bradycardia and dyspnea on exertion. An electrocardiogram showed a complete or two‐to‐one atrioventricular block. Her heart contractions were normal without any features of cardiomyopathy. After informed consent was obtained, we planned to implant an LBBAP.First, we performed His bundle potential (HBP) mapping using the C315 His sheath and select secure™ 3830 lead  as an angiographic marker. Subsequently, the 3830 lead was used as the LBBAP lead and screwed on the ventricular septum with a well‐fixed sheath contact approximately 2.4 cm apical from the HBP‐recorded site. We performed deep screwing with terminal monitoring of pQRSd and stimulus‐to‐left ventricular activation time (Stim‐LVAT) in lead V6. The pQRSd and Stim‐LVAT were gradually shortened. Finally, the pQRSd and Stim‐LVAT at the deep site of the septum were 122 and 62 ms, respectively. We assumed that the lead reached the LBB area Figure .Two‐step abrupt QRS morphological changes were observed during the threshold test using unipolar pacing from the lead tip electrode Figure . The firTwo‐step QRS morphology changes based on pacing output can be observed during the threshold test of LBBAP. A possible mechanism of this unusual observation is that unipolar pacing from the LBBAP lead tip was captured at multiple sites of the LBB–Purkinje system and/or left ventricular septal (LVS) myocardium. A previous report showed that the paced QRS axis changes depended on the pacing output.In clinical practice, some cases in which the LBBAP is considered successful could have distal LBB–Purkinje system pacing. A previous study showed that the success rate of strict LBBP was approximately 30% for LBBAP.In conclusion, this is the first report of two‐step paced QRS morphology changes during the LBBAP threshold test, which suggests that capturing occurs at multiple sites of the LBB–Purkinje system. This may be due to the complex networks of LBPF regions. Some cases of LBBAP might include capturing a distal site of the LBB–Purkinje system.The authors have nothing to disclose.Approval was obtained from the local ethics committee.The authors obtained consent from the patients.N/A.N/A.


Thermoclostridium stercorarium subspecies strain RKWS1  and the hemicellulolytic Thermoanaerobacter species strain RKWS2 . Discovery of indigenous thermophiles in plant biomass suggests that high-temperature microorganisms are more ubiquitous than previously thought.Reported here are complete genome sequences for two anaerobic, thermophilic bacteria isolated from wheat straw, i.e., the (hemi)cellulolytic  Thermoclostridium stercorarium subsp. strain RKWS1 and Thermoanaerobacter sp. strain RKWS2 were isolated from the wheat straw culture; each consumed a variety of soluble and insoluble saccharides found in plant biomass -cellobiose medium, starting with an indigenous mixed culture from wheat straw in LC medium . Colony dnaA gene predicted by Prodigal v2.6.3 (Reads were initially trimmed as part of the Guppy base-calling workflow. High-quality reads were subsampled using NanoFilt v2.8.0, with 500-bp length and quality score Q10 read quality cutoff values . Flye v2l v2.6.3 . Read mal v2.6.3 . Furtherl v2.6.3  and Medal v2.6.3 , Pilon (l v2.6.3 , and Chel v2.6.3  (Table 1l v2.6.3 . Averagel v2.6.3 , 17, avel v2.6.3 , and thel v2.6.3  using 16CP014673.1, CP014672.1, CP003992.2, NR_126170.1, CP016502.1, CP021850.1, CP001348.1, CP002403.1, ON077455.1, CP000924.1, CP009170.1, CP000923.1, CP002032.1, AE008691.1, LT838272.1, CP017237.1, CP001393.1, and CP020921.1.BioProject, BioSample, and genome accession numbers for RKWS1 and RKWS2 assemblies are given in


Toxoplasma gondii is a ubiquitous zoonotic parasite that can infect warm-blooded vertebrates, including humans. Felids, the definitive hosts, drive T. gondii infections by shedding the environmentally resistant stage of the parasite (oocysts) in their feces. Few studies characterize the role of climate and anthropogenic factors in oocyst shedding among free-ranging felids, which are responsible for the majority of environmental contamination. We determined how climate and anthropogenic factors influence oocyst shedding in free-ranging domestic cats and wild felids using generalized linear mixed models. T. gondii oocyst shedding data from 47 studies were systematically reviewed and compiled for domestic cats and six wild felid species, encompassing 256 positives out of 9,635 total fecal samples. Shedding prevalence in domestic cats and wild felids was positively associated with human population density at the sampling location. Larger mean diurnal temperature range was associated with more shedding among domestic cats and warmer temperature in the driest quarter was associated with lower oocyst shedding in wild felids. Increasing human population density and temperature fluctuation can exacerbate environmental contamination with the protozoan parasite T. gondii. Management of free-ranging domestic cats could lower the burden of environmental oocysts due to their large population sizes and affinity with human settlements. Toxoplasma gondii is a generalist zoonotic pathogen that can infect any warm-blooded vertebrate, including humans . Sa. SaT. goT. gondii in feces from wild felids were conducted in North America (n = 7), with two studies conducted in Asia and one study in South America (Domestic cat studies were primarily conducted in North America (n = 11), Asia (n = 11), and Europe (n = 7), with fewer studies conducted in Africa (n = 4), South America (n = 3), and Oceania (n = 2). The majority of shedding studies with confirmed  America . The majT. gondii oocyst shedding in domestic cats  and four variables for wild felids . A total of twelve linear models were considered to explain confirmed T. gondii oocyst shedding prevalence in a global dataset of free-ranging domestic cats and six wild felid species , not the prevalence of T. gondii infection, which is more easily captured due to presence of antibodies due to lifelong infection. Prior studies of T. gondii in wild mammals found a positive association between mean annual temperature and seroprevalence, however, infection in intermediate hosts and shedding by definitive hosts are related but separate phenomena that may be impacted differentially by climate, especially at different time scales [T. gondii infection prevalence in humans and livestock [T. gondii oocyst shedding should be assessed in longitudinal studies at the local scale in order to gain a clearer picture of this relationship.Oocyst load in the environment is not sufficient by itself to estimate infection risk because host and environmental factors can interact to influence location-specific risk. Our measured outcome was the prevalence of e scales . Climatee scales . While oivestock , 42, andivestock . MechaniT. gondii). In two of the original studies [T. gondii oocyst shedding are geographic and species-based sampling bias, and inconsistency of detection methods used to determine oocyst shedding. Due to the small sample size and large confidence interval for effect size in wild felids (62–1856), we caution that our wild felid results may be incredibly sensitive to sampling bias and may need further investigation to determine the true association with human population density. Despite the diversity of T. gondii genotypes present in South America, oocyst shedding in this region remains critically understudied. Studies are also sparse in Africa and Asia, even though both regions have multiple wild felid species and high projected human population growth [T. gondii oocyst contamination as it is relevant to human and wildlife health [Toxocara infections in the boroughs of New York City [T. gondii seropositivity among lower SES groups in the US [T. gondii and congenital toxoplasmosis in Brazil among lower SES groups [T. gondii transmission in regions that are relatively low risk for T. gondii exposure and an ambiguous picture where high-quality data may actually be more valuable to protect human public health and vulnerable animal populations.One limitation of our analysis is that many primary literature sources did not perform serological testing to discriminate between animals with previous evidence of exposure  and/or more holistic landscape-based interventions  that can reduce transport of oocysts [T. gondii is highly endemic but understudied such as South America and Africa, the utilization of molecular methods paired with microscopy to verify parasite identity and characterize oocyst genotypes, and more sampling of wild felid species. Most regions are under sampled for wild felids, despite the fact that a higher proportion of these animals are thought to be shedding at any given point in time. We do not attempt to prove a causal relationship between human population density and oocyst shedding prevalence, however, the association between human density and oocyst shedding suggests that focusing on free-ranging feral cats as the source of environmental oocyst contamination is an important disease management strategy. Feral cat management can serve to reduce the risk of toxoplasmosis in humans, livestock, and wildlife, and will also have added benefits for reducing predation of native wildlife species [T. gondii oocyst shedding prevalence in free-ranging domestic cat and wild felid populations. Human-caused climate change and urbanization may create more ideal environmental conditions for a generalist pathogen and its disease ecology, which can be relevant for other infectious diseases that are influenced by human population growth and increased urbanization.Identification of predictive and/or protective factors for oocyst shedding in free-ranging wild and domestic felids is highly relevant for stakeholders and policymakers to make informed decisions for human and animal health. Our results reinforce the role that free-ranging domestic cats have in contributing to biological contamination of  oocysts , 53. Ove species . We emphS1 File(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


