Gastric and esophageal cancers are malignant diseases with rising importance in Western countries. To improve oncologic outcome after surgery, it is essential to understand the relevance of germline mutations. The aim of the study was to identify and distinguish clinically relevant single-nucleotide polymorphisms (SNPs).In total, 190 patients with curative oncological resections of gastric and distal esophageal adenocarcinomas at Heidelberg University Hospital were eligible for this study. Outcome differences were determined for each SNP by analysis of clinical variables, survival, and mRNA expression levels.MGMT, p = 0.045) and rs9972882  were independent and significant survival predictors along with R status and pT/pN category. Group TT of rs12268840 had the highest rate of second primary carcinoma , lowest expression of MGMT based on cis-eQTL analysis in normal gastroesophageal tissue (p = 1.99 × 10−17), and worst oncologic outcome. Group AA of rs9972882 had the highest rate of distant metastases pM1 , highest expression of PGAP3 (p = 1.29 × 10−15), and worst oncologic outcome.Significant survival differences were found on univariate analysis for usual prognostic variables (such as pTNM) and for six SNPs. On multivariate survival analysis, the SNPs rs12268840  and malfunction of GPI anchoring (PGAP3) are distinct mechanisms that are relevant for tumor progression and relapse.Two intron variant SNPs of The online version contains supplementary material available at 10.1245/s10434-021-10771-y. Preexisting GERD leads to a 6.2-fold risk for GEJ,3 while antireflux therapies, physical activity, breastfeeding, nonsteroidal antiinflammatory drugs (NSAIDs), and HMG-CoA reductase inhibitors (statins) are inversely associated risk factors. Similar risk factors could be identified for gastric cancer (GC) as GERD bears a two- to fourfold risk for GC.5 Furthermore, it is known that chronic infection with Heliobacter pylori is another risk for GC.Cancers of the stomach and esophagus are devastating diseases accounting for nearly 9% of all malignancies.6 Most studies utilize high case numbers and hundreds of thousands of SNPs, but do not address specific clinical parameters other than the rough differentiation between case and control groups. However, it is necessary to establish ties between genotyped data and oncological outcome by making use of systematic follow-up on a well-differentiated cohort. While the number of allegedly relevant SNPs increases, it becomes harder to maintain an overview of those SNPs, which might also have a clinical significance and lead back to molecular pathways that are actually relevant. By correlating genotyped and imputed data with oncologic outcome parameters, we aim to identify clinically relevant loci.Genome-wide genotyping is a frequently used technique that has been widely practiced in the past. Recently, various single-nucleotide polymorphisms (SNPs) were identified that associate with either GERD, BE, GEJ, or GC.Eligible subjects of this study were patients with GC or GEJ who underwent oncological resection of gastric or esophageal cancer between 2008 and 2017 at Heidelberg University Hospital, Department of General Surgery. All patients provided written consent for data collection and analysis. Patients were excluded who deceased during the early postoperative phase or in-hospital as oncologic outcome was assessed. The trial protocol was approved by the ethics committee at the University of Heidelberg and was performed in accordance with the Declaration of Helsinki and Good Clinical Practices as well as local ethics and legal requirements.Prior to oncological resection, genomic DNA was extracted from peripheral blood leukocytes. Genotyping was performed by collaborating partners at the Department of Human Genetics, University of Bonn, Germany. It was accomplished for all patients using Infinium OmniExpress, Infinium OmniExpressExome, and Infinium Omni2.5Exome BeadChips (by Illumina) according to the manufacturer’s protocol at the University of Bonn. The overlap of the SNP content was subjected to initial quality control performed with PLINK v1.90b6.6. Samples with genotype call rate less than 97%, discrepancies in sex, divergent ancestry from the CEU HapMap 2010 population, and related samples were excluded from further analysis.All unambiguous SNPs with SNP call rate >95%, minor allele frequency (MAF) >1%, and Hardy–Weinberg equilibrium (HWE) > 0.001 were used for imputation. Imputation was performed with IMPUTE2 based on 1,000 Genomes Phase 3 as reference panel. As post-imputation quality control, we excluded all variants with an information score (INFO-score) < 0.8, HWE < 0.001, MAF < 1%, and SNP missing rate > 5% for best-guessed genotypes at posterior probability greater than 0.9 from further analysis.7 In-hospital mortality and postoperative complications were recorded during hospital course. To acquire long-term survival data, all patients were systematically followed up via continuous surveys. The relevant survival data for this patient collective were gathered until October 2020.In all cases, the diagnosis of gastric and esophageal adenocarcinoma was histopathologically confirmed and the resected specimens were analyzed at the Department of Pathology, University of Heidelberg. The histopathological staging was performed according to the 8th UICC edition, including TNM categories, R status, and grading.α = 0.05 was generally considered as statistically significant. For multivariate survival analysis, we established a Cox proportional hazards model with a level of significance of α < 0.2. All clinical and SNP parameters from the univariate studies with a p-value < 0.2 were included. The preoperatively assessed cTNM categories were excluded since a precise histopathology was available via the final pTNM histology.We extracted the genotype information from the initial PLINK files by using Gtool v0.7.5. The data were collocated using Microsoft Office Excel, and statistical tests as well as plots were calculated thereafter via GraphPad Prism 9 for Mac OS X. We defined overall survival from time of first diagnosis until death or until the most recent follow-up using the Kaplan–Meier method. Differences in survival were analyzed via Mantel–Cox (logrank) comparisons on STATA/SE 15.0 for Mac. A level of significance of n = 122, 42%), “esophageal neoplasm” , “gastroesophageal reflux” , or “Barrett’s esophagus”  showed a total result of 293 original articles. All reported SNPs were searched, and those that were available in the genotyped and imputed datasets were further analyzed.We performed a MEDLINE search via PubMed for SNPs that were reported as being responsible for or associated with BE, GERD, GEJ, or GC in previous genome-wide association studies. Search items varied and included terms such as “genome wide association study,” “gastric cancer,” “esophageal cancer,” “gastroesophageal reflux,” “Barrett’s esophagus,” “SNP,” and others. As of January 2021, a MeSH search with terms “genome wide association study” in combination with either “gastric neoplasm” . All local genes +/− 1 Mb of the selected SNPs were extracted based on data provided by the GENCODE project. We retrieved the summary statistics of linear regression results via GTEx eQTL Calculator and generated violin plots via GTEx eQTL Dashboard with layout modifications via Adobe Illustrator CC 2018 (Version 22.0.0).For cis expression quantitative trait loci (cis-eQTL) analyses in normal tissue, we accessed the Genotype-Tissue Expression (GTEx) database . GEJ patients had a higher mean BMI of 26.9 kg/m2 compared with GC patients . Besides neoadjuvant therapy , there were no significant differences between GEJ and GC patients regarding usual survival predictors. The results are demonstrated in Table p = 0.9417) and disease-free survival (p = 0.3893). The corresponding Kaplan–Meier curves are demonstrated in Supplementary Fig. 1a, b.In total, 61 out of 190 patients (32.1%) were diagnosed with GEJ (Siewert type I–III) and 129 patients (67.9%) had GC. Among GEJ patients, 82.0% were male, as opposed to 58.1% of GC patients (p = 0.0045), cN (p = 0.0079), and cM category (p < 0.0001) were significant predictors of survival. Neoadjuvant therapy (p = 0.0229) and type of operation (p < 0.0001) were also clinical variables that were shown to have statistical significance. Finally, the histopathological parameters pT (p < 0.0001), pN (p < 0.0001), pM (p < 0.0001), and R status (p < 0.0001) were significant for outcome. All results are summarized in Supplementary Table 1A,B.Median follow-up duration was 49.08 months (73.25 months for living cases and 20.74 months for deceased cases). The preoperative variables cT . Six SNPs showed significant differences on univariate overall survival (OS) analysis. The SNP IDs, genetic information, and median survival time (in months) are summarized in Table MGMT), 101 patients were genotyped as CC with a median OS of 62.1 months, 65 patients as TC , and 23 patients as TT with a median OS of 29.7 months . For rs9972882, 14 patients were genotyped as AA with a median OS of 20.3 months, 65 patients as AC with a median OS of 85.6 months, and 111 patients as CC with a median OS of 57 months  compared with groups AC  and CC , though not significant (p = 0.0675). Likewise, there were no significant differences in disease-free survival curves of groups TT , TC , and CC  of SNP rs12268840, with none of the groups showing any outstanding tendency according to the logrank test (p = 0.5625).Figure p = 0.020) and pN category (p = 0.001) as well as R status (p = 0.006) were statistically significant as well as the SNPs rs12268840 (p = 0.033) and rs9972882 (p = 0.041). In a confirmative Cox regression model, the parameters pT category (p = 0.009), pN category (p < 0.001), R status (p < 0.001), rs12268840 (p = 0.045), and rs9972882 (p = 0.030) were significant survival predictors.To further evaluate the significance of the identified SNPs, a multivariate regression analysis was performed including the