In this work, we introduce a generalized measure of cumulative residual entropy and study its properties. We show that several existing measures of entropy, such as cumulative residual entropy, weighted cumulative residual entropy and cumulative residual Tsallis entropy, are all special cases of this generalized cumulative residual entropy. We also propose a measure of generalized cumulative entropy, which includes cumulative entropy, weighted cumulative entropy and cumulative Tsallis entropy as special cases. We discuss a generating function approach, using which we derive different entropy measures. We provide residual and cumulative versions of Sharma–Taneja–Mittal entropy and obtain them as special cases this generalized measure of entropy. Finally, using the newly introduced entropy measures, we establish some relationships between entropy and extropy measures. X, the differential entropy is given byX and “log” stands for the natural logarithm, with The uncertainty associated with a random variable can be evaluated using information measures. In many practical situations in lifetime data analysis, experimental physics, econometrics and demography, measuring the uncertainty associated with a random variable is very important. The seminal work on information theory started with the concept of Shannon entropy or differential entropy introduced by Shannon 1948) [948 [1]. X with distribution function X. Asadi and Zohrevand (2007)  gavefined asE(X)=−∫0∞i 2009)  introduc. (2016) , is defir (2017)  introduch (2015) . Some ad  introduh (2015) ,11,12.Recently, Kharazmi and Balakrishnan (2020)  proposedX, the generalized Tsallis entropy of order X, Chakraborty and Pradhan (2021)  sub17  introet al. 20 [22] havIn the present work, we define a generalized cumulative residual entropy and study its properties in n uniquely determines the distribution function. Consequently, some characterizations of particular probability models have been obtained from this general result. Di Crescenzo and Toomaj (2017)  aX byf is the density function of X. Then, the cumulative hazard function Using integration by parts, from , we obtassion in  becomeso et al. 004 [2].The weighted cumulative residual entropy defined in  can be whow that  becomesNext, we show that the cumulative residual Tsallis entropy of order Tα(X) is CRTα(X)= ϕ(x)=x,  becomesChakraborty and Pradhan 2021) [21 [20] eα in  as10)WWα in  .In this sub-section, we introduce a generalized cumulative entropy and discuss some of its properties.Definition 2.Let X be a non-negative random variable with absolutely continuous distribution function F. Further, let where w and ϕ can be chosen arbitrarily under the existence of the above integral such that For the choices of  ϕ(x)=x,  reduces , (CE(X) . SimilarCE(X) in  reduces X, denoted by The reversed hazard rate function of tropy in  can be etropy in  for the  entropy  for the X, at time x, is defined asNext, we show that the cumulative Tsallis entropy of order CE(X) in . The meaessed as CTα(X)=E ϕ(x)=x,  yieldsGgiven by (12)WCTαx)=12x2,  yieldsGNext, we derive expressions for Next, let us consider the standard uniform distribution with pdf . (2022) .We now introduce generating function related to generalized entropy measures discussed in the preceding sections.Definition 3.Let X be a non-negative random variable with absolutely continuous distribution function F. Further, let t, we can interpret t once, we obtaint would similarly give rise to generalized cumulative residual entropies of orders 2, 3 and so on. For example, the generalized cumulative residual entropy of order two is given byBeing a function of asure in . We, theiven by1Gfre(2)=∫In a similar manner, we define the generating function for the generalized cumulative entropy as follows.Definition 4.Let X be a non-negative random variable with absolutely continuous distribution function F. Further, let t once and setting t would give rise to generalized cumulative entropies of orders 3, 4 and so on.Once again, differentiating  with resasure in . From   can be osure in (Gfce(2)=∫In this section, we introduce the cumulative  versions of Sharma–Taneja–Mittal (STM) entropy. We then show that these are indeed special cases of the generalized residual and the past entropy introduced in Sharma and Taneja (1975)  and MittSα,β=1β−α entropy , as a sp entropy  along wi entropy .In this sub-section, we introduce the cumulative residual version of the STM entropy.Definition 5.Let X be a non-negative random variable with absolutely continuous survival function Definition 6.Let X be a non-negative random variable with absolutely continuous survival function Next, we show that consider∫0∞(F¯(x)e obtain∫0∞(F¯(x)ConsiderSRα,β=1β−=x, from , we obtaBy following the same steps as performed for , the cum/2, from , we arriSRWα,β=1βIn this sub-section, we introduce cumulative and weighted cumulative STM entropies, and then show that they are indeed special cases of the generalized entropy.Definition 7.Let X be a non-negative random variable with absolutely continuous distribution function F. Then, the cumulative STM entropy is defined asIn this case, the weighted cumulative STM entropy is defined as follows.Definition 8.Let X be a non-negative random variable with absolutely continuous distribution function F. Then, the cumulative weighted STM entropy is defined asOf course, in the special cases of en, from , we obta¯α−1(X),SPWα,β=1β¯α−1(X),SPWα,β=1βX. Using the new entropy measures introduced in the preceding sections, we establish some relationships between entropy and extropy measures in this section.Apart from entropy, extropy and its properties have also been studied for quantifying the uncertainty associated with a random variable X, extropy is defined as [For a non-negative random variable fined as J(X)=−12i et al.  defined fined as (29)CJ(Xi (2022)  discusseRecently, Balakrishnan et al. , Bansal and Nair  subseque may see . Sudhees may see  proposedX is defined as Sathar and Nair  defined u (2021)  introducand Nair  defined i (2022)  expresseX, we haveNext, we establish some connections between different entropy and extropy measures. For the choice of s, using , we haveFor the choices of x), from  and 32)w(u)=−F=∫2wtropy in .Z is defined asZ is the weighted average of the dynamic cumulative extropy in =∫0tropy in .In this work, we have introduced two general measures of entropy, viz., generalized cumulative residual entropy and generalized cumulative entropy. Several entropy measures known in the literature were all shown to be special cases of these generalized measures. Cumulative residual entropy, weighted cumulative residual entropy, cumulative residual Tsallis entropy and weighted cumulative residual Tsallis entropy are all special cases of the generalized cumulative residual entropy. Cumulative entropy, weighted cumulative entropy, cumulative Tsallis entropy and weighted cumulative Tsallis entropy are all special cases of the generalized cumulative entropy.We have presented a generating function approach to obtain generalized measures of higher-order. We have shown that the generalized cumulative residual entropy of order two reduces to the weighted cumulative residual entropy of Mirali et al. (2016) . MoreoveY, given the value of another random variable X. Conditional entropy, as a measure of information, can be defined through any measure, such as the Shannon entropy measure (denoted by In information theory literature, conditional entropy is the amount of information required to describe the outcome of one random variable


Purpose: The diagnosis of autism spectrum disorder (ASD) is reliant on evaluation of patients’ behavior. We screened the potential diagnostic and therapeutic targets of ASD through bioinformatics analysis.Methods: Four ASD-related datasets were downloaded from the Gene Expression Omnibus database. The “limma” package was employed to analyze differentially expressed messenger (m)RNAs, long non-coding (lnc)RNAs, and micro (mi)RNAs between ASD patients and healthy volunteers (HVs). We constructed a competing endogenous-RNA (ceRNA) network. Enrichment analyses of key genes were undertaken using the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes database. The ImmucellAI database was used to analyze differences in immune-cell infiltration (ICI) in ASD and HV samples. Synthetic analyses of the ceRNA network and ICI was done to obtain a diagnostic model using LASSO regression analysis. Analyses of receiver operating characteristic (ROC) curves were done for model verification.Results: The ceRNA network comprised 49 lncRNAs, 30 miRNAs, and 236 mRNAs. mRNAs were associated with 41 cellular components, 208 biological processes, 39 molecular functions, and 35 regulatory signaling pathways. Significant differences in the abundance of 10 immune-cell species between ASD patients and HVs were noted. Using the ceRNA network and ICI results, we constructed a diagnostic model comprising five immune cell-associated genes: adenosine triphosphate-binding cassette transporter A1 (ABCA1), DiGeorge syndrome critical region 2 (DGCR2), glucose-fructose oxidoreductase structural domain gene 1 (GFOD1), glutaredoxin (GLRX), and SEC16 homolog A (SEC16A). The diagnostic performance of our model was revealed by an area under the ROC curve of 0.923. Model verification was done using the validation dataset and serum samples of patients.Conclusion:ABCA1, DGCR2, GFOD1, GLRX, and SEC16A could be diagnostic biomarkers and therapeutic targets for ASD. Autism spectrum disorder (ASD) is a group of heterogeneous neurodevelopmental disabilities characterized by an early onset of impaired social function, abnormal stenosis, and repetitive behaviors and interests . WorldwiBehavioral and educational interventions remain the most popular treatment measures for ASD. Early behavioral interventions improve the core symptoms of ASD patients, as well as their verbal and cognitive ability and social adaptability . SeveralA microRNA (miRNA) is a non-coding RNA with a length of ∼22 nucleotides. miRNAs can inhibit the translation of target messenger (m)RNAs by binding to partially complementary sequences within them . The seqWe aimed to screen the potential diagnostic markers and therapeutic targets of ASD from the perspective of a ceRNA network. We downloaded ASD-related datasets from the Gene Expression Omnibus (GEO) database to screen differentially expressed mRNAs, lncRNAs, and miRNAs in ASD patients to construct a ceRNA network. Based on analyses of differences in the infiltration of immune cells between samples from ASD patients and healthy volunteers (HVs), and combining analyses of the ceRNA network, diagnostic marker genes were obtained by least absolute shrinkage and selection operator (LASSO) regression analyses. Finally, we validated the diagnostic model through validation datasets and samples of peripheral blood from ASD patients .www.ncbi.nlm.nih.gov/geo/), 19 datasets were found pertaining to ASD (Homo sapiens): five brain-sample datasets, 11 blood-sample datasets, and three stem cell- or fibroblast-sample datasets. Considering the tissue differences in gene expression, we chose four brain datasets  for analyses (GSE102741 was eliminated because it focused mainly on genes related to the histamine system). mRNA expression data were acquired from the GSE38322 dataset | >0.5 to obtain genes with significantly different