Both secondary tricuspid regurgitation (STR) and heart failure with preserved ejection fraction (HFpEF) are relevant public health problems in the elderly population, presenting with potential overlaps and sharing similar risk factors. However, the impact of severe STR on hemodynamics and cardiorespiratory adaptation to exercise in HFpEF remains to be clarified.To explore the impact of STR on exercise hemodynamics and cardiorespiratory adaptation in HFpEF.We analyzed invasive hemodynamics and gas-exchange data obtained at rest and during exercise from HFpEF patients with severe STR (HFpEF-STR), compared with 1:1 age-, sex-, and body mass index (BMI)- matched HFpEF patients with mild or no STR (HFpEF-controls).2) and 12 HFpEF-controls patients were analyzed. HFpEF-STR had higher (p < 0.01) right atrial pressure than HFpEF-controls both at rest (10 ± 1 vs. 5 ± 1 mmHg) and during exercise (23 ± 2 vs. 14 ± 2 mmHg). Despite higher pulmonary artery wedge pressure (PAWP) at rest in HFpEF-STR than in HFpEF-controls , PAWP at peak exercise was no more different (28 ± 2 vs. 29 ± 2). Left ventricular transmural pressure and cardiac output (CO) increased less in HFpEF-STR than in HFpEF-controls . This latter was due to lower stroke volume (SV) values both at rest  and at peak exercise . Despite these differences, the two groups of patients laid on the same oxygen consumption isophlets because of the increased peripheral oxygen extraction in HFpEF-STR (p < 0.01). We found an inverse relationship between pulmonary vascular resistance and SV, both at rest and at peak exercise .Twelve HFpEF with atrial-STR (mean age 72 years, 92% females, BMI 28 Kg/mSevere STR complicating HFpEF impairs SV and CO reserve, leading to pulmonary vascular de-recruitment and relative left heart underfilling, undermining the typical HFpEF pathophysiology. The development of transcatheter interventions might enlarge the number of candidates to tricuspid regurgitation (TR) repair, including elderly patients with several comorbities and secondary TR (STR) –4. AmongThis study was approved by the Ethics Committee of the Istituto Auxologico Italiano . All patients signed a written informed consent to allow the use of their clinical data for research purposes.We analyzed the cohort of patients who underwent an elective, clinically indicated cardiac catheterization at rest and during exercise at Istituto Auxologico Italiano between January 2018 and January 2022.2).Cases were the patients with HFpEF and severe STR (HFpEF-STR). HFpEF was defined based on the presence of signs and/or symptoms of heart failure, a left ventricular (LV) ejection fraction (EF) >50%, associated with invasive hemodynamic demonstration of HFpEF. Invasive diagnosis of HFpEF was established in patients with an end-expiratory pulmonary artery wedge pressure (PAWP) at peak exercise equal or higher than 25 mmHg, and/or PAWP/ cardiac output (CO) slope higher than 2 mmHg/L/min) –18. FromWe excluded patients with LV EF < 50%, secondary forms of HFpEF , more than mild left-sided valvular heart disease, congenital heart disease, pulmonary vascular diseases , pulmonary hypertension due to lung disease and/or hypoxia, severe comorbidities. Additionally, we excluded patients with congenital or acquired primary TR, cardiac implanted electronic device-related TR, and previous tricuspid surgery. Among the cases, we distinguished between atrial- and ventricular-STR, based on the detection of invasive pulmonary artery systolic pressure lower or higher than 50 mmHg .Echocardiography was performed according to current recommendations of the European Association of Cardiovascular Imaging and the American Society of Echocardiography by experienced cardiologists on the same day of the right heart catheterization , using a2) equation as follows: CO = VO2/C(a-v)O2, where C(a-v)O2 is the oxygen arteriovenous difference. Furthermore, to evaluate the relative contribution of the elements of the Fick equation to exercise capacity, we plotted CO as a function of C(a-v)O2 on which we represented VO2-isophlets  to directly measure gas-exchange data and ventilation . A 7-F fLV trans-mural pressure (LVTMP), as a measure of LV preload, independent of right heart filling and pericardial restraint, was calculated as PAWP–RAP . We plot2 was measured as the highest 30-s value obtained at the end of the effort. The slope of the relationship between minute ventilation and carbon dioxide production (VE/VCO2 slope) was calculated over the linear component of VE vs. VCO2  was applied to compare the continuous variables between HFpEF-STR and HFpEF-controls, while Chi-square test (or Fisher test) was used to compare the categorical variables. For each hemodynamic variable measured during exercise, an ANOVA model for repeated measures was fitted, considering an unstructured variance-covariance matrix to take into account the correlation among measurements on the same subjects. The included covariates in each model were group (HFpEF-STR or HFpEF-controls), time  and their interaction. The statistical significance of interaction term suggested a different trend of the hemodynamic variables between groups. Moreover, we tested the least square means (LS-means) differences among groups at each time by means of unpaired t-test applying the False Discovery Rate (FDR) approach to control the inflation of the type I error.Continuous variables are reported as mean and standard deviation (SD) or median and interquartile range if the data did not follow a normal distribution. The categorical variables are shown as absolute frequencies and proportions. For data at rest, unpaired The relationship between continuous hemodynamic variables at specific time-point was investigated by means quadratic B-spline with 1 knot. The goodness of fit of the model was estimated by means of R2. This value, ranging from 0 to 1, represents the proportion of total variance of an independent variable explained by a dependent variable.P-values were two-sided.All analyses were performed using SAS version 9.4 software . Statistical significance was set at the 0.05 level. All Patients' selection flowchart is depicted in p < 0.01). The rhythm at the time of right heart catheterization was atrial fibrillation in 83% of HFpEF-STR and 8% of HFpEF-controls. Among the other cardiovascular comorbidities, arterial hypertension was the most common affecting 75% of HFpEF-controls and 83% of HFpEF-STR patients (p = 1.000).The clinical characteristics of our study groups are summarized in Representation of HF signs and symptoms did not significantly differ between HFpEF-STR and HFpEF-controls . Brain np = 0.067). As expected, because of the incidence of atrial fibrillation, patients with HFpEF-STR presented larger LA and RA volumes that were, respectively, 43 and 84% larger than in HFpEF-controls . No patient with HFpEF-STR presented with leaflet tenting.LV geometry and function were similar between the two groups, while RV dilation was non-significantly more frequent in HFpEF-STR than in HFpEF-controls  and PAWP  than HFpEF-controls, consistent with mild post-capillary pulmonary hypertension (PH). PH, defined by a mean pulmonary artery pressure >20 mmHg, was present in 83% of HFpEF-STR and in 25% of HFpEF-controls. SV was lower in HFpEF-STR than in HFpEF-controls , which was compensated by higher heart rate  to maintain cardiac index, which resulted on average at the lower limit of normal in HFpEF-STR . PVR was slightly increased in HFpEF-STR patients but not significantly higher to that of HFpEF-controls . RAP was twice as high in HFpEF-STR than in HFpEF-controls , with tall V waves  and a high RAP/PAWP ratio .At rest, patients with HFpEF-STR had higher mean pulmonary artery pressure . RAP V waves displayed an opposite behavior, increasing more in HFpEF-STR patients than in HFpEF-controls , with persistently higher RAP and RAP/PAWP ratio in HFpEF-STR patients than in HFpEF-controls . Accordingly, LVTMP increased only in HFpEF-controls during exercise , coherent with LV underfilling in HFpEF-STR patients.During exercise , PAWP in2, was not different at peak exercise between HFpEF-STR and HFpEF-controls, who reached the 71 and 77% of predicted values, respectively (p = 0.446), as shown in 2 . In particular, HFpEF-STR and HFpEF-controls roughly lay roughly on the same VO2 isophlets at each step of exercise, with HFpEF-STR rightward and downward shifted . Exercise hyperventilation (VE/VCO2 slope) did not differ between HFpEF-STR and HFpEF-controls.Exercise capacity, as evaluated through peak VOown in 2  behaved  shifted . Accordip-value = 0.018). In particular, at peak exercise, it resulted 40 mL lower in HFpEF-STR (p = 0.03). The rate of increase of SV in the two groups however did not significantly differ  suggested high heterogenity in the relationship.The SV was lower in HFpEF-STR than in HFpEF-controls (group y differ . Similary differ . However2 extraction) to maintain exercise performance; (iii) pulmonary vascular derecruitment and LV underfilling, leading to lower than expected increase in left heart filling pressure during exercise  marked RA dilation and RA hypertension, both at rest and during exercise; (ii) reduced SV and CO reserve, with patients relying on heart rate and on peripheral compensation . The etiology of severe STR was atrial-functional in the great majority of these patients, on whom we decided to focus our analyses. Of note, 83% of patients with atrial-STR had a hemodynamic diagnosis of PH at rest according to the new definition %. The et, 8–15 reCoherent with the non-negligible prevalence of atrial-STR in our hemodynamically-proven HFpEF population, both atrial-STR and HFpEF share common denominators, such as aging and atrial fibrillation. First, the prevalence of STR increases with age . Similar2 did not relevantly differ between HFpEF-STR patients and HFpEF controls. Nonetheless, HFpEF-STR systematically presented with lower SV, and had to rely more on heart rate and peripheral O2 extraction [despite this latter being slightly lower than normal . All patients signed a written informed consent to allow the use of their clinical data for research purposes.CB contributed for conceptualization, data curation, investigation, methodology, visualization, and writing the original draft. SC contributed for conceptualization, data curation, investigation, methodology, supervision, and visualization and writing the original draft. GC contributed to data curation, investigation, methodology, and writing—review and editing. DS contributed for data analysis and writing—review and editing. AZ contributed for data analysis and writing—review and editing. CD contributed for supervision and writing—review and editing. MG, FH, MT, NR, and FP contributed to writing—review and editing. GPe contributed to supervision and writing—review and editing. J-LV contributed to conceptualization, supervision, and writing—review and editing. GPa contributed to supervision and writing—review and editing. LB and DM contributed to conceptualization, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.


