This study investigated the factors associated with the high susceptibility of perimenopausal women to depression.A total of 66 perimenopausal women participated in this study. The Zung self‐rating depression scale (SDS) was used to evaluate the intensity of depressive symptoms. The modified Kupperman index (KI) was used to assess common perimenopausal symptoms. Psychosocial factors were assessed via the Eysenck Personality Questionnaire, attitudes toward menopause checklist, and metacognition questionnaire (MCQ). Levels of serum estradiol, testosterone, and progesterone were determined.β = .361, p = .001), years of education , and F3 cognitive self‐consciousness score of MCQ .There were statistically significant associations between SDS standard score and the KI scale score (High susceptibility to depression of perimenopausal women may be related to lower educational level, more severe perimenopausal symptoms, and altered metacognition. The perimenopause is a window of susceptibility to depression for women. The causes of the high susceptibility of perimenopausal women to depression remain unclear. High susceptibility to depression of perimenopausal women may be related to lower educational level, greater extraversion and unstable personality characteristics, more severe perimenopausal symptoms, and altered metacognition. The inclusion criteria were as follows: (1) women from 45 to 55 years old, (2) who met the criteria for perimenopause according to the Stages of Reproductive Aging Workshop System  is calculated as the raw score × 1.25 (range: 25–100). An SS of 50 has been used as a criterion value for depression in screening work. SS values of <50, 50–59, 60–69, and >70 are typically used to denote depression severity as none, mild, moderate‐to‐severe, and very severe, respectively  was used to evaluate 13 common perimenopausal symptoms. Each symptom was assessed on a four‐point Likert scale , and each item was weighted when calculating the total sum score (range: 0–63). Scores 0–6, 7–15, 16–30, and >30 defined the degree of severity as none, mild, moderate, and severe, respectively. These 13 symptoms were classified into 3 subscale factors, namely, urogenital symptoms , somatic symptoms , and psychological symptoms   was used to assess the personal characteristics Gong,  and fourAttitudes toward menopause (ATM) were assessed using the ATM checklist, a self‐rating questionnaire developed by Neugarten et al.  to invesThe 30‐item metacognition questionnaire (MCQ‐30) was used to assess 3 domains of positive and negative metacognitive beliefs, metacognitive monitoring, and judgments of cognitive confidence . Subjects’ serum estradiol, testosterone, and progesterone levels were assessed during their appointments. Fasting blood samples were collected from all participants in the early morning. The serum concentrations of these sex hormones were measured using the Siemens ADVIA Centaur XP Immunoassay System  in the Department of Clinical Laboratories in the Third Affiliated Hospital of Sun Yat‐sen University.2.5p < .2 in the simple regression model were selected as independent variables for the multiple stepwise regression analysis with the dependent variable of SDS SS. The significant level of multiple regression analysis was set as .05.All statistics were obtained using the Statistical Package for the Social Sciences . The simple regression was used to analyze the relationships among the SDS SS and demographic, serum sex hormones, KI scores, EPQ scores, ATM scores, and MCQ scores, respectively. FDR  approach was used for the multiple testing corrections. Variables with 33.1All participants completed the SDS and the mean SS = 44.74, SD = 9.84; range: 26.0–65.0. Regarding depression intensity, 42 (63.64%) persons had no depressive symptoms, 16 (24.24%) reported mild depression, and 8 (12.12%) reported moderate or severe depression.3.23.2.12 with a mean value of 24.24 (SD 2.84) kg/m2. According to the results of the simple regressions, variables including education years , BMI , and child numbers  were selected as an independent variables for the multiple regression. After the FDR correction, SDS SS was negatively related to education years (q = .047). See Table In total, 66 women participated in this study . They had an average of 7.78 (SD = 1.83) years of education (range: 5–12). Twenty (30.3%) participants had one child, and 44 (66.7%) had two or more children. Approximately half of the participants had a family income of more than 3000 RMB per month. Sixteen (24.2%) women reported none or mild work pressure, and 5 (7.6%) persons felt severe pressure. Body mass index (BMI) ranged from 19.53 to 32.05 kg/m3.2.2F = 1.911, p = .172) was selected as one of the independent variables for the multiple regression.Table 3.2.3The positive screening rates (percentages of the item score ≥1) of symptoms from high to low were: 63.6% for Fatigue, 60.6% for arthralgia/myalgia, 57.5% for headache, 54.5% for vertigo, 50.0% for nervousness, 47.0% for sexual complaints, 42.4% for paresthesia, 36.4% for sweating/hot flushes, 28.8% for palpitation, 25.8% for insomnia, 22.7% for formication, 15.2% for melancholia, and 9.1% for urinary tract infection. Symptoms of fatigue, arthralgia/myalgia, headache, vertigo, and nervousness were common, with incidences of more than 50%. Regarding the severity level of KI, 39.4% of participants reported no symptoms, 47.0% reported mild symptoms, and 13.6% reported moderate‐to‐severe symptoms.β = .386, p = .001, FDRq = .021) was significantly related to the SDS SS and was selected as an independent variable for the multiple regression. See Table As the simple regression results, the total KI score , N , and L  subscale scores of EPQ; and F1  and MCQ F3  subscale scores of MCQ. The relationships between SDS SS and ATM negative subscale score , MCQ F2 score , and MCQ F4 score  were not significant, but these three p values were lower than .2. Thus, all these variables were selected as independent variables in the multiple regression analysis. Results of FDR corrections showed that the N score was positively correlated with SDS SS (q = .021), and the L score was negatively correlated with SDS SS (q = .038).As shown in Table 3.3β = .361, p = .001), years of education , and F3 cognitive self‐consciousness scores of the MCQ  and lower lying (L) scores were at a relatively high risk of depression. These findings accord with a study of sex differences in vulnerability to depression that found neuroticism, as evaluated by EPQ, was higher in women than in men and contributed to women's high vulnerability to depression  diathesis factors, including high neuroticism and aberrant metacognitive processes; (2) unfavorable developmental circumstances, namely, limited educational experience in childhood or adolescence; and (3) current harmful somatic complaints associated with severe perimenopausal symptoms. According to the S‐REF model, stressful events in early life foster aberrant metacognition  and dysfunctional schema (Kraft et al., 5The high susceptibility to the depression of perimenopausal women was associated with aberrant metacognitive features, lower educational levels, and more severe perimenopausal symptoms.Hongying Han, Xiaowei Xia, Xianglan Wang, and Ximei Zhi; designed the study and wrote the protocol. Hongying Han and Xiaowei Xia; managed the literature searches and analyses. Huirong Zheng, Zhiyong Zhong, and Chongbang Zhao; helped to gather research data, and Hongying Han wrote the first draft of the manuscript. All authors contributed to and approved the final manuscript.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.https://publons.com/publon/10.1002/brb3.2826.The peer review history for this article is available at


Grape pomace is one of the most abundant by-products generated from the wine industry. This by-product is a complex substrate consisted of polysaccharides, proanthocyanidins, acid pectic substances, structural proteins, lignin, and polyphenols. In an effort to valorize this material, the present study focused on the influence of extraction conditions on the yield and physico-chemical parameters of pectin. The following conditions, such as grape pomace variety (Fetească Neagră and Rară Neagră), acid type , particle size intervals , temperature , pH , and extraction time  were established in order to optimize the extraction of pectin. The results showed that acid type, particle size intervals, temperature, time, and pH had a significant influence on the yield and physico-chemical parameters of pectin extracted from grape pomace. According to the obtained results, the highest yield, galacturonic acid content, degree of esterification, methoxyl content, molecular, and equivalent weight of pectin were acquired for the extraction with citric acid at pH 2, particle size interval of ≥125–<200 µm, and temperature of 90 °C for 3 h. FT-IR analysis confirmed the presence of functional groups in the fingerprint region of identification for polysaccharide in the extracted pectin. D-galacturonic acid  residues linked by α- chain [Pectin is a natural polymer found in the plant cell walls, mainly in fruits, such as apples, citrus fruits , and vegetables ,2. Pecti4) chain . An esse4) chain ,6. Gener4) chain . The ext4) chain ,9.Presently, other vegetables and fruits residues represent a new source of pectin with impressive food utilization as valorizing innovative products and as functional component with health-promoting benefits . CurrentVitis vinifera varieties (Fetească Neagră and Rară Neagră) as an unconventional source for pectin extraction. Fetească Neagră is a dark-skinned grape variety cultivated mainly in several Romanian regions and also in the Republic of Moldova. The Fetească Neagră grapes are medium to large, cylindrical-conical with spherical, medium-sized berries, dark purple skins and have a weight of 190–230 g [Based on these remarks, we proposed in present study the application of grape pomace of two different 90–230 g . The Rar90–230 g .Vitis vinifera varieties (Fetească Neagră and Rară Neagră) using conventional method of extraction and establish the optimal conditions of grape pomace pectin extraction. Moreover, the other objective of this