clinical prognostic factors Table . The hisp = 0.4966), pN (p = 0.1675), and pM category (p = 0.5984) or the R status (p = 0.6419) were not different between the three groups CC, TC, and TT for SNP rs12268840. However, we found that rates of second primary carcinomas (SPC) were significantly higher in the TT group (average rate 30.4%) as compared with 9.2% in the TC group and 4.0% in the CC group (p = 0.0003). Furthermore, the three groups differed significantly in preoperatively detected Laurén type (p = 0.0243).Prognostic parameters such as the pT (p = 0.0117). The remaining histological parameters pT (p = 0.1029) and pN category (p = 0.1226) as well as the R status (p = 0.6976) were not significantly different between the groups, either. All results for SNPs rs12268840 and rs9972882 are summarized in Supplementary Table 3A,B.In total, 42.9% of patients in group AA of SNP rs9972882 had distant metastases (pM1) on final histopathological workup as opposed to 12.3% in group AC and 13.5% in group CC  analysis for all local genes in normal gastric and esophageal tissue . The results are presented in Supplementary Table 4A,B.MGMT in all upper gastrointestinal tissue types. This is supported by the normalized effect size (NES) of −0.60 in gastroesophageal junction tissue (p = 1.5 × 10−23), meaning that the alternative allele  leads to a significantly lower expression of MGMT as compared with the reference allele C. Likewise, in esophageal mucosa NES was −0.64 (p = 3.5 × 10−35), in esophageal muscularis −0.52 (p = 3.0 × 10−28), and in gastric tissue −0.56 (p = 2.0 × 10−17) , in esophageal muscularis −0.17 (p = 3.4 × 10−11), and in gastric tissue −0.23 (p = 1.3 × 10−15). In all normal upper gastroesophageal tissue types, rs9972882 did not prove to be a significant eQTL for STARD3 and ERBB2 (Her2/neu) . NES in gastroesophageal junction tissue was −0.15 , highest mRNA expression of PGAP3, and also worst outcome. The results are consistent with previous works and open a new perspective on clinically relevant germline SNPs that could directly affect carcinogenesis and tumor relapse in upper gastrointestinal cancer.In this study we retrieved SNPs from the current literature that were reported to be associated with either BE, GERD, GEJ, or GC. We then analyzed theses SNPs via genotyped and imputed data of curatively resected patients at our medical center. We were able to show that two intron variant SNPs of 8 The authors analyzed 11 SNPs that were previously identified as high-evidence genetic susceptibility markers for GC in a former meta-analysis.9 Sung et al. were not able to find significant associations of these 11 SNPs with overall survival. However, the authors found subtype-specific associations for GC of the cardia region and diffuse-type GC.The SNPs that were selected by this literature-based approach have been identified as relevant for esophageal and gastric adenocarcinoma according to previous genome-wide association studies (GWAS) on mostly large collectives. Similar attempts of correlating SNPs with clinical outcome parameters were made by Sung et al. in 2017.MGMT) and rs9972882 (intron variant of STARD3), were significant on multivariate analysis and will therefore be further discussed. According to the Cox regression model, both SNPs were independent survival predictors next to pT/pN categories and R status.In our study, we confirmed usual prognostic factors such as pTNM category and R status on univariate survival analysis and all variables (except pM category) on multivariate analysis. Based on these results, we assume that this study’s validity is ensured concerning the identification of further survival predictors. Only two SNPs, namely rs12268840 (intron variant of MGMT that encodes for the O-6-methylguanine-DNA methyltransferase (abbreviated as MGMT). DNA methylation is one of the most important types of epigenetic modification and plays a crucial role in carcinogenesis.10 MGMT is a DNA-repair enzyme that protects cells from carcinogenic effects by removing DNA adducts of alkylating agents from the O-6 position of guanine.11 Thus, studies have shown that loss of MGMT leads to an increased carcinogenic risk in mice.12 For gastric cancer, Zhang et al. were able to demonstrate that MGMT silencing via promoter hypermethylation was significantly associated with an increased risk of GC.13 Other works have shown that reduced expression of MGMT in esophageal adenocarcinoma organoids led to sensitivity towards temozolomide and taxane agents.14 The study by Doecke et al. that initiated the inclusion of rs12268840 to this literature-based approach found that homozygous carriers of rs12268840  with frequent acid reflux had a significantly higher risk of GEJ.15SNP rs12268840 is located on chromosome 10 and is known as an intron variant of 16 Patients in group TT had the highest rate of SPC, while patients in group TC and CC had lower rates of SPC and significantly higher median OS. We interpret these results as group TT possibly having had a lack of MGMT to fight malignant diseases in the past medical history as well as in the postoperative follow-up. This may have led to a worse oncologic outcome in general.Our cis-eQTL analysis has shown that, in upper gastrointestinal tissues of healthy probands, group TT expressed the least MGMT mRNA in comparison with groups CC and CT Fig. a. FurtheSTARD3, it proved to be a significant cis-eQTL for PGAP3 according to mRNA expression in normal upper gastrointestinal tissue. In contrast, rs9972882 was not a significant eQTL for neighboring genes STARD3 or ERBB2 (Her2/neu).Although SNP rs9972882 most likely qualifies as an intron variant of PGAP3 transcribes the post-GPI attachment to proteins phospholipase 3, which is an enzyme for fatty acid remodeling of glycosylphosphatidylinositol (GPI)-anchored proteins (abbreviated as GPI-APs). GPI-APs are glycoproteins that are anchored to the outer layer of the plasma membrane and therefore exposed on the cell surface. Precisely, PGAP3 is responsible for the removal of unsaturated fatty acids at the sn-2 position of GPI-APs. Germline mutations of PGAP3 lead to a subtype of hyperphosphatemia and intellectual disabilities, also known as Mabry syndrome type 4.17 Kwon et al.18 have found that PGAP3 is co-amplified  with ERBB2(Her2/neu) in esophageal and gastric cancer. Murakami et al.19 were able to show that PGAP3 knockout mice had enhanced responses to alloreactive and antigen-specific stimuli. It is clear that GPI-APs play important roles in many biological processes such as signal transduction and cell–cell interaction. Our data could be interpreted that overexpression of PGAP3 within group AA of rs9972882 represents a malfunctioning of GPI anchoring.20 The differentiation of molecular subtypes could be especially relevant for the prediction of potential susceptibility for (neo-)adjuvant treatment.21 So far, the CIN subtype was encountered more frequently in the gastroesophageal junction and cardia,22 which was also the most frequent anatomic location of gastric adenocarcinoma in this study group. Due to its role as a DNA-repair enzyme, MGMT and its dysfunction may represent another aspect of the CIN subtype demonstrated by TCGA. However, tumor tissue and sequencing patterns would be necessary to properly investigate the effect of MGMT expression  on actual prognosis for the separate TCGA subtypes. For this reason, genomic and tumor-specific data analysis will be necessary to infer that these results are generally applicable to all gastroesophageal cancer subtypes.The classification of gastric cancer by The Cancer Genome Atlas (TCGA) Research Network into Epstein–Barr virus (EBV)-positive, microsatellite instability (MSI)-high, genomically stable (GS), and chromosomally instable (CIN) subtypes has drawn attention to a general molecular differentiation of gastric cancer.23 This circumstance may also be in accordance with the insights by TCGA that distant esophageal adenocarcinoma and chromosomally unstable gastric adenocarcinoma (the most frequent TCGA subtype of gastric cancer) share many genomic amplifications and could possibly be considered a single disease entity.24This study is based on a data collection of numerous clinical variables to enable a profound analysis of essential prognostic factors accompanied by reliable survival data from a systematic follow-up. Moreover, our analyses allow a linkage and translational approach to investigate histopathological, oncological, and genotyped features of upper gastrointestinal tumors. The ultimate goal of this study was to identify those genetic alterations that actually have a clinical implication in terms of a significantly different prognosis. However, it is necessary to remark that there may be a potential selection bias concerning those SNPs that could be of interest but were not investigated due to missing reports in the literature. Another limitation of this study is that both GEJ and GC were analyzed in the same collective in terms of testing for specific SNP markers. However, we were able to prove beforehand that both groups did not differ in decisive outcome parameters. Last but not least, the separate analysis of both groups GEJ and GC did not show any significant differences, neither for overall nor for disease-free survival. Previous works by our own group have demonstrated the comparability of both cancer entities regarding long-term oncological outcome.MGMT and PGAP3.By correlating the above-mentioned SNPs with our clinical data, we identified two clinically relevant SNPs. Our data suggest that the DNA-repair enzyme MGMT is relevant in gastroesophageal cancer and that PGAP3 is a potentially novel agent in carcinogenesis. In the future, it will be necessary to reproduce these results for larger and more homogeneous samples. Eventually, in vitro studies are required to comprehend the molecular mechanisms of Supplementary file1 (JPG 211 kb)Supplementary file2 (JPG 375 kb)Supplementary file3 (XLSX 63 kb)Below is the link to the electronic supplementary material.