Immune milieus play an important role in various types of cancer. The present study focuses on the effect of Th1 cytokines on pediatric acute lymphoblastic leukemia (ALL). The reaction of ALL cell lines and patient-derived xenografts (PDX) to the most important Th1 cytokines TNF-α  and IFN-γ (interferon gamma) is analyzed and correlated with the respective cytokine receptors and the intracellular signaling molecules. ALL cell lines and ALL PDX display a great heterogeneity in cell death after incubation with TNF-α and IFN-γ. Several samples show a dose-dependent and additive induction of cell death by both cytokines; others do not react at all or even display an increased viability. Apoptosis is the main type of cell death induced by Th1 cytokines in ALL cells. Over all leukemia cells analyzed, IFN-γ receptor (IFNGR) shows a higher expression than both TNF-receptors, resulting in higher phosphorylation of STAT1  compared to phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) in the TNF pathway. The activation of STAT1 correlates with the amount of cell death after stimulation with Th1 cytokines. TNF-α and IFN-γ lead to heterogeneous reactions in ALL cell lines and ALL PDX but are able to induce cell death by apoptosis in the majority of ALL blasts. The correlation of a high expression of IFNGR and following activation of STAT1 with cell death indicates an important role for IFN-γ signaling in this setting. A high initial peripheral blast count correlates negatively with IFN-γ expression. These findings imply suppression of host Th1 immunity caused by leukemia [There is evidence that immune mediators are associated with outcome in pediatric leukemia –3. IL-10TNF-α is considered to be a major pro-inflammatory mediator, with an optional capacity to induce apoptosis . The parIn non-Hodgkin’s lymphoma, acute and chronic myeloblastic leukemia and chronic lymphocytic leukemia high levels of TNF-α and its receptors are associated with poor outcome and therapy resistance , 7.In ALL, TNF-α has been reported to exert either proliferative or cytotoxic effects on primary blast cells ex vivo . TNFR-1 Comparing the plasma levels of TNF-α of children suffering from B-ALL at diagnosis with healthy controls, higher levels were detected in ALL patients. Furthermore, a positive correlation between TNF-α levels with blast cell and white blood cell count was observed. The TNF-α levels in ALL patients were negatively correlated with S-phase leukemic cells and apoptotic cells but not with treatment response or survival .Analyzing the role of the TNFRs in leukemia, a retrospective analysis of serum concentration of soluble TNF receptors (sTNFR-1 and sTNFR-2) in adult patients showed significantly elevated levels in AML (acute myeloblastic leukemia) and ALL. Moreover, in AML patients, a negative correlation between the sTNFR-1 levels and disease-free survival and overall survival was observed. In ALL, no such correlation was detected .Alongside TNF-α, IFN-γ is the second major proinflammatory Th1 cytokine . GenotypSimilar to TNF-α, both pro-tumorigenic and pro-apoptotic properties of IFN-γ have been described .Senescence, a permanent arrest in G1/G0, is an important endogenous defense mechanism against an early stage of neoplasms in various tissues such as oncogene-induced senescence in lymphoma development . The indNovel immune-therapeutics used against ALL have become an important pillar of therapy. Bispecific antibodies or CAR-T-cells (chimeric antigen receptor T-cells) can lead to release of large amounts of pro-inflammatory cytokines, with levels of TNF-α rising tenfold and levels of IFN-γ rising up to 1000-fold –21. The In the present study, we describe the reaction of ALL cell lines and ALL PDX to the Th1 cytokines TNF-α and IFN-γ and analyze the expression of the respective cytokine receptors and the main intracellular signaling pathway.MHH-CALL-4, Nalm-6, Nalm-16, Reh, RS4;11 and KOPN-8 were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) and cultured according to instructions.Prkdcscid IL2rgtmWjl/Sz (NSG) mice were purchased at The Jackson Laboratory and maintained under specified pathogen-free conditions in the research animal facility of the University of Tuebingen, Germany. All experimental animal procedures were conducted according to German federal and state regulations . Patient-specific leukemia was induced in NSG mice as described before [NOD.Cg-d before . The stuPatient characteristics of the samples used to generate the patient-derived xenografts (PDX) are displayed in Table Flow cytometry was performed on an LSRII by BD Biosciences. Viability of cells was assessed in the forward scatter versus sideward scatter (FSC/SSC) plot. The percentage of specific cell death was determined using the following formula: specific cell death = (cytokine induced cell death − spontaneous cell death)/(100 − spontaneous cell death) × 100%.We used the FITC Annexin V Apoptosis Detection Kit with 7-AAD by Biolegend (Cat. 640922) according to manufacturer’s instructions. Staurosporine was used as a positive control.The Apo-ONE® Homogeneous Caspase-3/7 Assay by Promega (#G7790) was used according to the manufacturer’s instructions. Staurosporine was used as a positive control.The antibodies detecting TNF-α receptors were purchased from R&D Systems: TNFR-1 (CD120a)-APC (FAB225A), TNFR-2 (CD120b)-APC (FAB226A) and IFNGR (CD119)-PE were purchased from BioLegend (Cat. Nr. 308704).The antigen binding capacity of the cytokine receptors was assessed by flow cytometry using Quantum simply cellular by Bangs Laboratories α-mouse IgG (Cat. 815).For cell cycle analysis, we used the BrdU Flow Kit Cat. No. 559619 by BD Biosciences according to the manufacturer’s instructions. Incubation time with BrdU was chosen according to the doubling time of the cell line .The antibodies against the phosphorylated downstream signaling molecules were purchased from BD Biosciences: pNF-kBp65-PE-Cy7 (56035) and pSTAT1-BV421 (562985) as were the buffer solutions. All were used according to the manufacturer’s instructions.p value of < 0.05 were considered statistically significant.Data were analyzed using Microsoft® Excel, Version 16.12 and IBM® SPSS Statistics Version 22 for Windows and GraphPad Prism9. Differences between subgroups were described by two-way ANOVA; correlation analysis was performed using the Spearman's nonparametric measure of rank correlation. Results with a In MHH-CALL-4 the incubation with TNF-α and IFN-γ leads to a decrease in cells in S-phase and a simultaneous increase in cells in subG1. Most other ALL cell lines show little or no changes in cell cycle after incubation with Th1 cytokines . The positive control, a melanoma cell line, goes into senescence with a strong reduction of S-phase and high increase in resting cells, as shown previously by Braumüller and colleagues  The induction of cell death in six B-cell precursor ALL cell lines  incubated with different concentrations of TNF-α  and IFN-γ  was examined alone and in combination at multiple points in time . Four cell lines show a dose-dependent induction of cell death by both cytokines, the highest amount of cell death induction detected in MHH-CALL-4. The two cell lines Nalm-6 and Nalm-16, however, did not undergo cell death under incubation with Th1 cytokines. Nalm-16 even showed a slight tendency toward increased viability under certain concentrations , TNF-α receptor type 2 (TNFR-2), IFN-γ receptor (INFGR) and membrane bound TNF-α. The expression of TNF receptors and TNF-α is expressed at relatively low levels, especially in the PDX samples. The ALL cell lines display a higher level of all three receptors analyzed. IFNGR is generally expressed at a much higher level with large intra-individual differences and with a less clear separation between cell lines and PDX Fig. a. The exAfter assessing the main cytokine receptors for TNF-α and IFN-γ we analyzed the intracellular signal transduction via phosphorylation of the main downstream effectors of both cytokines. For TNF-α, we focused on the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) and for IFN-γ on phosphorylation of STAT1 . The activation of the signaling transduction pathways by TNF-α and IFN-γ was quantified by comparing the phosphorylation state of the transcription factor in medium control with the phosphorylation state after incubation with the respective cytokine.The phosphorylation level of NF-κB after incubation with TNF-α was extremely diverse and varied largely between cell lines and PDX, some samples displaying a strong phosphorylation, while others did not show any increase in phosphorylation in the presence of TNF-α compared with medium control Fig. a. The exThe reactions of ALL cell lines and PDX to incubation with the pro-inflammatory cytokines TNF-α and IFN-γ were highly variable, as was the expression of the cytokine receptors. In our analysis, the amount of TNF receptors positively correlate with each other. ALL cells with high amounts of TNFR-1 also have higher amounts of TNFR-2 and of TNF-α.TNFR-1 is a typical death receptor triggering apoptotic and necroptotic signaling, whereas TNFR-2 lacks a death domain. TNFR-2 induces NF-κB signaling via several pathways, can attenuate TNFR-1-induced classical NF-κB signaling and sensitize cells for TNFR-1-induced cell death. But TNFR-2 can also activate the alternative and classical NF-κB pathway which upregulates anti-apoptotic proteins and proliferation promoting factors . The surWe see a significant positive correlation between the expression of TNFR-2 and the activation of NF-κB as well as a negative correlation between