FXIII SNPs were genotyped in 660 Black, apparently healthy South African women. Alarmingly, 16 and 45% of the women presented with deficient and insufficient 25(OH)D, respectively. Total fibrinogen and maximum absorbance (as a measure of clot density) correlated inversely, whereas γ' fibrinogen correlated positively with 25(OH)D. γ' fibrinogen increased whereas maximum absorbance decreased over the deficient, insufficient, and sufficient 25(OH)D categories before and after adjustment for confounders. 25(OH)D modulated the association of the SNPs regarding fibrinogen concentration and clot structure/properties, but did not stand after correction for false discovery rate. Because only weak relationships were detected, the clinical significance of the findings are questionable and remain to be determined. However, we recommend vitamin D fortification and supplementation to reduce the high prevalence of this micronutrient deficiency and possibly to improve fibrinogen and plasma clot structure if the relationships are indeed clinically significant. There is a need for large cohort studies to demonstrate the relationship between vitamin D and cardiovascular and inflammatory risk factors as well as to uncover the molecular mechanisms responsible.The role of 25-hydroxyvitamin D [25(OH)D] in reducing the risk of cardiovascular disease (CVD) has been recognized, but the mechanisms involved are unclear. Researchers have discovered a link between vitamin D and fibrinogen. Until now, data on the relationship between vitamin D and the γ' splice variant of fibrinogen and fibrin clot characteristics remain unexplored. In this study, 25(OH)D, total and γ' fibrinogen, as well as turbidimetrically determined plasma clot properties, were quantified, and fibrinogen and  Howevproposed –12. Lessproposed , downregproposed , 15, enhproposed , 17, proproposed  and inflproposed , 20.2 and D3 and the precursor of the active form 1,25-dihydroxyvitamin D3 D)—is the hallmark of assessing vitamin D status. The relationships between 25(OH)D concentrations and inflammatory markers, including the glycoprotein fibrinogen, have been investigated in numerous studies, with most sampling small or specific patient groups  as well as those of FXIII (FXIII SNPs might interact with 25(OH)D to modulate total or γ' fibrinogen concentration as well as the kinetics of clot formation, structural clot properties and fibrinolysis and warrant consideration.As vitamin D can be acquired through diet and cholecalciferol synthesized in a chemical reaction when the skin is exposed to sunlight, circulating concentrations of 25-hydroxyvitamin D-−25(OH)D, the sum of vitamin Dt groups –24. Datat groups , 25. Whet groups , 27, inft groups –30. Studt groups –33. Factt groups , 35. Furof FXIII . TherefoFGA, FGB, FGG and FXIII genes. To provide possible explanations for the observed relationships we considered the inflammatory marker C-reactive protein (CRP) and glycated hemoglobin A1c (HbA1c) which is a long-term indicator of glucose control. By controlling for CRP and HbA1c we explored whether the underlying mechanisms were related to inflammation or to vitamin D's effect on glucose control D determined in a group of ostensibly healthy Black women with total and γ' fibrinogen and fibrin clot properties. Second, we determined how these associations are influenced by specific genetic variations within the  control , 17. ThiThe Prospective Urban and Rural Epidemiology (PURE) study investigated the development of chronic lifestyle diseases, especially CVDs, by stratifying populations at different levels of urbanization . For thiEthical approval of the study protocol was obtained for the larger study as well as this nested study from the Health Research Ethics Committee of the Faculty of Health Sciences, North-West University , honoring the guidelines laid down in the Declaration of Helsinki of 1975 and revised in 2000.Blood pressure—systolic and diastolic—was measured twice using the OMRON HEM-757 automatic apparatus  with the cuff on the left arm after 5 min of sitting in a calm environment. The second measurement was recorded.th rib and iliac crest. Hip circumference was assessed at the level where the protrusion of the buttocks was greatest.While participants were lightly clothed and their arms hung freely at their sides, their body weights were recorded twice. Heights were measured twice using stadiometers with volunteers' heads in the Frankfort plane, bodies fully extended while inhaling. The means of the participants' weight and height measurements were used, to compute body mass index. Waist circumference was determined at the narrowest point between the 10Blood samples were collected between 07:00 and 11:00 from fasting participants. Sodium citrate-treated (3.8%) whole blood was centrifuged at 2,000 x g for 15 min to yield a buffy coat for DNA extraction and plasma samples to quantify coagulation markers. Ethylenediamine tetraacetic acid-treated whole blood was collected for HbA1c determination. Coagulated blood was collected to yield serum for lipids, CRP, and γ-glutamyl transferase (GGT) analysis. Aliquots were frozen on dry ice pellets, stored in the field at −18°C and then, after 2–4 days, at −80°C until analyzed.Lipograms , high sensitivity CRP and GGT concentrations were assayed using a Sequential Multiple Analyzer . Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula for triglyceride concentrations below 400 mg/dL. HbA1 concentrations were determined using the D-10 Hemoglobin testing system . Serum total 25(OH)D concentrations were quantified using a Roche Elecsys 2010 COBAS system . Whole blood was used for a rapid first response HIV card test 1–2.0 , and the outcome was confirmed with a Pareeshak test .2 (17 mmol/L), tPA  and phospholipid vesicles . Kinetics of clot formation , and structural clot properties (maximum absorbance representing clot density) were computed from the turbidity curves. The coefficient of variation for all assays was <10%.Total fibrinogen concentrations were established with the adapted Clauss method . Fibrinogen γ' was quantified with an ELISA using a 2.G2.H9 mouse monoclonal coating antibody against the human γ' sequence  for antigen capture and a goat polyclonal antibody against human fibrinogen . Turbidimetric analysis (A405 nm) was used  with modParticipants responded to various validated interviewer-administered questionnaires in a language of their choice to obtain demographic and lifestyle information.FGB-rs7439150, rs1800789 (1420G/A), rs1800791 (−854G/A), rs1800790 (−455G/A), rs1800788 (249C/T), rs1800787 (−148C/T), rs4220, rs4463047, FGA-rs6050, rs2070011 (2224G/A), and FGG rs2066865 and rs1049636 (9340T/C) as well as FXIII His95Arg A/G (rs6003) and Val34Leu, C/A (rs5985)], and two novel FGB SNPs in the promoter region (rs2227385 and rs2227388) identified via sequencing a subpopulation were genotyped. The Thermo Fischer Scientific® Taqman based assay, Illumina® VeraCode GoldenGate assay technology using a BeadXpress® platform and competitive allele-specific polymerase chain reactions (KASP) were used for genotyping.For details on the genetic analyses, please refer to Cronjé, Nienaber-Rousseau . In shorStatistica® version 13.3  and R version 3.5.0  were useFXIII gene polymorphisms in predicting total and %γ' fibrinogen concentration and clot properties, general linear models  with interactions were adopted. Since the frequency of carrier homozygote variant carrier status was low, we group the genotypes using a dominant genetic mode of action, that is, combining the heterozygotes with those that were homozygous for the variant alleles (0/1/1). Only interactions with a p-value < 0.05 are presented; however, for multiple testing and false discovery rates, the statistical threshold for significance was p < 0.005 [1/(16*3)(0.25)] when the false discovery rate was set at 25% D differed between the rural and urban women, with the former presenting with higher concentrations. Total and %γ' fibrinogen, time to clot formation indicated by lag time and the rate of lateral aggregation as indicated by slope also differed. Total fibrinogen and slope were higher in the rural than urban participants, whereas %γ' fibrinogen and lag time were higher in the urban than rural living participants. Clot density as indicated by maximum absorption and CLT did not differ between the urbanization subgroups. No difference in 25(OH)D concentration, fibrinogen, % γ' fibrinogen, lag time, slope, maximum absorbance, or CLT was detected between HIV-seropositive and -negative women.p < 0.001 with fibrinogen) as well as HbA1c , only the correlations between γ' fibrinogen and lag time remained and, after further adjustment with fibrinogen, only lag time remained worth mentioning.In p > 0.05).In FGA, FGB and FGG) and FXIII genes SNPs and their associations with total and %γ' fibrinogen concentration and clot properties have been described elsewhere D and total fibrinogen than those harboring the variant allele, resulting in an interaction. 25(OH)D did not correlate with lag time in those with homozygote carrier status for the wildtype allele. In contrast, when harboring the variant allele at rs5985 on the FXIII gene, a positive association was detected that resulted in an interaction. However, after additional adjustment for fibrinogen, the gene-diet interaction disappeared. An FGB gene (rs2227388)–vitamin D interaction was observed concerning maximum absorbance that became stronger after additional adjustment for fibrinogen. In women homozygous for the wildtype allele at rs2227388, no association between 25(OH)D and maximum absorbance was evident. Contrastingly, in those with one or two variant alleles, an inverse correlation was detected. 25(OH)D interacted with FGA: rs2227385 in relation to CLT before and after additional adjustment for fibrinogen. However, none of the interactions reached the significance required for adjusting for a multiple testing false discovery rate.In those carrying the p < 0.05 level, but did not stand after correction for false discovery rate.The role of vitamin D in reducing CVD risk has been recognized . In partdoogh, containing 500 IU vitamin D3 and 150 mg calcium/250 mL) and calcium plus vitamin D3-fortified doogh . The reduction was seen in both groups that also received calcium; future research is therefore needed to tease out the effects of vitamin D supplementation per se. Soare et al. D and fibrinogen before adjusting for other variables. Our study and the results obtained are in line with those of Parikh et al. , who inve et al.  failed te et al. . Vitamine et al. . To summRegarding γ' fibrinogen and clot properties, our study is the first to allude to a relationship with vitamin D. Here we expressed γ' as a percentage of total fibrinogen concentrations. In individuals with varying total fibrinogen concentration, the γ' fibrinogen concentration will likely also vary as its concentration is dependent on the total amount of fibrinogen produced. When expressing it as % of total fibrinogen, the potential confounding effect of differing total fibrinogen concentrations is excluded. Previously we reported our observations that while γ' was high when total fibrinogen concentration was high; a decreased γ'/total fibrinogen ratio was observed at the highest total fibrinogen concentrations . This fiIn vitro evidence demonstrated that the active metabolite of vitamin D, 1, 25(OH)D, and its analogs could couple to the vitamin D receptor and subsequently downregulate several inflammatory cytokines, including tumor necrosis factor-α, and interleukin 1 and 6 D or its metabolized product, 1, 25(OH)D, influences the expression of the fibrinogen genes, ytokines ; becauseytokines –49, thisSome limitations that could be addressed by future research must be acknowledged. As women are more likely to develop skeletal disorders associated with low 25(OH)D status than men and the study budget being constrained, we only included women. Our study's power to detect interactions with SNPs was low because we had access to a relatively small subset. Its strengths include that we investigated an ostensibly healthy population of Black South Africans known to present with high fibrinogen and low vitamin D owing to skin pigmentation, thereby increasing our chances of detecting an association should it exist. While total fibrinogen concentrations here reflect functional fibrinogen, the γ' fibrinogen levels are antigen based. Even though the absolute percentage may be slightly different if one measured total fibrinogen with an antigen assay, all samples were analyzed with the same methods, thus allowing comparison between individuals with low or high γ' fibrinogen. We, furthermore, report % γ' fibriniogen for correlations and relationships since the ratio is thought to be more physiologically relevant than the absolute concentration. Currently, our report is the only one of its kind to have considered the γ' fibrinogen molecule and plasma clot properties.Low vitamin D , 52, 53 lanthe.kruger@nwu.ac.za.The data analyzed in this study is subject to the following licenses/restrictions: Raw data were generated at the North-West University. Obtained data supporting the findings of this study are available from the PI of the study after obtaining the necessary ethical approval. Due to Ethical restrictions and the POPI act data are not publicly available. Requests to access these datasets should be directed to The studies involving human participants were reviewed and approved by Health Research Ethics Committee of the Faculty of Health Sciences, North-West University. The patients/participants provided their written informed consent to participate in this study.Conceptualization: CN-R. Methodology: PR, CN-R, ZL-L, IK, and MP. Statistical analysis and writing—original draft preparation: PR and CN-R. Resources and funding acquisition: IK and MP. Data curation and project administration: IK. Writing—review and editing: ZL-L, IK, and MP. All authors contributed to the article and approved the submitted version.This research was funded by SANPAD [South Africa–The Netherlands Research Program on Alternatives in Development (08/15)], the South African National Research Foundation (NRF GUN No. 2069139 and FA2006040700010), self-initiated research grants from the South African Medical Research Council, North-West University, Potchefstroom, South Africa, the Population Health Research Institute, Ontario, Canada and the Academy of Medical Sciences UK (Newton Fund Advanced Fellowship Grant [AMS-NAF1-Pieters to MP].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.