The results showed that NNSs significantly suppressed the planktonic growth, acid production, and biofilm formation of S. mutans or S. sanguinis compared with sucrose in single-species cultures. Additionally, decreased S. mutans/S. sanguinis ratio, less EPS generation, and higher pH value were observed in dual-species and saliva-derived multispecies biofilms with supplementary NNSs. Collectively, this study demonstrates that NNSs inhibit the cariogenic potential of biofilms by maintaining microbial equilibrium, thus having a promising prospect as anticaries agents.Nonnutritive sweeteners (NNSs) are sugar substitutes widely used to reduce the negative health effects of excessive sugar consumption. Dental caries, one of the most prevalent chronic diseases globally, results from a pathogenic biofilm with microecological imbalance and frequent exposure to sugars. Some research has shown that certain NNSs possess less cariogenic potential than sucrose, indicating their putative effect on oral microbiome. To uncover the alterations of acidogenic pathogens and alkali-generating commensals, as well as the biofilm cariogenic potential under the influence of NNSs, we selected four common NNSs  and established single-, dual-, and multispecies  Sugar is the generic name for sweet-tasting, soluble carbohydrates. For most people, “sugar” refers to the granulated household flavoring to enhance sweetness, whose main ingredient is sucrose, one of the most commonly consumed sugars in human diet . AlthougStreptococcus mutans and Lactobacillus spp.) and alkali-generating commensals  in dental plaque are closely associated with the initiation and development of caries [Dental caries is one of the most prevalent chronic diseases worldwide, characterized by local destruction of dental hard tissues. It is a multifactorial disease that occurs in the presence of a pathogenic biofilm and frequent exposure to sugars . Considef caries , 7.in vitro biofilm models established on glass beads or enamel slabs, with one or more bacteria strains inoculated [Sugar substitutes have been identified to contribute in caries prevention, as many of them cannot be utilized efficiently by oral bacteria to produce acids . Certainoculated –11. Howeoculated .S. mutans and S. sanguinis after they were cultured alone or in dual- and multispecies biofilm models and comparatively evaluated the results of NNSs groups with reference to sucrose of the same sweetness.This study is aimed to assess the effect of four NNSs  on the cariogenic potential of biofilms, which are extensively applied and considered safe for consumption by the United States Food and Drug Administration (FDA) . We measBrain Heart Infusion (BHI) was purchased from BD Difco (USA). Acesulfame-K, crystal violet (CV), Tris hydrochloride (Tris-HCl), Ethylenediaminetetraacetic acid (EDTA), and lysozyme were purchased from Sigma (USA). Saccharin, sucralose, sodium dodecyl sulfate (SDS), and formamide were purchased from J&K scientific (China). Aspartame was purchased from APExBIO (USA). Alexa Fluor 647-labeled dextran conjugate, SYTO 9 green fluorescent nucleic acid stain (485/498 nm), and phosphate-buffered saline  were purchased from Thermo Fisher Scientific (USA).S. mutans UA159 and S. sanguinis ATCC10556 were commercially obtained from the American Type Culture Collection (ATCC). S. mutans and S. sanguinis were grown at 37°C and 5% CO2 in BHI medium. They were cultured overnight and adjusted to optical density (OD) 0.5-0.6 at 600 nm (1 × 108 CFU/ml based on the OD600nm versus CFU/ml curve of each strain) and further 1 : 100 diluted in the growth medium for inoculation.The liquid medium containing one of the NNSs  was prepared to a concentration which has the same sweetness degree as sucrose according to information published by FDA . For plaS. mutans and S. sanguinis were inoculated separately in six sterile 15 ml centrifuge tubes, each of them containing 5 ml different growth media . After incubation at 37°C and 5% CO2 for 24 h, we suspended the cultures and measured their OD600nm using Nanodrop One  with cuvettes (light path = 10 mm). The cultures were then recollected, centrifuged at 5000 g for 10 min to remove the precipitation. The pH values of culture supernatants were then recorded by pH electrode InLab Expert Pro .Overnight cultures of S. mutans or S. sanguinis grown in media containing different NNSs was observed after CV staining. Overnight cultures of each strain were inoculated in a 96-well microtiter plate, each well containing 200 μl medium . After cultivation for 24 h, we aspirated the culture supernatant, washed each well three times with PBS, fixed the biofilms with methanol for 15 min, and stained with 0.1% (w/v) CV for 10 min. The redundant CV was removed by PBS rinses until the rinse solution appeared colorless. The images of the wells were captured by a stereo microscope . The biofilms were then destained by adding 200 μl 33% acetic acid to each well and shaking the plate at 37°C (100 r/min) for 30 min. The destaining solution of each well was added to another blank 96-well microtiter plate before the OD595nm value was measured. The experiment was carried out with triplicate samples and replicated three times, with data normalized by the blank wells.The biofilm formation of μl sterile saliva to each well and incubated the plate at 37°C for 2 h to coat the glass coverslips. After the saliva was aspirated, the growth medium was added into each well. . Overnight cultures of S. mutans and S. sanguinis were inoculated on saliva-coated glass coverslips (inoculum ratio = 1 : 1).Dual-species biofilm model was established as described previously . Whole uS. mutans and the saliva-derived multispecies bacteria were resuspended into BHI to OD600nm of 0.5-0.6, respectively, and mixed in a 1 : 10 volume ratio. The mixtures were then 1 : 100 diluted in fresh BHI and added to a 24-well plate with sterile saliva-coated glass coverslips.Saliva-derived multispecies biofilm model was established according to Guo et al.  with modBoth dual- and multispecies biofilm samples were grown at 37°C for 24 h before further analyses. The culture medium was collected to measure pH values after 24 h cultivation.μM Alexa Fluor 647-labeled dextran conjugate was added to the culture medium at the beginning of incubation. The labeled dextran serves as a primer for Glucosyltransferases and can be simultaneously incorporated during EPS synthesis in biofilm development [μM SYTO 9 green fluorescent nucleic acid stain (485/498 nm) through 15 min staining after cultivation. The imaging was performed using a confocal laser scanning microscope  equipped with a 60x  oil immersion objective lens. The scan speed, photomultiplier detector gain, and pinhole aperture were kept constant for all image stacks. Representative images were demonstrated using Imaris software (v7.0). The quantities of bacteria and EPS were based on 5 image stacks in each group obtained from three independent tests and measured with COMSTAT2 image analysis program [The bacteria and their EPS production were determined by bacteria/EPS staining as described previously . Brieflyelopment . The bac program  as a pluin situ hybridization (FISH) were either designed using ARB software packages [S. mutans specific probe (5′-ACTCCAGACTTTCCTGAC-3′), and S. sanguinis specific probe (5′-GCATACTATGGTTAAGCCACAGCC-3′) were labeled with Alexa Fluor 405, Alexa Fluor 488, or Alexa Fluor 594, respectively. The biofilms on glass coverslips were fixed in 4% paraformaldehyde for 6 h, rinsed with PBS, and dried. For probe penetration, the biofilm samples were treated with 500 μl lysis buffer  at 37°C for 20 min. The biofilms were then rinsed, serially dehydrated in ethanol , and dried. For probe hybridization, each bead was covered with 50 μl of hybridization buffer  containing 3 oligonucleotide probes (2 μM each) and incubated at 46°C for 90 min in a closed cassette with a piece of paper towel soaked by hybridization buffer to maintain humidity. Subsequently, the beads were washed with buffer , incubated in the washing buffer at 46°C for 15 min, and rinsed in 4°C nuclease-free water. The images were captured and analyzed with similar tools in the EPS staining experiment.The sequences of oligonucleotide probes in fluorescence packages  or acquipackages . UniversP < 0.05 was considered to be statistically significant.The statistical analysis and graphing of the data were performed using the GraphPad Prism software (v8.0), with all data demonstrated as mean ± standard deviations (SD). Group mean values were compared by one-way ANOVA and Tukey post hoc test. A two-tailed S. mutans cultures containing NNSs exhibited a significantly lower level of OD600nm and a significantly higher level of pH value than that containing 1% sucrose . However, analysis of FISH-labeled multispecies biofilms showed no significant difference among all groups in the biomass ratio of S. sanguinis to universal bacteria  and suppress the less aciduric commensal residents , driving the shift of microbial community to a more cariogenic consortium consequently. This positive feedback leads to a continuous local pH decline below the critical value which results in the demineralization of tooth tissues and eventually the development of caries lesion [S. mutans for biofilm formation and enamel demineralization based on carious lesion model in vitro [S. mutans, S. sanguinis, and S. mitis on glass beads [Dental caries results from polymicrobial infection in which imbalance between acidogenic pathogens and alkali-generating commensals in plaque biofilm plays a crucial role , 20, 21.s lesion , 22. Sugs lesion . Some rein vitro , 11. In ss beads . HoweverS. mutans, but also that of S. sanguinis, indicating the necessity to investigate the influence of NNSs on interactions between different species and the collective cariogenic potential of oral biofilms. Since the phenomenon of antagonism between S. mutans and S. sanguinis is a typical in vitro microcosm of interactions between acidogenic and alkali-generating bacteria [S. mutans and S. sanguinis to study bacteria interplay. The results indicated that the metabolism of dual-species biofilm to produce acids was active in high sucrose condition, and the aciduric S. mutans made competitive predominance over S. sanguinis (with their biomass ratio close to 2). Additionally, the pH value of dual-species biofilms with NNSs was higher than that of sucrose, and the S. mutans/S. sanguinis ratio significantly declined to about 1, suggesting the inhibitory effects of NNSs on biofilm acid production and S. mutans advantage. These outcomes demonstrated the ecological benefit of NNSs on the perspective of the in vitro competition between acidogenic and alkali-generating bacteria.Initially, we observed that on pure cultivation, NNSs not merely prohibited the growth and acid production of bacteria , 25, we S. mutans and other acidogenic organisms can metabolize fermentable sugars to produce acids which are responsible for cariogenesis [S. mutans are considered as essential virulence factors of dental caries, as they form the core of the scaffold of cariogenic biofilms, provide abundant primary binding sites, and enhance bacteria cohesion [S. sanguinis which is more sensitive to acidic condition [S. sanguinis is capable of producing alkali to gain a competitive advantage over S. mutans, thus preserving pH homeostasis and protecting against caries [S. mutans/S. sanguinis ratio, as well as increasing biofilm pH. Although the decline of S. mutans/S. sanguinis ratio primarily