expression.The mRNAs, lncRNAs, and miRNAs that showed different expression between ASD patients and HVs were screened in the corresponding datasets using “limma 3.5.1” within R . The screening thresholds were http://mirwalk.umm.uni-heidelberg.de/). In the Comparative Toxicogenomics Database , 24,785 genes related to ASD were retrieved using “Autism” as the keyword. At the intersection of predicted target mRNAs and ASD-related genes in the CTD, mRNAs that showed differential expression in the GSE38322 dataset were obtained using Venn (http://bioinformatics.psb.ugent.be/webtools Venn/). Subsequently, miRNA–mRNA pairs conforming to the regulation principles of miRNA and mRNA in the ceRNA network were obtained.The predicted target mRNA was obtained by predicting the target mRNA of differentially expressed miRNA in the GSE59286 dataset using the miRWalk database (https://diana.e-ce.uth.gr/lncbasev2/home/) was used to predict the target lncRNAs of differentially expressed miRNAs obtained in the GSE59286 dataset, and a threshold score >0.6 was set. Venn was employed to obtain the intersection of predicted lncRNAs and differentially expressed lncRNAs in the GSE36315 dataset. According to the functional mechanism of ceRNAs, differentially expressed miRNAs that had a regulatory relationship with the obtained lncRNA were acquired to construct miRNA–lncRNA pairs. Finally, according to the miRNA–mRNA pairs and miRNA–lncRNA pairs obtained, the lncRNA–miRNA–mRNA associations with the same miRNA as the core were screened to construct the ceRNA network, and Cytoscape (https://cytoscape.org/) was used to visualize the results.The LncBaseV3 database (http://bioinfo.life.hust.edu.cn/web/ImmuCellAI/). Then, Student’s t-tests were undertaken using “ggplot2” in R to analyze the different immune cells between samples from ASD patients and HVs. The correlation between significantly different immune cells and mRNAs in the ceRNA network was analyzed by the Spearman method using the “ggstatsplot” in R. The relationship between different immune cell–mRNA pairs was screened using a threshold of p < 0.05 and correlation coefficient |r| > 0.8.Analyses of the infiltration of immune cells were undertaken on the brain-tissue samples of 18 HVs and 18 ASD patients from the GSE38322 dataset using the ImmucellAI database (https://cran.r-project. org/web/packages/glmnet/index.html/) was used for the LASSO screening of diagnostic genes for ASD. The parameters were set as family = “binomial” and n fold = 20 . The eigenvalue of each sample was calculated based on the regression coefficient of screened genes and expression of the genes in the GSE38322 dataset using the formula i denotes the LASSO regression coefficient of the NO. i gene, xi denotes expression of the NO. x gene, and n denotes the number of diagnostic markers in the diagnostic model. The receiver operating characteristic (ROC) curve was plotted using “pROC” within R to assess the predictive efficacy of the diagnostic model and diagnostic markers for the disease.Based on the obtained mRNAs associated with immune cells and their expression in each sample of the GSE38322 dataset, “glmnet” v4.0-2 in R (http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes  database, respectively, using “cluster Profiler” in R. The relevant biological processes, molecular functions, cellular components, and regulatory signaling pathways involved were obtained using a significance threshold of p < 0.05. Gene set enrichment analysis (GSEA) was done for each diagnostic marker gene using GSEA v4.0.3 (www.gsea-msigdb.org/gsea/index.jsp/). Enrichment analyses using GO and KEGG databases were conducted using GSEA for high and low expression of the diagnostic biomarker genes. An FDR q-value < 0.05 was considered to denote significant enrichment.Analyses of the enrichment of the function and signaling pathways of mRNAs in the ceRNA network were done using the Gene Ontology database . Written informed consent was obtained from all participants.Blood samples from 10 ASD patients were obtained from the Liaoning Disability Service Center  and blood samples from 10 HVs were obtained from Shengjing Hospital of China Medical University between January 2022 to March 2022. −ΔΔCT method, and the gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control for normalization.We isolated the serum from whole blood. Then, we extracted the total RNA from serum using TRIzol™ Reagent . Reverse transcription was undertaken using the SweScript RT I First Strand cDNA Synthesis Kit . PCRs were carried out using the 2× universal Blue SYBR Green qPCR Master Mix Kit (Servicebio) according to manufacturer instructions. The reaction conditions were 95°C for 1 min , 95°C for 20 s (denaturation), 55°C for 60 s , and 72°C for 30 s (extension) and 40 cycles in total. Relative quantification was undertaken with the 2The primer sequences  used were: 5′-CTGGGAAGGTGGTTGTGTT-3′ and 5′-GTGGTTGGTGGCTGTGATA-3′ for GLRX; 5′-ACCCGTCCATCCTACATC-3′ and 5′-TCACCTCGTCTCTCTTCCGTC-3′ for SEC16A; 5′-GCGGGGAGAACTACTGGGAT-3′ and 5′-GTGGAGAAGGTGGCGAGAGA-3′ for DGCR2; 5′-GACTGCCTGTATGCCTTGT-3′ and 5′-CCGTCTGGCTGGACCTCTT-3′ for GFOD1; 5′-TCCTCTTTCCCGCATTATCT-3′ and 5′-GTCCATTTCTTGGCTGTTCT-3′ for ABCA1; 5′-CCCATCACCATCTTCCAGG-3′ and 5′-CATCACGCCACAGTTTCCC-3′ for GAPDH.t-test was used to compare gene expression among serum samples, and p < 0.05 was considered significant.Statistical analyses were undertaken using R 3.6.2. The Student’s p < 0.05 and |log2 FC| >0.5), 662 differentially expressed mRNAs were obtained in the GSE38322 dataset, of which 295 had upregulated expression and 367 had downregulated expression in the samples from ASD patients and HVs, respectively , induced regulatory T cells (iTregs), type-1 helper T cells (Th1), Th2 cells, central-memory cells, effector-memory cells, natural killer T (NKT) cells, monocytes, CD4− T cells, CD8− T cells, and 10 types of immune cells were significantly different between the two groups. Significantly more CD4− naïve, central-memory, effector-memory, monocyte, and CD4− T cells infiltrated in samples from ASD patients than in samples from HVs, whereas significantly fewer iTregs, Th1, Th2, NKT, and CD8− T cells infiltrated in ASD patients than in HVs 4-initial cells , DiGeorge syndrome critical region 2 (DGCR2), glucose-fructose oxidoreductase structural domain gene 1 (GFOD1), glutaredoxin (GLRX), and SEC16 homolog A (SEC16A). Based on the regression coefficient of each gene, the sample eigenvalue was calculated as:We undertook LASSO regression analyses on 23 mRNAs associated with immune cells under 20-fold cross-validation  and screFeature = ABCA1ch × −1.719843535 + DGCR2 × −0.003835302 + GFOD1 × −0.277741202 + GLRX × 0.01064725 + SEC16A × −1.697826627.ABCA1 of 0.883, whereas for DGCR2 it was 0.836, for GFOD1 it was 0.824, for GLRX it was 0.759, and for SEC16A it was 0.849. Hence, single genes could also be used as diagnostic biomarkers  of the model was ≤0.923: the diagnostic performance of the model was good. Single-gene analyses of the ROC curve revealed an AUC for omarkers .After GSEA for screened ASD-associated diagnostic marker genes, the top-10 pathways (according to GO and KEGG databases) for each diagnostic marker gene ranked by FDR q-values were obtained . These rABCA1, DGCR2, GFOD1, GLRX, and SEC16A) was analyzed in the GSE28475 dataset and the serum samples collected from study participants , and individuals who carry rare Sequencing has been the most promising strategy to reveal the pathogenesis and diagnosis of ASD . ThroughAn approach using bioinformatics analysis can be helpful in this scenario. A weighted gene co-expression network was constructed to analyze the genes known to be related to ASD, and to find intersections of these risk genes to discover novel diagnostic and therapeutic options . We attemiRNAs are at the core of a ceRNA network. There have been many reports of abnormal expression of multiple miRNAs in the brain tissue and peripheral blood of ASD patients . Given tWe constructed a ceRNA network comprising 49 lncRNAs, 30 miRNAs, and 236 mRNAs by processing the differentially expressed miRNAs, mRNAs, and lncRNAs obtained from targeting predictions in databases. Through analyses of functional enrichment, we discovered that the top-10 biological processes and molecular functions involved in the genes in this network were mainly related to mitochondrial-energy metabolism, cellular communication, transcriptional regulation, and glial-cell fate, whereas the main signaling pathways involved were associated with energy metabolism and neurodegeneration. These findings are consistent with the underlying pathological changes in ASD: examinations using functional magnetic resonance have demonstrated abnormal activation/inhibition of several brain regions involved in social interaction and information integration in the brains of ASD patients. Moreover, neuropathological examinations have revealed extensive abnormalities in synaptic development/structure and brain function in ASD patients. The brain is a region of high energy metabolism whose neurons are highly dependent on mitochondrial energy metabolism to support neurogenesis, signaling, and synaptic remodeling . FurtherStudies have demonstrated immune dysregulation, inflammation, and endogenous production of autoantibodies due to abnormalities of innate and adaptive immune systems in ASD patients . Autoantregs as well as Th1, Th2, NKT, and CD8− T cells in ASD patients than in HVs. Infiltration of more CD4−initial, central-memory, effector-memory, monocytes, and CD4− T cells was noted, which suggested that cellular immunity was also associated with ASD development. Mostafa et al. showed a reduction in the number of CD4+CD25high Tregs in ASD children  but theABCA1, DGCR2, GFOD1, GLRX, and SEC16A. Analyses of ROC curves revealed that the diagnostic model based on a combination of these five diagnostic genes had an AUC of 0.923. This value is higher than the AUC (0.910) of the three-methylation-markers diagnostic model identified using the same algorithm  resulted in a fourfold increase in ASD risk  (Little research has been done on ric acid . Increasdisorder . Trent eand ASD) , but theSEC16A encodes an endoplasmic-reticulum exit-site scaffolding protein, which is an important component of coat protein complex II (COP II) vesicles and mediates the transport of substances from the endoplasmic reticulum to the Golgi apparatus (SEC16A may be involved in ASD development through the immune system.pparatus . COPII ipparatus , and defpparatus . COPII ipparatus . Hence, in vitro studies, but the sample size of our validation dataset was small.Rapid advancement of sequencing technology has led to great progress in screening for the diagnostic markers of ASD, but it had two main limitations in our study. First, the sequencing results for mRNAs, lncRNAs, and miRNAs arose from three datasets and belonged to different patients, which brings uncertainty to the diagnosis of ASD. Second, the results of bioinformatics analysis must be validated by clinical and We constructed an ASD-associated ceRNA regulatory network. This network will provide a new perspective for explaining the biological processes of ASD. Furthermore, we identified five immune cell-associated potential molecular marker genes of ASD, which could be novel targets for therapy and immunotherapy strategies against ASD.


