Supplemental Digital Content is available in the text The impact of aldosterone blockade using eplerenone on hypertensives with obesity has not been clarified. We compared the efficacy and safety between eplerenone and trichlormethiazide in hypertensives with overweight or obesity.2] evaluated by ambulatory blood pressure (BP) measurement. Patients were randomly assigned to receive 50 mg of eplerenone (n = 102) or 1 mg of trichlormethiazide (n = 102), each of which were administered once every morning. Primary efficacy endpoints were systolic and diastolic BPs and biomarkers of glucose metabolism after 6 months of treatment.A prospective, randomized, open-labeled, blinded-endpoint design, multicenter trial enrolled 204 hypertension-treated outpatients with obesity [body mass index (BMI) ≥25 kg/mP < .001). The efficacy of SBP reduction after adjustment for age, sex, and BMI was significantly greater in the eplerenone group than the trichlormethiazide (P = .034), although the efficacy of DBP reduction was marginally significant (P = .072). Especially, the efficacy of BP reduction was more effective for aged over 65 years than less than 65 years. However, biomarkers of glucose metabolism were not significantly different between these 2 groups.At baseline, BPs were comparable between the two groups. Systolic/diastolic blood pressure (SBP/DBP) were reduced from 153.9 ± 12.6/84.6 ± 11.8 to 129.8 ± 14.2/73.7 ± 12.2 mm Hg by eplerenone therapy and from 152.2 ± 12.5/85.2 ± 10.9 to 133.8 ± 12.6/76.1 ± 8.6 mm Hg by trichlormethiazide therapy (all; The eplerenone therapy was more effective in BP lowering in hypertensives with overweight or obesity than the trichlormethiazide therapy, especially in the elderly. However, it is well known that thiazide diuretics have unfavorable effects in glucose metabolism.Hypertension with obesity is prevalent in Japanese population, which is crucial health problems since both hypertension and obesity are risk factors for cardiovascular events. The 10-year prospective study demonstrated that plasma aldosterone levels predicted the development of insulin resistance in a general population.Aldosterone plays a central role in hypertension, and hypertension is prevalent in patients with insulin resistance. We have previously investigated the significant relationship between plasma aldosterone levels and insulin resistance in 564 nondiabetic participants.Based on the findings from the prospective study, we hypothesized that the aldosterone blockade, eplerenone, is effective in hypertensives with obesity for lowering blood pressure (BP) as well as improving glucose metabolism compared to thiazide diuretics.22.12, currently on antihypertensive medication, and assessed the effects of eplerenone versus trichlormethiazide on biomarkers of blood pressure, heart rate, and glucose metabolism over 6 months. The present study was approved by the Ethics Committee of Kurume University School of Medicine (protocol No. 12138) and registered in the University Hospital Medical Information Network .We enrolled Japanese obese patients with BMI ≥25 kg/m1. Registration criteria2; 3) Patients treated with one or more antihypertensives , calcium channel blocker (CCB), alpha-blocker, or beta-blocker),(1) Inclusion criteria as follows: 1) Patients with SBP ≥140 mm Hg and/or DBP ≥90 mm Hg despite on antihypertensives; 2) Patients with BMI ≥25 kg/m1.If patients were on drug for diabetes, the types and doses of the drugs were stable during 3 months prior to enrollment2.Patients who provided written informed consents3.Japanese males and females who were more than or equal to 20 years old at the time of giving informed consent(2) Exclusion criteria as follows: 1) Patients on eplerenone or spironolactone during 3 months prior to enrollment; 2) Patients on diuretics including trichlormethiazide or combination drugs during 3 months prior to enrollment; 3) Patients who experienced myocardial infarction or stroke during 30 days prior to enrollment; 4) Patients who had cardiac surgery or percutaneous coronary intervention (PCI) during 30 days prior to enrollment; 5) Patients with serum potassium ≥5.0 mEq/L; 6) Patients with severe liver dysfunction; 7) Patients with moderate or severe renal dysfunction (Ccr < 50 ml/min); 8) Patients with grade III hypertension (SBP ≥180 mm Hg and/or DBP ≥110mm Hg); 9) Patients diagnosed as secondary hypertension; 10) Patients with hypersensitivity with eplerenone or sulfonamide derivative; 11) Patients who were contraindicated for use in eplerenone or trichlormethiazide; 12) Patients who were on medications contraindicated for use with eplerenone or trichlormethiazide; 13) Women who were either pregnant, lactating or of childbearing potential; 14) Patients who were considered not appropriate to enroll into this trial by the investigator.2. Study treatments(1) ScreeningA. Pre-screening1)Measure height and weight to calculate BMI2)Measure SBP and DBP at office with seated positionB. Main-Screening2 and SBP ≥ 140 mm Hg and/or DBP ≥ 90 mm Hg based on the pre-screening measurement.Conduct screening with following items on the patients who were BMI ≥ 25 kg/m(1)Obtain informed consent(2)Patient background:a. Age, b. sex, c. history of smoking, d. previous medical history (including duration of hypertension), e. other appreciable medical conditions(3)Complications(4)Concomitant medicationsa. ARBs and/or ACE-Is, b. CCBs, c. beta-blockers, d. diuretics, e. DRIs, f. Other concomitant medications (including drugs other than antihypertensives)(5)Concurrent therapy(6)Physical findings (7)Clinical laboratory tests C. After assignment of 2 groups1. Eplerenone group1)Keep the baseline medication(s) and administer eplerenone 50 mg/day.2)If SBP was not controlled under 140 mm Hg or DBP was not controlled under 90 mm Hg, the dose of eplerenone was to be titrated up to 100 mg/day with careful monitoring for serum potassium level.3)If necessary, antihypertensive medications such as ARB/ACE-I, DRI, CCB, alpha/beta-blockers could be added after 3 months of randomization.4)Diuretics were not allowed to prescribe in this group.5)If patients were prescribed cytochrome (CYP) 3A4 inhibitors , dosage of eplerenone should be kept as 25 mg/day and should not exceed the amount.6)If serum potassium level increased to 5.5 mEq/L, patients should stop eplerenone. Restart taking eplerenone with lower dose only if serum potassium level dropped to less than 5.0 mEq/L.7)As a general rule, the amount of concomitant medications should not be added, increased, or decreased during the study period, unless the investigators needed the modification.2. Trichlormethiazide group1)Keep the baseline medication(s) and administer trichlormethiazide 1 mg/day.2)If SBP was not controlled under 140 mm Hg or DBP not controlled under 90 mm Hg, the dose of trichlormethiazide could be titrated up to 2 mg/day.3)If necessary, antihypertensive medications such as ARB/ACE-I, DRI, CCB, alpha/beta-blockers could be added after 3 months of randomization.4)Eplerenone and/or spironolactone were not allowed to prescribe in this group.5)As a general rule, the amount of concomitant medications should not be added, increased, or decreased during the study period, unless the investigators needed the modification.3. Concomitant medications and concurrent therapies1)All medications and therapies used during the study period were considered as concomitant medications and concurrent therapies. All such practices were documented.2)Following medications are not allowed to use during the study period.SpironolactonePotassium canrenoatePotent CYP3A4 inhibitors and/or CYP3A4 revulsant D. Adverse event reportingWhen adverse events were observed in patients participated in this study, the investigators reported them to the data center  in accordance with the rules for reporting adverse events in usual clinical practice of the respective institutions.E. Study periodPatient entry period: November 1, 2012 to March 31, 2016 (including screening period); Protocol amended on September 11, 2013.Follow-up period: 6 months after enrollment F. Endpoints1) Primary endpoints:Change from baseline in office SBP/DBP after 24 weeks%Change from baseline in insulin sensitivity (homeostasis model assessment insulin resistance (HOMA-IR)) after 24 weeks1c (HbA1c) after 24 weeks%Change from baseline in hemoglobin A2) Secondary endpoints:Changes from baseline in high sensitivity C-reactive protein (hsCRP), eGFR, adiponectin, and the use of potassium supplement1.Safety parameters:(1)Serum potassium for up to 24 weeks(2)Adverse events, laboratory values, physical examinations, and vital signs for up to 24 weeks2.Observation