exhibited as an inclination (only acesulfame-K group significantly distinguished from sucrose group), the results of our experiments collectively provided elementary evidence for NNSs' effect of diminishing biofilm cariogenic capacity.To further identify the influence of NNSs on overall biofilm cariogenic potential, we established the saliva-derived multispecies biofilm model. Acid production, acidogenic/alkali-generating bacteria biomass ratio, and EPS generation are critical evaluation indicators for the cariogenic ability of biofilms, and each of them has a close association with others. ogenesis , 27. EPScohesion . EPS andcohesion . The aciondition . In contt caries , 31. ThiS. mutans biofilms was higher than that of S. sanguinis, while coculture and culture with saliva-derived bacteria did not show obviously increased growth of S. mutans relative to S. sanguinis. We speculate one of the reasons is that the stronger capacity for EPS production of S. mutans made the single-species biofilms more adhesive against PBS rinses to remove the excess CV. Moreover, since the interaction between different bacteria strains may influence the ultimate effect of external substances, it is necessary to apply the biofilm model comprised of a more complex microbial community. However, it is still important to highlight that the results of the present study were attained by in vitro approach. Despite the fact that the multispecies model derives from human saliva and consists of much more species than selected bacteria [It is worth noting that there were some differences in the results of NNSs' effect on bacterial growth between the single-species biofilm model and the multispecies biofilm model. When cultured separately with NNSs, the biomass of bacteria , the batbacteria –35. Therbacteria , 37, theS. mutans or S. sanguinis in planktonic culture and single-species biofilm. These sweeteners decrease S. mutans/S. sanguinis ratio and induce less acid/EPS production than sucrose in dual- and multispecies biofilm models. The benefit of NNSs for bacterial equilibrium in biofilm may have a promising prospect in caries management.Taken together, this study demonstrates that NNSs  suppress the growth and biofilm formation of


Although vitamin D and dentition status are each associated with frailty, their combined effects on frailty have not been studied. This study aimed to evaluate the combined effects of vitamin D and dentition status on frailty in old Chinese adults.Baseline data were obtained from the 2011–2012 wave of the Chinese Longitudinal Healthy Longevity Survey. A total of 1074 participants ≥65 years who were non-frail or prefrail at baseline were included; follow-up was conducted in the 2014 wave. Frailty was assessed by a 40-item frailty index (FI) and classified into frail (FI > 0.21), prefrail (FI: 0.1–0.21), and non-frail (FI ≤0.1). Vitamin D was assessed by 25-Hydroxyvitamin D (25(OH)D) and categorized into quartiles and dichotomies . The presence of ≥20 natural teeth was defined as functional dentition, otherwise as non-functional dentition. We used bivariate logistic regression and restricted cubic splines to examine the association between vitamin D, dentition status, and frailty. We created a multiplicative interaction between vitamin D and dentition status to test for their combined effect.A total of 205 (19.1%) incident frailty were identified during the 3-year follow-up. Participants with the lowest quartile of plasma 25(OH) D were more likely to be frail  than those in the highest quartile. Older adults with the lowest quartile of 25(OH) D and non-functional dentition had the highest odds of frailty . We also observed that a lower vitamin D level was associated with an increased risk of frailty with a threshold of 40.37 nmol/L using restricted cubic spline models. However, vitamin D levels were not significantly associated with frailty among participants with functional dentition.Low vitamin D levels were associated with an increased risk of frailty in older adults. Functional dentition modified the association of vitamin D with frailty.The online version contains supplementary material available at 10.1186/s12877-022-02857-3. Population aging is a major public health challenge globally . One of Vitamin D maintains serum calcium and phosphate homeostasis to promote strong bones, regulates the innate immune system, cell differentiation, and cell maturation, and influences cellular metabolism of skeletal muscle , 15. OldThe purpose of the present study is to evaluate the combined effects of low vitamin D levels and dentition status on the risk of frailty among old adults. We also investigated whether the association between vitamin D and frailty is modified by dentition status.The Chinese Longitudinal Healthy Longevity Survey (CLHLS) is an ongoing prospective, longitudinal study with the largest sample of the oldest old in China. It aims to identify the determinants of health and longevity among Chinese older adults. Half of the counties and municipalities were randomly selected in 23 of the 31 provinces, covering 85% of the national population. The baseline survey was conducted in 1998; alive participants were re-interviewed in follow-up surveys conducted every two or 3 years. The response rate was over 80%. Baseline data of the present study were obtained from a biomarker sub-study conducted in eight longevity regions in 2012: Laizhou City of Shandong Province, Xiayi County of Henan Province, Zhongxiang City of Hubei Province, Mayang County of Hunan Province, Yongfu County of Guangxi Autonomous Area, Sanshui District of Guangdong Province, Chengmai County of Hainan Province, and Rudong County of Jiangsu Province . These eThe inclusion criteria were the older people who were ≥ 65 years old and provided blood samples. The follow-up was conducted in the 2014 wave. Participants with any of the following conditions were excluded: (1) frailty at baseline; (2) missing data on key variables (25(OH) D level and/or natural tooth number); (3) lost to follow-up or death. The flow chart is shown in Supplementary Fig. S1, Additional file The outcome was the incident frailty during follow-up. Frailty status at baseline and follow-up was measured by the frailty index (FI)—a commonly used tool for assessing frailty. Following a standard procedure , we consVitamin D was assessed by plasma 25(OH)D. Trained medical personnels collected blood samples using heparin anticoagulant vacuum tubes from subjects who had fasted overnight. They centrifuged blood samples at 2500 rpm at 20 °C for 10 min, and measured plasma 25(OH) D levels by enzyme-linked immunosorbent assay . Plasma 25(OH) D was categorized into quartiles . Stratified analysis was analyzed at a dichotomous cut-off point of 25(OH) D deficiency recommended by the Endocrine Society Guidelines  [The number of remaining natural teeth was assessed at baseline by trained personnel based on a question: “How many natural teeth do you have?” Functional dentition was defined as the presence of ≥20 teeth which can assure an acceptable level of oral function , and we 2) is calculated as weight in kilograms divided by height in meters squared. We classified BMI (kg/m2) into four categories  [Based on the literature –35, we c8 kg/m2) . Plasma Continuous variables with skewed distribution were described using median . Categorical variables were expressed as frequency (percentage). Kruskal-Wallis test for continuous variables and chi-square test for categorical variables were applied to compare the differences in baseline characteristics.Bivariate logistic regression analyses were used to assess the association of vitamin D and dentition status  with frailty. Multiplicative interaction was tested to evaluate effect modification by including a cross-product term for dentition status and plasma 25(OH) D in two different ways  in the regression model. To observe the combined effect of vitamin D and dentition status on frailty, we created eight-level joint groups according to dentition status  and vitamin D quartiles. The subgroup analyses of the association of vitamin D with frailty were conducted by dentition status. Age, sex, baseline frailty status (non-frail and prefrail), residence, marital status, education level, economic independence, smoking status, drinking status, regular exercise, sleep time, BMI, calf circumference, and season of blood draw, albumin, and hs-CRP were included as covariates in adjusted models. The validity of the regression model was evaluated by calculating the tolerance and variance inflation factor (VIF) for multicollinearity diagnosis. Tolerance > 0.1 and VIF < 10 are used to indicate that there is no multicollinearity between the independent variables. Tests for trend across vitamin D quartiles were examined using ordinal values in separate models.We used restricted cubic splines to evaluate the pattern and magnitude of associations between 25(OH) D levels and frailty risk in all participants and participants stratified by dentition status . The 75th percentile of 25(OH) D level was treated as the reference, and the 5th, 25th, 50th, 75th, and 95th percentiles of 25(OH) D were treated as the 5 knots for spline. The likelihood ratio test tested the potential nonlinearity.P value less than 0.05 was considered statistically significant. The analyses were performed using Stata 13.0 .The results were presented as odds ratios (ORs) with 95% confidence intervals (CIs). A two-tailed n = 446) were prefrail and 58.5% (n = 628) were non-frail at baseline. The participants with higher plasma 25(OH) D levels were more likely to be younger, male, married, have a higher level of education and live in rural areas . Table A total of 205 (19.1%) incident frailty were identified during the 3-year follow-up. Participants with low vitamin D levels had a significantly higher frailty risk than those with normal vitamin D levels  D and non-functional dentition were 3.67 times more likely to develop frailty than those with the highest quartile of 25(OH) D and functional dentition Table . HoweverIn this nationwide cohort study of 1, 074 Chinese older adults, we observed that the odds of frailty in the lowest quartile of plasma 25(OH) D was higher than that in the highest quartile, and participants with the lowest quartile of 25(OH) D and non-functional dentition had significantly higher odds of frailty. In addition, we found functional dentition modified the association of vitamin D with frailty. Specifically, among participants with non-functional dentition, low vitamin D concentrations were associated with increased frailty risk, whereas there were no significant relationships among those with functional dentition.n = 4, 203) population-based study of older men aged 70–88 years, the risk of frailty increased by 50% during 5 year-follow up for participants who were not frailty at baseline and had low levels of vitamin D (< 52.9 nmol/L) [Our study was in agreement with previous studies that showed an inverse correlation between vitamin D and frailty , 37, 38. nmol/L) . In anot nmol/L) , which m nmol/L) , 41. Vit nmol/L) . Increas nmol/L) . HoweverNatural tooth number is a risk indicator of frailty as well as vitamin D , 44. A pFrailty is likely to be even more acute in China, which has the largest aging population in the world. In China, studies have shown that the prevalence of pre-frailty and frailty among people aged 60 years and above is 51.2 and 7.0%, respectively . FrailtyTo our knowledge, this was the first study to examine an interactive effect of vitamin D levels and