N2 stage disease constitutes approximately 20%–30% of all non-small cell lung cancer (NSCLC). Concurrently, surgery remains the first-choice treatment for patients with N2 NSCLC if feasible. However, the role of pneumonectomy in N2 NSCLC has rarely been investigated and remains controversial.We enrolled 26,798 patients with T1–4N2M0 NSCLC (stage IIIA/IIIB) from the Surveillance, Epidemiology, and End Results (SEER) database between 2004 and 2015. We compared the overall survival (OS) and cancer-specific survival (CSS) between patients who received pneumonectomy and those who did not receive surgery. The Kaplan–Meier method, Cox regression analyses, and propensity score matching (PSM) were applied to demonstrate the effect of pneumonectomy.p-values <0.001. Subgroup analysis demonstrated concordant results stratified by demographic or clinicopathological variables. In sensitivity analysis, no significant difference was observed between patients receiving single pneumonectomy and chemoradiotherapy without surgery in OS and CSS both before  and after PSM  with all p-values >0.4.Patients receiving pneumonectomy had a significantly better OS and CSS than those without pneumonectomy both before  and after PSM  with all For patients with T1–4N2M0 NSCLC (stage IIIA/IIIB), pneumonectomy is an independent protective factor of OS and should be considered when applicable. Although annual mortality has steadily declined for years, lung cancer remains one of the most common and deadly malignancies . As the Pneumonectomy is performed in patients with central giant masses or tumors involving the main bronchus or large blood vessels, particularly for N2 NSCLC patients. There are many controversies over pneumonectomy for N2 patients . Pneumonhttps://seer.cancer.gov/resources/). Patients diagnosed with T1–4N2M0 NSCLC using the American Joint Committee on Cancer (AJCC) eighth TNM classification from 2004 to 2015 were enrolled in this research. The following variables were downloaded: “Year of diagnosis”, “Age”, “Race recode”, “Sex”, “Marital status”, “Laterality”, “Primary Site—labeled”, “Histologic Type ICD-O-3”, “Grade”, “RX Summ–Surg Prim Site”, “Derived AJCC T, 6th ed (2004–2015)”, “Derived AJCC N, 6th ed (2004–2015)”, “Derived AJCC T, 7th ed (2010–2015)”, “Derived AJCC N, 7th ed (2010–2015)”, “Survival months”, “Vital status recode”, “SEER cause-specific death classification”, “Regional nodes positive”, “Regional nodes examined”, and “Sequence number of tumor”. The AJCC TNM classification was transformed into the eighth version. The exclusion criteria were as follows: (a) patients who have more than one malignant tumor; (b) patients under the age of 18 years old; (c) patients treated with surgery other than pneumonectomy; (d) patients who received a diagnosis of NSCLC based on autopsy/death certificate merely or were diagnosed without being pathologically confirmed; and (e) patients with unknown data of variables needed by our research. We collected data from the SEER database, which included 18 cancer registries of the National Cancer Institute, using the SEER*Stat software .http://www.empowerstats.com) and R software . p-value <0.05 was regarded as statistically significant. Categorical variables were presented as number (proportion). Chi-square test or Fisher’s precision probability test was performed to compare categorical variables, as appropriate.Two statistical software were used in analyses: EmpowerStats  and the pneumonectomy group (n = 865). Before PSM, there was a significant difference between the two groups in all included variables, namely, year of diagnosis, age, gender, race, marital status, laterality, primary site, histologic type, differentiation, T, regional nodes examined, regional nodes positive, and radiotherapy or chemotherapy . However, we conducted the PSM to match paired patients and decrease differences in baseline characteristics. After PSM, a total of 782 pairs of patients were included, with no statistical difference in variables used for PSM .As shown in p < 0.0001; We compared the OS and CSS between the no-surgery group and the pneumonectomy group, shown in p < 0.05). Multivariable analysis demonstrated that surgery, year of diagnosis, age, gender, race, marital status, primary site, histological type, differentiation, T classification, and radiotherapy or chemotherapy were independent risk factors for OS of T1–4N2M0 NSCLC patients; the same results were found for CSS except that laterality was also independently related to CSS. The unadjusted-HR for pneumonectomy versus no surgery before  and after PSM  with all p-values <0.001 and adjusted-HR for pneumonectomy versus no surgery before  and after PSM  with all p-values <0.001 showed better survival outcome of patients who underwent pneumonectomy.We conducted the univariable and multivariable analyses to screen the risk factors for OS and CSS of T1–4N2M0 NSCLC patients 3. Beforp = 0.089 and 1.464 (1.055–2.031) for CSS with p = 0.022. The adjusted-HR (95% CI) of regional nodes positive >5 compared with 0 was 1.874 (1.371–2.562) for OS and 2.211 (1.562–3.130) for CSS with all p-values <0.001.To analyze the influence of regional nodes examined/positive on OS and CSS of patients receiving pneumonectomy, we also conducted univariable and multivariable analyses Table 4.p-values < 0.05). Only the HR for unknown marital status, >23 regional nodes examined, and >5 regional nodes positive group was not statistically significant.We compared the OS and CSS of the no-surgery group and the pneumonectomy group in subgroups of different variables Figure 2n = 13,803) or single pneumonectomy (n = 222). The survival analysis demonstrated no significant disparity between chemoradiotherapy and single pneumonectomy in OS and CSS both before and after PSM , as shown in p = 0.832 before PSM and 0.988 (0.799–1.222) with p = 0.913 after PSM (p = 0.413 before PSM and 0.938 (0.744–1.182) with p = 0.586 after PSM  . The treSimilar to a previous study , we condNevertheless, pneumonectomy should be applied after comprehensive preoperative evaluation with strict surgical indications. Only 865 patients received pneumonectomy in this study, accounting for 3.2% of the entire study cohort. A study summarized the application of pneumonectomy for primary lung cancer in the Netherlands, and they demonstrated that the mean postoperative mortality was 7.1% . An earlp < 0.001 .The dataset supporting the conclusions of this article is available in the SEER*Stat software  and Shanghai “Rising Stars of Medical Talent” Youth Development Program: Outstanding Youth Medical Talents.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.


Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of “candidate ciliopathy genes.” From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes  PCD results from an inability of motile cilia to transport mucus through the respiratory tract and nasal sinuses resulting in persistent infection1. Situs abnormalities are characterized by lack of normal left–right axis asymmetry. Within the embryonic node there are monociliated cells that beat, moving extracellular fluid and morphogens in order to establish normal left–right axis asymmetry. These developmental abnormalities are also a result of altered cilia motility2. Male infertility is caused by an abnormal flagellum, itself a modified motile cilia, that affects the progressive and non-progressive motility of sperm4. Male infertility can also be caused by loss of motile ciliary action within the efferent ductules of the testes and/or the ciliated epithelial lining of efferent epididymal ductules that are responsible for moving spermatozoa to the epididymis for final maturation5.The pathophysiology of ciliopathies reflects abnormalities in the function of both primary and motile cilia. Primary cilia dyskinesia (PCD) is a multi-syndromic disorder caused by defects in motile cilia, including chronic otosinopulmonary disease, 6. Each renal tubular epithelial cell contains a single primary cilium that can act in mechano- and chemo-sensation. Polycystic kidney disease (PKD) and nephronophthisis (NPHP) are diseases in which normal renal architecture is replaced by cysts caused by abnormalities in the primary cilium of renal epithelial cells7. Mutations of proteins that are components of the primary cilium, for example PKD1, PKD2, and PKHD1, are associated with PKD, while mutations of the ciliary proteins NPHP1 and INVS are associated with NPHP6.Two hallmark features of abnormal structure and function of primary cilia include aberrant kidney function and retinal degeneration8. While single gene mutations exist that are unique to each syndrome, several syndromes can share a single genetic basis. For example, CEP290 has been linked to NPHP, BBS, SLS, and JBTS12. CEP290 is a known centrosomal protein that is found in renal epithelial cells and retinal photoreceptors13. Outside of retinal and renal phenotypes, skeletal anomalies such as polydactyly can also develop as is the case with the ciliopathy Meckel–Gruber syndrome (MGS)14. The clinical spectrum of ciliopathies can include hydrocephalus, intellectual disability, craniofacial defects, cardiac anomalies, lung disease, skeletal abnormalities, liver and pancreatic cysts, kidney disease, and reduced or total infertility.Ciliopathies that commonly have symptoms of retinal degeneration include Leber Congenital Amaurosis (LCA), NPHP, Senior-Loken Syndrome (SLS), Joubert Syndrome (JBTS), and Bardet–Biedl Syndrome (BBS)17. These studies have shed light on new disease-causing or disease-contributing genes associated with ciliopathies17. However, in the discovery of such genes it is useful to validate predicted ciliopathy proteins or interacting proteins in an in vivo model system. Knockout mice that express a null “loss of function” allele for a gene can determine if mutations in predicted genes lead to the expected phenotype and are useful diagnostically in clinical cases of human ciliopathies, including forms of pediatric ophthalmic pathology.Considerable effort has been directed toward understanding the scope of proteins interacting with cilia, both directly and through secondary protein interactions, in order to more fully understand potential disease causing mutations that encompass ciliopathies18. To date, production and comprehensive phenotyping has been completed for 6,440 unique genes in IMPC data release 11.0 queried 29 April 2020. Cohorts of KO mice  are analyzed and studied using a sequential set of phenotyping tests starting at 4 weeks of age and a set of terminal tests at 16 weeks of age that includes necropsy, tissue collection, and histology using standardized protocols20. Standardization and harmonization of testing protocols and data analysis are performed by the IMPC Data Coordination Center (DCC) to identify and confirm phenodeviants22. Given the characteristic presentation of ciliopathies with functional or morphological abnormalities in eyes, kidneys, and reproductive tract, we queried the IMPC database for all mouse lines with abnormal ocular traits and at least one other ciliopathy-associated system: renal or reproductive.The International Mouse Phenotyping Consortium (IMPC) is a mouse and phenotype data resource for single gene knockout (KO) mouse lines. Since its inception it has expanded to 20 research centers with expertise in high throughput KO mouse production and phenotypingWe present novel genes in KO mouse lines that exhibit phenotypes consistent with ciliopathies. Further, we present evidence indicating how loss-of-function mutations in these genes are related to the dysfunction of primary or motile cilia. Candidate ciliopathy genes presented here are of potential interest for further investigation of their association to the cilium and to confirm their association with clinical ciliopathies, particularly those responsible for pediatric eye disease.The IMPC database contained KOs for 748 genes which exhibited abnormal ocular phenotype(s) identified by eye examination, optic imaging, and/or histopathology (“ocular genes”). Of these, 256 demonstrated abnormal renal morphology at necropsy (“kidney morphology genes”). 132 had abnormal values for blood urea nitrogen (BUN) and 110 had abnormal plasma creatinine (CRE); collectively . Of the total 229 renal function genes, 13 were found to have altered levels of both BUN and CRE. Abnormal reproductive tract morphology was identified at necropsy and/or histopathology (“reproductive morphology genes”) in 223 lines. Abnormal reproductive phenotypes was identified by fertility analysis (“reproductive function genes”) in 378 lines. These groups are summarized in Fig. Abce1, Abhd11, Abi2, Ap4e1, Aplnr, Atp8b1, Bambi, Chsy3, Cops7a, Dnase1l2, Edc3, Fndc3b, Folr1, Gcgr, Gkn1, Gpa33, Ica1, Mink1, Mmachc, Nacc1, Pgbd1, Ptp4a1, Pygo2, Rspo1, Rxfp2, Sik3, Spred3, Sra1, Sun1, Tmem160, Tmem30b, and Wdr62. Detailed phenotype abnormalities can be found in Table S2.Query of the IMPC database for five different categories of traits associated with ciliopathies led to five distinct gene sets: “ocular genes”, “kidney morphology genes”, “kidney function genes”, “reproductive tract morphology genes”, and “reproductive tract function genes”. To identify “potential ciliopathy genes” with a particular focus on those associated with congenital blindness syndromes, all “kidney morphology genes”, “kidney function genes”, “reproductive tract morphology genes”, and “reproductive tract function genes” were then assessed for concomitant ocular trait abnormalities. If a line had one ocular and any renal or reproductive system abnormality then it was considered to be a “potential” ciliopathy gene. A total of 140 unique potential ciliopathy genes were identified , Mlh1, and Rxfp2 , as well as capsular thickening and wrinkling in the cortical region of the lens  showed expression of LacZ under control of the Prkab1 promoter in the photoreceptor outer segments  have a decreased total retinal thickness , JBTS, and BBSAbi2, Ap4e1, Cdc42, Cdk4, Celsr1, Cops7a, Cyba, Dtnbp1, Dync1li1, Ehmt1, Folr1, Herc1, Klhl5, Mapt, Mib2, Myo7a, Prkab1, Ptp4a1, S1pr3, Sik3, Sugp1, Ube2a, Wsb2, and Xbp1) based on STRING protein interaction analysis , Mlh1, Rxfp2, and Wdr62.  with capsular thickening and wrinkling in the cortical region of the lens  contained round bodies in the lumen with few spermatozoa. This suggests a failure in the maturation of sperm and subsequent immune mediated degradation of the sperm leading to testicular atrophy  and efferent ductules of the epididymis and primary cilia of the testis, rete testis, epididymis, and prostateAp4e1 KO mice have been shown to specifically have retinal abnormalities of marked reordering of the outer plexiform layer. Other retinal abnormalities of Ap4e1 KO mice include reduced responses in both the scotopic a-wave, which represents photoreceptor function, as well as the scotopic b-wave on ERG38. Here we recapitulate a different retinal phenotype for Ap4e1 mutants consisting of decreased POS thickness  had abnormal morphological features detected by imaging that are consistent with the spectrum of phenotypes observed in ciliopathies. Wdr62 and Dyncli1 have previously described roles in vertebrate ciliary biology, while Abi2, Ap4e1, and Prkab1 do not. Abi2, Ap4e1, and Prkab1 therefore have strong evidence to be novel candidate ciliopathy genes. These candidate genes require validation in unsolved human cases with systemic features pertinent to ciliopathy. If relevant in human disease, these mouse KO may serve as valuable models for study of this group of ciliopathies.In summary, we present in vivo analytical findings for 32 candidate ciliopathy genes based on phenotypic changes identified in KO mice and predicted protein–protein interactions with known human cilia genes. One of the 32 has been shown previously to have function in human cilia, leaving 31 new candidate human ciliopathy gene candidates. Twenty-five of these are previously undescribed candidate vertebrate ciliary genes. Five of these genes predicted to have ciliary interaction  adult knockout female (n ≥ 7) and male (n ≥ 7) mice between 4 and 16 weeks of age and compared to contemporaneous data on age, sex , and genetic background (C57BL/6 N)-matched wildtype control mice. Strict ethical review and guidelines of accrediting authorities were followed by all International Mouse Phenotyping Consortium (IMPC) centers specific to their national and regional legislation and local institutional guidelines . The IMPC Consortium collects data from international member institutes who collect phenotyping data guided by their own ethical review panels, licenses, and accrediting bodies that reflect the national and/or geo-political constructs in which they operate. We have captured this data via an ethical and funding survey from each contributing institute, this can be found at http://www.mousephenotype.org:8858/wp-content/uploads/2020/02/EthicalInfo2014.pdf. A comprehensive overview of the IMPC mouse phenotyping protocol consistent with ARRIVE guidelines55 can be found at https://www.mousephenotype.org/about-impc/animal-welfare/arrive-guidelines/ and is published previously21.Detailed methods for mutant mouse production has been published previously56. Fertility testing of both sexes was performed at 8–14 weeks, renal morphology was assessed during necropsy at 16 weeks, and clinical chemistry testing was done on a serum sample collected prior