Enterobacter sp. FN0603 alone and in combination of both. Wheat seeds were soaked in silicon and bacterial solutions and sown in pots containing artificial saline-alkaline soils to compare the effects among all treatments. The results showed that the treatments with silicon and FN0603 alone significantly changed plant morphology, enhanced the rhizosphere soil nutrient content and enzyme activities, improved some important antioxidant enzyme activities  and the contents of small molecules  that affected osmotic conditions in the top second leaves. However, treatment with silicon and FN0603 in combination significantly further increased these stress tolerance indexes and eventually promoted the plant growth dramatically compared to the treatments with silicon or FN0603 alone (p < 0.01), indicating a synergic plant growth-promoting effect. High relative abundance of strain FN0603 was detected in the treated plants roots, and silicon further improved the colonization of FN0603 in stressed wheat roots. Strain FN0603 particularly when present in combination with silicon changed the root endophytic bacterial and fungal communities rather than the rhizosphere communities. Bipartite network analysis, variation partitioning analysis and structure equation model further showed that strain FN0603 indirectly shaped root endophytic bacterial and fungal communities and improved plant physiology, rhizosphere soil properties and plant growth through significantly and positively directing FN0603-specific biomarkers (p < 0.05). This synergetic effect of silicon and plant growth-promoting microorganism in the mitigation of saline-alkaline stress in plants via shaping root endophyte community may provide a promising approach for sustainable agriculture in saline-alkaline soils.Although microorganisms and silicon are well documented as factors that mitigate salt stress, their effect mitigating saline-alkaline stress in plants remains unknown. In this study, wheat plant seeds were treated with silicon,  SiliconTriticum aestivum L.) is one of most important cereal crops because it nourishes approximately 4.5 billion people, is planted extensively and has the potential for high global productivity  assess effects of silicon, Enterobacter sp. FN0603 and the combination of both on the wheat plant growth and physiobiochemical characteristics, rhizosphere soil physicochemical properties and bacterial and fungal community structures in rhizospheric soils and in roots and (2) elucidate the mechanisms of the saline-alkaline stress mitigation by silicon and the PGPR strain.The present study treated wheat seeds with silicon, a PGPR strain 2.2.1.The wheat cultivar employed was Yongliang No. 4 spring wheat, which is widely planted in Northwest China where saline-alkaline stress reduces plant growth and limits crop production. Our previous experiments  showed tZygophyllum xanthonylon and Haloxylon ammodendron and tested their plant growth promoting properties, including nitrogen fixation, solubilization of inorganic phosphorus, siderophore production, 1-Aminocyclopropane-1-Carboxylate (ACC) deaminase activity, indole-3-acetic acid (IAA) production, biofilm formation and exopolysaccharide (EPS) production. A strain isolated from the rhizosphere soil of Haloxylon ammodendron , and designated FN0603, which displayed all of these plant growth-promoting traits and high salinity-alkalinity tolerance (Enterobacter and had the highest 16S rRNA gene sequence similarity (99.87%) with Enterobacter hormaechei EN-114T , FN  and FN_Si . Wheat seeds were treated by soaking them in the appropriate solution  solutions containing silicon alone, FN0603 alone, both silicon and FN0603 or no silicon or FN0603) for 20 min. CMC-Na was employed to enhance adhesion to the seed surface.Four treatments were set up in this experiment, i.e., NU , Si , the culture was shaken with 160 rpm/min for 3 days then centrifugated to obtain the cells. The resulted cells were resuspended to an OD600 of 2.0 in sterilized 0.1% CMC-Na solution and immediately used to soak the seeds.An isolated single colony of FN0603 was inoculated into sterilized 1/2 R2SiO3 for the Si treatment, in an FN0603 cell suspension for the FN treatment and in a mixture of Na2SiO3 and FN0603  for the FN_Si treatment. After the excess liquid was removed, the seeds were immediately sown in pots.Healthy wheat seed grains were soaked in sterilized 0.1% CMC-Na solution for the NU treatment, in a sterilized 0.1% CMC-Na solution containing 0.8% Na2.3.+ concentration to 100 mM and the pH to 9.0 with Na2CO3 and NaCl: firstly adjusted pH to 9.0 by Na2CO3, then calculated Na+ concentration and replenished by NaCl to 100 mM), until the volume escaping from the bottom of the pot did not change within 2 h. The resulting soil was considered completely saturated by the saline-alkaline solution. After the first irrigation, 10 seed grains were sowed in each pot and covered with 1 cm of the same soil as in the pot. Twenty replicate pots for each treatment were prepared. The pots were cultivated under conditions of 5,000 Lux light-intensity, a light/dark cycle of 14/10 h and a temperature of 25 ± 2°C in a room without sunlight. During cultivation, pots were regularly irrigated with 185 mL of sterile distilled water per pot when the leaves appeared to be wilting. Water overflew from pot bottom was poured back into the corresponding pot the next day to ensure a stable salt content. The saline-alkaline fields planted wheat probably enriched halotolerant microorganisms and they may interact with silicon and strain FN0603 to rapidly help wheat in responding the stress. The saline-alkaline fields are far away from our laboratory and moreover the soils are just needed minor as source. Thus, the pot experiment was designed to add minor saline-alkaline source soils.The surface soil (top 10 cm) of fields near Huhhot City  and Barynoer City  were collected as neutral bulk and saline-alkaline source soils, respectively. The collected soils were screened through a 2 mm sieve to remove debris such as roots and stone. The sieved bulk soils were mixed well with quartz sand in a 2:1 volume ratio and a small amount of sieved saline-alkaline source soils  were selected at 30 days after emerged, the entire plant was sampled, and the corresponding rhizosphere soil, leaves and roots were taken for further analysis. After the roots were carefully dug out, large soil chunks were removed by shaking and the residual soil brushed off, then the roots were quickly frozen in liquid nitrogen and stored at −80°C until ready for the rhizosphere soil analysis . After t2.5.Wheat growth parameters including morphology, seedling emergence, tiller number, shoot length, root length, shoot dry weight and root dry weight were observed and measured. Germination rate was determined by the formula  described by where TDW- total (shoot + root) dry weight.2.6.649, OD665, and OD470, respectively, and calculated using the Equations  contents and the activities of the reactive oxygen species-scavenging enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). For chlorophyll and carotenoids analysis, 0.5 g fresh leaves were homogenized in 50 mL ethanol to extract pigments and determined ODquations  (2), (3)where V- the final volume of the supernatant and W- fresh weight of the leaf.kits were G0101F, G0105F, G0107F, G0109F, and G0124F for SOD, CAT, POD, MDA and root activity, respectively.Fresh leaves of 0.1 g were crushed homogenously in 1 mL the particular extract liquid, and then determined using spectrometer methods followed the protocols of manufacturer . The used 2.7.2Cr2O7-H2SO4 volumetric dilution heating method. Available nitrogen (AN) was determined by the alkaline diffusion method. Available phosphorus (AP) was extracted with 0.5 M NaHCO3 (pH 8.5) and measured at 880 nm. Rhizosphere soil and distilled water  were mixed and shaken for 30 min, then read the electrical conductivity (EC) of the filtered leach solution using conductivity meter. Soil and distilled water  were mixed and shaken for 30 min, then measured the pH of the filtered leach solution using pH meter.Rhizosphere soil was air-dried and screened through 1 mm, then determined properties following the method according to kits of Suzhou Grace Biotechnology Co., Ltd. The used kits were G0314W, G0305F, G0301W, G0302W, G0303W and G0307F for ALPT, ALP, UE, SC, CAT and DHA, respectively.For soil enzyme activity assays, rhizosphere soil was air-dried and screened at 60 mesh. Following the particular protocols, the activities of soil alcalase protease (ALPT), alkaline phosphatase (ALP), urease (UE), sucrase (SC), catalase (CAT) and soil dehydrogenase (DHA) were determined using Soil properties 2.8.® PowerSoil® Pro Kit  and the FastDNA® Spin Kit for Soil , respectively. The fungal ITS1 regions were amplified by PCR using primers ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC). The V5–V7 regions of the root endophytic bacterial 16S ribosomal RNA gene were amplified by PCR using the primer pair 799F (AACMGGATTAGATACCCKG) and 1193R (ACGTCATCCCCACCTTCC), the V3-V4 regions of the rhizosphere bacterial 16S ribosomal RNA gene were amplified by PCR using the 338F primer (ACTCCTACGGGAGGCAGCAG) and the 806R primer (GGACTACHVGGGTWTCTAAT) by an ABI GeneAmp® 9700 PCR thermocycler . The concentration and purity of the DNA was assessed using a NanoDrop® ND-2000 spectrophotometer . A NEXTFLEX® Rapid DNA-Seq Kit  was used to build a library and Illumina’s Miseq PE300 platform  was used for high-throughput sequencing. All amplicon sequencing was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. .According to the manufacturer’s protocols, DNA was extracted from the rhizosphere soil and the clean roots using the DNeasyα- and β- diversity indices were calculated as described by The Fastp software2.9.p < 0.05) that had significant differences in abundance. Referred to r| > 0.6, p < 0.05) using the R package “psych”  effect size (LEfSe) analysis 3 was use “psych” . The axi “psych” .2.10.A Mantel test (based on Bray–Curtis) of the physiologic properties of the wheat and soil and all of the root-associate microbiome was conducted using the R package “linkET”  after sc2.11.The results of the Mantel test and network indicated that most of soil properties, wheat physiology and wheat growth parameters were poorly correlated with the rhizosphere communities, so the structural equation model (SEM) and variation partitioning analysis (VPA) were only focused on the root endophytic communities.