dentition status on frailty. Our longitudinal study used the frailty index to assess the relationship between vitamin D, dentition status, and frailty as it evaluates a broader range of diseases (such as cognitive function) than a frailty phenotype and may provide additional information to explore correlations. Nevertheless, we recognize several limitations of this study. First, our participants were Chinese older than 65 years of age. Our results have limited generalizability to other ethnic groups. Second, dentition status and 25(OH) D levels were assessed at baseline only. Thus, tooth loss and 25(OH) D concentration changes during the observation period may confuse the results. In future studies, it will be essential to examine the effect of these changes. Third, because CLHLS is not explicitly designed for dental research, other information related to oral health is lacking. The number of teeth was self-reported, and the reliability of the numbers was limited. When exploring the deeper relationship between oral health and frailty, dentition status may not be an adequate proxy for oral function. Finally, as with any analysis, residual confusion due to non-measured factors cannot be ruled out.We found that low vitamin D levels are associated with an increased risk of frailty. We also found that functional dentition modified the association between vitamin D levels and frailty. These results suggest that 25(OH) D levels and dentition status can be early warning indicators of frailty. It would lead to more aggressive interventions to prevent and treat frailty in older people who have a combination of significant tooth loss and vitamin D deficiency. Additional research is needed to investigate how to maintain functional dentition through oral care, combined with nutritional interventions aimed at optimizing plasma 25(OH) D concentrations, to potentially reduce the occurrence of frailty.Additional file 1.


To determine the efficacy of widely available subtype clinical tests to characterize evaporative dry eye disease (EDED) related to meibomian gland dysfunction (MGD) compared to normal and to validate those clinical cut points in an independent sample.  A diagnostic accuracy study (52 subjects), an investigator-masked study, was followed by a larger independent sample (364 subjects) analysis to confirm efficacy in normal and EDED subjects. All subjects were 18 years of age and older and were classified using a battery of clinical tests for dry eye that included symptoms, tear meniscus height, tear stability, ocular staining, evaporative-specific tests, and the Schirmer I test. n = 26) and EDED (n = 26) subjects completed the efficacy study. The global tests of tear breakup time, staining, and symptoms all produced AUCs ≥ 0.70, representing acceptable discrimination. EDED-specific tests of eyelid marginal signs, gland secretion quality, and gland loss did not demonstrate acceptable test efficacy or differences between normal and EDED subjects. In a larger, independent sample of normal and EDED subjects, gland secretion quality and eyelid marginal score achieved acceptable diagnostic levels: AUCs of 0.789 (CI: 0.734–0.844) and 0.729 (CI: 0.648–0.810), respectively, but not lipid interferometry grade or lower eyelid gland dropout estimated using meiboscopy.  Normal (nondry eye;  Meibomian gland secretion quality is an efficient and useful functional indicator in EDED and should be incorporated into core outcome sets for this dry eye subtype. Dry eye disease (DED) is a common clinical condition, affecting 5 to 50% of the population, depending on the sampling approach and diagnostic criteria . Of all As a common subtype of dry eye, EDED is a condition with major health and quality of life impacts that requires diagnostic methods that can also monitor treatment. In medical specialties, sets of clinical outcome criteria including those important to patients are developed by consensus groups into core outcome sets (COSs) . COSs arThe 2011 MGD workshop report (diagnostic subcommittee ) suggestIt was the purpose of this investigation to determine the test efficacy (sensitivity and specificity) of widely available clinical tests to characterize EDED related to MGD compared to normal and to validate those preliminary clinical cut points in an independent sample.This was a two-part investigation: an initial rigorous efficacy study , as shoParticipant flow and investigator masking for diagnostic efficacy samples. The index test was the summed MGD score (as shown in the text) at a cut point of 5.4 on the 0–12 scale. The reference standard was clinical diagnosis of normal or EDE via the third masked examiner.Major inclusion criteria were normal or dry eyes as determined by global DED tests, over age 18, and willingness to discontinue topical ocular drop use on the day of the assessment. Subjects were included if their dry eye management was stable for 30 days prior to enrollment. EDED was classified using either the lower eyelid gland secretion score ≥1.0  or glandμl of 1.0% NaFl; yellow filter used, mean of three values), corneal (fluorescein) and conjunctival (lissamine green) staining with NEI and Oxford schemes, gland secretion  or the specific meibomian gland data . Symptoms , eyelid–3 scale  using a –3 scale , and the–3 scale . For the–3 scale  custom aThe classification scheme used to assign subjects as normal or EDED was identical to that reported previously . In briea priori sample size estimate was made to compare normal and EDED subjects relative to a cumulative score comprised of functional and morphological assessments (index test) [Statistical analysis was undertaken using Minitab version 18 . An ex test) . The comRelative to a receiver operating characteristic (ROC) curve, an area under the curve (AUC) of 0.80 (excellent discrimination) vs. the chance level of 0.50  was the pvalues were two-tailed and adjusted for multiple comparisons with p < 0.05 considered significant. 95% confidence intervals were constructed for each comparison. For ordinal data, age, gender, and dry eye subtype were compared using odds ratios and 95% CIs; those CIs that included the null value of 1.0 were not considered significant.For both the index test and the independent sample analyses, multiple regression analysis (continuous variables) and polytomous logistic regression  were undertaken to examine the effects of age, sex, and dry eye subtype  on the parameters of interest. Comparisons of normal vs. EDED were controlled for age and sex if significant from the continuous or ordinal analysis. For continuous data, ANOVAs including age, gender, and dry eye type were conducted, with Tukey pairwise tests for significant factors between normal and EDED subjects. The The ROC curve analysis for AUC and optimum cut point  was undertaken for the normal and EDED data.p=0.184). The ADDE subject data were eliminated from the data set so that only normal and EDED subjects were compared.Twenty-six normal, 10 ADDED, and 26 EDED subjects completed the study. The mean ages (±SD) were 53.2 (±14.9), 55.7 (±6.9), and 61.0 (±17.1) for the normal, ADDE, and EDED subjects, respectively. No statistical difference was found for age in the three groups . The global dry eye tests of symptom questionnaires, TBUT, and staining scores achieved test sensitivities near or above 0.70, considered an acceptable level for an effective dry eye test [AUCs, cut points, sensitivity, and specificity values were derived from ROC analysis . The indAdditional data were available from several prospective studies conducted concurrent with the efficacy study using identical test methods and classification criteria. The available data varied by test measure, up to a maximum number of 364 charts from clinically normal subjects and subjects having EDED. The EDED test data of this independent sample are summarized in p values were <0.001 for marginal signs and secretion quality, and p=0.034 for gland dropout).Regression analysis was undertaken for marginal signs (0–5), gland secretion quality (0–3), and gland dropout (0–4), all from the lower eyelid of the right eye. This demonstrated a significant association with age for all three parameters (Receiver operating characteristic (ROC) analysis was undertaken for the five EDED-specific clinical tests. Comparisons between normal and EDED subjects are presented in Gland secretion: AUC 0.789 (95% CI: 0.734–0.844), cut point 1.1 ; sensitivity = 0.79, specificity = 0.78; and n = 136 normal, 228 EDED subjects Marginal eyelid signs: AUC 0.729 (95% CI: 0.648–0.810), cut point 4.5 ; sensitivity = 0.79, specificity = 0.63; and n = 94 normal, 93 EDED subjects MGD-specific questionnaire: AUC 0.603 (95% CI: 0.494–0.713), cut point 40.5 ; sensitivity = 0.51, specificity = 0.71; and n = 57 normal, 88 EDED subjects Interferometry: AUC 0.556 (95% CI: 0.484–0.629), cut point 2.1 ; sensitivity = 0.57, specificity = 0.57; and n = 85 normal, 190 EDED subjects Gland atrophy (meiboscopy): AUC 0.378 (95% CI: 0.308–0.449), cut point 0.95 ; sensitivity = 0.54, specificity = 0.36; and n = 100 normal, 229 EDED subjects Cut points were determined on the basis of maximal values sensitivity and specificity.95% confidence intervals, diagnostic scale cut points, and subject sample sizes are summarized in text.It was the purpose of these investigations to determine whether routinely available clinical methods  can reliably identify EDED related to MGD by examination of functional and morphological changes in the lower eyelid. Under rigorous study design (such as double investigator masking), only the global clinical tests of symptom questionnaires, TBUT, and staining scores demonstrated adequate test efficacy (sensitivity and specificity >70%)  and the However, in the independent sample, gland secretion, using the Bron scale , 19 withn = 26 normal and MGD subjects). In the independent sample data  does not appear to be a viable indicator of EDED given the low AUCs in both samples Tables . This meWhite-light tear interferometry of the tear lipid layer has been recommended by the DEWS II diagnostic subcommittee  to assisRelative to developing core outcome sets (COSs) for clinical trials in dry eye, it appears that a combination of global and specific tests for EDED is necessary. The present and recently reported data suggest that the global tests of symptoms, osmolarity, tear stability, and corneal and conjunctival staining are effective in establishing the diagnosis and severity of EDED. However, given the high test efficacy of both tear stability measurement and staining, it does not appear necessary to also include tear osmolarity as an additional global indicator of EDED. The cost of this test also mitigates against its widespread use. Assessment of gland secretion using the 0–3 scale  in 0.1 uThese studies suffered from selection bias, or the use of the test under consideration to classify the condition, which tends to overestimate its efficacy . EffortsIn summary, it appears from the independent sample using clinical tests that only meibomian gland secretion quality is a useful specific indicator of the evaporative subtype of dry eye. Gland morphology assessed using meibography appears necessary to diagnose and quantitate changes associated with evaporative dry eye disease. Additional work should endeavor to more comprehensively assess the test efficacy of gland expressibility directly with the several available clinical methods and indirectly via the effect of gland patency on lipid layer thickness to establish cut points for diagnosis and treatment monitoring.