the antigen binding capacity of TNFR-2 and the level of annexin V positive cells after incubation with Th1 cytokines.Despite a clear positive correlation between the expression of TNFR-1 and TNFR-2 with membrane bound TNF-α, we see no association between the antigen binding capacity of either TNF receptor or the membrane bound ligand TNF-α with the induction of cell death after incubation with Th1 cytokines.The IFN-γ receptor showed a high antigen binding capacity on all ALL samples, a finding which has previously been shown in AML . SimilarThe level of cell death after incubation with Th1 cytokines in pediatric ALL strongly correlates with the expression of IFN-γ receptor and subsequently also the phosphorylation of STAT1 in pediatric ALL (cell lines as well as PDX). Our results indicate that in pediatric ALL the IFN-γ signaling pathway plays a major role in the cell death induction after Th1 cytokine exposure.TNF-α and IFN-γ have been shown to synergistically induce cell death in different cell types such as murine hepatoma cells , a myeloBut the induction of senescence by TNF-α and IFN-γ in several solid tumors may be part of the explanation . Th1 cytMany ALL samples showed an additive or even synergistic effect of both Th1 cytokines on specific cell death, but others showed no increase in cell death at all. The expression of IFN-γ receptor and the consecutive activation of STAT1 were the main reason identified in this work but is surely not the only answer to the differential reaction of ALL cells to exposure to TNF-α and IFN-γ.As mentioned in the introduction, several studies have investigated germline polymorphisms in cytokine genes such as the association of TNF and IL-10 genotypes with adverse outcomes in solid and lymphoid malignancies  or gene Individual oncological therapies are available including TNF-α and IFN-γ. The combination of TNF-α, IFN-γ and melphalan in an isolated limb perfusion setting shows impressive remission rates in irresectable soft tissue sarcomas  and melaApart from the local therapeutic possibilities in solid tumors mentioned above, the systemic application of exogenous TNF-α and IFN-γ does therefore not constitute a realistic anti-tumor therapy in leukemia. Many novel immunotherapeutic agents, however, lead to an increase in endogenous Th1 cytokines.The T cell engaging CD19/CD3-bispecific antibody blinatumomab has become a standard treatment in relapsed ALL. After the first infusion a transient release of cytokines including IFN-γ and TNF-α can be observed . In an iAnother potent immunotherapeutic option for relapsed or refractory ALL patients is CD19-targeted CAR (chimeric antigen receptor)-T-cell therapy. After infusion, a massive expansion of CAR T-cells leads to secretion of vast amounts of IFN-γ and TNF-α and further stimulates bystander immune cells and endothelial cells . Cytokin−/− tumors [For immune checkpoint inhibition, it has been shown that gene expression profiles from tumor tissue samples of different solid tumors who responded to anti-PD-1 therapy had higher expression of IFN-γ-related genes . In mous− tumors .In our analysis, the expression of IFNGR and phosphorylation of STAT1 was clearly correlated with specific cell death of ALL samples after incubation with Th1 cytokines. The expression level of IFNGR might be a prediction marker for the clinical response to immunotherapeutic approaches in ALL, similar to the results described above for solid tumors.The limitation of our study is the mere in vitro character and the moderate number of individual leukemia samples analyzed. Statistical significance also does not necessarily reflect causal relationships. This work nevertheless demonstrates that the Th1 cytokines TNF-α, and IFN-γ can lead to apoptosis in BCP-ALL cells, especially IFN-γ and its receptor seem paramount for the induction of cell death in this setting. The strong heterogeneity of reaction between different ALL cells to the Th1 cytokines warrants further investigation and might lead to future tailored immunotherapeutic concepts.


LINC00669, in lung adenocarcinoma (LUAD) by analyzing the TCGA and GEO databases. Pan‐cancer analysis indicated significantly upregulated LINC00669 across 33 cancer types. GSEA revealed a tight association of LINC00669 with the cell cycle. We next attempted to improve the prognostic accuracy of this lncRNA by establishing a risk signature in reliance on cell cycle genes associated with LINC00669. The resulting risk score combined with LINC00669 and stage showed an AUC of 0.746. The risk score significantly stratified LUAD patients into low‐ and high‐risk subgroups, independently predicting prognosis. Its performance was verified by nomogram (C‐index = 0.736) and decision curve analysis. Gene set variation analysis disclosed the two groups' molecular characteristics. We also evaluated the tumor immune microenvironment by dissecting 28 infiltrated immune cells, 47 immune checkpoint gene expressions, and immunophenoscore within the two subgroups. Furthermore, the risk signature could predict sensitivity to immune checkpoint inhibitors and other anticancer therapies. Eventually, in vitro and in vivo experiments were conducted to validate LINC00669's function using qRT‐PCR, CCK8, flow cytometry, western blot, and immunofluorescence staining. The gain‐ and loss‐of‐function study substantiated LINC00669's oncogenic effects, which stimulated non‐small cell lung cancer cell proliferation but reduced apoptosis via activating the Wnt/β‐catenin pathway. Its oncogenic potentials were validated in the xenograft mouse model. Overall, we identified a novel oncogenic large intergenic non‐coding RNA (lincRNA), LINC00669. The resulting signature may facilitate predicting prognosis and therapy responses in LUAD.Lung cancer poses severe threats to human health. It is indispensable to discover more druggable molecular targets. We identified a novel dysregulated long non‐coding RNA (lncRNA),  We identified and validated a novel oncogenic lincRNA of LINC00669. The resulting signature based on LINC00669‐assoicated cell cycle genes may facilitate predicting prognosis and therapy responses in LUAD. The incidence and associated death exponentially increase for most types of cancer among populations ≥65 years old, which is partially attributed to growing somatic mutations with aging.LncRNA MARCKSL1‐2 was remarkably downregulated in docetaxel (DTX)‐resistant lung adenocarcinoma cells, while overexpression of this lncRNA in tumor cells reversed DTX resistance.SCAMP1‐AS1, LINC00892, LINC01481, and AC004812.2, AC099522.2) could be potentially used as diagnostic biological markers for screening N2 metastasis in pT1 lung adenocarcinoma (LUAD).LCIIAR foreseen a poor prognosis in lung cancer.LINC00669, is overexpressed and associated with unfavorable prognosis in LUAD. Only one research with experimental data on LINC00669 has been published, indicating that LINC00669 accelerated nasopharyngeal cancer cell proliferation and invasion through sequestering the JAK/STAT suppressor SOCS1.Long non‐coding RNAs (lncRNAs) are an essential class of ubiquitously expressed genes regulating various biological functions,c‐MYC and CYCLIN D1).The aberrant activation of the wingless related‐integration site (Wnt)/β‐catenin signaling pathway contributes to tumor progression and drug resistance in NSCLC.LINC00669 is strongly associated cell cycle. The signature out of LINC00669‐associated cell cycle genes robustly predicted patient prognosis, intratumoral immune cell infiltration, and immunotherapy response in LUAD. Furthermore, experimental evidence indicated that LINC00669 promoted lung cancer progression through the Wnt signaling pathway.Here, our bioinformatics analyses showed that 22.12.1.1https://nci.nih.gov/tcga/), along with clinical information. We first pinpointed 282 dysregulated lncRNAs between 517 tumors and 59 normal tissues , which was validated with a GSE19804 LUAD cohort. This dataset contained 60 pairs of lung tumors and adjacent normal tissues, among which RNA‐seq data from 56 LUAD tumors and matched adjacent normal lung tissue specimens were used for this study.LINC00669, associated with survival, was chosen for the study. Moreover, the TCGA database enabled us to evaluate the expression levels of LINC00669 across 33 types of cancers.The RNA‐Seq dataset of the lung adenocarcinoma cohort was downloaded from The Cancer Genome Atlas (TCGA)  database (https://sites.broadinstitute.org/ccle) to acquire the RNA‐seq data of 119 NSCLC cell lines. The median expression level of LINC00669 was used to divide NSCLC cell lines into LINC00669low and LINC00669high subsets. All analyses were performed with the TCGA‐LUAD cohort thereafter without notification.Gene set enrichment analysis (GSEA) was conducted to explore the function of 2.1.3LINC00669‐correlated genes were obtained from the TCGA‐LUAD cohort . We also secured 14,162 differentially expressed genes in LUAD . Besides, 1056 cell cycle genes were retrieved from the CancerSEA database (http://biocc.hrbmu.edu.cn/CancerSEA/). A Venn diagram approach was used to select LINC00669‐related cell cycle genes that are differentially expressed in LUAD. The univariate Cox regression analysis further filters out genes without significant association with OS. A prognostic signature was constructed with the remaining significant genes using the multivariable Cox regression model. After generating the signature, the expression levels of genes of interest and risk coefficients were integrated to appoint each patient a risk score as published previously.J statistic (J = sensitivity + specificity −1) was