study was to define the structure of pectin samples using FT-IR analysis and scanning electron microscopy (SEM).Concerning the extraction of pectin from grape pomace, only a few studies were found in the literature ,22,23. DVitis vinifera varieties (Fetească Neagră and Rară Neagră) from a 2019 harvest, cultivated in the Bugeac area, Republic of Moldova. The grape pomace was dried in an oven with air circulation Zhicheng ZRD-A5055  at 50 °C until constant weight was achieved. Then, dried pomace was powdered using a food processor  and separated it on the following particle size intervals: <125 µm, ≥125–<200 µm and ≥200–<300 µm using an analytical sieve shaker Retsch AS 200 Basic .Grape pomace was collected by processing two different m-hydroxydiphenyl, potassium hydroxide, sodium tetraborate, D-galacturonic acid, and ethyl alcohol were acquired from Sigma-Aldrich .Nitric acid, sulfuric acid, citric acid, sulfamic acid, hydrochloric acid, sodium hydroxide, w/v) acquired by adding nitric acid, sulfuric acid, and citric acid to ultrapure (Milli-Q) water until a pH 2 was achieved. For each acid, three different mixtures were prepared according to the particle size intervals. Then, the mixtures were kept in a weather bath Precisdig  at the temperature of 90 °C for 3 h.Initially, a sample of 10 g grape pomace powder was mixed with 100 mL of solvent  was added to supernatants in order to achieve 1:1 ratio (v/v). The mixtures were kept at 4–6 °C for 12 h to accomplish the precipitation. The precipitated pectin was separated by centrifugation at 2320 g and 22 °C for 30 min. The pectin was washed 3 times with ethyl alcohol  and dried in an oven with air circulation Zhicheng ZRD-A5055  at 50 °C until constant weight was achieved.After extraction, the mixtures were cooled to room temperature, around 20–22 °C. Firstly, the solid material was segregated by centrifugation at 2320 g and 22 °C for 35 min. Then, the obtained supernatants were put through a clean strainer and placed into the neck of a DuranPectin yield was calculated using Equation (1):wder (g) ,24.m-hydroxydiphenyl method developed by Filisetti-Cozzi and Carpita [m-hydroxydiphenyl solution in 0.5% sodium hydroxide (w/v) was added and vortex mixed for 5 s. The absorbance was read at 525 nm against the reagent control using a UV-Vis-NIR spectrophotometer .The galacturonic acid content  of pectin was estimated using the sulfamate/ Carpita  and Melt Carpita . Sample  Carpita . 20 mg oD-galacturonic acid calibration curve was prepared for each batch of samples.A The degree of esterification (DE) of pectin was determined by the titrimetric method described by Franchi  and Wai The DE of pectin samples was measured in triplicate.The equivalent weight (EW) of pectin samples was determined by Ranggana , as follThe EW of pectin samples was measured in triplicate.The neutral solution was collected from the determination of EW and 12.5 mL of 0.25 M sodium hydroxide were added in a stoppered flask. The mixed solution was stirred thoroughly and kept for 30 min at room temperature around 20–22 °C. Then, 12.5 mL of 0.25 M hydrochloric acid was added and titrated with 0.1 N sodium hydroxide to the same end as before [The MeO of pectin samples was measured in triplicate.w/w) were filtered through 0.45 μm membranes before injection. The calibration was performed using pullulan standards . The LC solution software version 1.21  was utilized to collect the data.Molecular weight (Mw) was determined by high-performance size-exclusion chromatography using a HPLC system  equipped with a LC-20 AD liquid chromatograph, SIL-20A auto sampler, a Yarra 3 µm SEC-2000 column  and coupled with a RID-10A refractive index detector . The samples were made according to Dranca et al. . Elutionab) and chroma (C*ab) were obtained from the reflection spectra of the samples with illuminant D65 and 2° observer.The color of the pectin samples was analyzed in triplicate at 25 °C with a CR-400 chromameter . CIE L*; hue  was used to display the spectra. The samples were placed on the ATR crystal, and the spectra were recorded in triplicate.Fourier-transform infrared spectroscopy (FT-IR) analysis was made using a Nicolet i-20 spectrophotometer . The spectra were recorded in transmission mode using the attenuated total reflectance (ATR) system within the wave number range of 4000–400 cmThe microstructure of the pectin samples was examined by scanning electron microscopy . Dried pectin powder was fixed to the sample table with conductive double-sided adhesive carbon tape and analyzed using an accelerating voltage of 30 kV at different magnifications . The Vega software  was utilized to display the structural morphology.p < 0.05) with Fisher’s least significant difference (LSD) procedure.Results were submitted to analysis of variance (ANOVA) using XLSTAT software . The ANOVA test was used to evaluate the difference between means at the 95% confidence level .The yield and physico-chemical characteristics of pectin depend on the source, extraction method, and different factors used  ,33. The Additionally, Limareva et al.  did not ab) were affected by the grape pomace variety (p < 0.0001). Galacturonic acid and methoxyl content define the quality of pectin [ab) value (39.27) and chroma (C*ab) value (11.26) in comparison with FN grape pomace pectin . Thus, pectin samples were presented a color ranging from red to red-purple according to CIE chromaticity diagram. Grape pomace extracted pectin color was predominantly due to tannins and anthocyanins, which are the main polyphenolic compounds responsible for color in red grape cultivars [ab and C*ab), some relevant pictures of pectin samples extracted under the influence of acid type with fixed conditions: pH 2, particle size of ≥125–<200 µm, 90 °C, and extraction time of 3 h are showed in The physico-chemical parameters, such as GalA, MeO, and color measuring parameters  were not significantly influenced by acid type (p > 0.05). Thus, the pectin yield presented the following values, 5.80%, 5.84%, and 6.01% for sulfuric, nitric, and citric acid extraction, respectively. However, Raji et al. [The use of mineral acids  for pectin extraction has been reported to environmental consequences and increased costs ,38. Regai et al.  reportedi et al.  did not p < 0.01). Therefore, the GalA presented the following values: 31.45 g/100 g for sulfuric, 38.95 g/100 g for nitric, and 52.01 g/100 g for citric acid of pectin from grape pomace. As well, Ma et al. [Prunus domestica). Considering the whole range, citric acid and hydrochloric acid extraction exhibited the highest EW (1020 g/mol) and the lowest EW (833.3 g/mol), respectively [w was significantly influenced by the acid type (p < 0.0001). The Mw ranged from 5.25 × 104 g/mol for nitric acid to 5.34 × 104 g/mol for citric acid. The highest value of L* (48.33) was obtained by pectin extraction with citric acid, while the lowest L* (40.44) was obtained by nitric acid extraction. This can be explained by the main phenolic compounds, such as phenolic acids which are found in grape pomace and retained after extraction. Thus, Manasa [The GalA and EW were significantly influenced by the acid type . The grape pomace pectin yield presented the following values, 4.64%, 5.71%, and 7.31% for particle size intervals of <125 µm, ≥200–<300 µm and ≥125–<200 µm, respectively. Huang et al. [w (5.35 × 104 g/mol) from grape pomace pectin was obtained for particle size interval of ≥125–<200 µm. The MeO of pectin from grape pomace showed the following values, 3.98%, 4.60%, and 6.18% for particle size intervals of ≥200–<300 µm, <125 µm, and ≥125–<200 µm, respectively.The particle size increases the extraction efficiency and improves the functional properties of pectin, such as solubility, gelation, emulsification, texture, and viscosity ,45. The g et al.  reportedg et al. . Additiog et al.  presenteab, and C*ab) were not significantly influenced by particle size (p > 0.05). The DE and EW of pectin from grape pomace presented a range of 71.31–74.92% and 540.38–565.70 g/mol, respectively. Geerkens et al. [ab (36.80) was obtained by pectin extraction with <125 µm particle size, while the lowest L* (40.44) and h*ab (34.08) by ≥200–<300 µm of grape pomace. Thus, pectin samples from grape pomace were presented a color ranging from red-orange to red according to CIE chromaticity diagram.The DE, EW, and color parameters  were significantly influenced by the pH of acid solution (p < 0.0001). The grape pomace pectin yield presented the following values, 6.14%, 7.56%, and 12.43% for pH of 3, 2, and 1, respectively. The similar results were obtained by Yapo [The strength of mineral or organic acid used to extract pectin from different fruits and vegetables had significant effect on the pectin yield. Moreover, the yield of pectin decreased with the increasing of acid strength ,46,47. T by Yapo  in orderw of pectin from grape pomace presented a range of 70.39–78.92% and 5.28 × 104–5.39 × 104 g/mol, respectively, for different pH values. The higher the pH, the higher the value of DE of pectin. Additionally, Yapo [p < 0.01). The GalA of pectin presented the following values: 39.40 g/100 g, 61.67 g/100 g, and 45.57 g/100 g for pH of 1, 2, and 3, respectively. Yapo [ab and C*ab) were influenced by pH value of extraction pectin from grape pomace. The highest values of L* (59.92), h*ab (42.48), and C*ab (12.37) were obtained at pH 3 and the lowest values of L* (23.89), h*ab (25.06), and C*ab (4.41) at pH 1. This can be explained by the fact that the stability of polyphenols is pH-dependent; the lower the pH, the more stable the polyphenols during extraction.However, Colodel and Petkowicz  reportedly, Yapo  establisly, Yapo  reportedly, Yapo . Wathonily, Yapo  related ly. Yapo  reportedly. Yapo ,50. The The increasing time extraction using citric acid is correlated with an enhancement of the pectin yield . The infw, L*, and h*ab were significantly influenced by the time of extraction (p < 0.0001). The pectin yield presented the following values, 5.38%, 5.77%, and 7.50% for 1, 2 and 3 h, respectively. Thus, the results indicated that the yield of grape pomace pectin increased with enhancement of extraction time. Kulkarni and Vijayanand [w/v). Additionally, they reported that pectin extracted for 30 min had a higher MeO (9.84 g/100 g) and EW (839.1); in this case, MeO and EW decreased with increase of extraction time. Irrespective of these results, the highest value of MeO (6.54%) and EW (618.42 g/mol) were obtained using citric acid at pH 2, for 3 h of extraction for the pectin from grape pomace. The highest DE of pectin from grape pomace (77.77%) was obtained at extraction time of 3 h and the lowest for 1 h (67.17%). Chan and Choo [w ranging from to 5.25 × 104 to 5.38 × 104 g/mol at pH 2.Thereby, the yield, DE, Mjayanand  studied and Choo  reportedp < 0.001). Thus, the GalA presented the following values: 33.69 g/100 g, 39.04 g/100 g, and 46.98 g/100 g for 1, 2, and 3 h, respectively. Kulkarni and Vijayanand [ab), the highest values of L* (71.52), and h*ab (46.71) were presented for grape pomace pectin extracted with citric acid pH 2, at 90 °C for 3 h. Berardini [The GalA was significantly influenced by the time of extraction (jayanand  obtainederardini  reportedab were significantly influenced by the temperature of extraction (p < 0.0001). The grape pomace pectin yield presented the following values: 4.23, 6.02, and 7.30% for 70, 80, and 90 °C, respectively. Chan and Choo [Citrus unshiu fruits pectin. They reported that temperature below 40 °C kept the pectin structure more intact. The highest value of GalA was obtained at 90 °C (62.21%). Contrastingly, Chan and Choo [Extraction temperature is a major characteristic affecting the structure of the pectin. The increasing temperature significantly enhanced the yield of pectin using citric acid extraction ,51,54. Tw of pectin extracted from grape pomace were 608.30 g/mol, 6.75%, and 5.32 × 104 g/mol, respectively, for time extraction of 90 °C. The DE was not significantly influenced by the temperature and presented a range of 74.65–81.40% (p > 0.05). Wai et al. [A lower temperature caused less degradation to pectin structure. Moreover, the highest values of EW, MeO, and Mi et al.  achievedi et al. . The L* i et al. .−1, which was attributed to intermolecular hydrogen bonding of O(6)H∙∙∙O(3) [−1. The presence of this shift was assigned to –CH stretching vibration peak [−1. This peak was attributed to the C–H bond [The information on the functional groups presents in the pectin from grape pomace of two varieties Fetească Neagră and Rară Neagră  extracted in the following conditions: pH 2, particle size of ≥125–<200 µm, 90 °C, and extraction time of 3 h, was evaluated by FT-IR analysis. The attenuated total reflectance FT-IR spectra of grape pomace pectin are illustrated in H∙∙∙O(3) . Pectin ion peak . As wellC–H bond , S–H or C–H bond , which iC–H bond . MoreoveC–H bond .−1 represents the major chemical functional groups in pectin [−1 was associated with phenolic esters of pectin [−1 corresponded to the protein amide in the pectin molecules [−1 suggested –CH bending of –CH2 groups [−1 and 1000 cm−1 showed the presence of alcohols, esters, ethers, and carboxylic acids (C–O stretch) [−1 was attributed to the asymmetrical stretching of the COO- and C–O group [−1. The bands in the region 1200–850 cm−1 were produced by stretching vibration of C–O, C–C, and ring structures [−1 were as a result of a high amount of homogalacturonan in pectin. A less intense peak at around 1132 cm−1 was due to C–O stretching vibrations [−1 was assigned to C–O and C–C stretching vibrations [−1 and 842 cm−1 corresponded for β-glycosidic linkages and CH2 bending, respectively [−1 can be attributed to cyclic –CH bending and to the C-C deformation vibrations of pectin ring [The FT-IR spectra in the region between 2000 and 1000 cmn pectin ,60,61. Tolecules . The pea2 groups . The peastretch) . The banructures . Intensebrations . The peabrations . The banectively . Peak aptin ring .Scanning electron microscopy (SEM) is an affective technique in analysis of different materials (organic and inorganic) on a nanometer to micrometer (µm) scale . The morThe novelty of the present work is that only a few studies are about the pectin extraction from grape pomace. The mechanism of pectin extraction was studied under the following conditions: grape pomace variety, acid type, particle size, extraction temperature, pH, and time. The results showed that extraction conditions have a huge impact on the structure and properties of pectin. The highest yield, GalA, DE, MeO, and EW of pectin from both grape pomace varieties (FN and RN) were obtained for citric acid extraction at pH 2, particle size interval of ≥125–<200 µm, and temperature of 90 °C for 3 h. The obtained results are in agreement with other studies, in which it was established that citric acid at pH 2 is a great alternative to the different mineral acids. Overall, we conclude that grape pomace is not just a waste generated in wine industry, but a by-product with commercially important fibres, such as pectin.


Lactobacilli always contain carbohydrate polymers, which interfere with the analysis of the exopolysaccharide (EPS) produced by the bacteria. In this investigation, a novel medium formulation that could avoid such interference was successfully developed. The beef extract, yeast extract, and peptone used in this formulation were subjected to the removal of polysaccharides before use. The factors affecting the EPS production were optimized by a single factor test, Plackett–Burman design, and Box–Behnken design. The optimum formula was ascertained as: 7.5 g L–1 yeast extract, 12.5 g L–1 beef extract, 10 g L–1 peptone, 21.23 g L–1 maltose, 5.51 g L–1 yeast nitrogen base, 2 g L–1 K2HPO4, 5 g L–1 anhydrous sodium acetate, 2 g L–1 ammonium citrate, 0.58 g L–1 MgSO4⋅7H2O, 0.25 g L–1 MnSO4⋅H2O, and 1 mL L–1 Tween 80. The initial pH of the medium was 6.5. The optimized conditions for fermentation of the strain to produce EPS were as follows: seed size 1%, culture temperature 37°C, and culture time 20 h. Optimum results showed that EPS yield was 496.64 ± 3.15 mg L–1, being 76.70% higher than that of unoptimized conditions (281.07 ± 5.90 mg L–1). The EPS was mainly comprised of glucose and guluronic acid, with a weight average molecular weight of 19.9 kDa; it was also characterized by Fourier transform infrared spectroscopy and UV analysis. EPS was found to significantly enhance the phagocytic capacity, promote the NO, TNF-α, IL-1β, and IL-6 secretion, and improve mRNA expression of cytokines in RAW 264.7 macrophages, indicating its considerable immunomodulatory activity. Western bolt and immunofluorescence results demonstrated that the EPS was able to increase p65 nuclear translocation in the macrophages, indicating that EPS enhanced immunity via the NF-κB signaling pathway. EPS investigated in this work has potential as an attractive functional food supplement candidate for the hypoimmunity population.The traditional media used for the fermentation of  Lactobacilli organisms, which was generally regarded as safe (GRAS), has been widely used in food-, medicine-, and cosmetics-related industries (Lactobacilli EPS possesses remarkable immunostimulatory effects with no obvious side effects (The exopolysaccharide (EPS) secreted by dustries . Recentl effects . EPS mai effects ; enhanci effects –6.Lactobacilli. Beef extract, yeast extract, and peptone are often used as organic carbon and nitrogen sources in those media for the fermentation of Lactobacilli. A lot of polysaccharide-analogs derived from those components cannot be utilized by the microorganism and would precipitate together with EPS when using the alcohol precipitation method to prepare EPS  under accession number NO. CCTCC M 2020325. Macrophage RAW264.7 cells were kindly provided by the College of Pharmaceutical Science, Zhejiang University. Beef extract, yeast extract, and peptone were purchased from Hangzhou Microbial Reagent Co., Ltd. . Glucose, galactose, fructose, sucrose, maltose, lactose, lipopolysaccharide (LPS), and neutral red were obtained from Sigma Chemical Co., Ltd. . Yeast nitrogen base (YNB) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . The kits for MTT, NO, and total protein assays were purchased from Nanjing Jiancheng Bioengineering Institute . ELISA kits for the mouse IL-1β, IL-6, and TNF-α assays were supplied by Lanpai BIO . All other chemical reagents used in this study were of analytical grade.MRS agar . The strL. rhamnosus ZFM216 was revived twice by incubating on MRS agar at 37°C for 48 h before use. A single colony was inoculated in 10 mL of MRS broth and cultivated at 37°C for 24 h, and then the bacterial suspension  was transferred into an MRS broth (100 mL) for 32-h cultivation at 37°C. Isolation of polysaccharides was carried out according to a published protocol with some modifications  for 12 h at 4°C, followed by centrifugation  to remove protein. A quadruple volume of pre-chilled anhydrous ethanol was then added and allowed to stand for 12 h at 4°C. The precipitate was obtained by centrifugation , then redissolved in 10 mL of ultrapure water and dialyzed (MW cut off 2000 Da) against deionized water for 72 h. The dialyzate was concentrated and then underwent freeze-drying to obtain polysaccharides. The polysaccharide content was monitored by measuring the total sugar content  Removal of polysaccharide-analogs in the three main components of the medium  before the preparation of the new medium. In brief, beef extract, yeast extract, and peptone were dissolved in pure water , a quadruple volume of pre-chilled anhydrous ethanol was then added, and allowed to stand for 12 h at 4°C. After centrifugation , the supernatant was concentrated and used for the preparation of the medium. (2) Replacement of glucose by other sugars as a carbon source by comparison . (3) Addition of yeast nitrogen base as nitrogen source. (4) Optimization of dosage of nitrogen and carbon sources .The traditional MRS was composed of 5 g LL. rhamnosus ZFM216, single factor tests were first performed to investigate the effects of culture time (0–32 h), culture temperature , seed size , initial pH , carbon source , and the dosage of nitrogen source  on the EPS yield.To optimize the culture conditions for the EPS production by The Plackett–Burman experimental design, an economical and effective