χ2 tests, the comparative fit index (CFI) and the root mean square error of approximation (RMSEA). Low χ2 values (p > 0.05), high CFI (>0.90) and low RMSEA (<0.05) indicated a well-fitting model. VPA determined the explanatory degree among strain FN0603, specific biomarkers, root endophytic bacterial communities, root endophytic fungal communities, rhizosphere soil properties, wheat physiology and wheat growth.A SEM was implemented with the software IBM SPSS AMOS (Ver. 23) with maximum-likelihood estimation and visualized by the affinity designer software . Model fitness was examined using the 2.12.p < 0.05 or p < 0.01 and labeled with uppercase or lowercase letters, respectively. Column diagrams of the physiologic parameters of the wheat and soil were drawn with the R package “EasyStat”  and the results were considered significant at the levels of asyStat” .3.3.1.Enterobacter strain FN0603 is more effective than with silicon. Obviously, both silicon and FN0603 can promote wheat growth under saline-alkaline stress conditions but the presence of both more effectively mitigates the stress.Thirty days after emergence, several commonly used plant growth parameters were analyzed. Compared with NU, the Si and FN treatments both evidenced remarkably different morphologies , with thp < 0.01). The contents of the pigment chlorophyll and the root activities were significantly enhanced in Si, FN and FN_Si compared to NU as well. The magnitude of the effects followed the order FN_Si, FN and Si.Physiological parameters of the functional wheat leaves were determined, including the antioxidase activities, chlorophyll content, carotenoid content and root activities, which are commonly responsible for the plant antioxidant capacity, as well as the proline and the total soluble protein contents, which indicate the osmotic regulation capacity under salt stress . The res3.2.p < 0.01).Significant differences in the improvements of the physiochemical parameters and enzyme activities of the rhizosphere soil were observed in the presence of silicon and FN0603 alone as well as in the combination treatment compared to NU . In majo3.3.α-diversity box plots showed that the Chao1 and Shannon indexes were approximately one order of magnitude greater for rhizosphere microorganisms than for the corresponding root endophytes; nevertheless the α-diversity of the root endophytes was much higher for the treatments compared to the rhizosphere microorganisms. The treatments did not show striking differences in the Chao1 and Shannon indexes of the rhizosphere bacteria, but a significant increase in the Chao1 index of the rhizosphere fungi was seen, particularly for FN_Si, which was 10.43% higher compared to NU (p < 0.05) of the root endophytic bacteria and fungi; Silicon and FN0603 alone reduced the Shannon indexes of the root endophytic bacteria and fungi, and the combination extremely significantly (p < 0.01) decreased the Shannon indexes up to 42.5 and 42.3%, respectively , Pseudomonas (2.68–4.56%) and Enterobacter (0.18–1.09%) for the rhizosphere bacterial community (Enterobacter (4.12–47.95%) was dominant and its abundance increased with treatment in the order Si, FN and FN_Si  and Actinobacteriota (27.28–28.92%) were greatest . Only fiommunity . For thend FN_Si . AdditioNeocosmospora and Fusicolla, and decreased Fusarium , respectively; and they included 14 genera shared by the two groups. Among 14 genera, the relative abundances of four genera , Enterobacter, one unclassified , Fusarium, and Myrmecridium, were significantly increased in FN_Si over Si and FN treatments, suggesting that the four genera with high abundance may have a stronger interaction between strain FN0603 along with silicon and wheat plant under saline-alkaline stress.For the rhizosphere microorganisms, the numbers of biomarkers were higher in Group Si  and FN (17 and 5) than in Group FN_Si (7 and 3); but for the root endophytes, the numbers were greater in Group FN_Si  and FN (39 and 13) than in Si (35 and 3) . A corre3.5.p < 0.01), but EC, pH and MDA had negative effects on these parameters (p < 0.01) and significant correlations with a few soil properties, wheat plant physiology and growth  and wheat physiology ; soil properties were directly and positively affected by the root endophytic fungal community  and bacterial communities (not significant)  (Group Si shown in λ = −0.647) and the combination of FN0603 and silicon (Group FN_Si shown in λ = −0.616) showed endophytic bacterial community have an significant and direct but negative effects on the endophytic fungal community (p < 0.001).SEM was employed to evaluate the direct and indirect effects of treatment with silicon alone, FN0603 alone and silicon and FN0603 combined on the microbial communities, soil properties, wheat physiology, and wheat growth. In all groups defined by the LEfSe assay above, all of the models fit the data well. Wheat growth was directly, positively and significantly affected by rhizosphere soil properties  –C. Silicλ = −0.764, p < 0.001), but the endophytic biomarkers induced by and significantly correlated with FN0603 had extremely significant, direct and positive effects on the endophytic bacterial  and fungal communities . FN0603 directly affected endophytic biomarkers in Group FN  and Group FN_Si . Besides indirect effects through mediating the entophytic bacterial and fungal communities, the significant and positive direct effects of endophytic biomarkers on soil properties were observed with λ values of 0.530 and 0.550 (p < 0.05) in Group FN and Group FN_Si, respectively. The models of Group Si, FN and FN-Si explained 37.6–58.4% of the endophytic biomarkers of the variation in the root endophytic bacteria and 84.8–99.7% of the variation in the other items.In Group Si, endophytic biomarkers  had significant, direct and negative effects on the endophytic bacterial community  enhanced activities of the reactive oxygen species-scavenging enzymes SOD, CAT and POD, and increased the proline content and reduced the MDA content in the wheat leaves in the presence of saline-alkaline stress. However, treatment with a combination of silicon and FN0603 effectively improved the antioxidant capability by further increasing the activities of the reactive oxygen species-scavenging enzymes and the proline content and decreasing the MDA content, compared to treatment with either silicon or FN0603 alone, the differences were highly significant (p < 0.01). Unexpectedly however, the leaf carotenoids level of treated wheat plants under saline-alkaline stress were lower, and the combination treatment even reduced those levels by 20.5% compared to the untreated control in this study. This result was distinctly different from the common finding that carotenoids are significantly increased under salt stress 2 solubilization, which positively affected the nutrient cycle and may have importantly contributed to the protection of wheat under saline-alkaline stress. These observations suggest that the interaction between silicon and FN0603 is synergistic and the combining silicon and PGPR treatments is a more effective approach to mitigate saline-alkaline stress in plants.Seed-priming by inoculation with PGPR activates the rhizosphere soil by improving nutrient levels, such as available nitrogen and phosphorus and organic matter, as well as the activities of soil enzymes . These oEnterobacter mitigates salt stress and promote plant growth by various processes, such as the production of ACC deaminase, IAA biosynthesis, phosphorus solubilization, nitrogen fixation, siderophore production, EPS secretion and biofilm formation , although the solely inoculated of silicon did not affect the abundance of the strain FN0603, suggesting that silicon promoted the colonization of FN0603 in wheat roots under saline-alkaline stress. As far as we know, this is the first report of such a function for silicon. According to the abundance-occupancy concept  and the highest abundance accounted for 14.84% of the inoculation with FN0603 in the present study. Some Gibberella could colonize wheat roots and significantly promote plant growth under water stress  can be found at: FFX and FYF designed the study. FFX completed the experimental content, data analysis, and wrote the first draft of the manuscript. FYF revised the manuscript. YGL contributed some to community assays. XBW, YZG, and KT commented on the revision of the manuscript. All authors contributed to revising, read, and approved the submitted version.This work was financially supported by the National Natural Science Foundation of China (31960021) and Key Technology Research Project of Inner Mongolia Autonomous Region (2021GG0360).FFX is a student at Inner Mongolia Agricultural University.The remaining 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.