determined for the time‐dependent ROC curve with the biggest AUC and used as a cutoff to define patients with different risks. A Kaplan–Meier survival analysis was carried out with the R package rms, followed by uni‐ and multivariable Cox regression analyses.With Pearson's correlation analysis, 15,868 2.1.4Next, a Cox‐based nomogram was generated, coupled with calibration curves. We also performed decision curve analysis (DCA) to quantify the net benefit for the nomogram. This analysis assisted the understanding of the clinical benefit of the risk model.2.1.5Gene set variation analysis (GSVA) was adopted to interrogate the differences between the two subgroups concerning molecular features. The relevant gene sets were down from HALLMARK and Kyoto Encyclopedia Genes and Genomes (KEGG) databases, and the R package of GSVA was chosen for the analysis.2.2Intratumoral infiltrating immune cells account for a crucial component of the tumor microenvironment (TME).https://tcia.at/) in 2017 by integratively analyzing the immune landscapes and antigenomes across 20 solid tumors.In addition, Charoentong and colleagues developed The Cancer Immunome Atlas  were used to achieve stable expression or knockdown of LINC00669 (NR_024391.1) in target cells.We purchased human bronchial epithelial (HBE) cells and NSCLC cell lines, H1975, H1299, H358, and A549, from ATCC. HBE cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with the addition of 10% fetal bovine serum (Sigma) at 37°C and 5% COLINC00669 cDNA sequence were constructed as described previously.LINC00669. The sequences of shRNAs targeting LINC00669 were shRNA‐1: 5′‐TTTAGATTAAGCACTATCC‐3′, shRNA‐2: 5′‐TTGATGTTGAAGAACTTTG‐3′, shRNA‐3: 5′‐TAATTTACTAGAGCTTACC‐3′. H1299 cells were infected with empty vectors or lentiviruses overexpressing LINC00669. In parallel, H358 cells were used for silencing LINC00669 by LV‐sh shRNAs. Cells infected by lentiviruses underwent selection with puromycin to establish stable cell lines.Lentiviral vectors (pLVX‐Puro) carrying human 2.3.2LINC00669 (Forward primer: 5′ GACATCAGCATCTCAGTTCTC 3″ and Reverse primer: 5′ T'GGTCAACCTCTTCTAATCC 3″). Data were analyzed with ABI Prism 7300 SDS software, using The 2−ΔΔCt method to assess relative gene expression. Expression analyses of LINC00669 were normalized to the housekeeping gene (GAPDH: Forward Primer: 5′ AATCCCATCACCATCTTC 3′ and Reverse primer: 5’ A'GCTGTTGTCATACTTC 3′).After discarding the culture medium, we harvested NSCLC cells, and total RNAs were isolated using the Trizol‐based protocol (Thermo Fisher Scientific). The RNA concentration of each sample was measured with a Nanodrop1000 spectrophotometer (NanoDrop). Complementary DNAs (cDNAs) were produced from 1 μg of RNA template using the Thermo Scientific™ RevertAid™ First Strand cDNA synthesis kit. The SYBRGreen mix (Thermo Fisher Scientific) was employed to accomplish quantitative PCR (qPCR) on an ABI Applied Biosystems 7300 platform. Amplification reaction yielded a PCR product of 271 bps for 2.3.3LINC00669 affects NSCLC cell proliferation. Cell suspensions were added to a 96‐well tissue culture plate (3 × 103 cells per well) and then grew in a 5% CO2 humidified incubator at 37°C. The assay was performed in triplicate. After incubation for 24, 48, and 72 h, the medium was removed, and the CCK8 assay was conducted following the manufacturer's direction. Finally, cell proliferation was presented as optical density (OD) measured by an ELISA plate reader at the wavelength of 450 nm.We used a cell counting kit‐8 kit  to determine whether the gain or loss of function of 2.3.4LINC00669 stimulates NSCLC cell proliferation by upregulating the Wnt/β‐catenin pathway. H1299 cells stably overexpressing LINC00669 were treated with XAV939 (MedChemExpress LLC), an inhibitor of the Wnt/β‐catenin pathway.NSCLC cells were stained with the Annexin V‐FITC/PI Apoptosis Detection Kit (Beyontime Biotechnology). Cells were counted on a CytoFLEX Flow Cytometer (Becton Dickinson). In the case of investigating whether 2.3.5NSCLC cells were harvested and lysed according to routine laboratory protocol. After using the BCA protein assay kit (Thermo Fisher Scientific) to determine total protein concentrations, an SDS‐PAGE was run to separate the identical amounts of total proteins, which were next transferred onto a nitrocellulose membrane. The blots were preincubated with 5% nonfat dry milk and reacted with the indicated primary antibody. The primary antibodies used in this study were as follows: antibodies against PCNA, Ki67, Survivin, Cleaved Caspase 3 (Abcam), H3 (Proteintech), GAPDH (Proteintech), β‐catenin . Secondary HRP‐conjugated antibodies (SolelyBio), detecting mouse‐ or rabbit‐derived primary antibodies, were used when appropriate. We finally visualized blots with Enhanced Chemiluminescence (Thermo Fisher Scientific).2.3.6n = 6 for shCtrl vs. shLINC00669). H358 cells with stable knockdown of LINC00669 or control cells (2 × 106) suspended in PBS were subcutaneously implanted into the left flanks of mice. Tumor length and width were measured every 3 days using digital calipers (ProSciTech Pty Ltd.). Mice were sacrificed on the 33 days from the beginning of cell injection, and tumors were collected and weighed. Tumor specimens were sequentially fixed in formalin and embedded in paraffin, followed by histological analysis. Immunofluorescence (IF) staining was performed on tumor sections to determine the expression levels of Ki67 and proliferating cell nuclear antigen (PCNA). Primary antibodies for Ki67 (Abcam), PNCA (Proteintech), and Alexa Fluor 488‐labeled anti‐rabbit IgG (H + L) (Beyotime Biotechnology) were used for IF staining.The animal protocol was approved by the Institutional Review Board of Harbin Medical University Cancer Hospital. Female athymic nude mice at 5–6 weeks of age were used in this study . We kept mice under specific pathogen‐free conditions. Mice were randomly assigned into control or experimental groups  was employed to compare the differences among groups ≥3. The significant results were subjected to post hoc tests. Moreover, a two‐way ANOVA was applied as needed. All analyses were two‐sided. The nominal 0.05 significance level was adopted throughout the study.We conducted all statistical analyses using the SPSS version 22 . The Student's 33.1LINC00669, an intergenic lncRNA, was upregulated in both LUAD cohorts and associated with OS and progression‐free interval (PFI)  Figure . Pan‐canr Figure . These r3.2LINC00669's oncogenic potential, we interrogated its functional relevance in LUAD by performing GSEA with RNA‐seq data of the TCGA‐LUAD cohort and 119 NSCLC cell lines from the CCLE database. GSEA revealed that LINC00669‐correlated genes were significantly associated with the cell cycle pathway in both datasets , which were termed LINC00669‐associated cell cycle genes   Figure . The tim) Figure . Using t3.3LINC00669  may largely determine tumor prognosis and therapeutic efficacy.s Figure . Moreove4 Figure . IPS, th4 Figure .3.6A previous study indicated that IPS is an effective predictor of response to ICP blockade.We also investigated the risk score‘s ability to predict response to commonly used drugs for NSCLC. By analyzing RNA‐seq data with the pRRophetic algorithm, the resulting log (IC50) unveiled that the high‐risk group was significantly more sensitive to paclitaxel, doxorubicin, and etoposide than the low‐risk group Figure .3.7LINC00669 in lung cancer, its expression levels were enhanced significantly in the indicated NSCLC cell lines than in normal HBE  cells  selection, LINC00669 expression was lower in H358 cells with stable shLINC00669 transduction and higher in H1299 cells with stable expression of LINC00669 than in corresponding control cells  and survival (survivin) makers, as well as increased apoptosis maker (cleaved‐Caspase 3) in LINC00669‐depleted H358 cells. Again, the forced expression of LINC00669 showed reverse effects in H1299 cells ,LINC00669 overexpression inhibited and activated the Wnt pathway, respectively. XAV939 treatment could attenuate LINC00669‐induced nuclear translocation of β‐catenin and TCF‐1 upregulation .However, the overall survival rates for NSCLC remain unsatisfying, especially in advanced diseases. Therefore, research is called for continuously improving the understanding of lung cancer biology and mechanisms of tumor progression, and discovering more novel therapeutic targets. Besides coding RNA, genomes are now well‐known to be transcribed and generate thousands of lncRNA, which have essential roles in gene regulation. Their regulatory mechanisms are closely linked to subcellular localization, involving the modulation of chromatin conformation, the assembly and function of membrane‐less nuclear bodies, the half‐life time and translation of cytoplasmic mRNAs, and the activity of signaling pathways.LINC00669 as a novel oncogenic gene in NSCLC. The signature derived from LINC00669‐related cell cycle genes could effectively predict prognosis, TIME, and antitumor drug sensitivity. Moreover, in vitro and in vivo analyses demonstrated that LINC00669 boosted NSCLC growth by arousing the Wnt signaling pathway.This study identified LINC00669 and the cell cycle, suggesting it may regulate lung cancer cell growth. Its potential cell cycle regulation inspired us to construct a predicting signature by integrating LINC00669 and cell cycle genes. Traditionally, single genes are used to predict prognosis and drug sensitivity in cancer. However, cancer is a complex disease, which is hardly recapitulated by any single molecule. The identified single biomarkers are helpful but