two-level experiment design method, can effectively screen out the main factors affecting the yield of EPS. According to the results of single factor tests (nine factors) maltose, beef extract, yeast extract, peptone, YNB, time, temperature, pH, and inoculum size were chosen to conduct Plackett–Burman design. Each factor was defined at two levels, a high level being denoted by (1) and a low level being denoted by (−1) . The resY is the predicted yield of EPS, β0 is the model intercept, βi is the linear coefficients, Xi is the independent variable, and k is the number of the factors. Nine assigned variables were screened in 12 runs; all experiments were carried out in triplicate and the average EPS yield was taken as a response. Design-Expert software  was first used for the first-order model polynomial coefficient calculation and analysis, and then the significant variables were selected for further statistical analysis.where Box–Behnken design was conducted to further optimize the culture pH, the amount of maltose, and YNB for the EPS production. The design with 17 runs and the corresponding results are presented in Y is the predicted EPS yield, β0 is the model constant; βi, βii, and βij are linear, quadratic, and interaction coefficients, respectively; Xi and Xj are the independent factors; and k is the number of the factors. The second-order polynomial coefficient calculation and analysis were performed using Design-Expert software.where L. rhamnosus ZFM216 was performed using R-MRS , S-MRS (R-MRS containing 3 g L–1 of YNB), and M-MRS (the modified MRS medium under the optimum conditions), respectively. The method for the preparation of EPS was the same as that described in Section “Monitoring the Change in Polysaccharide Content in the Culture Medium During Fermentation.”The fermentation of L. rhamnosus ZFM216 was determined by the phenol sulfuric acid method  according to a published method with minor modifications . EPS sam3 (0.2 M) and NaH2PO4 (0.01 M) was added to the scale line and ultrasonicated. The ultrasonic dispersion was used for molecular weight determination. GPC system (Waters 1525 Isocratic HPLC Pump) was equipped with a PL aquagel-OH MIXED 8 μm chromatographic column and a differential detector (Waters 2414 Refractive Index Detector). Ultrapure water containing NaNO3 (0.2 M) and NaH2PO4 (0.01 M) was used as the mobile phase, the flow rate was 1 mL min–1, and the column temperature was 30°C. The standard sample was narrow distribution polyethylene glycol provided by Polymer Standards Service-USA, Inc., with molecular weights (Mp) of 330,000, 176,000, 82,500, 44,000, 25,300, 20,600, 12,600, 7130, 4290, 1400, 633, and 430, respectively, and the data were analyzed using Agilent GPC software.The molecular weight of EPS was analyzed by gel permeation chromatography (GPC) . EPS sam–1) were obtained by recording on a microplate reader  in the range of 200–800 nm  using a KBr pellet method scanning in the range of 400 to 4000 cm–1 .2.Murine monocyte-macrophage RAW264.7 cells were incubated in a DMEM medium, supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum, at 37°C in an incubator  containing 5% CO4 cell/well) were seeded on a 96-well microplate and incubated at 37°C for 24 h. The supernatant was removed and the cells were washed gently with 200 μL of PBS twice. Then, the cells were treated with different concentrations of EPS solution  for 24 h. MTT (10 μL) was added and incubated for another 4 h, and the medium was then removed. DMSO (100 μL) was added to dissolve the insoluble formazan, and the absorbance of the resulting solution was measured at 570 nm. DMEM containing LPS (1 μg mL–1) served as a positive control, and DMEM without EPS served as a control. All assays were performed in quintuplicate.Cell viability of RAW264.7 was investigated using the MTT assay . RAW264.5 cell mL–1) were treated with different concentrations of EPS solution  in a 96-well microplate and incubated at 37°C in a 5% CO2 atmosphere for 24 h. LPS (1 μg mL–1) was used as a positive control. After removal of the supernatant, the cells were washed gently with PBS twice, and neutral red  was then added to each well and continued to incubate for 1 h. Subsequently, the neutral red was removed and the cells were washed with PBS twice, followed by the addition of lysis buffer . After standing at room temperature for 2 h, the optical density was recorded at 540 nm.In this study, uptake of neutral red was used to assess the effect of EPS on the phagocytic activity of RAW 264.7 cells using a published protocol with minor modifications . RAW264.5 cell mL–1) were treated with different concentrations of EPS solution  in a 24-well microplate and incubated at 37°C for 24 h. LPS (1 μg mL–1) was used as a positive control. The concentrations of NO, IL-1β, IL-6, and TNF-α in the supernatant of the RAW264.7 cells were measured using a commercial NO assay kit and the corresponding ELISA kits according to the manufacturers’ instructions.RAW264.7 cells  were treated with different concentrations of EPS solution  or LPS (1 μg mL–1) in a 24-well microplate and incubated at 37°C for 24 h. The cells were then washed with cold PBS twice and used for RNA extraction. The total RNA from the cells was extracted using commercial E.Z.N.A.® Total RNA Kit II according to the manufacturers’ instructions. The quality and quantity of RNA were assessed by measuring the A260/A280 absorbance ratio using a NanoDrop 2000 spectrophotometer . RNA was reversed into cDAN using Hifari® III 1st Strand cDNA Synthesis SuperMix  according to the manufacturer’s instructions. Then, the cDNA encoding iNOS, TNF-α, IF-1β, and IF-6 genes were analyzed by RT-qPCR using Hieff® UNICON Blue qPCR SYBR Green Master Mix  according to the manufacturer’s instructions. The relative gene expression was calculated by the 2–ΔΔCt method, and the housekeeping gene β-actin gene was used as an internal control. Primer sequences for the genes were listed in The mRNA expression of inducible nitric oxide synthase (iNOS) and cytokines were assessed by RT-qPCR . RAW264.5 cell mL–1) were treated with different concentrations of EPS solution  or LPS (1 μg mL–1) in a six-well microplate and incubated at 37°C for 24 h, thereafter the total protein was extracted with 100 μL of lysis buffer (PMSF protease inhibitor: phosphatase inhibitor: RIPA lysis and extraction buffer = 1:1:100), the protein concentration was determined by BCA protein quantitative kit according to the manufacturers’ instructions, and stored at −20°C for standby. After denaturation in a boiling water bath, the proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The PVDF membranes were blocked with 5% BSA for 2 h, and then incubated with the corresponding primary antibodies against p65 (1:1000), p-p65 (1:1000), and GAPDH (1:500) at 4°C for 12 h, then incubated with the secondary antibodies IgG (1:8000) for 1 h at 37°C. The bands were visualized with a FluorChem HD2 system  after treatment with ECL luminous Kit. ImageJ software was used to determine the gray value for quantitative analysis.Western blot analysis was performed according to a published method with minor modifications . In brie5 cell mL–1) were treated with EPS solution  or LPS (1 μg mL–1) on a laser confocal dish and incubated at 37°C for 24 h. The cells were then fixed with 4% paraformaldehyde for 12 h, further treated with 0.5% Triton X-100 for 20 min and blocked with 5% BSA at room temperature for 1 h. The cells were incubated with the p65 primary antibody (1:100) at 4°C for 12 h and then incubated with the Anti-rabbit IgG (H + L) F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) at 37°C for 1 h in the dark. The nuclei were stained with DAPI for 5 min, and the cells were observed and photographed with a laser confocal microscope .The immunofluorescence staining analysis of nuclear translocation of NF-κB was performed according to the literature with minor modifications . RAW264.p < 0.01, the differences were considered statistically significant. All the data were expressed as mean ± standard deviation (SD).One-way analysis of variance (ANOVA) was performed using SPSS version 25  to compare the results. Duncan’s multiple range test was used for analyzing the significance of the differences. If L. rhamnosus ZFM216 decreased over time, and the final polysaccharide content was 582.30 ± 4.85 mg L–1, while the polysaccharide content in MRS broth without any strain remained almost unchanged (964.49 ± 3.96 mg L–1). The result indicated that some polysaccharide-analogs were metabolized by the strain. The polysaccharides presented in MRS broth with L. rhamnosus ZFM216 included the EPS produced by the strain and polysaccharide-analogs derived from the medium. Vaningelgem et al.  was used for bacterial culture, the growth state of the bacteria was comparable to that of the traditional MRS medium , was lower than that in the traditional MRS broth , and theS medium , and the–1) as carbon source, and BE (12.5 g L–1), YE (7.5 g L–1), Pe (10 g L–1), and YNB (3 g L–1) as nitrogen source. The seed size was found to have no obvious effect on the yield of EPS, thus 1% of the seed size was used in the following investigation.The effects of temperature, time, initial pH, seed size, carbon source, and the dosage of nitrogen source on the yield of EPS were investigated by the single factor tests. As illustrated in A Plackett–Burman design analysis was performed for each factor at two levers for 12 trials, and the corresponding EPS production are presented in 1, X2, X3, X4, X5, X6, X7, X8, and X9 represent dosages of maltose, BE, YE, Pe, and YNB, time, temperature, initial pH, and seed size, respectively.where Xp-value (<0.01) was low, implying the extreme significance of the model. The high determinant coefficient R2 of the model (0.9979) indicated that the model simulated the test process well. The influences of maltose concentration (X1), YNB (X5), and pH (X8) were extremely significant (p < 0.01) on EPS production, while the effects of BE (X2), YE (X3), Pe (X4), and temperature (X7) were significant (p < 0.05).Statistical analysis of the EPS production showed that the A), YNB (B), and pH (C) were selected for further optimization by