Common mental disorders such as depression, anxiety, and somatic symptoms are a major public health concern because it is prevalent and chronic, and its impact on physical health, psychological and economic consequences is very serious. Evidence on the prevalence and predictors of common mental disorders is very limited in Ethiopia. This study aims to determine the prevalence and associated factors with common mental disorders.A community-based cross-sectional study was conducted among 731 south Gondar zone residents recruited with a multistage sampling method. Data were collected by face-to-face interviews on socio-demographic, clinical, and psychosocial factors. Common mental disorders (CMD) were assessed using a self-reporting questionnaire (SRQ-20). A-List of Threatening Experiences and the Oslo social support instruments were used to identify the factors. We used bivariate and multivariable binary logistic regressions to identify factors associated with common mental disorders. Statistical significance was declared at P-value < 0.05.The prevalence of common mental disorders over the last four weeks was found to be 29.7% with 95% of confidence interval (CI) (26.4–33.1). After adjusting possible confounders, female sex, , poor social support , family history of mental illness , rural resident , current use of khat  current use of tobacco  and unemployment  were significantly associated with common mental disorders.The prevalence of common mental disorders was high, especially in Female sex, current substance use (khat chewing (leaves) and tobacco smoking), unemployment, rural residence, family history mental illness, and poor social support are the main determinants of common mental disorders. Early detection and appropriate intervention for common mental disorders in the community level should be promoted. Governmental strategies should be focused on implementing substance rehabilitation centers to treat Khat and tobacco might be helpful to minimize the burden of CMD in Ethiopia. Mental disorder is a global public health concern. According to the recent global data, one in five individuals were suffered with common mental disorders in the last year and 29.2% of individuals had episodes of common mental disorders (CMDs) in their lifetime . The othIn sub-Saharan Africa, the total burden of mental and substance use problems is estimated to rise by 130%, and approximately 20 million YLDs to 45 million YLDs could be experienced in the year 2050. This estimation significantly affected health and productivity in the region [Different studies showed that the prevalence of common mental disorders varied across the world. In Great Britain , 24.6% of study participants were suffered from CMD . AnotherIn Africa, the estimated prevalence of CMD in Tanzania ranged from 24–28.8% , 10. AndA community-based cross-sectional study was used to determine the prevalence of common mental disorders and their associated factors among residents of south the Gondar zone from May 3 to June 3, 2018.Debre Tabor town is the capital city of the south Gondar zone which is 666 km far from Addis Ababa  and 99 km far from Bahir Dar . The zone is divided into 15 districts. According to the 2007 population census report; the total population size of South Gondar is estimated at 2,051,738. From those, 1,041,061 are men and 1,010,677 women. From the total population, 9.53% of the population is urban residents. This zone had seven primary and one referral hospital providing health services during the data collection period.All adults whose age is 18 years and above in south Gondar zone, Northwest, Ethiopia.Adults who were living in south Gondar zone selected districts/woredas during the study period.We used a multistage sampling technique to select 731 participants. From 15 districts/woredas we randomly select 3 districts by simple random sampling. After selecting the districts, we selected three sub-districts/kebeles in each of the selected districts. To reach households of each sub-districts, simple random sampling was employed. In each of the areas, household lists were obtained from sub-district offices and health extension workers. We proportionally allocated the sample size to each district and further to the sub-district. The selected household members were further sorted for interviews. In the case of more than one adult study participant in a household, we selected one of them by lottery method .Socio-demographic characteristics: age, educational status, sex, ethnicity, marital status, employment status, and residence.Clinical factors: family history of mental illness, and comorbid medical/surgical illness.Psychosocial factors: social support and stressful life events.Behavioral factors: ever and current use of substances .Common mental disorders: assessed using self-reporting questionnaire (SRQ-20), a score of 6 or more considered having CMD [ving CMD .Social support: measured using Oslo social support scale (OSS-3), a score of 3–8, 9–11, and 12–14 categorized as poor, moderate, and strong social support respectively [ectively .Individual stress levels: were measured using a 12 item List of Threatening Experiences (LTE), if a study participant experienced one or more stressful life events for the last six months [x months .Current and ever use of substance: assessed using adopted alcohol, smoking, and substance involvement screening test (ASSIST). If a study subject using at least one of the specified substances in the last 3 months and lifetime considered as current and ever of use substance respectively [ectively .Comorbid physical illness: to assess comorbid physical illness, study subjects were asked “Did you have any comorbid physical or surgical illness?” with a response of “Yes” considered having comorbid medical/surgical illness.Family history of mental illness: was measured by asking “Did you have a family history of mental illness?” with a response of “Yes” considered having a family history of mental illness.Data were collected by face-to-face interviews using a semi-structured questionnaire that contained socio-demographic, social support, clinical factors, and substance-related factors.Common mental disorders were assessed using a self-reporting questionnaire (SRQ-20). A 20-item mental disorder screening instrument was developed by WHO to screen CMD. The tool measures depression, anxiety, and psychosomatic symptoms, known as CMD. Each item of SRQ has rated on a two-point scale (yes/no), “0” indicates the absence of the symptom and “1” indicates the presence of the symptom. A score of 6 or more in the self-reporting questionnaire was considered as having common mental disorders in the last one month. The tool was validated in low-and-middle-income countries with a sensitivity 78.6% and a specificity 81.5% , 22.Social support was measured by using three items of the Oslo social support scale (OSS-3). The sum score of Oslo social support ranges from 3 to 14 with higher score indicating strong support and lower score indicating lower support. The instrument categorized as scoring of 12–14 considered “strong support”, the score of 9–11 “moderate social support” and score of 3–8 “poor social support” .Individual stress levels were measured by using a 12 item List of Threatening Experiences (LTE). The tool measures the individual level of stress for the last six months. Each item of LTE has rated on a two-point scale (yes/no), “1” indicates the presence and “0” indicates absence of stressful life events over the last six months . To asseWe recruited degree holder psychiatry professionals for data collection and supervised by Master holder psychiatry professionals. The training was given on the data collection instrument and sampling procedure. Additionally, the questionnaire was designed in English and translated to Amharic language . The supervision was held regularly during the data collection period.Data were entered into Epi-data 3.1 after checking completeness and consistency and then exported to SPSS—version 20 for analysis. Factors associated with CMDs were selected during bivariate analysis with a value of p ≤ 0.2. In multivariable regression analysis variables with P-value, less than 0.05 at 95% confidence interval with its adjusted odds ratio were considered as statistically significant.Ethical approval was obtained from the ethical review committee of Debre Tabor University. Ethical clearance was also obtained from the ethical review committees of the University (Ref. No. DTU/RE/1/P5/2017). Permission was obtained from the respective district administration.A total of 731 respondents were interviewed with a response rate of 98.5%. The majority of the respondents, 386 (52.8%) were male; two hundred forty-two (40.2%) were in the age range of 26–40 years; four hundred (54.7%) were married; two hundred sixty-eight (36.7%) were grade 9–12; almost half (50.6%) of respondents were residing in urban areas; 693 (94.8%) were Orthodox Christian, and seven hundred eighteen (98.2%) Amhara by ethnicity. Regarding occupation, more than half (73.6%) were employed  had a family history of mental illness, sixty-five (8.9%) had a comorbid physical illness. Regarding social support, nearly one-third (31.9%) had poor social support and 31142.5%) had strong social support  were consumed alcohol, and 576(78.7%) drinking alcohol at the moment; 121(16.6%) were using khat (leaves), and 107(3.4%) were smoked currently .P-value, less than 0.2 were entered into the multivariable logistic regression model to control confounding effects.To determine the association of independent variables with CMDs, bivariate, and multivariate binary logistic regression analyses was carried out. In the bivariate analysis factors associated with CMDs at a p-value less than 0.05.The result of the multivariate analysis showed that female sex, current use of khat (chewing leaves), rural residence, social support, and current use of smoking, unemployment, and family history of mental illness was significantly associated with common mental disorders at a Individuals who use khat currently were 1.7 times more likely to have common mental disorders than individuals who didn’t use khat currently . Respondents who had poor social support were 2.3 times more likely to develop CMDs compared with those who had strong social support . Similarly, unemployment had 1.7 times more likely to have common mental disorders when compared to employed individuals . The likelihood of developing CMDs was 2.1 times higher among respondents who had a family history of mental illness compared with those who had no family history of mental illness . The female sex was 2.4 times more likely to develop CMDs compared with the male sex . Participants residing in rural were 2.0 times high likely to have CMDs than participants who live in urban . Participants smoking at the moment were 1.7 times high likely for CMDs compared to ever use of tobacco (smoking)  . Our finding was consistent with previous studies conducted in Ethiopia, 32.4% , in Sout.4, 33.1.This study showed that the female sex had a 2.4 times higher risk of CMDs compared with the male sex. This might be due to various of factors, including hormonal differences, the effects of childbirth, psychosocial stressors (high household responsibility), as a result of physical abuse, and behavioral models of learned helplessness. In addition to this low socio-economic status of females might have predisposed them to a higher risk for common mental disorders. This finding was supported by studies carried out in India , Brazil Regarding employment status, common mental disorders were significantly associated with unemployment. The significant association between unemployment and CMDs in the present study was similarly reported in previous community-based studies carried out in Ethiopia , 32, ChiIn the present study, participants with poor social support were more likely to have common mental disorders when compared with participants with strong social support. This finding was consistent with a previous Ethiopian study . This isOur study design prevented us from concluding the cause-and-effect relationships of the associations. recall bias might be also one of the other limitations. Since the data collection method was a face-to-face interview which might lead individuals to respond in socially acceptable ways during the process, especially in cases of substance-related questions.The magnitude of common mental disorders was found to be high. Female sex, current substance use (khat chewing (leaves) and tobacco smoking), unemployment, rural residence, family history mental illness, and poor social support were significantly associated with common mental disorders. Therefore; we recommend common mental disorders  focused on early regular screening by trained health professionals and linkage with mental health service providers. It is necessary to give emphasis to individuals with a family history of mental illness, women, and a history of mental illness. Governmental strategies should be focused on implementing substance rehabilitation centers to treat Khat and tobacco might be helpful to minimize the burden of CMD in Ethiopia.