Aegilops spp. is the closest genus to wheat (Triticum spp.), which makes Aegilops great candidates to exhibit precursors of wheat features. Aegilops cylindrica Host displays excellent salt tolerance. In the current study, biochemical and phytochemical compounds in the leaves of two wheat cultivars, one hyper-salt tolerant Ae. cylindrica genotype and their amphidiploids (derived from “Chinese Spring” × Ae. cilindrica and “Roshan” × Ae. cylindrica), grown under control and saline field conditions, were assessed. These compounds included total protein content, proline content, electrolyte leakage, total flavonoid content, total phenolic content, DPPH radical scavenging activity, and reducing power. In addition, phenolic components were also identified using HPLC analysis. Chlorogenic acid, ellagic acid, ferulic acid, syringic acid, vanillic acid, p-coumaric acid, caffeic acid, and gallic acid were the most abundant phenolic acids. Luteolin, apigenin, and rutin were the most abundant flavonoids in the leaves. Salt stress significantly increased all biochemical variables, with the exceptions of reducing power and p-coumaric acid. Interestingly, amphidiploid genotypes exhibited intermediate levels of most of the detected phenolic compounds between the two parental species. As demonstrated by bivariate correlations luteolin, chlorogenic acid, caffeic acid and apigenin could predict inhibition percentage by DPPH assay, suggesting a possible role in the cellular defense against oxidative stress in wheat. The amphidiploids and their wild parent performed significantly better than wheat cultivars on phenolic constituents, flavonoids, and maintaining redox homeostasis under salt stress conditions. Aegilops cylindrica Host is a wild species, sharing a common genome (D) with common wheat (Triticum aestivum L.). Several authors  derived from cross between “Chinese Spring” and “Roshan” wheat cultivar with Ae. cylindrica as the male parent.The purpose of this study was to determine how salt stress affects the leaf polyphenol composition, flavonoids, and cellular redox responses to oxidative stress in two wheat cultivars (including “Chinese Spring” and “Roshan”), one Ae. cylindrica genotype (no. 56), and two of their interspecific hybrids (amphidiploids) were used in this study. Two common wheat (Triticum aestivum L.) cultivars (“Chinese Spring” and “Roshan”) as the female parents were crossed with a highly salt-tolerant genotype . Subsequently, the colchicine treatment was carried out at the 4–5 tiller stage and the treated plants were kept under greenhouse conditions until they were harvested. Full details of the hybridization protocols, including emasculation, pollination, growth hormone treatment, embryo rescue, and colchicine treatment of amphihaploid plants, have been described in an unpublished article.Five genotypes, including two wheat cultivars (“Chinese Spring” and “Roshan”), one genotype  of Aegil–1. For each of two experiments [control and saline (250 mM NaCl)], a randomized complete block design replicated three times was used. Each plot was three rows, 1 m long, with a row spacing of 30 cm. In the control experiment, plants were irrigated with freshwater (EC = 1 dS m–1). In the saline experiment, fresh water was applied until the four tillers stage  but later shifted to saline water. The experimental plots received equal irrigation, monitored by flow meters, in the control and saline conditions. The saline experiment was irrigated with irrigation water connected to the upstream tank that delivered a concentrated solution of sodium chloride (1 M NaCl) to maintain the desired concentration of salinity (250 mM NaCl) using a calibrated flow meter. The plots were irrigated when the soil moisture reached above 80% of field capacity (ψ = –0.06) in the root zone. The soil ECe, in 0 – 40 cm soil depth, was measured in all plots at harvesting stages. The average soil ECe values were 2.4 and 15.4 dS m–1 for the control and saline field conditions, respectively.The seeds of amphidiploid genotypes and their parents were planted in the research farm of the Isfahan University of Technology, Lavark, Iran  in 2017-2018 growing season. The soil in the top 60-cm layer is a clay loam (pH 7.5) with an electrical conductivity (EC) of 2.4 dS m–1).Total protein content of leaf samples was determined at the grain filling stage according to –1), and centrifuged for 10 min at 8,500 rpm. Then, 2 ml of the supernatant was added to a mix soluble of 2 ml ninhydrin reagent and 2 ml acetic acid and kept in a water bath (100 °C) for 1 h. Subsequently, 4.0 mL of toluene was added to the samples and absorbance was recorded at 520 nm. Proline concentration was calculated using a standard curve according to the following formula:Free proline was extracted from the fresh leaves, derivatized with acid ninhydrin and absorbance read, according to First, 1.25 g of the dried leaf samples was mixed with 25 mL of 80% methanol in an orbital shaker (150 rpm) at 25°C for 24 h. Total phenolic content (TPC) of the methanolic extract solution was determined using Folin–Ciocalteu method as described by 2 solution. The mixture was then allowed to stand for 5 min before adding 150 μl of aluminum chloride (10%) solution. After that, 750 μl of NaOH solution (1 M) was added and the final volume of the mixture was adjusted to 2500 μl by deionized water. After an incubation period of 15 min, the absorbance was read at 510 nm using a spectrophotometer. The TFC has been expressed as mg quercetin equivalent (QE) per g dry weight (DW).The aluminum chloride assay was used to determine total flavonoid content of the methanolic extract solution . An aliqA) and corrected for the absorbance of blank reagent (AB) at the same wavelength. A DPPH-methanol solution containing 80% methanol was used as a negative control (AB). The synthetic antioxidant reagent, butylated hydroxytoluene (BHT), was used as a positive control. The IC50 value (μg ml–1), the concentration in μM that inhibits DPPH absorption by 50%, was calculated by linear regression analysis.The DPPH radical scavenging activity of the leaf samples was determined by the procedure described by –1) was added to 2.5 ml of 1% potassium ferricyanide and 2.5 ml 0.2 M phosphate buffer (pH = 6.6). The mixture was incubated at 50°C for 20 min, and 2.5 ml of trichloroacetic acid (10%) was then added. The homogenate was centrifuged at 3000 rpm for 10 min, and 2.5 ml of the supernatant was mixed with 2.5 ml of ferric chloride  and deionized water. The absorbance of the solution was read at 532 nm against a blank. The antiradical activity was expressed as IC50 (mg ml–1). The IC50 was calculated using a linear regression equation.The reducing power of the leaf samples was determined according to the method described by ® C18 column  and a UV absorbance detector was used. The extracts were filtered using a 0.45 μM nylon membrane filter , and 20 μL was injected into the analytical column. Eluent A was 0.1% formic acid in water (v/v), and eluent B was 0.1% formic acid in acetonitrile (v/v). The flow rate was 0.8 mL min–1, and the following linear gradient program was used: 10% to 26% solvent B for 20 min, 65% solvent B for 40 min, and finally to 100% solvent B for 45 min. Detection was performed at 200–400 nm using the absorbance detector. Individual phenolic acids, flavonoids, and flavanols in the leaf samples were determined and quantified by the assessment of their relative retention times to the authentic standards. The data have been expressed as milligrams phenolic acid per 100 g of DW (mg 100 g–1 DW).Individual phenolic acids of the leaves were analyzed using the high-performance liquid chromatography (HPLC) system. An HP 1090 series HPLC  equipped with a Waters SymmetryElectrolyte leakage (EL) was used to assess membrane permeability. The EL was determined at the grain filling stage using an electrical conductivity (EC) meter as described by n = 10) and expressed as g per plant.At maturity, ten plants from the middle of each plot were harvested. Grain yield was recorded per plant .The data were analyzed by analysis of variance (ANOVA) using the PROC GLM of SAS . The means were further analyzed by Fisher’s protected least significant difference (LSD) test at p < 0.01) to the biochemical traits in the leaf. The genotypes were significantly different for these traits. Also, significant genotype × salt stress interaction was found for all biochemical traits except for total protein content , which are much higher than those found for the “Chinese Spring” and “Roshan” wheat cultivars. Interestingly, the interspecific hybrid (amphidiploid) plants displayed an intermediate phenotype between male (Ae. cylindrica) and female (wheat) parents   .Ae. cylindrica derived amphidiploids showed less yield loss than wheat cultivars (“Roshan” and “Chinese Spring”). In contrast, Ae. cylindrica genotype produced the least loss of yield (data not shown).Grain yield was affected significantly by salt stress, genotype, and salt stress × genotype . Salt stAe. cylindrica genotype (21.7 mg TAE g–1 DW) and “Chinese Spring” × Ae. cylindrica amphidiploid genotypes (18.5 mg TAE g–1 DW) (Ae. cylindrica amphidiploid) in salt stress conditions. TFC was likewise increased significantly by salt stress when compared with the control conditions. In contrast to the female parents (wheat cultivars), the male parent (Ae. cylindrica) displayed the highest TFC content (7.74 mg QE g–1 DW) under salt stress conditions. Although, “Chinese Spring” × Ae. cylindrica amphidiploid genotype showed responses intermediate between two parents however, no significant difference was found between “Roshan” × Ae. cylindrica amphidiploid genotype and its female parents  . On the  parents .