Box–Behnken design. The results and the ANOVA of the models are presented in F-value of the model was high (115.02) and the p-value was extremely low (<0.0001), suggesting that the model was adequate. The value of the regression coefficient R2 was 0.9933, implying that the model fitted the test process well, while R2adj (0.9846) explained more than 98% of the reliability in EPS production. The p-value for lack of fit (0.2908) indicated the insignificance of lack of fit compared with the pure error. All the results showed a good consistency with the predicted yield of EPS, suggesting the validity of the mathematical model. The linear coefficients , the interaction term coefficients (AB and BC), and the coefficients of the quadratic term  had a significant effect on the yield of EPS (p < 0.01).According to the results of the Plackett–Burman design, the three factors which had an extremely significant effect on the EPS yield, namely maltose :–1, YNB 5.51 g L–1, and pH 6.5, and the predicted maximum production of EPS reached 500.16 g L–1. Comprehensively, the optimized composition of the M-MRS was as follows: 7.5 g L–1 yeast extract, 12.5 g L–1 beef extract, 10 g L–1 peptone, 21.23 g L–1 maltose, 5.51 g L–1 YNB, 2 g L–1 K2HPO4, 5 g L–1 anhydrous sodium acetate, 2 g L–1 ammonium citrate, 0.58 g L–1 MgSO4⋅7H2O, 0.25 g L–1 MnSO4⋅H2O, 1 mL L–1 Tween 80, and initial pH was 6.5. The optimized culture conditions for the production of EPS by L. rhamnosus ZFM216 were ascertained as follows: seed size 1%, culture temperature 37°C, and time 20 h. Under these conditions, the actual EPS yield was 496.64 ± 3.15 mg L–1, which was close to the predicted value, indicating that the model could be used to optimize the production of EPS from the strain. Furthermore, the EPS yield under the optimized condition was 76.70% higher than that in the unoptimized conditions (281.07 ± 5.90 mg L–1). The improvement of fermentation strategies helps to enhance EPS yield. Therefore, it is essential to improve or develop novel fermentation techniques for microbial EPS production. Moreover, the use of statistical models in fermentation helps to obtain the maximum EPS yields   of 32.2 kDa and a number average molecular weight (Mn) of 19.9 kDa, with a polydispersity index (PDI) of 1.61. This result agrees with some previous studies, in which the Mw of EPS from o 105 Da , 29, buta (7 Da) .No obvious absorptions at 260, 280, and >400 nm in the UV spectrum of EPS  indicate–1 is ascribed to the hydroxyl group  in the pyranose ring . In the report of Li et al. (36) a polysaccharide isolated from the Antarctic bacterium Pseudoalteromonas sp. S-5 insignificantly affected the viability of RAW 264.7 cells over the entire concentration range of 2.5–500 μg mL–1. Polysaccharides from different sources may have different effects on the growth of macrophages. Li et al. . The finding in the present work is similar to that of Wang et al. . The NO production reached a maximum at EPS concentrations of 500 and 600 μg mL–1, being 24.04 and 24.07 μM respectively, which was close to that in LPS-treated group (p > 0.05). Such an increase in NO production suggests that EPS may activate the tumoricidal and bactericidal activities of macrophages and play a vital role in the host defense system. Polysaccharides from Elaeagnus angustifolia L. pulp were found to be able to significantly increase the production of NO in the concentration of 25 to 400 μg mL–1. Both contents of TNF-α and IL-1β significantly increased with the increase of EPS concentration up to 600 μg mL–1 (p < 0.01), thereafter the values decreased (p < 0.01); while the content of IL-6 significantly increased with the increase of EPS concentration up to 500 μg mL–1 (p < 0.01), thereafter the value remained unchanged. The maximum content of TNF-α observed in the EPS group (153.30 pg mL–1) was still lower than that of positive control LPS (175.22 pg mL–1). The maximum contents of IL-1β and IL-6 of the EPS group  were close to the corresponding values of LPS . It can be concluded that EPS can stimulate RAW 264.7 cells and promote their secretion of cytokines, thereby exerting immune-enhancing effect. Zhou et al. . Immunomodulatory EPS upregulated mRNA expression of related enzymes  , TNF-α, IL-1β, and IL-6 were quantitatively analyzed using RT-qPCR. As shown in  enzymes . NO is a enzymes . Zhang e enzymes  also fouμg mL–1) .Nuclear factor kappa-B (NF-κB) is a family of proteins composed of complex polypeptide subunits. It usually forms a homologous or heterodimer from p65 and p50 and is inactivated in the cytoplasm due to the combination with the inhibitory protein complex IκB-α . The NF-Western blot analysis showed that the ratio of key proteins phosphorylated p65 (p-p65) to p65 was significantly increased compared to the blank group , suggest–1). In conclusion, EPS could activate macrophages through the NF-κB pathway . The EPS investigated in this work possessed good effects in enhancing the proliferation, phagocytosis, and the release of NO and cytokines of RAW264.7 macrophages, suggesting the good immunomodulatory activity of EPS. Upregulating the mRNA expression of iNOS, TNF-α, IL-1β, and IL-6 could be the causative immunomodulatory mechanism of EPS. EPS was also found to initiate and activate the immune signal transduction through the NF-κB signaling pathway. EPS could be a potential candidate for a functional food supplement for the hypoimmunity population.A novel culture medium for EPS-producing The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.LC designed and performed the experiments, conducted the data analysis, and wrote—original draft of the manuscript. QG involved in the supervision of the study and resources. TZ contributed to conceptualization, supervision, funding acquisition, and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


When silver nanoparticles were added, it exhibited strong resonance Rayleigh scattering (RRS) activity and surface-enhanced Raman scattering (SERS) effect. This new FeMOF nanocatalytic trimode indicator reaction was combined with the glyphosate aptamer reaction to establish a new SERS/RRS/FL trimode biosensor for glyphosate. The sensor can be used for the analysis of environmental wastewater, and a new method for detecting glyphosate content in wastewater is proposed. The linear range of the sensor is 0.1–14 nmol/L, the detection limit is 0.05 nmol/L, the recovery is 92.1–97.5%, and the relative standard deviation is 3.6–8.7%.The stable and highly catalytic Fe metal–organic framework (FeMOF) nanosol was prepared and characterized by electron microscopy, and energy and molecular spectral analysis. It was found that FeMOF strongly catalyzed the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) by H The method is simple and selective, with a linear range of 2 × 10−6–3 × 10−4 mol/L and a detection limit of 4.5 × 10−7 mol/L [2O2 to produce FL, a glucose oxidase coupled with FeMOF, the nanoenzyme FL method was proposed for the assay of glucose [2O2, for the detection of glyphosate.Metal–organic frameworks (MOFs) are crystalline porous materials with periodic network structures formed by the self-assembly of transition metal ions and organic ligands. It has the advantages of high porosity, low density, large specific surface area, regular pore channels, adjustable pore size, diversity, and tailorability of the topological structure, and is widely used in gas storage, pharmacology, chemical sensing, pollutant separation and chemical catalysis ,2,3,4. T9 nmol/L . Wang deatalysts . The Fe-−7 mol/L . Based o glucose . As far 2O. The linear range was 0.79–13.2 nmol/L, and the detection limit was as low as 0.72 nmol/L [2O2 and the regulation of Apt, Feng et al. constructed an analytical platform for SERS detection of acetamiprid (ACT). The linear range was 0.01–1.5 μg/L and the detection limit was 0.006 μg/L [2O2–3,3′,5,5′-tetramethylbenzidine (TMB), a SERS/FL dual-mode detection method for K+ was constructed. The linear ranges were 2–1000 nmol/L and 10–300 nmol/L, respectively. The detection limits were as low as 1.6 nmol/L and 9.4 nmol/L, respectively [Apt can bind to a variety of target substances with high specificity and selectivity; thus, it is widely used in the field of biosensors ,14,15,16006 μg/L . Compareectively .3+, and the regulation of aptamers. The linear range was 0.1–16 ppm, and the DL was as low as 8.75 ppb [2O2–TMB indicator reaction and its coupling with highly selective aptamers to establish trimode detection. In this study, a novel trimode SERS method for the determination of trace GLY was developed by combining the highly selective aptamer reaction with the sensitive FeMOF nanocatalytic H2O2–TMB trimode indicator reaction. It has the advantages of convenient operation, good selectivity, high sensitivity, and can provide trimodal options. The SERS method was applied to the determination of GLY in wastewater with satisfactory results.Glyphosate (GLY) is a non-selective and non-residue herbicide, which is very effective for perennial root weeds and is widely used in rubber, mulberry, tea, orchard, and sugarcane fields . It main8.75 ppb . Songa e8.75 ppb . Some of8.75 ppb . The triThe resonance Rayleigh scattering spectrum was scanned by Hitachi F-7000 fluorescence spectrophotometer , and the absorption spectrum was scanned by Tu-1901 dual-beam ultraviolet–visible spectrophotometer . The DXR smart Raman spectrometer  has a laser wavelength of 633 nm, a laser power of 0.5 mw, a slit of 2.5 μm, and an intelligent background scanning mode to obtain SERS spectra, which includes a scanning electron microscope  and a transmission electron microscope . An FD-1C-50 vacuum freeze dryer  was used to prepare solid samples, and the infrared spectra were obtained by scanning with a Fourier transform infrared spectrometer . The size spectra of nanomaterials were obtained by Nano-2s nanoparticles . Other instruments include an HH-S2 electrothermal constant-temperature water bath , used for heating the reaction system, and a KQ3200DB numerical control ultrasonic cleaner with ultrasonic power of 150 w and operating frequency of 40 KHz , used for cleaning the required equipment for the experiment.3 ; trisodium citrate ; NaBH4 ; 