In patients with acute ischemic stroke, hemorrhagic transformation (HT) is a common complication after intravenous thrombolysis (IVT). In this study, we evaluated the relationship between the ratio of C-reactive protein to albumin (CAR) before thrombolysis, HT, and functional outcomes in patients with acute ischemic stroke.We retrospectively analyzed data from 354 patients who received thrombolytic therapy at the Second Affiliated Hospital of the Wenzhou Medical University in China between July 2014 and May 2022. CAR was measured on admission, and HT was identified by cranial computed tomography (CT) within 24–36 h after treatment. Poor outcome was defined as a score on the modified Rankin Scale (mRS) > 2 at discharge. The multivariate logistic regression model was used to investigate the association between CAR, HT, and poor outcome after thrombolysis, respectively.p < 0.001), and the 131 patients (37.0%) who experienced poor outcome than in those who did not . Multivariate logistic regression indicated that CAR was an independent risk factor for both HT and poor outcome. The risk of HT was significantly higher among patients whose CAR fell in the fourth quartile than among those with CAR in the first quartile . Patients with CAR in the third quartile were more likely to experience poor outcome , as were those in the fourth quartile , compared to patients with CAR in the first quartile.A total of 354 patients were analyzed, and their median CAR was 0.61 . CAR was significantly higher in the 56 patients (15.8%) who experienced HT than in those who did not (0.94 vs. 0.56, High ratio of C-reactive protein to albumin in individuals with ischemic stroke is associated with an increased risk of HT and poor functional outcomes after thrombolysis. Intravenous thrombolysis (IVT) with recombinant tissue plasminogen activator (rt-PA) effectively improves functional outcomes in patients with acute ischemic stroke (AIS) . HoweverInflammation plays a pivotal role in vascular injury. Post-ischemic inflammatory responses that disrupt the blood–brain barrier may be responsible for hemorrhagic transformation, which then further worsens the prognosis of patients with acute ischemic stroke . C-reactThe present study investigated whether the ratio of C-reactive protein to albumin might be associated with HT as well as poor functional outcomes in acute ischemic stroke patients after thrombolysis.Our study retrospectively analyzed patients with acute ischemic stroke who received intravenous thrombolysis in the Second Affiliated Hospital of Wenzhou Medical University from July 2014 to May 2022. Any patient who received alteplase at 0.9 mg/kg until a maximum total dose of 90 mg within 4.5 h of stroke onset was included in the study unless (1) C-reactive protein and albumin levels at admission were unavailable, (2) computed tomography was not performed within 36 h after intravenous thrombolysis, (3) patients had an immune disorder or severe infection, defined as a history of cancer, hematologic disease, use of an immunosuppressant or the presence of sepsis on admission, (4) low-dose alteplase was used, or (5) thrombolysis was interrupted for reasons other than HT.This study was approved by the Ethics Committee of the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University. The Committee waived the requirement for written informed consent because patients or their legal guardians, at the time of treatment, consented for anonymized medical data to be analyzed and published for research purposes.The following data were extracted from medical records: the demographic factors of age and sex; vascular risk factors, including hypertension, diabetes, hyperlipidemia, and current smoking; comorbidities such as atrial fibrillation, coronary artery disease, and stroke history (defined as previous ischemic stroke events); antithrombotic therapy before thrombolysis, blood pressure and stroke severity on admission, with severity assessed using the National Institutes of Health Stroke Scale (NIHSS) score. Blood test results also were collected, including levels of C-reactive protein, albumin, hemoglobin, fibrinogen, D-dimer, creatinine and glucose, white blood cell count and platelet count, international normalized ratio (INR), activated partial thromboplastin time (APTT); and cranial computed tomography (CT). Stroke was classified based on possible etiologies according to the TOAST classification .Venous blood was analyzed using an automatic hematology analyzer  and an automated biochemical analyzer . Blood was sampled before treatment in the emergency department. CAR was calculated by dividing C-reactive protein level (mg/L) by albumin level (g/dL), both of which were determined in the same blood sample.All patients underwent CT scanning once on admission and again within 24–36 h after intravenous thrombolysis. For the present study, CT images were retrospectively evaluated by two experienced neurologists who were blinded to the patient’s clinical data, and disagreements were settled by discussion. HT was defined as intracranial hemorrhage that was detected on the second but not first, CT. HT was subclassified as hemorrhagic infarction (HI) of type 1 or 2, or as parenchymal hemorrhage (PH) of type 1 or 2, based on European Cooperative Acute Stroke Study criteria . In addiNormally distributed continuous variables were expressed as mean ± SD, skewed continuous variables as median , and categorical variables as frequency (percentage). In univariate analysis, differences between continuous variables in two groups were assessed for significance using the t test or U-test as appropriate, while differences between categorical variables in two or more groups were assessed using Pearson’s chi-squared test or Fisher’s exact probabilities test as appropriate.p < 0.1 in the univariate analysis.Unadjusted models and multivariate logistic regression were used to analyze the association of CAR with HT or poor outcome after thrombolysis. The model included CAR but not its individual components because of their cross-correlation. CAR was modeled either as a continuous variable, in which case odds ratios (ORs) were calculated per 1-SD increase; or in quartiles, in which case ORs were calculated relative to the first quartile. In multivariate logistic regression, model 1 was adjusted for age and sex, while model 2 was adjusted for age, sex and other variables that were associated with We calculated areas under receiver operating characteristic curves (AUCs) to assess the ability of CAR to identify patients who experience HT or poor outcome. The optimal cut-off CAR was calculated by the Youden’s index, defined as the value that gave the greatest sum of specificity and sensitivity.p < 0.05 were considered statistically significant.All statistical analyzes were performed using SPSS 26.0 for Windows . Results associated with two-tailed A total of 451 acute ischemic stroke patients who received intravenous thrombolysis treatment were screened for eligibility from July 2014 to May 2022. Based on the protocol , 97 patip < 0.05; p < 0.05; After thrombolysis, HT was observed in 56 patients (15.8%) and poor outcome was observed in 131 (37.0%) patients, respectively. HT was associated with a significantly higher incidence of atrial fibrillation, NIHSS score, diastolic blood pressure at admission, international normalized ratio, D-dimer level, and C-reactive protein level (p < 0.001). Patients in the highest quartile had a significantly higher incidence of HT than those in the lowest quartile  across all patients, and HT was significantly associated with higher CAR . Patients were then stratified according to whether they suffered HT or not. Subgroup analysis also showed a positive association between CAR and poor outcome risk among those who experienced HT  relative to the first quartile, 95% CI 2.62–20.50, p < 0.001; CAR in patients who experienced poor outcome was significantly higher than those who did not . At the optimal cut-off of 0.42, CAR was able to differentiate between patients in our study who experienced poor outcome or not with a sensitivity of 82.4%, specificity of 49.8%, and AUC of 70.9% .In addition, receiver operating characteristic curve (ROC) analysis indicated a significant association between the risk of HT and a CAR cut-off value of >1.38, with a sensitivity of 44.6%, specificity of 81.5%, and AUC of 65.8%  and Clinical Scientific Research Foundation of the Second Affiliated Hospital of Wenzhou Medical University (SAHoWMU-CR2017-01-212).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.https://www.frontiersin.org/articles/10.3389/fnagi.2023.1109144/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


Beauvaria bassiana silica nanoparticles (Si NPs) as a new approach to increase cotton (Gossypium hirsutum L. Merr.) defense against an insect herbivore, Spodoptera littoralis. AM and non-AM cotton plants were infested with S. littoralis and then sprayed with a biopesticide [B. bassiana Si NPs] or a chemical insecticide (Chlorpyrifos).Plant-herbivorous insects are a severe danger to the world’s agricultural production of various crops. Insecticides used indiscriminately resulted in habitat destruction due to their high toxicity, as well as disease resistance. In this respect, the development of a sustainable approach to supreme crop production with the least damage is a crucially prerequisite. As a result, the current study was carried out to understand the potential effect of arbuscular mycorrhizal (AM) fungi along with B. bassiana Si NPs fungal extract showed that the major constituents identified were Oleyl alcohol, trifluoroacetate, 11-Dodecen-1-AL and 13-Octadecenal, (Z)-(CAS). Besides, results revealed a highly significant decrease in growth parameters in S. littoralis infested plants, however, with AM fungal inoculation a substantial improvement in growth traits and biochemical parameters such as protein and carbohydrates contents was observed. In addition, stimulation in proline and antioxidant enzymes activity and a decrease in malondialdehyde content were observed after AM inoculation.The gas chromatography-mass spectrometry (GC–MS) analysis of AM fungi mitigate the harmful effects of herbivorous insects by strengthening the cotton plant’s health via enhancing both morphological and biochemical traits that can partially or completely replace the application of chemical insecticides. Beauvaria bassiana metabolites applied with silica nanoparticles (Si NPs) in biocontrol of herbivorous insects of cotton plants are first studied.• Interactions between arbuscular mycorrhizal (AM) fungi and • Oleyl alcohol, trifluoroacetate, 11-Dodecen-1-AL and 13-Octadecenal, (Z)-(CAS) were the major compounds in ethyl acetate extract detected by Gas chromatography-mass spectrometry (GC–MS) analysis.Spodoptera littoralis insect.• Inoculation of cotton plants with AM fungi promotes growth traits of cotton plants and enhances their tolerance to • S. littoralis caused a reduction in AM colonization rates; besides with Chlorpyrifos application, a higher decrease was reported.B. bassiana Si NPs this increase is reduced greatly.• The cotton leafworm causes stimulation of lipid peroxidation in cotton leaves, and with AM fungi and/or S. littoralis infestation.