–1 in the most salt-tolerant genotype of Ae. cylindrica to 2890 μg ml–1 in “Chinese Spring” the most sensitive wheat cultivar. Likewise, a similar trend was evident for the scavaging of DPPH in salt stress conditions.Plants can withstand saline conditions by various mechanisms for reducing oxidative damage and maintaining redox homeostasis. DPPH free radical scavenging activity (DPPH-RSA) of the leaves was influenced significantly by salt stress, genotype, and salt stress × genotype interaction . ScavengAe. cylinrica genotype and amphidiploids) was influenced significantly by salt stress, genotype, and salt stress × genotype interaction (Ae. cylindrica genotype containing the lowest IC50 (IC50 = 0.72 mg ml–1) under salt stress conditions, whereas it did not differ significantly from the amphidiploids. Moreover, wheat cultivar (“Chinese Spring” and “Roshan”) recorded the lowest activity under salt stress conditions. Although, IC50 value also exhibited similar trends for all genotypes in the control conditions, however, BHT was found to possess the most potent activity in both control and salt stress conditions.Reducing antioxidant power in the tested genotypes  was found in the leaves of the salt-tolerant, Ae. cylindrica genotype (ANOVA results of the non-flavonoid polyphenol (phenolic acids) and flavonoid compounds detected by HPLC are summarized in genotype . On the genotype .Ae. cylinrica and amphidiploids  than the wheat cultivars under salt stress conditions (Ae. cylindrica was seen in chlorogenic acid (Ae. cylindrica control conditions (data not shown).Clearly, nditions . Ellagicnic acid , vanillinic acid , gallic nic acid , and rutnic acid  contentsnic acid , as well–1 DW, respectively) of apigenin, while Ae. cylindrica had the lowest (10.01 mg 100 g–1 DW)  showed the highest content (44.48 and 33.56 mg 100 g g–1 DW) . In cont) –1 DW,  and flavr = 0.76∗∗) contributed the most toward the variation in yield loss.A backward stepwise regression analysis was performed to examine which combination of variables best predicts yield loss due to salt stress. The results showed that proline followed by TPC and DPPH were the strongest predictors of yield loss under salt stress conditions . Overallr = −0.66**). A negative correlation was found between TFC and DPPH scavenging rate (r = –0.58*) and also reducing power (r = −0.66∗∗). These results show that phenolic compounds (TPC and TFC) are positively associated with antioxidant activity. The correlation coefficients between the phenolic acids and the biochemical traits showed a positive between chlorogenic acid and DPPH scavenging rate (r = 0.58∗). Moreover, a positive correlation exists between ferulic acid content and TPC (r = 0.85∗∗).r = 0.54∗) and DPPH (r = 0.65∗∗); while negatively correlated with protein content (r = –0.56∗), proline content (r = –0.55∗), TPC (r = –0.93∗∗), and TFC (r = –0.59∗). These relationships were consistent with observations in salt-tolerant genotypes having higher protein content, proline content, TPC, and TFC as well as, lower EL and yield loss (r = 0.78∗∗), luteolin (r = 0.91∗∗), caffeic acid (r = 0.70∗∗), and apigenin (r = 0.75∗∗). A positive correlation was also found between luteolin and chlorogenic acid (r = 0.75∗∗); apigenin and ferulic acid (r = 0.77∗∗); as well as luteolin and apigenin (r = 0.76∗∗).The results of correlation coefficients between yield loss and other traits are in eld loss , 2. StroThe leaf proteins are vastly critical in the growth, reproduction, and ultimate grain yield of the plants. The reduction of protein content in the plant leaves due to salt stress is not surprising since it is well established that the initial targets of ROS are proteins in biological systems. The chloroplast is one of the primary targets of ROS, which causes marked alterations in a wide variety of proteins such as thylakoid and stromal, including degradation and inactivation of Rubisco . TherefoP5CS) in Ae. cylindrica may correlate with salt tolerance. This finding is in agreement with Increased accumulation of compatible solutes like proline has been suggested to enhance salt tolerance. Eventually, proline, likewise other osmolytes, modulate redox potential by conferring osmotic adjustment, protecting cellular membranes, and stabilizing enzymes under abiotic stress . ArabbeiAe. cylindrica and amphidiploid genotypes under stress conditions.In plant cells, the preservation of plasmid membrane integrity is a critical adaptive strategy against free radicals . In the These findings support the idea that plasma membrane may represent a promising strategy for improving the efficiency in regulating transmembrane ion and metabolite fluxes during the stress. The results of the current study are consistent with those of Zea mays L.) cultivars.There are many biochemical mechanisms that protect plants against the harmful influences of salt stress. Phenolic compounds are not only the most abundant secondary metabolites in the plant kingdom but also are the most crucial antioxidants for scavenging the excessive ROS that is generated by the majority of stressors. Flavonoids, as a group belong to phenolic compounds, are also known to have antioxidant properties . Our resOver the past several decades, researchers have consistently found that the strong association between polyphenols and abiotic-stress tolerance is an excellent predictive of the extent of patience, and hence can be used as an indicator of maintenance of the redox state in the cells . While tAe. cylindrica  had higher antioxidant activity than female parents (wheat cultivars) in both control and salt stress conditions. Further, the results also show that in comparison with female parent, amphidiploid plants had higher antioxidant activities expressed by DPPH (IC50). Specifically, the research presented here is not only motivated but also supported our initial works bestowing genotypic differences for antioxidant enzymes, malondialdehyde (MDA), and H2O2 in the same set of genotypes (The results of the current study clearly show that wheat and Aegilops genotypes differ for the accumulation of the polyphenols (TPC and TFC) in their leaves. These compounds were significantly increased in response to salt stress. enotypes .–1). The plant samples with stronger reducing powers are capable of donating their electrons to the ROS and induce degeneration. In this way, Ae. cylindrica and two amphidiploids were a superior group of genotypes with the lowest IC50 values.The reducing power is another protective mechanism of oxidative stress, which reveals the electron-donating ability of the natural extracts and cellular redox homeostasis. Non-enzymatic antioxidants can be defined as inactivation of oxidants by reductants in redox reactions in which one reactive species is reduced at the expense of the oxidation of another . The assAmaranthus tricolor L. (a leafy vegetable) in response to salt stress has already been found, including caffeic acid, chlorogenic acid, ferulic acid, gallic acid, 4-hydroxybenzoic acid, p-coumaric acid, salicylic acid, sinapic acid, and vanillic acid  and flavanols (rutin) identified in the genotypes. Increased accumulation of phenolic acids in the leaves of lic acid . Intereslic acid . Moreovelic acid . Ma et a–1 mM NaCl significantly decreased grain yield. This study support the hypothesis that there is a trade-off between reproduction and stress tolerance  had the most substantial inhibition on the DPPH radical among the genotypes studied. These findings are partially consistent with those of Origanum vulgare), which showed that increase in chlorogenic acid correlates significantly with the DPPH scavenging responses evoked by water stress.In addition, negative and strong correlation coefficients of TPC and TFC with IC50 of DPPH radical indicated that the phenolic compounds contribute to antioxidant potential in the studied genotypes. Likewise, a positive correlation has been reported between RSA and TPC in cereals . We alsoFerulic acid is a hydroxycinnamic acid and a precursor of lignin and vanillic acid biosynthesis with good antioxidant and anti-aging functions. It involves not only an inhibitor of the enzyme catalyzing the formation of free radical species and an enhancer of scavenger enzyme activity but a free radical scavenger. Therefore, salt stress might activate lignification inhibitors leading to accumulation of ferulic acid . AssociaAe. cylindrica), moderately salt-tolerant (amphidiploids), and salt-sensitive (T. aestivum) genotypes. The vigorous antioxidant activity and robust accumulation of phenolic compounds in the leaves of the male parent (Ae. cylindrica Host) and amphidiploid derivates would imply greater sophistication in genetic diversity for the evolvement of defense-oriented strategies to prevent the accumulation of intracellular free-radicals generated under salt stress.To our knowledge, this is the first report that establishes the antioxidant and protective role of polyphenols against salt stress-induced oxidative stress and the differential response of genotypes, using highly salt-tolerant (The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AA conceived and designed the research. RK conducted the experiments under the supervision of AA and SAMMM. RK performed the overall data analysis, and also wrote down the draft of the manuscript with significant inputs from AA and SAMMM. All authors contributed to the article and approved the submitted version.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.


Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease. Intra-articular corticosteroids joint injection (IAJI), with triamcinolone hexacetonide (TH) or triamcinolone acetonide (TA), is an effective additional treatment for oligo and polyarticular JIA. Previous studies have shown the benefits of TH over TA; however, TA is still used in many pediatric rheumatology centers. Our unit has experience with both regimens, and therefore we aimed to compare the efficacy and safety of TA versus TH for JIA patients.Chart review of JIA patients who were randomly (based on drug availability) treated with TA or TH IAJI during 2010–2019. Primary outcomes for efficacy were defined as full recovery from arthritis one month after IAJI and a relapse rate of arthritis 3 months after IAJI. Primary outcome for safety was defined as the occurrence of adverse events (AEs) during the follow up period after IAJI.P = 0.232). However, rate of relapse after 3 months was significantly higher for TA treated joints (27 (20.1%) vs. 13 (8.8%), respectively, P < 0.01). No AEs were documented except minor scars at four joint injection sites.Overall, 292 joints of 102 JIA patients were treated (138 TA/154 TH joints). Complete recovery after one month was documented in 107 (69.6%) of TA treated joints and 96 (69.5%) of TH treated joints (The recovery from arthritis was similar (~ 70%) with both regimens, however relapse rate was more than double in TA as compared to TH injected joints. These findings are important due to a contemporary shortage of TH in the US market. Intra-articular corticosteroid joint injection (IAJI) is one of the most prescribed medications for oligoarticular JIA . In accoThis is a retrospective chart review. We reviewed medical charts of children who fulfilled the ILAR revised diagnostic criteria for persistent/extended oligoarticular or polyarticular JIA , and hadOur pediatric rheumatology unit used TA for IAJI in JIA patients until May 2016, when TA was replaced by TH in June 2016 due to its availability and financial considerations. An intra-articular injection was performed according to clinical decision by a pediatric rheumatologist using standard protocol: prepare the skin with an antiseptic solution, aspirate the joint fluid to ensure the proper positioning of the needle, and then inject TA or TH. We used recommended doses , 14. TH Clinical assessment of arthritis was performed  by a pediatric rheumatologist at baseline, during the injection procedure, at ~ 1 month and ~ 3 months after the procedure, and at each follow-up visit thereafter. Complete response was defined as the absence of arthritis, and partial response as an improvement of arthritis (decrease in swelling or joint tenderness and/or increased range of motion). No response was defined as no change or worsening of arthritis. Primary outcome for efficacy was defined as full recovery from arthritis 1 month post IAJI, and relapse rate of arthritis 3 months post IAJI was defined as flare of arthritis after achieving complete response. Primary outcome for safety was defined as the occurrence of adverse events (AEs) during the follow-up period after IAJI. The AEs severity was classified according to the OMERACT 8 drug safety workshop .The study was approved by the Research Ethics Board of Rabin Medical Center .The following parameters were analyzed: gender, age of disease onset, disease duration, JIA type, number of joints involved, type of joint injected, first/re-injection, and laboratory parameters at baseline (C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), antinuclear antibodies (ANA) positive result (ANA ≥1:40 titer using Immunofluorescence exam), anti-CCP antibodies (in patients with polyarticular JIA ≥ 7 years of age)), and concomitant therapy with NSAID’s, methotrexate (MTX), or anti-tumor necrosis factor (TNFα) such as etanercept or adalimumab.p values< 0.05 were considered statistically significant.All results were expressed as mean and standard deviation (SD), median; and minimum and maximum, or frequency and percentage. The differences in patients’ characteristics between the treatment groups were analyzed using chi-square test. Independent samples t-test or Mann-Whitney U test were used when normal distribution was not justified. The response rates for both treatment groups were compared using chi-square. The efficacy of intraarticular TA or TH injection was analyzed using the Kaplan-Meyer test, estimating the time of arthritis flare over a period of 40 months; missing data were censored. The analyses were performed by using SPSS software, version 25 , tests were two-tailed, and The demographic, clinical, and laboratory parameters of both study groups are summarized in Table Of 292 joints injected the majority were in knees (61.6%) and ankles (25%), with 198 (67.8%) joints injected for the first time. A total of 94 (32.2%) joints were re-injected, 25 of them first with TA and when flared, re-injected with TH.Both groups were comparable for age of onset, disease duration, gender, mean number of joints per patient, type of joint injected, JIA subtype, laboratory parameters, and concurrent systemic therapy with MTX and anti-TNFα during and after IAJI. The rate of NSAID’s given after IAJI was significantly higher in the TA group. Mean follow-up duration was 21.9 (range 4–95) months.One patient with polyarticular JIA was older than 7 years old. His anti-CCP antibodies level was normal.Patients with overall twenty eight joints (14 in each treatment group) were additionally treated with systemic MTX during the time of injection procedure. In 6/28 joints, the therapy was started 6–12 weeks prior the IAJI. Among them 2 joints had not relapsed in the study period. Patients with overall 6 joints (3 in each treatment group) were treated with anti TNFα during the time of IAJI procedure. in 3/6 joints the therapy was started 6–12 weeks prior the IAJI (18 months for 2 joints and 4 months for 1 joints). Among them 2 joints had not relapsed in the study period and 1 joint had relapsed 7 months after the injection.P = 0.232). Partial response, defined as an improvement of arthritis, was seen in 23.2 and 16.9% of patients in the TA and TH groups, respectively, P = 0.232. No response or worsening was seen in 7.2 and 11.7%, respectively, P = 0.232. As seen in Table P = 0.018).Table p < 0.02). Logistic regression model showed the odds ratio for relapse was 2.24  when using TA for IAJI.Kaplan-Meyer analysis, which compared the efficacy of intraarticular TA or TH injection over a period of 40 months Fig. , showed Mild adverse reactions, such as skin atrophy or hypopigmentation at the injection site, were seen in only 4/292 (1.4%) of injected joints, for two joints in each group. No other complications, such as joint infection or chemical synovitis, were noted.P = 0.018). Moreover, this trend was sustained during the 40-month follow up with an odds ratio to relapse more than double [2.24 (CI 1.39–3.58)] in TA injected joints.Although studies show better long-term efficacy of TH vs. TA, there is a paucity of data regarding the short-term efficacy of both drugs , 11–13. As previously noted, earlier studies have addressed the long-term efficacy of TH vs. TA in JIA patients who received IAJI. Zulian et al. noted, in a prospective study, a significantly higher rate of response with TH than with TA at 6 months, 12 months, and 24 months  . In addiIn our study, patients with overall 34 joints were treated additionally with systemic therapy of either MTX or TNFα during the IAJI time. However, groups were balanced (17 patients in TH and 17 in TA). Moreover, in sensitivity analysis, results were comparable with the study findings.p = 0.133).Interestingly, patients with TH injected joints were more likely to be treated with anti TNFα as compared to patient treated with TA , presumably due to better TNFα availability. None the less, these differences were not significant  differs from TA (molecular weight 434.49) by an alteration of one side chain, which presumably makes TH less water soluble as compared with TA. Compounds with lower water solubility maintain effective synovial levels, thus providing longer duration of effectiveness within the peripheral joint space . AdditioAlthough IAJI can be an effective and safe line of treatment, repeated intra-articular steroid injections may predispose to charcot’s arthropathy as a potential side effect . In our Our study has several limitations. First, it is a retrospective study. Second, although this was the largest cohort reported to date, the number of injected joints was relatively small and not sufficiently powered. Third, our study is composed of a heterogeneous group of patients including a large portion with re-injected joints and patients treated with MTX or biological therapy during the study period. Yet, subgroup sensitivity analysis has not demonstrated significant differences among groups. Fourth, validated scoring systems such as Juvenile Arthritis Disease Activity Score (JADAS), were lacking.The recovery from arthritis was similar (~ 70%) with both regimens, however relapse rate (defined as flare of arthritis after achieving complete response) was more than double in TA as compared to TH injected joints. These findings are especially important due to the contemporary shortage of TH in the US market, and usage of TA in many pediatric rheumatology centers for IAJI around the globe.