3,3′,5,5′-tetramethylbenzidine ; hydrogen peroxide ; trimethylaminomethane ; hydrochloric acid ; ferrous sulfate heptahydrate ; 1,3,5-benzenetricarboxylic acid ; sodium hydroxide ; anhydrous ethanol ; glyphosate . The sequence number of the glyphosate aptamer (AptGLY) is TGC TAG ACG ATA TTC CAT CCG AGC CCG TGG CGG GCT TTA GGA CTC TGC GGG CTT CGC GGC GCT GTC AGA CTG ATG TCA . The above reagents were analytically pure, and the experimental water was deionized water.AgNO3, 2.0 mL 100 mmol/L sodium citrate, 600 μL 30% H2O2, and 600 μL 0.1 mol/L NaBH4 were added in sequence while stirring, the black nanosilver colloid was quickly stirred to obtain a black color. The black nanosilver colloid was immediately transferred to the light wave furnace, and the orange-red transparent nanosilver colloid was obtained by 250 °C light wave for 10 min. After natural cooling, the water was fixed to 50 mL for standby. The concentration was 68 mg/L AgNPs. The reagents used were all analytical pure, and the experimental water was sub-boiling water.Into a conical flask, 44 mL water, 2 mL 1.7 g/L AgNO3btc was added to the flask in batches and dissolved by ultrasound. Subsequently, 1.5 g FeSO4·7H2O was weighed and dissolved in a 100 mL round-bottom flask with 27 mL H2O ultrasonically. Under ultrasonic stirring, FeSO4 solution was slowly added to the H3btc solution at room temperature, and continuously stirred for 12 h to obtain a red-brown suspension. The suspension was centrifuged at 12,000 r/min to obtain red-brown products, and then the products were washed three times with deionized water and anhydrous ethanol, respectively. Then, the red-brown solid was obtained by high-speed freezing centrifugation . Finally, the product was vacuum freeze-dried for 24 h to obtain red-brown powder, namely FeMOF. The preparation of CoMOF/CuMOF/NiMOF followed the same steps as that of FeMOF, except replacing 1.5 g FeSO4·7H2O with CoCl2·6H2O, CuSO4·H2O, and NiCl2·6H2O of the same mass. A 0.01 g/L FeMOF solution was prepared with water.First, 0.53 g NaOH was added into the round-bottom flask and dissolved in 13 mL water, after which 0.735 g HGLY, and a certain concentration of glyphosate solution were added. The test tube was then placed in an 80 °C water bath for 5 min to allow the Apt to combine with the catalyst, after which was then added 100 μL 0.5 mmol/L TMB solution, 90 μL 0.1 mmol/L H2O2 solution, and 90 μL 0.1 mol/L Tris-HCl buffer. Subsequently, the solution was diluted to 2.0 mL with water, and mixed. After bathing at 45 °C for 30 min, the reaction was terminated in ice water, and 400 μL 68 mg/L AgNPs were added. The solution was placed into a quartz cell, and the Raman spectrum was recorded under the condition of 1 mW light source power and a 25.0 μm slit. The SERS intensity at 1607 cm−1 was measured, and the blank (I1607 cm−1) 0 was not mixed with GLY. The value of ΔI1607 cm−1 = I1607 cm−1 − (I1607 cm−1) 0 was calculated.Into a 5.0 mL test tube, 150 μL 0.01 g/L FeMOF, 200 μL 0.1 μmol/L Apt2O2 to produce TMBox. TMBox can make AgNPs aggregate, and its SERS activity and RRS effect are greatly enhanced. When Apt is added, it can be adsorbed onto the surface of FeMOF to inhibit its catalytic effect. The TMBox generated in the system decreases, and the SERS/RRS/FL signals decrease. When the target molecule GLY is added, it forms a stable complex with Apt and leaves the nanosurface. Its catalytic effect is enhanced, and the TMBox generated by the system is increased, and the SERS/RRS/FL signal is linearly enhanced. Accordingly, a new Apt-mediated FeMOF catalytic amplification RRS/SERS/FL trimode quantitative detection method for GLY can be established using the Raman spectrometer (Raman) and fluorescence instrument (FM)  .−1, 1383.5 cm−1, 1109.8 cm−1, 1044.9 cm−1, 943.6 cm−1, 759.6 cm−1, and 710.2 cm−1, respectively. The broad peak, 3445 cm−1, belongs to the hydroxyl -OH peak, the peaks at 1622.1 cm−1, 1383.5 cm−1, 1109.8 cm−1, and 1044.9 cm−1 belong to the stretching vibration peak of C=O, the bending vibration peak of hydroxyl, and the stretching vibration peak of C-O, respectively. Peaks at 759.6 cm−1 and 710.2 cm−1 belong to the fingerprint peak of FeMOF and the structure of Fe3+ oxide, respectively. It was proven that FeMOF was successfully prepared. In order to prove that the synthesized material is FeMOF, the crystal structure was analyzed by X-ray powder diffraction (XRD). −1 μm.2O2 at 45 °C in a water bath. The catalytic product, TMBox, has a SERS effect  sol, TMBox can make AgNPs aggregate, and enhance its SERS activity and RRS effect. The large specific surface area of FeMOF provides a large number of reaction sites for the reaction between H2O2 and TMB. The porous structure of FeMOF can adsorb the reactants to the nanosurface of the nanomaterial, the electrons on the nanopores speed up the redox electron transfer to enhance the H2O2–TMB reaction. At the same time, Fe(II) on the catalyst surface will activate hydrogen peroxide to generate ·OH radicals and Fe(III) [2O2 to generate HO2· and Fe(II)MOF, and the circulation of Fe(II)MOF and Fe(III)MOF on the catalyst surface plays the role of a nanocatalyst  ,29, FeFe(I1607 cm−1 of the H2O2–TMB system, FeMOF was prepared by heating 0.398 g, 0.585 g, 0.735 g, and 0.815 g trimellitic acid to room temperature, and stirring for 12 h according to the method. The effect of trimellitic acid addition on the catalytic effect of H2O2–TMB system was investigated. With the increase in trimellitic acid content, SERS signal value first increased and then decreased. When the addition amount of trimellitic acid was 0.735 g, I1607 cm−1 was the largest. Therefore, the amount of trimellitic acid added was 0.735 g. The effects of 0.2 g, 0.36 g, 0.53 g, and 0.84 g sodium hydroxide on the catalytic effect of H2O2–TMB system were investigated. With the increase in sodium hydroxide content, SERS signal value first increased and then decreased. When the addition amount of sodium hydroxide was 0.53 g, I1607 cm−1 was the largest. Therefore, the addition amount of sodium hydroxide was 0.53 g. The effects of 0.6 g, 1.2 g, 1.5 g, and 2.1 g ferrous sulfate heptahydrate on the catalytic effect of H2O2–TMB system were investigated. With the increase in ferrous sulfate heptahydrate content, the SERS signal value first increased and then decreased. When the addition amount of ferrous sulfate heptahydrate was 1.5 g, I1607 cm−1 was the largest. Therefore, the addition amount of ferrous sulfate heptahydrate was 1.5 g.The preparation conditions of FeMOF were selected by single-factor transformation. The three factors of trimellitic acid, sodium hydroxide, and ferrous sulfate hexahydrate were investigated . Based o2O2 concentration on the SERS signal of the system was investigated. When 4.5 × 10−3 mmol/L H2O2 was added, ΔI reached the maximum; therefore, 4.5 × 10−3 mmol/L H2O2 was selected (GLY concentration on the SERS signal of the system was investigated. When 8 nmol/L AptGLY was added, ΔI reached the maximum, hence 8 nmol/L AptGLY was selected (The analytical conditions were examined. The effect of Hselected . The effselected . The effselected . The effselected . The effselected . The effselected .2O2–TMB–FeMOF–AptGLY–GLY–AgNPs SERS system, in the concentration range of 0.1–14 nmol/L GLY (1607 cm−1 was linear to the concentration of GLY, and the linear equation was ΔI1607 cm−1 = 722C − 49.2. The linear correlation coefficient, R2, was 0.9947, and the DL was 0.05 nmol/L. For the H2O2–TMB–FeMOF–AptGLY–GLY–AgNPs RRS system, in the concentration range of 1–10 nmol/L GLY, the change of RRS intensity ΔI at 370 nm was linear with the concentration of GLY. The linear equation was ΔI370 nm = 82.2C + 18.1, the coefficient R2 was 0.9950, and the DL was 0.5 nmol/L (410 nm = 68.9C + 12, the coefficient R2 was 0.9748, and the DL was 1.0 nmol/L (Under the selected experimental conditions, for the Hol/L GLY A, the SE5 nmol/L B. For th0 nmol/L C. With tAccording to the experimental method, the effect of interfering substances on the determination of 2.5 ng/L glyphosate was detected. The results presented in The wastewater sample was filtered by 100 mL, and the supernatant was diluted to obtain the sample solution. According to the experimental method, 100 μL sample solution was taken to determine glyphosate content . The SER2O2 to produce more TMBox probe molecules. Through the regulation of nucleic acid aptamers, an effective SERS/RRS/FL trimode detection method for GLY in soil and water environments was established. This method combines the characteristics of SERS detection with low DL and good accuracy of RRS detection, and can be applied to the detection of GLY in actual samples. The recovery rate is between 92.1% and 97.5%, and the results are satisfactory. In addition, a reasonable nanocatalytic mechanism was proposed, that the surface electrons on FeMOF nanopores enhanced the redox electron transfer efficiency.In this study, FeMOF prepared by aqueous phase method at room temperature exhibited good catalytic performance, which can facilitate TMB oxidization by H