fail to fully represent lung cancer's complex mechanisms. Fortunately, striking advances in high‐throughput mRNA profiling techniques have made RNA‐seq affordable today. Over the past years, the machine learning approach coupled with RNA‐seq data has generated diverse gene signatures to improve the prediction of prognosis or therapeutic benefit.Our GSEA unravels a tight linkage between LINC00669 alone only has weak prognostic power with an AUC of 0.547, which might be attributed to the failure to consider its functional relevance to lung cancer development. Instead, virtual information concerning gene regulation on cancer cell survival could better portray candidate biomarkers' molecular implications in cancer progression and improve their predicting accuracy.LINC00669‐related cell cycle genes achieved an AUC of 0.700. While combined with risk score and stage, the AUC of LINC00669 added up to 0.746. A growing body of evidence has shown success in establishing robust prognostic signatures by combining single molecular biomarkers and their biological relevance.It was not surprising that When we tried to dissect the differential molecular mechanism underneath the low‐and high‐risk group, GSVA disclosed high activity of some pathways in the high‐risk group, which are known to facilitate tumor cell survival and growth, including hypoxia, DNA repair, glycolysis, EMT, MYC targets V1, MYC targets V2, MTORC1 signaling, DNA replication, and regulation of autophagy. These dysregulated malignancy‐related molecular characteristics partially explain the inferior clinical outcomes in the high‐risk group.LINC00669‐related cell cycle genes was associated with the different TIMEs. It is well recognized that T cells  play a central part in antitumor battle by releasing interferon‐γ (IFNγ), granulysin (GNLY), perforin (PRF1) and granzymes (GZMs), as wells as other mechanisms.+ cytotoxic T cells, CD3+ cells, or CD45RO+ memory T cells have been reported to associate with favorable survival in various cancer.+TH1 cells also participate in antitumor response by synthesizing transcription factor T‐bet and secreting IFNγ.Furthermore, our results showed that the signature of Immunophenoscore is a comprehensive determinant of tumor immunogenicity, calculated by integrating four critical types of tumor immunogenicity determinants: antigen processing (MHC), immune checkpoints and immunomodulators, effector cells . IPS has been validated to predict response to inhibitors CTLA and PD1. Consistently, our analysis indicated that the low‐risk groups have higher IPS than the high group, suggesting that the tumors in the low‐risk group are more immunogenic. Moreover, we also found that the low‐risk group has a tendency to respond to inhibitors of CTLA4. Our results align with others.LINC00669 in NSCLC with in vitro and in vivo evidence. Qing et al. reported that LINC00669 was enhanced in NPC and related to a poor prognosis.LINC00669 triggered the JAK/STAT signaling pathway to stimulate NPC cell proliferation and invasion.LINC00669 was found to decoy SOCS1 to prevent it from interacting with STAT1, thereby stabilizing STAT1 and activating the JAK/STAT signaling pathway.LINC00669 in other tumors have not been reported, and its functions remain to be clarified. The present study indicated that LINC00669 accelerates NSCLC growth by activating the Wnt signaling pathway.We also validate the oncogenic role of LINC00669 on the proliferation and apoptosis resistance of lung cancer cells.The Wnt signaling tightly regulates lung development, regeneration, and disease progression, including lung cancer.LINC00669 is a key oncogenic lncRNA in lung cancer. The signature of the LINC00669‐related cell cycle gene could distinguish two groups of LUAD patients with different survival, TIME, and anticancer drug sensitivity. Our results endorse the potential application of LINC00669 as a biological marker and therapeutic target in lung cancer.In conclusion, Jinhong Zhu: Conceptualization ; data curation ; funding acquisition ; investigation ; methodology ; project administration ; supervision ; writing – original draft (lead). Kui Cao: Data curation ; methodology ; visualization . Ping Zhang: Investigation (supporting); validation (supporting). Jianqun Ma: Conceptualization (supporting); funding acquisition (lead); project administration (lead); writing – review and editing (lead).This study received financial support from the Natural Science Foundation of China (82172786), the Natural Science Foundation of Heilongjiang Province (LH2021H077), and the National Cancer Center Climbing Fund of China (NCC201908B06).The authors declare no conflict of interest.The protocol of animal studies received approval from the Institutional Review Board of Harbin Medical University Cancer Hospital.Figure S1.Click here for additional data file.Table S1.Click here for additional data file.


Black individuals and men are predisposed to an earlier onset and higher prevalence of hypertension, compared with White individuals and women, respectively. Therefore, the influence of race and sex on reactive oxygen species (ROS) production and superoxide dismutase (SOD) activity following induced inflammation was evaluated in female and male human umbilical vein endothelial cells (HUVECs) from Black and White individuals. It was hypothesized that HUVECs from Black individuals and male HUVECs would exhibit greater ROS production and impaired SOD activity. Inflammation was induced in HUVEC cell lines (n = 4/group) using tumor necrosis factor-alpha . There were no between group differences in ROS production or SOD activity in HUVECs from Black and White individuals, and HUVECs from Black individuals exhibited similar SOD activity at 24hr compared with 4hr of TNF-α treatment (p>0.05). However, HUVECs from White individuals exhibited significantly greater SOD Activity (p<0.05) at 24hr as compared to 4hr in the control condition but not with TNF-α treatment (p>0.05). Female HUVECs exhibited significantly lower ROS production than male HUVECs in the control condition and following TNF-α induced inflammation (p<0.05). Only female HUVECs exhibited significant increases in SOD activity with increased exposure time to TNF-α induced inflammation (p<0.05). HUVECs from White individuals alone exhibit blunted SOD activity when comparing control and TNF-α conditions. Further, compared to female HUVECs, male HUVECs exhibit a pro-inflammatory state. CVD is ytokines , vaccineytokines , or diffytokines  is possiImportantly, compared with HUVECs from White individuals, HUVECs from Black individuals have exhibited greater inflammation, as indicated by higher NO levels and higher Interleukin-6 (IL-6) concentrations . HUVECs Sex differences in inflammatory response following induced inflammation have also been noted in HUVECs . In umbiTherefore, the purpose of the current study was to investigate the influence of race and sex on ROS production and SOD activity in HUVECs following induced inflammation. We hypothesized that HUVECs from Black individuals would exhibit greater ROS production and lower SOD activity than HUVECs from White individuals following induced inflammation. Further, we hypothesized that female HUVECs would exhibit lower ROS production and greater SOD activity than male HUVECs following induced inflammation. TNF-α was utilized to induce inflammation due to its role in inflammation and cytokine production in the human body  and its All procedures were approved by the Institutional Review Board at the University of Maryland- College Park (IRB# 1713185) and conformed to standards set by the Declaration of Helsinki. All protocols were performed on anonymized, commercially obtained HUVEC samples, making the protocol exempt from direct human subjects informed consent, as determined by the IRB.HUVECs were obtained from a commercial company, Lonza, from young, healthy donors free of overt disease or pregnancy complications. Eight total cell lines were obtained: two cell lines of each race and each sex . For analyses of HUVECs from Black and White individuals Figs  and 2, eTNF-α, a pro-inflammatory cytokine, was used to induce inflammation in the HUVECs. First, optimization experiments were performed to determine the TNF-α concentration needed to elicit inflammation without unnecessary cell death. Pooled HUVECs (separate from those used in experiments) in endothelial basal media and 2% fetal bovine serum were exposed to increasing concentrations of TNF-α for 24 hours and cells were visually analyzed for cell death and detachment. ROS production was measured via CellROX green assay and cell count was assessed via Hoechst assay . Based on the results, the experimental TNF-α concentration was determined, as the greatest ROS production per viable cells occurred at 50 ng/ml.All experiments were performed in triplicate on Black, White, female, and male HUVECs treated identically and cultured in parallel. Based on estimated between group means and standard deviations of n = 6/group from TNF-α stimulation in Brown et al. , a powerTo determine ROS production at 4 hours and 24 hours following time-matched control or TNF-α stimulation, CellROX green assays  were performed according to manufacturer’s instructions. Cells were passed into 96 well plates with EGM-2 and 2% fetal bovine serum media and allowed to grow to 80% confluence prior to experimentation. On the day of experimentation, media with or without 50 ng/ml TNF-α was placed on the cells and allowed to incubate for 4 or 24 hours (separate plates used for each time condition). For the fluorescence assay, 5 μM of CellROX Reagent and 1 