• AM fungi increased total soluble protein, carbohydrates, proline content and antioxidant enzymes in cotton plant leaves as a result of  Gossypium hirsutum L.) is an important natural fiber and economic crop that provides substantial benefits to humans  oxy] phenyl] propane (1.22%) were also detected.Table B. bassiana Si NPs on larval mortality percentage as compared to the control. The highest mean of larval mortality percentages was 39.84% recorded at fraction No. 3 of B. bassiana Si NPs; while the lowest larval mortality (10.5%) was recorded at fraction No. 4.Data in Table B. bassiana Si NPs either in the presence or absence of S. littoralis. Infestation of cotton plants with S. littoralis had an inhibitory effect on growth parameters (Table p < 0.05) improved (4.9 g/plant) compared to non-AM ones (3.6 g/plant). Moreover, our findings revealed that with AM inoculation an increase in all growth parameters of cotton plants treated with B. bassiana Si NPs as compared to non-AM ones.Figure rs Table . In AM pS. littoralis infested cotton plants with Chlorpyrifos compared to infested control. Remarkably, there was an increase in shoot Fwt and root Dwt of cotton reared with S. littoralis with the dual application of AM fungi and B. bassiana Si NPs or Chlorpyrifos insecticide , vesicles (V), arbuscules (AR)] were observed in cotton roots  compared to control ones  application a significant increase was recorded. There are several causes for qualitative changes of sugars and proteins at the infection site, Kousar et al. [Sugars are the primary photosynthetic products that form the building blocks for all other chemical components of plants , as wellr et al.  that infr et al.  and Chenr et al.  reportedr et al. .Oryza sativa treated with insecticides, Acetochlor and Bensulfuron methyl. This reduction may be a result of an osmotic shock of pesticides resulting in a release of protein and loss of membrane transportability in the leaf cells. Also, Chlorpyrifos reduced the ratios of NAD and NADP, the electron transport system interfered and the ATP levels decreased, leading to the diminution in the carbohydrates content. Our findings conflict with what Metwally and Abdelhameed [Cucumis sativus plants treated with Agrothoate. However, with AM fungal inoculation, an increase in carbohydrates content was detected, this result could be attributed to enhancing photosynthesis and hydrolysis of starch to soluble sugar by AM and the sink effect of AM demanding sugars [The decrease of soluble carbohydrates and protein with Chlorpyrifos coordinated with what Shakir et al.  and Huanelhameed  stated ig sugars , 68.Lipids play an important role in maintaining the structural integrity of cells as a fundamental component of cellular membranes, and lipid peroxidation reveals the severity of oxidative stress . MalondiS. littoralis infestation and Chlorpyrifos and B. bassiana Si NPs applications significantly increase the MDA content of both AM and non-AM cotton plant leaves; this pesticide caused oxidative stress in cotton leaves by ROS production that caused peroxidation in membranous lipids and the formation of MDA [Vigna radiata treated with Chlorpyrifos. Also, Abd-ur-Rahman et al. [Brassica oleracea infested with S. litura after application of Bifenthrin, Emamectin Benzoate and Lufenuron insecticides.n of MDA , 71. Hown of MDA . This ren of MDA  in Vignan et al.  exhibiteS. littoralis infestation may be due to the enrichment in proline synthesizing enzymes and reduction in catabolizing ones or its circumscribed assimilation in protein synthesis, a further increase was recorded with AM fungal inoculation. Kaur et al. [B. bassiana Si NPs, the effectivity of AM fungi was higher under infested conditions and produce higher proline as osmoregulant. Ullah et al. [S. litura caused a significant increase in proline content in tomato leaves, and further augmentation in its content was recorded with the application of PGPR under both control and infested conditions.The accumulation of cellular osmolytes in plants helps in sustaining cellular function and physiological stability under stress including pathogen attacks , 74, 75.h et al.  affirmedS. littoralis infested cotton plants and treated with Chlorpyrifos showed a significant enhancement in the proline content, similar records were observed by Parween et al. [Vigna radiata in response to chlorpyrifos and by Wu et al. [Brassica oleracea infested with S. litura after application of Bifenthrin, Emamectin Benzoate and Lufenuron insecticides. These pesticides seem to have imposed oxidative stress on treated cotton plants, which is a rapid and sensitive response of plants to environmental stress.Additionally, n et al.  on Vignau et al.  on rice u et al.  noticed Defense responses are often induced in the plants following the attack by pathogens or herbivore feeding. Once attacked by pests, plants initiate sophisticated defense responses including the regulation of defense pathways and production of defensive compounds which result in local or systemic resistance , 77. AmoS. littoralis infestation was inconsistent with Mohamed et al. [S. littoralis and reported well-expressed CAT, POX and PPO in their shoots. Similarly, our findings are coherent with the previous studies of Kaur et al. [S. littoralis infection is a clear indication of the possible involvement of these enzymes in the plant detoxification mechanisms besides this anti-oxidative defense is a crucial part of the basic metabolism, empowering the plant to cope instantaneously with rapid environmental stresses.The increase in CAT, POX and PPO activities in cotton plant leaves with d et al.  who studr et al.  and Zhaor et al.  that disPOX plays a role in lignification and cell wall rigidity and protects the plants against infestation by herbivores . BesidesAllium cepa plants. These pesticides seem to have imposed oxidative stress on treated plants, which is a rapid and sensitive response of plants to environmental stress. There is some evidence of pesticide degradation by elevated activity of oxidoreductase enzymes [B. bassiana Si NPs, the effectivity of AM fungi was higher under infested conditions.Figure  enzymes , 51. NotB. bassiana Si NPs to S. littoralis infested cotton plants can lead to a higher ROS elimination that can denature different plant bio-molecules. Our results corroborate with Xu et al. [B. brongniartii Fe0 NPs enhanced the activities of CAT and POX. Similarly, a study by Sharma and Mathur [Brassica juncea infested with S. litura that lead to an enhanced immune system against herbivory. Also, Abd-ur-Rahman et al. [Brassica oleracea infested with S. litura after Bifenthrin, Emamectin Benzoate and Lufenuron insecticides application. As a result, the observed anti-oxidative enhancement in AM colonized cotton plants infested with S. littoralis is a mechanism to combat insect induced oxidative stress and help plants to maintain the ROS levels below their deleterious levels and mediate quick removal of toxic ROS so that metabolism remains stable [Also, the further augmentation in CAT, POX and PPO activities owing to the dual application of AM inoculation with Chlorpyrifos or u et al.  who repod Mathur  confirmen et al.  proclaims stable .B. bassiana Si NPs against S. littoralis infested cotton plants as a biological alternative solution to reduce the negative environmental consequences of chemical pesticides such as Chlorpyrifos. Our results showed that Oleyl alcohol, trifluoroacetate was the major compounds in ethyl acetate extract detected by Gas chromatography-mass spectrometry (GC–MS) analysis that acts as nematicide and pesticide. Also, the results exhibited the stimulatory effects of AM fungal inoculation on growth, biochemical parameters as well as the detoxifying mechanism in cotton plants treated with B. bassiana Si NPs or Chlorpyrifos under S. littoralis stress. From the above findings, it is suggested that AM fungi can potentially be used in environmentally friendly S. littoralis management. However, further work is required to determine the efficacy and persistence of AM fungi and B. bassiana Si NPs under field conditions.This study described the insecticidal bio-efficacy of AM fungi and S. littoralis (Boisd.) (Lepidoptera: Noctuidae). The original culture was obtained from a well-established culture reared at the Department of Cotton Leafworm, Plant Protection Research Institute, Sharkia Branch, Egypt. The maintained insect culture was reared under laboratory conditions of 26 ± 1 °C, 70 ± 5% humidity and 12 h/12 h of day/night cycle [Ricinus communis). Upon pupation, pupae were carefully collected. When adults emerged, they were sexed and kept in mating cages. Cotton wools soaked in 10% sugar solution were placed as wickers to provide sources of nutrition for moths and changed daily to avoid fermentation.The present study was carried out on the Egyptian cotton leafworm, ht cycle . The newB. bassiana was obtained from Assuit University, Mycology Center, Egypt and was cultured on potato dextrose agar (PDA) medium for 2 weeks at 28 ± 1 °C. Spores were harvested and the spore suspension was cleaned from hyphal debris [7 conidia/ mL [B. bassiana applied with Si NPs were prepared by inoculating 1 mL of B. bassiana spore suspension and 1 mL of Si NPs solution (500 ppm) in an Erlenmeyer flask (250 mL) containing 100 mL sterile liquid Dox broth medium, then incubated at 28 ± 1 °C and 50–60% humidity for 7 days. Cell filtrate and mycelial mat were separated by using Whatman filter paper No.1 under the biosafety cabinet; therefore, the cell-free supernatant (CFS) became ready for further use and for foliar application.The fungal isolate of l debris  and its idia/ mL . The metFunneliformis mosseae, Funneliformis constrictum, Gigaspora margarita and Rhizophagus irregularis together in pots filled with sterilized sandy clay soil were propagated on Sudan grass (Sorghum sudanenses Pers.) roots as an appropriate trap plant for inoculum production. AM inoculum consisted of AM spores, hyphae and colonized root fragments.Spores of AM fungi were isolated from rhizospheric soil from Sharkia Governorate, Egypt via wet sieving and decanting technique . ApproxiIt is an organophosphorus insecticide supplied by Dow Agro Sciences with a chemical name of O, O-diethyl O- phosphorothioate (IUPAC). It inhibits the acetylcholine esterase (AChE) enzyme. It is used at the recommended dose of 5 mL /L of water.Gossypium hirsutum L. Merr.) obtained from Agriculture Research Center, Giza, Egypt, were surface sterilized with 7% sodium hypochlorite (NaOCl) for 10 min, subsequently rinsed with sterilized water and sown in sterilized plastic pots  filled with 10 kg sterilized soil. The physicochemical characteristics of the soil were analyzed  in the Botany and Microbiology greenhouse Department, Faculty of Science, Zagazig University, Egypt. Seeds of a variety Giza 86 of cotton (1- Non-AM treatments: cotton seeds were planted in pots that received 100 g of sterilized soil per pot.2- AM treatments: cotton seeds were planted in pots and received 100 g Sudan grass-root fragments per pot at the sowing date (approx. 80 spores/g trap soil).B. bassiana Si NPs and Chlorpyrifos) were sprayed. Each treatment was divided into four groups according to infestation with S. littoralis as follows:Fifty days post sowing, plants were artificially infested using one egg mass of the cotton leafworm/ plant. When the 1st instar larvae emerged from egg masses on leaves, they were allowed to feed on plants till reached the 2nd instar larvae then the tested compounds (a- The 1st group was left without infestation (control).b- The 2nd group was artificially infested with S. littoralis and not sprayed either with Chlorpyrifos compound or B. bassiana Si NPs.c- The 3rd group was artificially infested with S. littoralis and then sprayed with Chlorpyrifos compound.d- The 4th group was artificially infested with S. littoralis and then sprayed with B. bassiana Si NPs.n = 5) for each treatment. The pots were arranged in a completely randomized design (2 × 4). The plants were harvested after 1-week spraying of the tested compounds to evaluate the morphological criteria and mycorrhizal status, and to conduct a variety of biochemical analyses.There were five replicates . The extract was dried by a rotary evaporator (60–65 °C) to get a crude extract which was then separated by thin-layer chromatography (TLC)  [S. littoralis using leaf-dip bioassay [Ricinus communis. The percentages of larval mortality were calculated after 24, 48 and 72 h post-treatment to determine the most bioactive fraction that was afterwards analyzed by GC–MS at the National Research Center, Cairo, Egypt using G/C/MS-QP − 1000– Ex gas chromatograph-Mass spectrophotometer (SHIMADZU-Japan)   using hebioassay  using trThe whole cotton plants were removed from the soil, their shoots were detached and their roots were washed separately to remove soil particles and their fresh weights (Fwt) were measured. Subsequently, after drying in an electric oven at 80 °C for 48 h, the shoot and root dry weights (Dwt) were registered. Fwt and Dwt were measured as the average in gram per plant (g/plant). Also, total Fwt and Dwt of cotton shoots and roots as well root/ shoot ratio (R/S) were calculated.B. bassiana Si NPs and Chlorpyrifos, the roots of AM colonized cotton plants from different treatments were washed separately with tap water then cut into small pieces of 0.5–1 cm in length, then cleared with 10% KOH. The colonization percentage was evaluated by staining with 0.05% trypan blue for 15 min at 90 °C [Gossypium hirsutum was calculated using the following equation:After 1 week of spraying of at 90 °C , 92. Forat 90 °C  accordinCarbohydrates content was estimated in 0.1 g Dwt of cotton leaves by phenol sulphuric acid method  at 490 nTotal protein content was estimated in 0.5 g Fwt of cotton plant leaves after homogenizing in 10 mL of 25 mM Tris – HCl buffer solution   and its Lipid peroxidation was determined by measuring malondialdehyde (MDA) formation by the thiobarbituric acid method designated by Ohkawa et al.  after hoProline content in a known Fwt of AM and non-AM cotton plant leaves was determined after homogenizing in 10 mL of 3% aqueous sulphosalicylic acid . Equal v*Where 115.5 is the molecular weight of proline.A known Fwt of cotton leaves (0.5 g) was homogenized in 4 mL of ice-cold 50 mM potassium phosphate buffer  containing 1 mM EDTA. The homogenate was centrifuged at 10000 rpm at 4 °C for 10 min and the supernatant was used for determining the activities of catalase (CAT) at 240 nm , peroxidp < 0.05 was considered to be significant.Each result was presented as the mean of 5 replicates. The significant differences between treatments were statistically evaluated by Tukey,s HSD test and two-way analysis of completely randomized (ANOVA) using Costat statistical software (2005) program.