To assess vitamin D receptor (VDR) gene polymorphisms and bone mineral density and to investigate the possible risk factors of osteoporosis and fracture in rheumatoid arthritis (RA).A total of 97 RA patients and 45 matched controls were enrolled. Serum vitamin D level, VDR genotyping, dual-energy X-ray absorptiometry (DEXA) scan, trabecular bone score (TBS), and fracture risk assessment (FRAX) in 10 years were assessed. Disease activity score (DAS28) and modified health assessment questionnaire (MHAQ) were measured.p < 0.0001) but the TBS and VDR genotyping were comparable . The vitamin D level was comparable with the control . None of the patients was receiving anti-osteoporotic therapy or biologic therapy. There was a significant association between the presence of osteoporosis and age, disease duration, menopause, and rheumatoid factor (RF) positivity. The TBS was significantly lower and FRAX higher in patients with positive RF and anti-CCP. FRAX was significantly related and the TBS inversely with the age, disease duration, serum uric acid, alkaline phosphatase, and MHAQ.The mean age of the patients was 47.9 ± 8.9 years; 85 females, 12 males (F:M 7.1:1) and mean disease duration 9.4 ± 6.2 years. DAS28 was 4.52 ± 1.04 and MHAQ 0.6 ± 0.4. There was a significant difference between cases and controls as regards DEXA and FRAX (Reduced BMD and increased tendency to fractures are remarkable in RA patients. Vitamin D level was decreased in patients and control, and VDR gene polymorphisms were not linked to RA. TBS and FRAX are effective tools to assess osteoporotic fractures in RA. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, with variable clinical expression. It arises more frequently in females than in males, being predominantly observed in the elderly. It can be ranging from mild self-limiting disorder to a very aggressive form, resulting in joint destruction and progressive disability . Bone isThe remodeling process is the result of interactions between these cells (osteoclasts and osteoblasts) and multiple molecular agents, including growth factors, hormones, and cytokines . AlteredThe fracture risk assessment (FRAX) in 10 years tool was developed to evaluate the 10-year risk of bone fragility fractures in females and males older than 40 years. This tool relies on selected clinical risk factors for fractures, where bone mineral density (BMD) was measured at the femoral neck. Adjusted versions were established, according to the local epidemiological data, and its availability for most countries in the world . OsteopoThe study included 97 RA patients fulfilling the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria  and 45 aPatients were subjected to full history taking including menstrual and menopausal age, diet, sun exposure, smoking, current medications, prior fractures, family history of hip fracture as well as clinical examination, and assessment of the body mass index (BMI). Visual analog scale (VAS)—pain , modifieLaboratory investigations included complete blood picture, erythrocyte sedimentation rate, C-reactive protein, liver and kidney functions, thyroid stimulating hormone (TSH), parathormone (PTH), alkaline phosphatase (ALP), glycated hemoglobin (HbA1c), lipid profile, rheumatoid factor, anti-citrullinated cyclic peptide, serum calcium, and 25-hydroxy vitamin D.A total of 2 mL of fresh whole blood was collected in EDTA-containing tubes and stored in − 80 °C. The genomic DNA was isolated using the isolation kit . DNA samples were stored at − 20 °C; DNA concentration was determined using a NanoDrop lite Spectrophotometer (Thermo Scientific). Genotyping of Fok-I (rs2228570) and Taq-I (rs731236) was performed using TaqMan SNP Genotyping Assay. Real-time polymerase chain reaction (PCR) was performed using 5 μL TaqMan Genotyping Master Mix (2 ×), 0.5 μL TaqMan SNP Genotyping Assay (TaqMan probes) (20 ×), 3.5 μL DNase Free Water, and 1 μL DNA (50 ng), to bring the final reaction volume to 15 μL. PCR consisted of a hot starts at 95 °C for 10 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. All assays were performed using TaqMan Genotyping Master Mix on 96-well plates.T-scores were calculated through the TBS iNsight software  version 3.0 and were performed on the same vertebrae as those used for the BMD measurements.The BMD was assessed using the DXA scan  at the lumbar spine L1–L4, left femoral neck, and forearm. BMD measurement at or below − 2.5 SD from the optimal peak bone density of healthy young adult of the same sex was considered osteoporotic . The pathttp://www.shef.ac.uk/FRAX  with 9 clinical variables: age, BMI, previous fracture, hip fracture in a parent, alcohol use, smoking status, systemic glucocorticoids, RA, secondary osteoporosis, and femoral neck BMD.The FRAX was computed using the algorithm available online at t-test, chi-square test, and one-way ANOVA test were used. The Spearman correlation test was considered to test the association between quantitative variables. Regression analysis was conducted to determine the independent risk factors predicting osteoporosis. ROC curve was analyzed for VDR gene expression as a predictor for RA. The p-value < 0.05 was considered significant.Data was collected and analyzed using the Statistical Package for the Social Sciences (SPSS) software version 22. Data was presented as numbers and percentages or mean and standard deviation. Normality of distribution was considered using the one-sample Kolmogorov–Smirnov test. For comparisons, the independent samples p = 0.18 and p = 0.88, respectively). Characteristics, osteoporotic risk factors, DEXA, TBS, and FRAX of patients and controls are presented in Table p < 0.0001) while 76.3% were married vs 91.1% (p = 0.01). Twenty-eight patients had their faces only exposed to the sun while 7 patients and 3 controls had their faces, arms, and legs exposed. The mean tender joint count (TJC) in the patients was 7.6 ± 6.9 and swollen joint count (SJC) was 1.6 ± 2.2. According to the DAS28, 61 (62.9%) had moderate, 27 (27.8%) high, and 6 (6.2%) low activity, while 3 (3.1%) were in remission.This study included 97 RA patients: 85 (87.6%) females and 12 (12.4%) males (7.1:1) with a mean age of 47.9 ± 8.9 years (35–70 years). The 45 controls were age (45.9 ± 8.1 years) and sex  matched (p = 0.4). None of the patients was receiving anti-osteoporotic therapy or biologic therapy.The laboratory investigations and mediations received by the patients are presented in Table p = 0.002 and p < 0.0001).A comparison of the characteristics of the RA patients according to the BMD is presented in Table p = 0.001, p = 0.026, and p = 0.008). Those with positive anti-CCP had a significantly lower BMD  and TBS and higher FRAX  compared to those negative .The FRAX for major OP # was significantly higher in those receiving steroids (5.6 ± 2.2) compared to those not ; the femur T-score was significantly lower in those on LFN (− 0.5 ± 1.2) compared to those not  and the T-score spine was lower in those receiving HCQ (− 0.9 ± 1.5) compared to those not .The TBS was significantly lower and FRAX (major and hip) higher in patients with positive RF  compared to those with negative   Fig. .Table 4Cβ 0.5, 95% CI 0.01–0.99; p = 0.046), the presence of metabolic syndrome , and dyslipidemia  with the risk of osteoporosis in RA. The VDR rs2228570 genotyping (FokI) could not predict RA disease from control  or patients with and without osteoporosis . Only the age at onset and receiving MTX would significantly predict the TBS in RA patients .There was a significant association between considering a diet rich in calcium (The coexistence of RA and osteoporosis is common and can lead to serious complications. The role of vitamin D and vitamin D receptor (VDR) gene polymorphisms in RA pathogenesis and clinical course of the disease has been investigated, with controversial data according to many factors including genetic, ethnicity, and environmental factors .The current study showed that there was a significant difference between patients and controls as regards DXA, TBS, and FRAX. A lower BMD in RA patients compared to controls has been shown with an increased prevalence of osteoporosis . The effThere was no significant difference between cases and controls as regards vitamin D level as most of them had deficiency. Similarly, other studies demonstrated that there was no significant difference in serum vitamin D level in RA and controls , 28. VitA series of polymorphisms in VDR gene have been reported. They include BsmI, ApaI, TaqI restriction sites, variable PolyA length, and FokI restriction site. Of these polymorphic sites, BsmI are substitutions on intron 8 whereas ForkI restriction enzyme identifies a polymorphic site in exon 2 at the 50 ends of the VDR gene. They have an effective role, a functional significance of these polymorphisms, and their potential effects on RA susceptibility . The curIt was detected in the current study that there was a significant difference relation of VDR genotype (Fok-I) and CRP levels among cases but not with other parameters. Virtually, all immune cells express VDR, making them susceptible to vitamin D-mediated modulation, so after binding to VDR, active vitamin D has a direct immunosuppressive effect on dendritic cells, reduces CD4 + cells proliferation, and differentiation into Th1 and Th17. VDR agonists were proposed to be selective inhibitors of Th1 cell development and found to inhibit Th1-type cytokines such as IL-2 and TNF-α directly . FurtherT-score was significantly lower in those on LFN and the T-score spine was lower in those receiving HCQ. The FRAX was significantly related and the TBS inversely with the age, disease duration, SUA, ALP, and MHAQ. The FRAX was further significantly related to the LDL level and the TBS negatively with the anti-CCP titre. In concordance, Haugeberg et al. showed that reduced BMD was associated with age, current use of corticosteroids, physical disability, and RF positivity in RA [There was a significant association between the presence of osteoporosis and age, disease duration, presence of menopause, and RF positivity. The TBS was significantly lower and FRAX higher in patients with positive RF. Those with positive anti-CCP had a significantly lower BMD and TBS as well as a higher FRAX. The FRAX for major OP # was significantly higher in those receiving steroids; the femur ty in RA . Lee andty in RA . Choi etty in RA  revealedty in RA .Kwok and others concluded that all men (aged ≥ 70 years) and women (aged ≥ 65 years) should receive universal DEXA assessment for osteoporosis and those with an increased FRAX should receive anti-osteoporotic treatment to prevent hip fractures .Among the study limitations is the relatively small sample size and assessing only two VDR genes for their association with the risk of bone loss. So, further multi-center research is necessary among larger cohorts, involving different geographical distribution.In conclusion, the reduced BMD and increased tendency to fractures are remarkable in RA patients. Vitamin D level was decreased in patients and control, and the VDR gene polymorphism Fok-I and Taq-1 were not linked to RA. TBS and FRAX are effective tools to assess osteoporotic fractures in RA. Bone loss and fragility fracture are related to age, disease duration, menopause, SUA, RF and anti-CCP positivity, function impairment, and steroids.