to termination.A comprehensive ophthalmic examination was performed on both eyes of each mouse at 15–16 weeks of age by experienced technical staff using ocular imaging equipment overseen by lead site scientists and/or expert ophthalmologists. Examiners were trained to identify and annotate background lesions  commonly but inconsistently observed in the C57BL/6 N strain57. Irises were pharmacologically dilated with 2.5% phenylephrine HCl : 1% tropicamide  to facilitate eye examinations. A 0.1 mm slit beam at the highest intensity setting was used to evaluate the anterior segment including cornea, anterior chamber, and lens followed by assessment of the posterior segment including the vitreous chamber. The fundus was examined via indirect ophthalmoscopy using a 60 diopter double aspheric handheld lens .As described in prior publications from the IMPC, phenotyping of mice was performed in a randomized fashion and technicians were blinded to the genotype (mutant or wildtype) of mice before and during examinationOCT imaging was performed (machine dependent on institution), after dilatation with both tropicamide 1% and phenylephrine 2.5%. Thickness of the total retina, inner nuclear layer, outer nuclear layer, were manually measured at a distance of ~ 0.2 mm from each side of the optic nerve using calipers in ImageJ software. Measurements from both sides of the optic nerve head and between left and right eyes were averaged for each animal.g at 8 °C. Plasma samples were analyzed optimally on the day of collection in a clinical chemistry analyzer either Beckman Coulter AcT Diff, Siemens Advia 2120 or Hemavet Multispecies Hematology Analyzer HV950FS .Blood urea nitrogen and creatinine concentrations were analyzed on ~ 160–200 μl of plasma separated from whole blood collected by retro-orbital puncture in a gel tube containing lithium heparin after centrifugation for 10 min at 5000×56. Macroimages were captured of abnormal gross findings when possible. All fresh tissue samples collected at necropsy were immediately immersed in fixative  and prepared for histopathological examination by a veterinary pathologist. Mild to moderate focal retinal dysplasia observed by histopathology were considered incidental background strain changes attributed to C57BL/6 N background and were excluded from genotype-associated phenotype calls. Parasagittal sections of eyes were sectioned at 5 µm, and stained with hematoxylin–eosin (H&E). Retinal layer measurements were taken of total retina, nerve fiber layer, retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor outer segment, retinal pigmented epithelium, and choriod were manually measured at a distance of ~ 0.2 mm from each side of the optic nerve using calipers in NDP.view2. (U12388-01) .A complete necropsy was performed and all abnormal findings were recorded and annotated using the standardized IMPC Gross Pathology ontologyWhen possible at some centers with the requisite instrumentation, advanced imaging techniques were used to document suspected abnormalities for follow-up and evaluation. In these cases, mice were first anesthetized with an intraperitoneal injection of ketamine/midazolam (50–75/1–2 mg/kg) and their eyes dilated using topical tropicamide 1% and phenylephrine 2.5% drops and lubricated with artificial tears containing methylcellulose. Anterior segment and fundus images were acquired with a Micron III or IV retinal imaging microscope .58. Abnormal reproductive morphology was identified by query of the database for “abnormal reproductive morphology”. Abnormal reproductive function was identified by searching for “infertility” and its synonym “abnormal fertility/fecundity”.The IMPC website’s data release 11.0 was queried for five common abnormalities in human ciliopathy syndromes: abnormal ocular traits, abnormal renal morphology, abnormal renal function, abnormal reproductive morphology, and abnormal reproductive function. For “abnormal ocular traits” the term “abnormal ocular phenotypes” was used to search the database. Abnormal renal morphologies were identified by searching “abnormal kidney/renal phenotypes”. Abnormal renal function was identified by searching for “abnormal blood urea nitrogen (BUN)” or “abnormal blood creatinine (CRE)”, both commonly used lab values to assess kidney function in clinical practice59  with a search term  to determine previous association between the respective trait abnormalities. The search was further modified to determine whether a KO mouse had been previously generated and analyzed. For candidate ciliopathy genes, gene names and the search terms “cilia” or “ciliopathy” were queried for previous implication in ciliary biology. In addition, MalaCards63 database was queried for each gene; those with publications supporting disease associations are summarized in Table Potential ciliopathy genes that had a predicted protein interaction with known ciliopathy genes either directly or through a secondary protein were considered to be “candidate” ciliopathy genes. All candidate ciliopathy genes were individually queried  adult knockout female (n ≥ 7) and male (n ≥ 7) mice between 4 and 16 weeks of age and compared to contemporaneous data on age, sex , and genetic background (C57BL/6 N)-matched wildtype control mice. Detailed methods for mutant mouse production has been published previouslyStrict ethical review and guidelines of accrediting authorities were followed by all International Mouse Phenotyping Consortium (IMPC) centers specific to their national and regional legislation and local institutional guidelines .All authors have agreed to the publication of this article.Supplementary Information 1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


In the published article, there was an error in In the published article, there was an error in the Results section, SCIG Administration Characteristics, Paragraph 1. These sentences previously stated:A correction has been made to “The mean infusion flow rate was 23.86 mL/h/site and was likewise lower in children and adolescents (4.19–16.25 mL/h/site). As a general trend, the dose of octanorm per kg, duration of infusions, infusion volume, and infusion flow rate increased with age (Table 2).”The corrected sentences appear below:“The mean infusion flow rate was 22.86 mL/h/site and was likewise lower in children and adolescents (14.19–16.85 mL/h/site). As a general trend, the dose of octanorm per kg, duration of infusions, infusion volume, and infusion flow rate increased with age (Table 2).”the Results section, Infusion Site Reactions, Paragraph 1. This sentences previously stated:A correction has been made to “No localized site reactions were observed for three-quarters  of analyzed infusions.”The corrected sentence appears below:“No localized site reactions were observed for three-quarters  of analyzed infusions.”the Discussion section, Paragraph 8. This sentence previously stated:A correction has been made to “In the current study, 0.23% of infusions were associated with an infusion site reaction.”The corrected sentence appears below:“In the current study, 23% of infusions were associated with an infusion site reaction.”the Disclosure section, Paragraph 1. This sentence previously stated:A correction has been made to “RK reports… …grants from Vietnam National Children and Hospital Hanoi, Vietnam…”The corrected sentence appears below:“RK reports… …grants from Vietnam National Children’s Hospital Hanoi, Vietnam…”The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.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.


Beta-hydroxybutyrate (βOHB), along with acetoacetate and acetone, are liver-produced ketone bodies that are increased after fasting or prolonged exercise as an alternative fuel source to glucose. βOHB, as the main circulating ketone body, is not only a G-protein coupled receptor ligand but also a histone deacetylases inhibitor, prompting the reexamination of its role in health and disease. In this study, we compared the effects of two commercial βOHB formulations an enantiomer R βOHB and a racemic mixture ±βOHB on induced pluripotent stem cell cardiac myocytes (iPS-CMs) electrophysiology. Cardiac myocytes were cultured in R βOHB or ±βOHB for at least ten days after lactate selection. Flouvolt or Fluo-4 was used to assay iPS-CMs electrophysiology. We found that while both formulations increased the optical potential amplitude, R βOHB prolonged the action potential duration but ±βOHB shortened the action potential duration. Moreover, ±βOHB increased the peak calcium transient but R βOHB reduced the peak calcium transient. Co-culturing with glucose or fatty acids did not ameliorate the effects, suggesting that βOHB was more than a fuel source. The effect of βOHB on iPS-CMs electrophysiology is most likely stereoselective, and care must be taken to evaluate the role of exogenous βOHB in health and disease. Energy consumption in mammals is derived largely from the consumption of three macronutrients; protein, carbohydrates, and fat . During The three ketone bodies are beta-hydroxybutyrate (βOHB), acetoacetate, and acetone . βOHB anTraditionally, elevations in blood ketones have been viewed as pathogenic by the medical community because of their association with the diabetic ketoacidosis . HoweverCompared to the brain, which normally relies on glucose as its main energy source, the heart in contrast is an omnivore, capable of utilizing amino acids, glucose, fatty acids, lactate, and ketone bodies to meet its energy demand . While tA growing consensus is that the increase in ketone body oxidation is adaptive . As for βOHB is a chiral molecule existing as two enantiomers, R/d and S/l. The R/d enantiomer is the predominant enantiomer in circulation. In contrast, the S/l enantiomer is a short-lived intermediary created by the final round of fatty acid β-oxidation that is mostly converted into the R/d enantiomer . BecauseDuring normal human development, after birth, βOHB levels rapidly rise until they gradually decrease after the first two to three days of lactation . During Therefore, in this study, we decided to test the effects of two commercially available different βOHB formulations, a pure R enantiomer and a stereoisomer mix on induced human pluripotent stem cell-derived cardiac myocytes (iPS-CMs) electrophysiology. In addition to being a human cell system, iPS-CMs are phenotypically identical to neonatal cardiac myocytes, making them the ideal reductionist model to study human cardiac development. We tested the hypothesis that culturing iPS-CMs with βOHB will metabolically mature iPS-CMs electrophysiology.iPS-CMs were generated similarly to the protocol described by Burridge et al. . iPS celiPS-CMs were fixed in formalin for 10 min before being washed 3X with PBS. Cells were then permeabilized with PBS containing 