μM Hoechst in EGM-2 and 2% fetal bovine serum was placed on the cells . Following a 30 minute incubation, the medium was removed and cells were washed three times with PBS. Fluorescence was immediately performed via a micro-plate reader  with excitation at 485nm and emission at 520nm for CellROX and excitation at 361nm and emission at 486nm for Hoechst.Cell count was assessed via Hoechst assay  performed according to manufacturer’s instructions. To determine the ROS produced per viable cells, a CellROX/ Hoechst ratio was calculated and used to compare ROS production between race and sex groups.SOD activity was subsequently determined in cell lysate samples via a commercially available SOD activity assay kit according to manufacturer’s instructions . Through xanthine oxidase dependent superoxide production, this assay indexes activity of all three SODs: SOD1 (cytoplasmic), SOD2 , and SOD3 (extracellular). One unit of SOD is the amount of SOD needed to elicit 50% dismutation of superoxide .On the day of assay, 200μL of the diluted radical detector was added to each well in the 96 well plate. Then, 10μL of serially diluted standard or diluted sample was added to each well. 20μL of xanthine oxidase was added to each well as quickly as possible. Following a 30 minute incubation on a plate shaker, absorbance was read between 440 and 460 nm using a plate reader. The absorbance of the known standards was used to create the absorbance-concentration equation. The determined equation was then used to calculate the concentration of each sample based on absorbance. Standards and samples were analyzed in duplicate and averaged across duplicates. SOD activity is normalized to protein content (U/mg).s; Formula: Hedges’s gs = s = 1 represents a 1 standard deviation difference in means between two compared groups. For Hedges’s gs, an effect size of 0.2 is considered a small effect size, 0.5 is considered a medium effect size, and 0.8 is considered a large effect size. All statistical analyses were performed in GraphPad Prism v9 .The present study evaluated the impact of race and sex on inflammatory responses and ROS clearance capacity in HUVECs following induced inflammation. All data were assessed for normality and outliers via Shapiro-Wilk test for normality and the ROUT 1% method  , respecteadsheet . Effect Fig 1A). For ROS production in the TNF-α condition, there was a significant main effect of time alone . In HUVECs from Black individuals, the effect size of ROS production at 24hr as compared with 4hr of TNF-α stimulation was Hedges’s gs = 0.73 (p = 0.079). In HUVECs from White individuals, the effect size of ROS production at 24hr as compared with 4hr of TNF-α stimulation was Hedges’s gs = 1.37 (p = 0.0025).For ROS production in the control condition, there were no significant model effects, suggesting similar ROS production within and between races during the time-matched control  and TNF-α conditions  . However, further analyses revealed that SOD activity normalized to protein content in HUVECs from Black individuals was similar between time points for both conditions (SOD activity (U/mg)- B control 4hr vs 24hr: p>0.05; TNF-α 4hr vs 24hr: p>0.05). SOD activity normalized to protein content in HUVECs from White individuals was significantly greater at 24hr as compared to 4hr in the control condition alone (SOD activity (U/mg)- W control 4hr vs 24hr: p = 0.0034, Hedges’s gs = 1.54; TNF-α 4hr vs 24hr: p>0.05).For SOD activity normalized to protein content (U/mg), there was a significant effect of time for both the control . Further investigation revealed female HUVECs exhibited significantly lower ROS production than male HUVECs at 24hr in the control condition . For ROS production in the TNF-α condition, there were significant effects of time and sex . Both female and male HUVECs exhibited greater ROS production at 24hr of TNF-α stimulation when compared to 4hr of TNF-α stimulation . Interestingly, female HUVECs exhibited significantly lower ROS production than male HUVECs at 24hr for the TNF-α condition .For ROS production in the control condition, there was a significant main effect of sex . For SOD activity normalized to protein content in the TNF-α condition, there was a significant effect of time . Further within sex analysis revealed female HUVECs exhibited significantly greater SOD activity normalized to protein content at 24hr as compared with 4hr for both conditions , and male HUVECS exhibited similar SOD activity (U/mg) between time points for both conditions (p>0.05).For SOD activity normalized to protein content in the control condition, there were significant effects of time and a significant interaction  HUVECs from White individuals alone experienced an increase in SOD activity with increased growth time that was abolished with TNF-α treatment; 2) female HUVECs exhibited significantly lower ROS production than male HUVECs in the control and TNF-α conditions; and 3) female HUVECs exhibited significantly greater SOD activity with increased exposure time to TNF-α.s = 1.37) as compared to HUVECs from Black individuals (Hedges’s gs = 0.73).Previous studies have shown that HUVECs from Black individuals have significantly higher basal protein expression of various superoxide-producing NADPH oxidases, suggesting higher basal ROS production –15. InteA plausible explanation for discrepancies in the present study as compared to the previous literature is the differential inflammatory pathways in HUVECs from Black and White individuals. Specifically, recent studies suggest HUVECs from Black individuals have greater inflammatory responses  to TNF-α stimulation as compared to HUVECs from White individuals , 24. HUVAs race is a social construct, a second potential explanation for the discrepancies in response to stressors in the present study versus previous studies could be social factors (beyond the scope of this study) whereby socially, Black individuals experience more chronic stress  than White individuals , and thuand total antioxidant capacity and SOD activity [SOD is an enzyme that primarily attenuates ROS levels by clearing superoxide. In the present study, when comparing the 4hr and 24hr control conditions, HUVECs from White individuals, but not HUVECs from Black individuals, exhibited greater SOD activity normalized to protein content. This may be due to the previously mentioned differential impact of the magnitude of the inflammatory stimulus used . The increase in SOD activity in HUVECs from White individuals in the control condition was absent in the TNF-α condition, suggesting a reduction or attenuation of the normally increased SOD activity when exposed to TNF-α in HUVECs from White individuals alone. In the present study, SOD activity in HUVECs from Black individuals did not significantly change with time in the control or TNF-α condition. Indeed, HUVECs from Black individuals have exhibited significantly lower SOD activity normalized to protein content basally and following 4 hours of TNF-α exposure when compared with HUVECs from White individuals , 13, 14.activity , 29.Sex differences in ROS production and SOD activity may be due to 1) male HUVECs being primed to exhibit a pro-inflammatory state due to higher androgen exposure in the umbilical cord , 2) maleSex differences in ROS production and SOD activity may be due to differences in androgen actions, total antioxidant capacity, or cellular stress responses. The concentration of estrogen in umbilical cord blood does not differ between fetal sex, and, thus, male and female HUVECs likely experience similar estrogen concentrations . Lower aThe present study’s results suggest a greater ability to clear ROS via greater SOD activity following induced inflammation in female HUVECs, as indicated by lower ROS levels as compared to male HUVECs yet increased SOD activity in female HUVECs alone. Indeed, previous literature suggests about a quarter of endothelial cell transcripts for genes are influenced by sex . Thus, tstem from disparities in socio-economic status, educational status, as well as other social determinants of health which a growing body of research has shown to be linked to structural racism” faced by the mothers (beyond the scope of the current study) (p. H2372) [These disparate physiological responses, especially in the HUVECs, could “. H2372) . The pur. H2372) . Therefo. H2372) . Further. H2372) .The current study includes some limitations. First, only SOD activity was measured, and, thus, a complete picture of ROS clearance capacity was not fully captured. Second, HUVECs are subject to venous circulation as opposed to arterial circulation, meaning HUEVCs undergo different hemodynamics and are in a different environment than arterial endothelial cells. Importantly, HUVECs are a commonly used endothelial cell model , 25, 31,in vitro research.HUVECs from Black and White individuals exhibit divergent responses to TNF-α-induced inflammation, with HUVECs from White individuals, but not HUVECs from Black individuals, exhibiting a blunted increase in SOD activity with increased exposure time to TNF-α. Interestingly, female and male HUVECS exhibited sex differences in ROS production and SOD activity; female HUVECs exhibiting significantly lower ROS production and significantly higher SOD activity than male HUVECs following TNF-α exposure, suggesting sex differences in susceptibility to induced inflammation in HUVECs. The current findings underly the importance of noting the race and sex (or indicating that race and sex are pooled if using pooled HUVECs) of HUVECs used in S1 Data(XLSX)Click here for additional data file.