The child’s groups were associated with mortality, both in the univariate  and multivariate ) analyses, also introducing Klotho and the TNF-α as dichotomic variables. However, the independent prognostic value of the Child’s groups was displaced by Klotho when only cirrhotics were considered; Klotho, over the median (574.4 pg/mL), was associated with higher mortality (with p = 0.04 and OR = 2.68 (1.06–6.84)). We conclude that Klotho is increased in liver cirrhosis. It is directly related to TNF-α, MDA, and to mortality in cirrhotics.α-Klotho (Klotho) is an antiaging hormone with anti-inflammatory and antioxidative properties. Some studies suggest that Klotho increases in response to enhanced oxidative damage and inflammation. Alcoholism is a proinflammatory condition. The aim of this study was to analyze the relationship between Klotho and the serum levels of the inflammatory markers in alcoholic liver disease and to assess its prognostic value. We included 184 alcoholics and 35 age- and sex-matched controls. We determined the serum levels of Klotho, the tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and malondialdehyde (MDA), and routine laboratory variables. Patients were followed-up with during 16 ± 18 months; 67 patients died. Klotho levels were higher among cirrhotics (with KW = 37.00 and  Alcoholism may be viewed as an inflammatory condition . SmolderKlotho is a membrane-bound protein that may act as a receptor for the fibroblast growth factor 23 (FGF23). The cleavage of this membrane-bound Klotho by proteases, such as a disintegrin and metalloproteinase (ADAM) 10 and ADAM 17, generates the soluble Klotho protein, a multifunctional hormone with powerful anti-inflammatory and antioxidant effects ,10, relaIn previous reports, we have found that, despite its “protective” effect, Klotho levels were higher in cirrhotics and were related to liver function impairment . We intep = 0.08) and sex  χ2 = 3.10; p = 0.08).This study included a total of 184 patients (12 women), aged 58.94 ± 11.27 years, drinkers of 189 ± 128 g of alcohol per day for 32 ± 13 years, who were consecutively admitted to the internal medicine service of our hospital. Klotho was also determined to 35 controls of a similar age  and clinical  and laboratory data . The study was reviewed and approved by the Institutional Review Board of our hospital (No. 2019-83).Patients (besides 1) were followed-up during 16 ± 18 months . During this period, 67 patients died. The causes of death included liver failure in 14 cases; sepsis in 20 cases; neoplasia in 10 cases; vascular causes (including sudden death) in 4 cases; unknown in 19 cases.All the patients underwent a routine laboratory evaluation. The serum albumin, prothrombin activity, and serum bilirubin were recorded to assess liver function and to calculate the Child–Pugh’s score (Child’s score) . This scAfter blood extraction, samples were placed in vacutainer tubes without an anticoagulant and allowed to clot (a maximum of 10 min at room temperature); then, they were centrifuged immediately at 3000 rpm at 4 °C for 5 min. After that, 200 µL of the serum was separated, aliquoted in an Eppendorf tube , and stored, frozen, at −87 °C until analysis. All serum freezing samples at −87 °C were stored at a time not exceeding 1 month for analysis and were later analyzed in batches.® Performance Assay , with a sensitivity of 0.29 pg/mL for the TNF-α, a sensitivity of 0.36 pg/mL for IL-6, and a sensitivity of 1.97 pg/mL for IL-8.Klotho levels were determined by ELISA . The interassay and intrassay variation coefficients ranged from 2.9–11.4% and 2.7–3.5%, respectively. The percentage of recovery ranged from 84.7 to 97.5%. The sensitivity was 6.15 pg/mL, and for the cross-reactivity with other molecules, such as osteopontin, the platelet-derived growth factor, or the vascular endothelial growth factor, it was <0.1%. The TNF-α, IL-6, and IL-8 were determined by a Luminexg for 10 min. Each one was placed in a 96 well plate and read at 535 nm in a microplate spectrophotometer reader . The calibration curve was prepared with authentic MDA standards. The detection limit of this assay was established in 0.079 µmol/L; the intra- and inter-assay of the coefficient of variations were 1.82% and 4.01%, respectively. The serum concentration of MDA was expressed in micromoles per liter. To avoid possible interferences of compounds that react or absorb at 532 nm, each sample was provided by our blank tube (sample without the TBA reagent), and the absorbance was subtracted from each sample tube [Serum MDA levels, referred to as the thiobarbituric acid-reactive substance (TBARS), were measured according to the method described by Kikugawa et al. , with sople tube . Furtherple tube .t-test was used with the variables with a normal distribution, whereas non-parametric tests, such as the Mann–Whitney’s U (Z) test, Kruskal–Wallis (KW), and Spearman’s (ρ) correlation tests, were used when the variables included in any of these analyses showed a non-parametric distribution. Qualitative variables were compared by means of a chi-square test. Survival was analyzed by means of Kaplan–Meier curves, comparing patients who died during the observation period with those who survived using a log-rank (LR) and Breslow tests. We also performed a stepwise Cox regression analysis, introducing variables that showed significant differences between survivors and non-survivors in the univariate analysis. Non-parametric variables, such as cytokines or MDA, were dichotomized according to the median values or, in the case of the TNF-α, according to detection level. All these analyses were performed with SPSS software (15.0) .The Kolmogorov–Smirnov test was used to explore if the variables showed a normal distribution or not. A Student’s p < 0.05 with the SNK test). Globally, the differences were highly significant , albumin (with ρ = −0.15 and p = 0.047), and bilirubin (with ρ = 0.26 and p < 0.001) but not to serum creatinine . Marked differences were observed when Klotho was compared among the three Child’s groups  than patients belonging to the Child A group. Klotho was also related to platelet count . Therefore, the Klotho values were higher among cirrhotics and were related to liver function impairment. Klotho was significantly related to prothrombin activity (with ρ = −0.39 and p < 0.001) and between Klotho and MDA (with ρ = 0.21 and p = 0.006) but not between Klotho and IL-6 or IL-8. The TNF-α values were significantly lower among patients with Klotho values below the median than among patients with Klotho values over the median (with Z = 2.85 and p = 0.004). Also, the MDA values were significantly higher among patients with α-Klotho values over the median (with Z = 3.012 and p = 0.003). Therefore, Klotho levels were associated with increased lipid peroxidation and increased serum TNF-α values.Significant relationships were observed between Klotho and the TNF-α (with ρ = 0.28 and p = 0.28 (NS). Moreover, the TNF-α levels were not related to albumin, prothrombin, bilirubin, or the Child–Pugh score. This was not the case for IL-8, which was significantly higher in cirrhotics (with Z = 4.06 and p < 0.001), especially in the more decompensated ones (with KW = 11.9 and p = 0.003 when IL-8 was compared among the three Child’s groups) and closely related to the prothrombin activity (with ρ = −0.21 and p = 0.006) or the platelet count (with ρ = −0.30 and p < 0.001). A somewhat weaker association with the liver function was observed with IL-6. When classified as a dichotomic variable, according to the median values, the IL-6 levels over the median were significantly associated with liver cirrhosis (with χ2 = 9.77 and p = 0.002), and the IL-6 values were inversely related to the prothrombin activity (with ρ = −0.30 and p < 0.001). In addition, IL-8 (with Z = 3.87 and p < 0.001) and MDA (with Z = 3.98 and p < 0.001) were significantly higher among patients with IL-6 values over the median. The TNF-α values were also higher in this group but did not reach a statistical significance (with Z = 1.60 and p = 0.11).As shown in p = 0.004), and the MDA values were strongly associated with liver function impairment  and with the platelet count (where ρ = −0.31 and p < 0.001). Also, significant relationships were observed between the MDA levels and IL-6 (with ρ = 0.31 and p < 0.001) and IL-8 (with ρ = 0.49 and p < 0.001) but not with the TNF-α (with ρ = 0.06 and p = 0.46). Therefore, lipid peroxidation is increased in liver cirrhosis and is related to liver function impairment. MDA is also related to the proinflammatory cytokines IL-6 and IL-8. Liver cirrhosis was significantly associated with IL-6 and IL-8 over the median but not with the TNF-α values.Relationships between MDA and liver function were stronger than those observed between cytokine levels and liver function. The MDA values over the median were associated with liver cirrhosis (χ2 = 8.36 and p = 0.012) and Klotho (with Z = 2.46 and p = 0.014) were higher among those who died. Those patients with Klotho levels over the median showed a marginally significant trend to a higher mortality  but not in cirrhotics only. The TNF-α was also related to mortality .Survival was also marginally related to MDA  but notp = 0.001, Breslow = 12.33, and p = 0.002; p = 0.67, Breslow = 0.16, and p = 0.69).The Child groups were strongly associated with mortality  when assessed by the Cox regression model, also introducing Klotho and the TNF-α as dichotomic variables. Performing the same analysis only in cirrhotics, we observed that Klotho classified in the medians displaced the Child’s score, where α-Klotho, over the median (574.4 pg/mL), was associated with higher mortality (with p = 0.04 and RR = 2.68 (1.06–6.84)).The Child–Pugh groups had the only variable that showed an independent relationship with mortality  and MDA levels, a finding shared by the vast majority of authors involved in research related to the alcoholism-associated inflammatory response. As commented, alcoholism is associated with increased intestinal permeability that leads to increased amounts of Gram- (and Gram+) bacteria in the portal blood. These bacteria activate liver macrophages and Kupffer cells, leading to increased cytokine secretion, especially the TNF-α and IL-6, and the production of ROS. This pathway importantly contributes to the proinflammatory status that can be observed in alcoholics. In liver cirrhosis, the distortion of the vascular architecture facilitates the escape of intestinal bacteria to the systemic circulation, stimulating many cells of the innate immune system that can secrete proinflammatory cytokines, such as the TNF-α. Therefore, theoretically, cytokine levels are higher, and the ROS production is increased among cirrhotics. In this study, we observed increased MDA in cirrhotics and also increased IL-6 and Il-8, but not increased TNF-α values. The lack of differences between cirrhotics and non-cirrhotics in the TNF-α levels is difficult to explain. An unexpected large proportion of patients (78%) showed a TNF-α below the detection levels, but all the TNF-α, IL-6 (20% of the cases below the detection level), IL-8 (in only one case below the detection levels), Klotho (in none of the cases below the detection level), and MDA (in none of the cases below the detection level) were determined in the same blood sample. In summary, Klotho, MDA, IL-8, and IL-6 behave similarly in patients with alcoholic liver disease, being more or less intensely related to disease progression and liver function impairment. This was not observed with the TNF-α, in contrast with the results of most studies .However, despite the lack of differences between cirrhotics and non-cirrhotics regarding TNF-α values, Klotho levels were directly related to the TNF-α values. There may be a theoretical basis to explain this relationship. The TNF-α may repress Klotho mRNA transcription . The KloKlotho has been considered a protective hormone, exerting antioxidant effects  and deprIn addition to the shortage imposed by the lack of differences in the TNFα values among cirrhotics and non-cirrhotics, there are other limitations of this study. The diagnosis of cirrhosis was made on clinical/ultrasonographic features. This may constitute a shortage of our study. Moreover, strictly speaking, the Child’s score should only be applied to cirrhotics, but for the sake of uniformity, we also used it for non-cirrhotic patients since it is an excellent tool to globally assess the derangement of the liver function and progression of liver disease. Following Prystupa et al. , we furtWe conclude that Klotho is increased in liver cirrhosis, being related to the TNF-α, lipid peroxidation, and especially to liver function impairment. Possibly, this association may explain the counterintuitive results regarding the relationship of Klotho with mortality, something observed not only in the whole group of alcoholics but also, especially, among cirrhotics. Further studies are needed to assess the behavior of Klotho in other instances characterized by an intense inflammatory response and to discern the mechanisms explaining the (possible) increase of Klotho levels in these conditions.