0.25% Triton X-100. After permeabilization, cells were incubated in a blocking buffer  for 1 h before being incubated with anti-troponin I (MilliporeSigma), Anti-Sarcomeric Myosin , and anti-alpha actinin (MilliporeSigma) overnight according to manufacturer’s instructions. Coverslips were then washed 3X in PBS before being incubated with Donkey anti-Mouse Alexa Fluor 488 (Thermo Fisher) for an hour before being washed 3X in PBS and stained with Dapi (MilliporeSigma). Coverslips were then imaged using an EVOS cell imaging system.After being metabolically selected, iPS-CMs were cultured in one of the following 6 media for a minimum of 10 days. See 2PO4, 1 mM MgCl2, 5.5 mM Glucose, 5.4 mM KCl, 1.8 mM CaCl2, 15 mM HEPES) with a 1:1000 dilution of the FluoVolt dye (component A) and 1:100 of the PowerLoad (component B) for 25 min at room temperature before imaging. For calcium recordings, Fluo-4 was loaded at a concentration of 10 µM dissolved in DMSO and 10% pluronic F-127 in Tyrode’s solution for 20 min at 37 degrees, followed by a 20-min washout in Tyrode’s solution at room temperature.Voltage and calcium recordings were made using an IonOptix recording system, as described previously by Han et al. . For volAfter dye loading, iPS-CMs were placed on an inverted microscope, perfused with Tyrode’s solution, and paced at 3 Hz using field stimulation. A 40X objective was used to image iPS-CMs. Because both FluoVolt and Fluo-4 emission/excitation spectrum work with the standard FITC settings, dye-loaded cells were excited using the IonOptix’s HyperSwitch Light Source (75W xenon-arc bulb) and the DP-Fluo Package (Excitation/Emission). To avoid phototoxicity, neutral density filters were used to limit the intensity of the light, and recordings were limited to 5 s in duration. iPS-CMs fluorescence emission wavelengths were recorded using a photomultiplier tube.Voltage and Calcium transients were analyzed using IonOptix’s IonWizard software. Only cells that followed 1:1 pacing were included. For voltage recordings, the upstroke velocity, peak amplitude, and action potential durations of 30, 50, and 90 percent  were measured. For calcium recordings, peak calcium, halfwidth, and reuptake times were calculated.p-value < 0.008 was considered statistically significant.Data were presented as the mean ± SE. Unpaired t-tests with a Bonferroni correction were used to test for statistical significance. A Next, we cultured iPS-CMs in 6 different conditions. The base media used was RPMI 1640 with/without glucose. The control media refers to RPMI 1640 with glucose (11.1 mM). This also served as the base media for glucose-containing fatty acid and βOHB media. While 11.1 mM is considered to be prediabetic, it is an almost universally accepted iPS-CMs culture basal media . RPMI meTo test the effects of βOHB on iPS-CMs electrophysiology, we cultured cells without glucose but with 5.5 mM ±βOHB or R βOHB as its primary carbon source. This amount was chosen since it approximates the steady state level of βOHB during a prolonged fasting . BecauseAs shown in However, in We next quantified the changes in the action potential duration. Compared to the control conditions, fatty acids prolonged the APD 30 and APD 50 A,B. InteAfter examining the effects of βOHB on the optical action potentials, we then decided to examine its effects on the calcium transient. Similar to the optical action potentials, culturing iPS-CMs in different media results in different calcium transient waveforms. Compared to optical action potentials, calcium transients have a slower upstroke time and longer decay time. While both the fatty acid media and ±βOHB increased the calcium transient amplitude, the R βOHB decreased it A–E. ThisAfter quantifying the amplitudes, we next examined the effects of βOHB on the calcium transient kinetics. Compared to the control condition, ±βOHB reduced both the half-width, defined as the time between 50% time to peak and 50% time to baseline. ±βOHB also shortened the reuptake time, defined as a 90% return to baseline. This was observed regardless of whether glucose and/or fatty acids were present in the media. Conversely, R βOHB had the opposite effect, and increased both the half-width and the reuptake time, regardless of the cultural situation A,B.The main findings of this study are that the R enantiomer of βOHB alters iPS-CMs electrophysiology differently in contrast to a racemic mix . While bNormally, circulating levels of βOHB are in the micromolar range. However, after periods of fasting or prolonged exercise βOHB can rise to 7 mM, or in the case of diabetic ketoacidosis, 20 Mm . While tFor over a century, it has been known that nutritional ketosis could successfully reduce and even eliminate seizures in patients with drug-refractory epilepsy . A ketogCorreia et al. discovered that culturing iPS-CMs with fatty acids as their primary carbon source increased the action potential amplitude, a reduction in the calcium transient amplitude, and alterations in the decay of the fluorescence signal for both the voltage and calcium signal . AssociaIn this study, while we found that fatty acids increased the AP amplitude and prolonged the APD 30 and APD 50, they did not prolong the APD 90, which contradicts their previously published results . LikewisWe recorded voltage and calcium transients from iPS-CMs paced at 3 Hz, the upper limit of the fetal resting heart rate, while Hu and Correia recorded their transients in iPS-CMs paced at 1 Hz, the extreme low end of the fetal resting heart rate. Excitation-contraction kinetics are rate dependent, and therefore is the most probable explanation for the discrepancies. Regardless, it highlights a limitation with using iPS-CMs.Currently, there are no consensus on which protocol should be used to generate iPS cells, which iPS cell lines should be used for research, how many iPS cell lines should be used for a given study, which iPS base media should be used, which differentiation protocols should be used to generate iPS-CMs, which supplements should be included in the iPS-CMs media, how many independent differentiations should be used in a study, and at which time point after differentiation to use iPS-CMs. While consensus statements are emerging on how to use this technology, they are currently recommendations and not obligatory.Irrespective, the metabolic selection of cardiac myocytes appears to be highly reproducible. Tohyama et al. study demonstrating that cultures of 99% cTnT positive cells can be obtained by replacing glucose with lactate has been cited 494 times according to Scopus at the time of this publication . LikewisFirst, it suggests that the chirality of βOHB is important for its biological function in the heart. Second, βOHB may play a role in cardiac development. Third, it highlights the fact that the racemic formulation of βOHB matters. Currently, there is no consensus on which formulation of βOHB is utilized in scientific studies. Moreover, many studies do not use commercially available formulations but custom-synthesize their βOHB .Therefore, we conclude that the βOHB enantiomer may be key for understanding the role of βOHB in cardiac development and understanding possible discrepancies in the reproducibility of studies involving nutritional ketosis achieved via exogenous supplementation versus a ketogenic diet. Studying cell metabolism under different culture conditions is what we are going to explore next. Understanding this information will be more conducive to the use of ketone bodies to fully utilize their physiological functions.


This study investigates the effect of amiodarone on ablation outcomes in ARVC. (2) Methods: The study enrolled patients with ARVC undergoing CA of sustained VT. In all patients, substrate modification was performed to achieve non-inducible VT. The patients were categorized into two groups according to whether they had used amiodarone before CA. Baseline and electrophysiological characteristics, substrate, and outcomes were compared. (3) Results: A total of 72 ARVC patients were studied, including 29 (40.3%) “off” amiodarone and 43 (56.7%) “on” amiodarone. The scar area was similar between the two groups. Patients “off” amiodarone had smaller endocardial and epicardial areas with abnormal electrograms. Twenty of 43 patients (47.5%) “on” amiodarone discontinued it within 3 months after CA. During a mean follow-up period of 43.2 ± 29.5 months, higher VT recurrence was observed in patients “on” amiodarone. Patients “on” amiodarone who discontinued amiodarone after CA had a lower recurrence than those without. (4) Conclusions: Patients with ARVC “on” amiodarone before CA had distinct substrate characteristics and worse ablation outcomes than patients “off” amiodarone, especially in those who had used amiodarone continuously. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy, characterized by fibro-fatty infiltration of the right ventricle owing to mutations in the desmosomal proteins . ConsequFurthermore, a recent study demonstrated that concomitant amiodarone therapy may affect outcomes in patients with ischemic VT who are undergoing CA . The aboFrom 2013 to 2020, we retrospectively screened patients with ARVC who had undergone successful CA of drug-refractory VT in three medical centers . Based on the 2010 modified Task Force criteria, all patients met the diagnosis of definite ARVC . The indIn previous publications, we described details of an electrophysiological study, substrate mapping, and ablation strategies . After oThe RV epicardial bipolar scar and LVZ were defined as areas with a peak-to-peak bipolar voltage of <0.5 mV and 0.5–1 mV, respectively. The voltage maps were manually edited to avoid intracavitary points. An average bipolar or unipolar voltage was calculated. The scar, LVZ, and areas with abnormal electrograms  were assessed by using the standard surface area measurement tool on the navigation system . When muPatients were followed up at the first, third, and sixth months after CA, as well as every three–six months thereafter. ICD interrogation, ECG, and Holter monitoring were performed. Recurrent VA, defined as sustained VT or VT/VF, requires ICD interventions .t-test was used to compare continuous variables, while chi-square tests with or without Yate’s correction for continuity or Fisher’s exact tests were used for categorical variables, as indicated. A multivariate Cox proportional hazards regression analysis was performed with variables with a p-value of less than 0.2 in the univariate analysis for a hazard ratio to predict the VT/VF recurrence. We considered a p-value of 0.05 to be statistically significant. The statistical analyses were conducted using the Statistical Package for the Social Sciences, Version 22.0 .The continuous and categorical variables were expressed