LVAT  and RVAT  were significantly shorter with LBBAP than with RVSP. Moreover, all the repolarization parameters studied were significantly shorter in LBBAP than in RVSP , irrespective of the baseline QRS morphology. LBBAP was associated with significantly better acute depolarization and repolarization electrocardiographic parameters compared with RVSP.Compared with conventional right ventricular septal pacing (RVSP), several studies have shown a net clinical benefit of left bundle branch area pacing (LBBAP) in terms of ejection fraction preservation and reduced hospitalizations for heart failure. The purpose of this study was to compare acute depolarization and repolarization electrocardiographic parameters between LBBAP and RVSP in the same patients during the LBBAP implant procedure. We prospectively included 74 consecutive patients subjected to LBBAP from 1 January to 31 December 2021 at our institution in the study. After the lead was placed deep into the ventricular septum, unipolar pacing was performed and 12-lead ECGs were recorded from the distal (LBBAP) and proximal (RVSP) electrodes. QRS duration (QRSd), left ventricular activation time (LVAT), right ventricular activation time (RVAT), QT and JT intervals, QT dispersion (QTd), T-wave peak-to-end interval (Tpe), and Tpe/QT were measured for both instances. The final LBBAP threshold was a 0.7 ± 0.31 V at 0.4 ms duration with a sensing threshold of 10.7 ± 4.1 mV. RVSP produced a significantly larger QRS complex than the baseline QRS (194.88 ± 17.29 ms vs. 141.89 ± 35.41 ms,  Physiological pacing has received significant interest in the last decade as a response to the growing evidence that conventional right ventricular pacing (RVP) is associated with a decrease in left ventricular function and heart failure, a condition termed pacing-induced cardiomyopathy . Two carThe aspect and duration of the paced QRS complex have been topics of significant interest over the years. The left bundle branch block (LBBB)-like pattern induced by RVP triggers and maintains inter- and intraventricular electrical dyssynchrony, which is the basis for progressive ventricular mechanical dysfunction over time. Studies using activation mapping have shown that LBBB induced by RVP may worsen conduction barriers in the left ventricle compared with baseline LBBB . Along wFrom an electrocardiographic point of view, most studies have focused on and reported measurements of ventricular depolarization. Therefore, little data have been published on repolarization during physiological pacing . ElectroThe hypothesis of this study was that LBBAP generates better electrocardiographic depolarization parameters (thus abbreviating known predictors for heart failure development and promoting left ventricular function preservation or improvement) and better repolarization parameters  compared with RVP.In order to test this hypothesis, we compared acute depolarization and repolarization electrocardiographic parameters between left ventricular septal pacing and right ventricular septal pacing in the same patients during the LBBAP implant procedure.This was a prospective, analytical, single-center study.All consecutive patients who underwent LBBAP, either for bradyarrhythmias or for cardiac resynchronization therapy, between 1 January and 31 December 2021, in the Cardiac Pacing Laboratory of the Brașov County Clinical Emergency Hospital in Romania, were eligible for inclusion in the study. For the patients with symptomatic bradyarrhythmias, the main criterion for choosing LBBAP over another pacing strategy was an expected high ventricular pacing burden that would be encountered in different atrioventricular node disorders . Moreover, patients less than 75 years old and patients with a decreased baseline ejection fraction, but without criteria for resynchronization therapy, and with an indication for cardiac pacing, were considered for physiological pacing. The inclusion criteria were definite proof of lead penetration to the left side of the septum, the adequate capture of both left and right sides of the septum (see below), and twelve-lead electrocardiogram (12-lead ECG) recordings throughout the procedure. In the end, 74 patients were enrolled in the analysis.The baseline demographic and clinical characteristics of the patients were recorded.The LBBAP procedure at our institution was performed as follows: either a Medtronic C315 His or C304 His catheter  was first placed at the atrioventricular junction, a Medtronic Select Secure 3830 lead  was used to record the His bundle electrogram, and the position of the His bundle was stored as a fluoroscopic reference. The catheter was then advanced in the right anterior oblique projection approximately 1.5–2 cm toward the apex and maintained perpendicular to the septum. The lead was advanced deep into the septum with clockwise rotations under continuous fluoroscopy monitoring in the left anterior oblique projection. Lead advancement was stopped when ventricular premature beats with a right bundle branch block morphology (RBBB) appeared on the 12-lead ECG. Successful LBBAP was defined according to the commonly used criteria in the literature as a paced QRS complex from the distal electrode of RBBB morphology and one of the following: a recorded left bundle branch potential, a left ventricular activation time less than 80 ms for baseline narrow QRS, and less than 90 ms for baseline wide QRS complex or proof of transition from nonselective to selective pacing with decreasing pacing amplitude [The depth of penetration was assessed with contrast injection delineating the right side of the septum, ensuring that the proximal electrode was in contact with the septum a.Using the programmer, unipolar pacing at the same rate (20 bpm above the baseline rate) and the same amplitude  was performed at the distal electrode to capture the left side of the septum (termed LBBAP for the purpose of this study), and then at the proximal electrode to capture the right side of the septum (termed RVSP for the purpose of this study). We recorded the 12-lead ECGs for both settings after 30 s of pacing b,c. AddiQRS duration (QRSd).Left ventricular activation time (LVAT)—during RVSP, from the first R wave notch in leads V5-V6 to the end of the QRS complex; during LBBAP, from the beginning of the QRS complex to the peak of R wave in leads V5-V6 .Right ventricular activation time (RVAT)—the difference between QRSd and LVAT .QT interval—longest QT interval measured in any lead.JT interval—the difference between QT and QRSd.QTd—the difference between the longest and shortest QT interval measured in any lead.Tpe—the mean of the intervals between the peak and the end of the T wave measured in all leads.Tpe/QT—the ratio between the Tpe and the QT interval.The 12-lead ECGs during the procedure were recorded using the Workmate Claris EP system . All electrocardiographic measurements were performed with the optimal augmentation at 50 mm/s sweep speed using the calipers provided by the system. The beginning of the QRS complex was considered the pacing artifact for RVSP and for nonselective LBBAP and the first deflection of the QRS complex in selective LBBAP when an isoelectric interval was recorded after the pacing artifact. The end of the T wave was determined using the tangent method . The leads without a clear T wave were excluded from the analysis. The following ECG measurements were performed for both RVSP and LBBAP:Patients were followed for a period of 12 months with pacing and sensing parameters, and complications were recorded at the end of the follow-up period.t-test or Wilcoxon test for dependent groups and for different groups using the t-test or the Mann–Whitney U test for independent groups according to the normality of distribution. A confidence interval of 95% was used for all tests and a p < 0.05 was considered statistically significant.Continuous variables are presented as mean ± one standard deviation or as median and interquartile range. Categorical variables are presented as frequencies and percentages. A statistical comparison of means within the same group was performed using the Statistical analysis was performed using SPSS software v 26.0 .This study complied with all aspects of the Declaration of Helsinki and was approved by the institutional ethics committee.All patients were informed and provided their written consent before the procedure.The baseline characteristics of the patients are presented in The final LBBAP threshold was 0.7 ± 0.31 V for a 0.4 ms pulse duration with a sensing threshold of 10.7 ± 4.1 mV and an impedance of 557.16 ± 158.5 Ohm. The fluoroscopy time was 10.44 ± 7.33 min, and the total procedural time was 118.36 ± 27.35 min. There were three intraprocedural septal perforations with lead migration into the left ventricular cavity managed with lead retraction and repositioning at another site, without further issues, and two cases of acute chest pain during lead fixation without signs of myocardial ischemia, which spontaneously resolved after the procedure.p = 0.014) and constant sensing parameters . There was one case of lead dislodgement, without any other lead- or procedure-related complications.The twelve-month follow-up showed an improved pacing threshold , while LBBAP did not significantly change the mean QRS duration . LBBAP was associated with significantly improved depolarization parameters in the entire study group (p < 0.001), LVAT , and RVAT  were significantly shorter with LBBAP than with RVSP.RVSP produced a significantly larger QRS complex than the baseline QRS  significantly better depolarization parameters, including shorter left and right ventricular activation times; and (ii) significantly better repolarization parameters, irrespective of baseline QRS morphology.The observation that the paced QRS complex is narrower in LBBAP than in RVP is supported by several published studies . The expOn the other hand, the activation of the right ventricle in LBBAP is not completely understood. There are two possible mechanisms involved: transseptal myocardial activation from the tip of the lead and rapid activation through the conduction system (retrograde conduction up the left bundle and then down the right bundle) . RegardlVentricular activation time is directly proportional to the conduction properties of the ipsilateral bundle. This explains why, irrespective of the baseline QRS complex (and left bundle conduction), the capture of the left bundle during LBBAP resulted in nonstatistically different LVATs. On the contrary, an interesting finding in our study was that RVAT was similar when LBBAP was performed in patients with baseline normal QRS and in patients with RBBB. Because the right bundle was slowly conducting or nonconducting in the latter patients at baseline, plausible explanations for this result could be the dominance of transseptal activation of the right ventricle, or recruitment, with a higher voltage, of dormant right bundle branch fibers during physiological pacing.The limited existing data showed better repolarization parameters in LBBAP patients than in RVSP patients . AlthougSeveral studies have questioned the relevance of QTd as a measure of regional repolarization heterogeneity . We thinTp-Te and Tp-Te/QT are considered to reflect the last part of repolarization, are measures of transmural dispersion of repolarization in the left ventricle, and have been shown to predict ventricular arrhythmias . PreviouThe final LBBAP procedural parameters were similar to those described in large registries from experienced centers. Moreover, in line with the existing data, the one-year follow-up showed constant pacing and sensing thresholds and no major postprocedural complications, except one lead dislodgement, which needed reintervention .One of the limitations of the study is the measurement of ventricular depolarization and repolarization times on the surface ECG, which may have led to less accurate values. Other techniques, such as noninvasive electrocardiographic mapping or invasive activation mapping, provide more accurate results but are more difficult to use intraoperatively. Nevertheless, reports using the latter techniques support our findings that LBBAP leads to rapid and synchronous ventricular activation .Another limitation of the study derives from the difficulty with surface ECG interval measurements, especially the correct identification of the J point and the T-wave offset. Nevertheless, because this was an intrapatient study, and the measurements were made using the same tools, the measurement and selection biases associated with comparing different groups of patients were minimized and likely had no impact on the final results. This includes differences in intervals due to baseline structural or electrical heart diseases or an increased ventricular mass .This was an acute intraprocedural study, and there is the possibility that the measured parameters could change over time. Unfortunately, once the permanent pacemaker is implanted, there is no possibility of pacing on the proximal electrode, so the reproduction of the study during follow-up, until the time for the box change, is impossible.An important issue with studies that include patients with RVSP is that the final position of the lead with fluoroscopic guidance may not be true septal, as previously proved with postprocedural echocardiography or computer tomography imaging . The strTo the best of our knowledge, this is the first study to compare the LBBAP and RVSP electrocardiographic parameters of depolarization and repolarization in an intraindividual setting. Previous studies also reported electrocardiographic differences related to different pacing modalities, but the study populations consisted of separate study groups for each pacing strategy, and they did not investigate both repolarization and depolarization parameters (including both left and right ventricular activation times) . The majLeft bundle branch area pacing is associated with significantly better acute depolarization and repolarization electrocardiographic parameters compared with right ventricular septal pacing.