Background: Mealtime television use has been cross-sectionally associated with suboptimal diets in children. This study aimed to assess the two-year prospective association between baseline mealtime television use and subsequent diets in young children, and identify socioeconomic differences. Methods: Parents reported their child’s television use at meals, and fruit, vegetable, and discretionary food intakes. Multivariable linear and logistic regression analyses assessed the association between baseline mealtime television use and follow-up diet outcomes. Differences were assessed by socioeconomic position. Results: Participants were 352 Australian parents of children aged six months to six years. Daily mealtime television use (average frequency/day) was associated with higher daily frequency of discretionary food intakes  0.07–0.67) at the 2-year follow-up. Individually, television use during breakfast and dinner (1–2 days/week compared to never) predicted higher daily intake frequency of discretionary food, β 0.36 (95% CI 0.12–0.60) and β 0.19 (95% CI 0.00–0.39), respectively. Similarly, 3–7 days/week of television use during breakfast and lunch predicted higher frequency of discretionary food intake, β 0.18 (95% CI 0.02–0.37) and β 0.31 (95% CI 0.07–0.55), respectively. Associations were not socioeconomically patterned. Conclusions: Investigating mealtime television use motivators across the socioeconomic spectrum could inform interventions targeting the high consumption of discretionary foods in children.  One of the major contributing factors to poor health outcomes in high-income countries is sub-optimal nutrition, beginning in early childhood. In Australia, children consume well above the recommended intakes for discretionary foods ,2, well A family’s socioeconomic position (SEP) is also an important correlate of children’s diets and obesity risk ,12. ChilMeals within the home are a convenient context to promote healthy diets to young children. Regardless of SEP, mealtimes present opportunities to improve children’s dietary outcomes, and the more mealtimes a child shares with their family, the higher the likelihood of them consuming more healthy diets ,17. DinnTelevision (TV) viewing during mealtimes is an important focus because it is a frequent and potentially modifiable behaviour. Around one third of young children in Australia eat while watching TV at least once every day , and thiBoth mealtime TV use  and dietGiven this, research aiming to understand what influences children’s diets and eating behaviours, and socioeconomic differences, is fundamental. Such research provides a better understanding of how to improve diets and overall health of both children and adults, and, in turn, the health of future generations. Therefore, in this study of Australian children aged six months to six years, we aimed to assess prospective associations between TV use during specific mealtimes  and fruit, vegetable, and discretionary food intakes at the subsequent two-year follow-up, and determine if associations were socioeconomically patterned. This study used data from the Family Meals with Young Kids Study, an online survey completed in 2014 , and folRecruitment was conducted online using social media to advertise the study on blogs, Facebook™, and online parent groups. Participants followed a link to an online screening tool and survey, hosted on SurveyMonkey™ . Follow-At baseline, mealtime TV use was assessed by asking ‘how often does (child’s name) watch television while eating (each of breakfast/lunch/dinner/snacks)?’ . Five reTo calculate average daily mealtime TV use, as conducted previously , a cumulAll dietary measures have been previously validated ,45,46 anFruit and vegetable intakes were assessed by one item each, asking about ‘how many serves of vegetables does (child’s name) usually eat per day?’ (with examples), and included five response options: my child does not eat these, <1 serve/day, 1 serve/day, 2 serves/day, 3 serves or more/day. Informed by the data distributions, intakes of fruit and vegetable were dichotomised for analysis as <2 and ≥2 serves per day, respectively. The baseline questionnaire also collected child age, child gender, location of meals, and family meal frequency. These variables have been considered as potentially influencing the association between diet and shared mealtimes ,49, and The education level of the responding parent at baseline was used to define SEP for this study. Previous research has found maternal education to be the strongest and most consistent socioeconomic predictor of children’s dietary intakes ,11,39,50Due to the large number of responding parents who were university educated in this sample (>70%), education was dichotomised to ‘university educated’ and ‘non-university educated’; comprising ≤ 12 year or equivalent, trade/apprenticeship , and certificate/diploma .A test–retest study was conducted at baseline to measure the reliability of survey questions. A subsample of 54 participants completed the same survey again, two weeks after initial completion. Intraclass correlations (ICCs) were performed for each ordered categorical variable, and Cohen’s Kappa was performed for each non-ordered categorical variable. Intraclass correlations assessing the reliability of dietary measures ranged from moderate (ICC 0.5) to good (ICC 0.81). The reliability of the mealtime TV use measure ranged from good (ICC 0.77) to excellent (ICC 0.96) . The intDescriptive analyses were conducted to explore the sample characteristics. Multivariable logistic or linear regressions were used to examine the prospective associations between mealtime TV use at baseline and intakes of fruit and vegetables (<2 and ≥2 serves per day), and discretionary foods (intake frequency per day), at the 2-year follow up, respectively. Separate models were conducted for each meal type , as well as average daily mealtime TV use. Crude models were conducted first with mealtime TV use as the exposure, and dietary intakes at follow-up as the outcome with adjustment for baseline dietary intakes. Adjusted models included the following covariates: child age, child gender, family mealtime frequency, SEP, and mealtime location. Pearson and Spearman correlations were conducted to test for potential multicollinearity between mealtime TV use and covariates. No significant correlation was found, so all covariates were included simultaneously in the adjusted model. Analyses to detect possible interaction between mealtime TV use and covariates were also performed by creating interaction terms (the product of two variables). The interaction between mealtime TV use and mealtime location was significantly associated with dietary intake; therefore, that interaction term was also included as a covariate in the adjusted model. p < 0.05. To test if the association between mealtime TV use at baseline and dietary intakes at follow-up differed by SEP, we conducted stratified analyses (crude and adjusted) by SEP, and the likelihood ratio test was used to test for differences between stratum-specific regression coefficients. For adjusted analysis, the model adjusted for all aforementioned covariates, except for SEP. All analyses were conducted in StataIC  with statistical significance set at Of 992 participants at baseline, 369 participants with complete data on mealtime TV use at baseline and dietary intakes at both baseline and follow-up were included in the crude model. Seventeen participants had missing data on covariates, resulting in a final sample of 352 included in the adjusted model . A compaAt baseline, most children (74–86%) had optimal eating locations for main meals . In contrast, most children (71%) had suboptimal eating locations for snacks. A large proportion of parents ate each meal type with their children 3–7 days per week (74–90%). Around one third of children ate breakfast, lunch, and/or dinner while watching TV at least once per week, and a similar number ate snacks while watching TV on 3–7 occasions per week.At baseline, 71% and 54% of children consumed ≥ 2 serves of fruits and ≥2 serves vegetables, respectively. At follow-up, 82% of children consumed ≥ 2 serves of fruits, and 69% consumed ≥ 2 serves of vegetables. The mean daily frequency of discretionary foods was 0.96 (standard deviation (SD) 0.88) at baseline, and 1.30 (SD 0.85) at follow-up.p < 0.01) (p = 0.03). Significant associations were also found between mealtime TV use during lunch (3–7 days per week), dinner (1–2 days per week), and snack times (3–7 days per week), and a higher frequency of discretionary foods. No significant associations were found between baseline mealtime TV use and vegetable intakes at follow-up, although dinner TV use 3–7 times per week showed a trend towards association with lower odds of consuming ≥ 2 serves of vegetables/day (odds ratio (OR): 0.56; p = 0.07). For fruit, mealtime TV use at lunch (1–2 days/week) and dinner (1–2 days/week) was associated with lower odds of consuming ≥ 2 serves of fruit per day, with odds ratios of 0.47 (p = 0.04) and 0.48 (p = 0.03), respectively.Crude analysis revealed that overall daily mealtime TV use at baseline was associated with a higher frequency of consuming discretionary foods at 2-years follow-up  . No evidIn the adjusted model , the sigp = 0.15–0.82). Stratified analyses by two SEP groups are presented in p = 0.01) and dinner , and 3–7 days per week for breakfast  and lunch , were significantly associated with discretionary food frequency. Additionally, watching TV during dinner 1–2 days per week was associated with lower odds of consuming ≥ 2 serves of fruit per day in this group . In both SEP groups, mealtime TV use was not associated with vegetable intakes (p < 0.05). In the multivariate analyses, parent education was not significantly associated with baseline mealtime TV use at any of the assessed mealtimes and prospective intakes of fruit, vegetables, and discretionary foods , and the association with discretionary food intakes was no longer significant. Eating in a location other than at a table/bench (such as in the lounge room or the bedroom) has been found to be cross-sectionally associated with reduced vegetable intake , poorer This study highlights the importance of understanding young children’s TV use during snacking, in the context of mealtimes. Given that snacks contribute substantially to young children’s overall diet intake and diet quality , but areAlthough this study found no significant associations between mealtime TV use and prospective intakes of fruit and vegetables, all results were in the expected direction, with intakes lower as TV use increased. Previous cross-sectional studies supporting this association have used measures such as four-day  or sevenThis study did not find a differential effect of SEP on associations between mealtime TV use and diet. The strengths of the associations are similar, suggestive of the stratified analysis by SEP groups resulting in the low SEP group being too small to detect true differences. The results suggest that mealtime TV use is prospectively associated with discretionary food intakes, irrespective of SEP. This supports some cross-sectional research that mealtime components, such as TV, may not be influenced by SEP . AlthougTo our knowledge, this is the first study to assess the prospective association between mealtime TV and intakes of fruit, vegetables, and discretionary foods. Given that the majority of mealtime research is cross-sectional, providing clarity of the temporal order of the outcomes measured is an important strength of this study. It is also the first study to assess a range of eating occasions across the day, including snacks. A potential limitation of this study may be the dietary measures, given their brevity, and that in the current study, child fruit and vegetable intakes were reportedly higher than the Australian average . Though This study identified that a higher frequency of mealtime TV use in young children was prospectively associated with a higher frequency of discretionary food intakes two years later, irrespective of SEP. Reducing mealtime TV use could be a promising intervention strategy in the pursuit of reducing young children’s discretionary food intakes, the promotion of optimal diet quality, and improving health outcomes for young children and future generations.