as mean ± standard deviation and percentage, respectively. The Student’s p = 0.027). Patients “off” amiodarone received more class Ic AAD medications (10 (34.5%) vs. 3 (7.0%); p = 0.004). In terms of the other parameters and Task Force criteria, there were no significant differences between the two groups.In total, we screened 106 patients who were diagnosed with definite ARVC and had received successful CA of VA . The anap = 0.001). The characteristics of the RV substrate are shown in p < 0.001; 211.2 ± 26.0 ms vs. 183.6 ± 24.2 ms, p = 0.002). In addition, the areas of abnormal electrograms within the endocardium and epicardium were significantly larger in patients “on” amiodarone . In the patients with amiodarone before CA, there was no significant difference in the substrate characteristics between patients with amiodarone durations of more than 6 months or not  patients, including 12 (41.4%) and 35 (81.4%) patients “off” and “on” amiodarone, respectively (iodarone  than thoiodarone . (Endo: s or not .p = 0.008). The mean cycle length of inducible VTs was 306.3 ± 66.1 ms. Of 112 inducible VTs, 61 VTs were mappable . Nine of the 61 mappable VT circuits were considered intramural because they lacked identifiable diastolic paths despite simultaneous epicardial and endocardial mapping. Three in the “off” amiodarone and six in the “on” amiodarone, (p = 0.731), all identified VT isthmuses and were correlated with areas with abnormal electrograms and abnormal substrates.During the electrophysiological study, 112 VTs (1.6 ± 0.8/patient) were induced, including 37 and 75 VTs in patients “off” and “on” amiodarone, respectively . In all patients, non-inducibility of VT was achieved following CA. There were no procedural complications.Each patient underwent ablation of the isthmus if mappable, and complete elimination of the endocardial and/or epicardial abnormal substrates. In patients “on” amiodarone, the total procedure duration and ablation time were significantly longer than in those “off” amiodarone  is the only independent predictor of VT/VF recurrence.After a follow-up period of 43.2 ± 29.5 months, two patients (2.5%) died of non-cardiovascular causes (pneumonia: 2), and 24 (33.3%) experienced VT/VF recurrences. The incidence of VT/VF recurrence was significantly higher in patients “on” amiodarone than those “off” amiodarone (19/43 (44.2%) vs. 5/29 (17.2%), log-rank According to our study, amiodarone use before CA in patients with ARVC is associated with a longer activation time and a greater area of abnormal electrograms within the RV endocardium and epicardium. Despite the similar ablation strategy and endpoints , VT recurrence was higher in patients “on” amiodarone before CA. Regardless of acute success, patients whose amiodarone use was discontinued 3 months after CA experienced fewer VT recurrences during subsequent follow-up. Amiodarone has been shown to prolong the total QRS duration and result in low amplitude signals in previous studies , which iIn contrast to the above-mentioned study regarding ischemic cardiomyopathy, we found that patients “on” amiodarone had larger areas of abnormal electrograms than those “off” amiodarone. Inconsistent findings as compared to ischemic substrates can be attributed to the following plausible explanations: (1) amiodarone prolongs conduction velocity heterogeneously in fatty-infiltrated myocytes and creates more abnormal electrograms in patients with ARVC; and (2) there is a diversity of disease progression and scarring in ARVCs, which could also contribute to nonuniform ablation outcomes. However, as a result of the study design, we cannot determine the cause-and-effect relationship between amiodarone use and the extent of abnormal electrogram extension in the present study. Further studies focusing on the effects of amiodarone on the diseased substrate in ARVC will be warranted. In this study, we found that patients who were “on” amiodarone had higher recurrences of VT than those who were “off” amiodarone before CA. These findings may be explained by the following reasons: First, a greater extent of diseased substrates has been observed in amiodarone users, suggesting a worse disease status and/or faster disease progression in these individuals. Previously, we found that the progression of the disease could also cause the expansion of abnormal substrates, which was linked to VT recurrences . The secTo the best of our knowledge, this is the first study to investigate the impact of amiodarone use before CA in patients with ARVC. Amiodarone use before CA can result in distinct substrate characteristics, such as longer activation times and larger areas with abnormal electrograms and was associated with worse ablation outcomes. According to the above findings, we suggest the physician withdraw amiodarone before CA if the patient can tolerate it. Despite the standard ablation strategies and endpoints, VT recurrences were still higher in patients without discontinuation, reflecting the importance of alternative strategies to assess the ablation endpoints in these patients who could not withdraw the amiodarone. Future studies are warranted to address this aspect. There were some limitations in the present study. This is a retrospective study in which only patients who had successfully undergone ablation were included. The prescription of amiodarone was based on the clinical physician’s discretion. In patients with ARVC before CA, it is difficult to standardize the use of AADs. Furthermore, the use of amiodarone after CA was also at the discretion of the physicians, which may have affected the present findings. In this retrospective study, it is difficult to determine whether the amiodarone or the need for amiodarone affects substrate characteristics and recurrence. Nevertheless, the bias can be minimized by multivariate analysis and collaboration between referral centers. Additionally, epicardial approaches were not performed in all patients. However, even with the above-mentioned differences, there are more patients “on” amiodarone who are undergoing epicardial ablation to achieve the same outcome. In this regard, we concluded that epicardial ablation was not the cause of the differences between the two groups. A prospective study is warranted to validate the impact of amiodarone on the ablation outcome in patients with definite ARVC.Despite similar ablation strategies and endpoints, amiodarone use before CA may be associated with a higher rate of VT recurrence in patients with ARVC. Higher VT recurrences were observed in ARVC patients who continued to take amiodarone despite the acute success.


To address the low Ca−induced growth inhibition of tomato plants, the mitigation effect of exogenous Si on tomato seedlings under low−Ca stress was investigated using different application methods. We specifically analyzed the effects of root application or foliar spraying of 1 mM Si on growth conditions, leaf photosynthetic properties, stomatal status, chlorophyll content, chlorophyll fluorescence, ATP activity and content, Calvin cycle−related enzymatic activity, and gene expression in tomato seedlings under low vs. adequate calcium conditions. We found that the low−Ca environment significantly affected (reduced) these parameters, resulting in growth limitation. Surprisingly, the application of 1 mM Si significantly increased plant height, stem diameter, and biomass accumulation, protected photosynthetic pigments, improved gas exchange, promoted ATP production, enhanced the activity of Calvin cycle key enzymes and expression of related genes, and ensured efficient photosynthesis to occur in plants under low−Ca conditions. Interestingly, when the same amount of Si was applied, the beneficial effects of Si were more pronounced under low−Ca conditions that under adequate Ca. We speculate that Si might promote the absorption and transport of calcium in plants. The effects of Si also differed depending on the application method; foliar spraying was better in alleviating photosynthetic inhibition in plants under low−Ca stress, whereas root application of Si significantly promoted root growth and development. Enhancing the photosynthetic capacity by foliar Si application is an effective strategy for ameliorating the growth inhibition of plants under low−Ca stress. Conversely, Si application effectively alleviated the decreases in the above parameters. In particular, we found that 0.1Ca + LSi treatment was significantly (p < 0.05) more effective than 0.1Ca + RSi treatment in alleviating Fv/Fm under low−Ca stress (p < 0.05) higher under low−Ca stress compared with those in CK, except for excess excitation energy [(1 − qP)/NPQ]; whereas, Si application significantly (p < 0.05) reduced the increasing trend of these parameters, with Si spraying being more effective (We found that the Fv′/Fm′ and Y(II) of tomato leaves were significantly  reduced by 48%, 32%, and 37%, and the Ca2+−ATPase activity was significantly (p < 0.05) reduced by 38%, 22%, and 28% after 0.1Ca, 0.1Ca + RSi, and 0.1Ca + LSi treatments, respectively, compared with those in CK. Low−Ca stress significantly (p < 0.05) reduced the activities of H+−ATPase and Ca2+−ATPase of tomato leaves, whereas Si application effectively alleviated these reductions; however, the alleviation effect of different Si application methods did not differ significantly (p < 0.05) reduced the ATP content, dropping to only 52% of that in CK after 0.1Ca treatment but was restored to the levels shown in CK at normal Ca levels after Si application  increased. The activities of Rubisco, TK, and Ru5PK in tomato leaves treated with CK + LSi were also significantly (p < 0.05) increased. However, the activities of Rubisco, RCA, SBPcase, TK, Ru5PK, NADP−GAPDH, FBPase, and PRK were significantly (p < 0.05) reduced in tomato leaves treated with 0.1Ca compared with those in CK (p < 0.05) increased after Si application compared with those after 0.1Ca treatment. We also found that the activities of SBPase after 0.1Ca + LSi treatment were significantly (p < 0.05) higher than those after 0.1Ca + RSi treatment, whereas those of TK and PRK were increased after root application of Si. These results indicate that low−Ca stress severely affected photosynthesis−related enzymatic activities, which were effectively alleviated after applying exogenous Si in plants under low−Ca stress. Moreover, we found that Si promoted photosynthetic carbon assimilation and alleviated growth inhibition in tomato plants under low−Ca stress by increasing the activities of Calvin cycle−related enzymes.We found that compared with CK, the activities of RCA and SBPcase in tomato leaves treated with CK + RSi and CK + LSi were significantly  reduced under low−Ca stress, with the gene expression of RCA showing the greatest decrease, as indicated by qRT−PCR. Conversely, the root application of Si significantly (p < 0.05) upregulated the expression of Rubisco and Ru5PK compared with that in CK (p < 0.05) upregulated the gene expression levels of Rubisco, RCA, SBPcase, TK, Ru5PK, NADP−GAPDH, and ATP. We concluded that the increased expression of genes related to the regulation of the activity of key photosynthetic enzymes after Si treatment might be one of the main reasons for enhanced photosynthesis.We found that compared with CK, CK + LSi treatment significantly increased the expression of related at in CK a,e. SimiCa is involved in many processes of plant growth, including the effective maintenance of plant cell structure . As Ca i2, the raw material for photosynthesis, is essential for facilitating the exchange of gases between the above−ground parts of the plant and the atmosphere [2+ [Chlorophyll is essential for converting light energy to chemical energy in plants, and its reduction limits the photosynthetic process . In addimosphere . Therefohere [2+ , which hhere [2+ . In our here [2+ ,58. In t2+ signaling pathway has been suggested to regulate the lutein cycle−dependent NPQ [2+ is also involved in forming activation sites as PSII−related oxidoreductase cofactors [Chlorophyll fluorescence parameters respond to the absorption, conversion, transfer, and distribution of light energy by plants and can be used to monitor the effects of environmental factors on plant photosynthesis. Fluorescence parameters, such as Y(II), Fv/Fm, Fv′/Fm′, qP, Y(NPQ), NPQ, 1 − qP/qN, and 1 − qP can accurately reflect the intensity of photosynthesis . The Ca2dent NPQ . Ca2+ isofactors , while tofactors . In our ofactors . Si protofactors ,62. In oofactors ,56,63.+ reduction, and forming ATP through photosynthetic phosphorylation, finally converting electrical energy into active chemical energy. H+−ATPase, which is indispensable for photosynthesis, is an ATP−driven proton pump that couples ATP hydrolysis with the generation of transmembrane electrochemical proton gradients [2+ enters the cytoplasm of endodermal cells through Ca−permeable channels on the cortical side of the Casparian band, requiring cytoplasmic Ca2+−ATPase or intracellular Ca2+/H+−antibody pumping from the cytoplasm, and by regulating the expression and activity of these transporter proteins, Ca2+ can be selectively transported to the xylem at a rate consistent with above−ground requirements [2+ in chloroplasts regulates the photosynthetic pathway and is the main source of energy supply for plant cells [2+−ATPase and H+−ATPase, as well as the ATP content, were closely related to Ca2+. Si’s alleviation of low calcium stress may be related to the promotion of calcium absorption and conversion by Si. The activities of Ca2+−ATPase and H+−ATPase were significantly reduced in tomato plants under low−Ca stress  and disinfected them with 2.5% sodium hypochlorite. We then soaked them in warm water for 6 h and left them to germinate in a dark incubator at 28 °C . Subsequently, uniformly germinated seeds were selected and sown in hole trays containing vermiculite. When both cotyledons of tomatoes were fully expanded, healthy, uniformly grown seedlings were selected and fixed in 1.5−L black plastic hydroponic boxes (2 plants per box). Seedlings were incubated for 7 d each with 1/8 and 1/4 times of Hoagland nutrient solution  was used as the Si source, Ca(NO3)2·4H2O  was used as the Ca source, and the missing N during Ca deficiency treatment was compensated with NH4NO3 . The experiment was conducted in an artificial climate chamber at 28/18 °C (day/night), 20,000 Lx light for 12 h, and 75% humidity. The nutrient solution was replaced every 3 d, and the pH was adjusted to 6.0 ± 0.2 with dilute hydrochloric acid.We selected uniform and full tomato seeds  for both spraying and root application. For root application treatment, sodium silicate was dissolved in the nutrient solution each time it was renewed. For foliar spraying treatment, 1 mM sodium silicate solution  was evenly sprayed on the front and back of leaves until saturation at 10 pm each night; distilled water was sprayed in the same way for other treatments. Based on the morphological characteristics of tomato seedlings, 1.0 mM Si was found to be the most effective in relieving moderate−Ca stress in tomato seedlings .Based on the screening results of experiments 1 and 2, and with reference to the root and spray application of experiment 2, four treatments were set up in experiment 3: (1) 4 mM Ca in Hoagland nutrient solution + root application of 1 mM Si (CK + RSi); (2) 4 mM Ca in Hoagland nutrient solution + foliar application of 1 mM Si (CK + LSi); (3) 4 mM Ca in Hoagland nutrient solution (CK); (4) 0.1 mM Ca in Hoagland nutrient solution (0.1Ca); (5) 0.1 mM Ca in Hoagland nutrient solution + root application of 1 mM Si (0.1Ca + RSi) (6) 0.1 mM Ca in Hoagland nutrient solution + foliar application of 1 mM Si (0.1Ca + LSi). Plant morphological indicators were measured after 7 d of treatment.Three plants were randomly sampled from each treatment, photographed, and their respective growth conditions recorded. The plant height  and stem diameter (at 1 cm from the root) of seedlings were measured using a straightedge and vernier caliper, respectively. Tomato seedlings were cut from the rhizome junction, weighed for above− and below−ground fresh weight (FW), then placed in an oven at 105 °C for 15 min, dried at 75 °C to constant weight, and weighed again for above− and below−ground dry weight (DW) .Fully developed functional leaves were selected, weighed (FW), completely immersed in a self−sealing bag filled with distilled water, and fully hydrated for 24 h. Then, after drying the excess water on the surface with absorbent paper, their post−hydration weight (TW) was obtained. Finally, the leaves were placed in an oven, dried at 70 °C to a constant weight, and weighed again (DW) .Leaf relative water content was calculated using Equation (1):Tomato roots from each treatment were scanned using an EPSON expression 11000XL scanner , and scanned photos were analyzed using the Win RHIZO 5.0 software  to obtain the total root length, root surface area, root volume, average root diameter, and the number of root tips and forks.Chlorophyll content was determined by spectrophotometry after 95 % ethanol extraction. Absorbance was measured at 665, 649, and 470 nm according to Anwar’s method .Under steady light intensity from 09:00 to 11:00 a.m., functional leaves of tomato seedlings at the same height were selected, and the gas exchange parameters of tomato leaves, such as Pn, Gs, Tr, and Ci, were measured using a Ciras−2 portable photosynthesizer .After incubation in the dark for 30 min, functional tomato leaves of uniform length were selected, and the Fv/Fm was calculated by measuring the initial fluorescence (Fo), maximum fluorescence (Fm), steady−state fluorescence (Fs), maximum fluorescence yield (Fm′), and initial fluorescence (Fo′) in the light using an IMAPING−PAM modulated fluorometer . Y(II) of PSII, qP , and Y for 30 min each. After drying, samples were sprayed with a layer of metal powder, and the stomata were observed and photographed using a scanning electron microscope  [Tomato leaves were cut into small slices (5 mm × 5 mm) (avoiding the main leaf veins) and placed in 10−mL centrifuge tubes, fully submerged in 4% glutaraldehyde fixative  for 2 h, and then rinsed with phosphate buffer  at 10 min intervals for 40 min. Subsequently, leaves were rinsed with a graded series of ethanol solutions  for 20 min each, soaked in 75% ethanol for 12 h, then dehydrated using another graded series of ethanol solutions  for 20 min each, and finally transferred thrice to 100% ethanol for 30 min each. After dehydration, the ethanol was removed from samples using a graded series of +−ATPase was determined using an H+−ATPase ELISA kit  and Ca2+−ATPase was determined using a Ca2+−ATPase activity kit .The ATP content of tomato leaves was determined using an ATP content determination kit ; HThe activities of Calvin cycle key photosynthetic enzymes, RuBPCase , FBPase, SBPase, NADP−GAPDH, TK, RCA, PRK, and Ru5PK were determined using ELISA kits  according to the manufacturer’s instructions.® 96 Real−Time PCR System  using SYBR Primer Ex TaqTM II . Reaction conditions were as follows: 40 cycles at 95 °C for 15 min, 95 °C for 10 s, 60 °C for 30 s, and 55 °C for 10 s. Relative gene expression was calculated using the 2−∆∆CQ method [Total RNA was extracted from tomato leaves using an RNA extraction kit . cDNA was obtained by reverse transcription using the Fastking RT kit ; corresponding primers were designed according to the CDS sequences of each gene in the tomato genome of Ensembl Plant using Primer 5.0 . All PCRQ method .To avoid sampling chance errors, the number of replicates was set to ≥3 for all experiments. The obtained data were analyzed by one−way ANOVA using the SPSS software . Graphical illustrations were created using Microsoft PowerPoint 2010, Origin Pro.9.0 , and Adobe Illustrator 2020.Studies have shown that an environment of lower Ca concentration causes serious harm to plant growth, which is visually manifested as growth restriction. In particular, Ca deficiency reduces chlorophyll content, affects chlorophyll fluorescence, limits stomatal opening, reduces Calvin cycle−related enzymatic activity, and inhibits the expression of photosynthesis−related genes. Si application improved growth indexes, such as stem diameter, dry and fresh weight, increased photosynthetic pigment content, improved gas exchange conditions, reduced excess light energy dissipation, promoted photosynthetic phosphorylation for the conversion of electrical potential energy into ATP, enhanced Calvin cycle−related enzymatic activities such as those of rubisco, SBPcase, and FBPcase, and promoted root growth and the expression of photosynthesis−related genes, hence protecting the photosynthetic system and alleviating the low−Ca stress−induced inhibition of plant growth. Most importantly, the beneficial effects of Si on the activities of enzymes related to root development and leaf photosynthesis in tomato seedlings were greater under low calcium stress than under normal conditions. We speculate that Si may play a certain role in promoting the absorption and transport of calcium in plants to alleviate the effect of low calcium stress on tomato growth. We will study this further in future work to clarify the exact mechanisms underlying Si’s effects. Foliar spray application of Si significantly enhanced plant photosynthetic performance, whereas root application of Si promoted lateral root development. These results provided insights for the application of exogenous Si in the practical cultivation of plants in calcium−deficient environments.