To explore a novel Immune-associated gene signature for overall survival (OS) in patients with oral squamous cell carcinoma (OSCC).  Expression profiles of genes and corresponding clinical materials of OSCC patients were obtained through the TCGA database. With a LASSO Cox regression model, a multigene signature was established to predict the OS of OSCC patients. Some molecular experiments including RNA interference, MTT, and Transwell assay were applied to verify the role of the risk gene FGF9 in OSCC. P < 0.05). According to multivariate Cox regression analyses, the risk score was considered an independent factor for OS prediction . ROC curve-based analysis revealed the signature's ability for prediction. According to functional analysis, the immune cell expression and immune function of the HG were significantly inhibited. After knocking down the high-risk gene FGF9, the migration, proliferation, and invasion capabilities of OSCC cells HSC6 were significantly suppressed (P < 0.05).  43 immune-related prognostic DEGs were identified in OSCC. A 17-gene signature was established to assign the patients to either a high-risk group (HG) or a low-risk group (LG). The HG presented a shorter OS than the LG ( A novel immune-associated gene signature was identified for predicting the prognosis of OSCC. These risk genes show great potential as targets for OSCC treatment. Oral and maxillofacial malignancies include oral squamous cell carcinoma (OSCC), glandular epithelial carcinoma, and basal cell carcinoma, among which OSCC is the most common. According to reports, the incidence of OSCC ranks sixth in malignant tumors, and its treatment includes surgery, radiotherapy, and chemotherapy. Recurrence and metastasis are found in about 50% of OSCC cases within 3 years after therapy. There are more than 145,000 cases of death each year, with a 5-year survival of approximately 50% .At present, the formulation of postoperative treatment protocols and prediction of OCSS treatment outcomes mainly rely on the tumor differentiation grading and preoperative TNM staging, which, however, are considered less satisfactory . A growiIn the present research, immune-associated prognostic genes were screened out based on OSCC samples in The Cancer Genome Atlas (TCGA) database to establish an immune-associated gene set with higher sensitivity and specificity for forecasting the outcome of OSCC. Molecular biology methods were used to verify the role of the core genes in the gene set in OSCC.The protocol was approved by the Ethics Committee of Beijing Stomatological Hospital, Capital Medical University (BSH39879).https://portal.gdc.cancer.gov/repository), the data about RNA sequence and corresponding clinical information of 413 samples were downloaded. The cohort covered 380 OSCC samples and 32 control ones. The expression profiles of genes were subjected to normalization by the scale method offered in the limma R package. Normalized read count values were applied. Due to the accessibility of TCGA to the public, our research was exempted from the ratification of local ethics committees. Our research followed the TCGA access policies and publication guidelines  to obtain immune-related DEGs in OSCC. With the STRING database (https://www.string-db.org), a PPI network was generated for the overlapping immune-related prognostic DEGs.R language CIBERSORT algorithm was applied to assess the expression and relationship of 22 immune cells in OSCC. The screening criteria of CIBERSORT analysis was P < 0.05 . R limmaTo minimize the overfitting risk, a prognostic model was developed by LASSO-penalized Cox regression analysis . ThroughPCA was conducted through the “prcomp” function of the “stats” R package. With t-SNE, the distribution of different groups was identified by the “Rtsne” R package. To analyze the survival of every gene, the “surv_cutpoint” function of the “survminer” R package was used to define the optimal cut-off expression. The “survival ROC” R package was used to perform time‐dependent ROC curve-based analyses for assessing the significance of the gene signature in prediction.∗P < 0.05 values were changed via the BH method. ssGSEA in the “gsva” R package was adopted to calculate the infiltrating score of 16 immune cells as well as the activity of 13 immune-associated pathways [The “clusterProfiler” R package was used for both GO and KEGG analyses on the basis of the DEGs between the high-risk group (HG) and low-risk group (LG). pathways .2) in DMEM  comprising 10% FBS  and 100 U/ml penicillin/streptomycin (Beyotime Institute of Biotechnology). siRNAs were adopted to transfect the cells via Lipofectamine® RNAiMAX transfection reagent . HSC6 cells (1 × 105 cells) were placed in 6-well plates in complete DMEM, followed by overnight incubation (37°C). The siRNA lipoRNAiMAX mixture was then transferred to incubation wells comprising cells suspended in 800 μl DMEM (cell density: about 30–40% (2 × 105 cells)). The control siRNA and FGF9 siRNA  were used at a concentration of 50 nM. After 6-h culture (37°C), the medium was changed to a fresh one (comprising FBS + penicillin/streptomycin), followed by 48-h continuous incubation (37°C) before later experimentation. HSC6 cells were assigned to four experimental groups: CG, HSC6 cells transfected with negative control siRNA or KD-FGF9 groups, HSC6 cells transfected with FGF9 siRNA group.The human OSCC cell strain HSC6 was acquired from the CCTCC  and subjected to incubation , GADPH .The total RNA of HSC6 cells was acquired using the TRIzol reagent , and its quality was guaranteed via the agarose electrophoresis. The RNA was then reverse-transcribed to acquire the complementary DNA (cDNA) using a corresponding kit supplied by Invitrogen . Subsequently, an SYBR Green PCR kit  was adopted to quantify the genes at an mRNA level. The relative miRNA was calculated using the 2−The total proteins of the cells were obtained using RIPA lysis buffer , and one BCA assay kit  was adopted for protein concentrations assay. The lysates were treated with 10% SDS-PAGE to separate target proteins based on their molecular weight, and the proteins were transferred to the PVDF films , followed by blocking in 5% defatted milk. The films were then treated with first antibodies against FGF9  and incubated overnight at 4°C, the GAPDH , and were then probed with the second antibody.μl of MTT  was supplemented after transfection or therapy, and a further 4-h culture was carried out, followed by supplementation of DMSO (200 μl) for dissolving the formazan. OD values (490 nm) were then tested, and the relative cell viability was computed : 100%.With the methods adopted previously, cell viability was tested. Totally, 20 5 cells/ml) till the formation of a monolayer. The scratch area was tested under one microscope  at 0 and 24 h. Under the microscope, the distance of cells migrating to the scratch area was measured, followed by analysis using ImageJ .Before the cell wound healing assay, the cells were placed in 6-well plates (5 × 105) were placed on the upper side of Matrigel-coated polycarbonate Transwell filters. For the determination of invasion, the cells were subjected to suspension in a medium without serum, and a serum-contained medium was added to the lower compartment. The cells were treated with 2-h incubation (37°C). The noninvasive cells in the upper compartment were wiped off using cotton swabs. The invasive cells on the lower membrane were treated by 10-min immobilization with 100% methanol, air-drying, and dyeing through crystal violet solution , followed by counting under one microscope.The cells  or SPSS (Version 23.0). If not specified above, P < 0.05 denotes statistical significance, and each Pvalue was two tailed. The data of western blot, qPCR, Transwell, and wound healing was evaluated with the Student's t-test.The Student's In OSCC, there was an infiltration correlation between immune cells. For example, CD8T cells and CD4 memory T cells activated were both bound up with resting infiltration positively; NK cells activated and mast cells resting infiltration positively correlated. M0 macrophages and CD4 memory T cells activated, the infiltration of CD8T cells, and M1 macrophages negatively correlated .246 immune-related genes and 71 prognostic-associated genes in total were identified. By taking the intersection of the two, 43 immune-related prognostic DEGs in OSCC were identified  43 genesn = 95) or LG (n = 95) based on median cut-off in the training set. PCA and t-SNE analysis revealed the distribution of patients from different risk groups in 2 directions . ROC survival curve analysis revealed that in the training set, the AUC area of the model was 0.806, 0.749, and 0.799 in the first to the third year, respectively, suggesting the high specificity and sensitivity of the model  or an LG (n = 95). PCA and t-SNE analysis revealed the distribution of patients from different risk groups in 2 directions  (Figures P < 0.001; test set: HR = 4.531, 95% CI = 2.120–9.682, P < 0.001; Figures Univariate and multivariate Cox regression analyses among the available variables were conducted for ascertaining whether the risk score was one independent factor for forecasting OS prognosis. In the former analysis, the risk score was markedly bound up with OS in the training set and test set (HR = 4.300, 95% CI = 2.371–7.798,  Figures . AccordiP < 0.05, Figures P < 0.05, Figures P < 0.05, Figures P < 0.05, Figures For illustrating the biological functions and pathways bound up with the risk score, the DEGs between HG and LG were applied for GO enrichment and KEGG pathway analyses. According to functional analysis, DEGs were primarily enriched in immune-associated GO terms in both the training and test sets, including immune response, humoral immune response, and B-cell-mediated immunity (P < 0.05, Figures P < 0.05, P < 0.05, P < 0.05, PCR and western blot results showed that siRNA successfully knocked down the expression of FGF9 in HSC6 cells (SCC is a prevalent pathological type of oral and maxillofacial tumor. The unique anatomical structure of the oral cavity leads to recurrence or invasive metastasis of oral squamous cell carcinoma after surgery . In receStudies have found that there are some important genes behind the differences in immune infiltration in the tumor microenvironment that regulate immune cells. Herein, 43 immune-related prognostic genes were identified in OSCC, and a new prognostic prediction model containing 17 genes was developed. Research has revealed a valuable contribution of these genes to cancer. For example, Yang et al. found that MIF can drive the malignant progression of pancreatic cancer . Zhang eIn the present research, the new prediction model can accurately distinguish high- and low-risk OSCC patients, with high sensitivity and specificity. Therefore, the novel model shows great potential as an auxiliary prognostic prediction target. Moreover, in the analysis of independent risk factors, the tumor staging of OSCC is one of the independent risk factors, which is consistent with the nature and characteristics of malignant tumors . Risk scAn OSCC prognosis prediction model was established based on immune genes with good prediction efficiency. The model risk genes show great potential as new targets for OSCC diagnosis and therapy and are worthy of in-depth study.