parameters(1)Patient background: Age, sex, height, weight, BMI, abdominal circumference, history of present illness, complications, past medical history, therapeutic situation, pretreatment(2)Office blood pressure/pulse: Systolic and diastolic blood pressure (sitting), heart rate(3)1c, insulin, adiponectin, cortisolBlood chemistry: complete blood count, blood urea nitrogen (BUN), creatinine (Cr), electrolytes , AST, ALT, gamma-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (ALP), total bilirubin, total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, remnant-like lipoprotein cholesterol (RLP-c), fasting plasma glucose, HbA(4)Insulin sensitivity: homeostasis model assessment insulin resistance (HOMA-IR)(5)Inflammatory markers: hsCRP(6)Renal function: eGFR (IDMS-MDRD equation method)(7)Renin-angiotensin system (RAS) components: (8)Electrocardiography (ECG) (on a voluntary basis)(9)Compliance status: Calculation is performed by considering cases where all prescribed doses are taken as 100%.(10)Safety: Serum potassium, serum Na+, hepatic function test3 design was used.Prospective, Randomized, Open, Blinded-Endpoint (PROBE)The registration process was as follows:1.Investigators confirmed that screened patients met the inclusion criteria and did not meet the exclusion criteria.2.Investigators informed to a preassigned investigational staff member about patients who met registration criteria.3.6 was employed in randomization.Eligible individuals were to be randomized to either of the eplerenone group or diuretic group with 1:1 proportion. To ensure allocation balance on baseline covariates , Pocock–Simon Minimization AlgorithmNext, analyses of primary endpoints, secondary endpoints and safety were performed in the following analysis sets.Per Protocol Set (PPS): subset of the patients in the full analysis set who completed a certain pre-specified minimal exposure to the treatment regimen had some minimum number of measurements of the primary variable (s) had no major protocol violationsFull Analysis Set (FAS): The FAS was defined as all randomized patients. The primary and secondary analyses were performed according to the randomized treatment.Safety Analysis Set: The Safety Analysis Set was defined as all patients who received at least one dose of study drug on their actual treatment received.P < .05. Time-trends of endpoints during entire study period will be examined by employing mixed-effects models where baseline measurement of SBP, DBP, and heart rate levels as well as age and sex were used as covariates. All data analyses were carried out based on the intention-to-treat approach.Changes of primary endpoints were compared between the two groups using Analysis of Covariance. Difference between two groups were regarded as statistically significant if Data were independently and strictly monitored and analyzed by the data center .44.1 to the eplerenone (n = 102) and trichlormethiazide therapies (n = 102)  Table . At base2) Table .4.2P < .001) and from 152.2 ± 12.5 to 133.8 ± 12.6 mm Hg in the trichlormethiazide (P < .001) . Mean DBP significantly reduced from 84.6 ± 11.8 to 73.7 ± 12.2 mm Hg in the eplerenone (P < .001) and from 85.2 ± 10.9 to 76.1 ± 8.6 mm Hg (P < .001) in the trichlormethiazide . Potassium levels in eplerenone therapy were significantly higher (P = .002) than trichlormethiazide therapy. The SBP and DBP lowered levels between eplerenone and trichlormethiazide stratified by age groups (≥65 and <65 years) were demonstrated in Fig. P = .015) and DBP (P = .024) levels in patients aged over 65 years of eplerenone therapy were significantly lower than trichlormethiazide therapy. However, glucose metabolism does not show a significant difference between the two groups.Figure 5This study provides the first evidence for the efficacy and safety of eplerenone therapy even in a relatively high age group, which demonstrated that 50–100 mg eplerenone therapy significantly lowered SBP in patients with hypertension and with overweight or obesity, especially in patients aged over 65 years, compared with 1–2 mg of trichlormethiazide.5.1 reported that the eplerenone may be useful add-on therapy in hypertensive patients. Eplerenone doses of 50–400 mg once daily are well tolerated and effective in reducing BP in patients with mild-to-moderate hypertension during a 24-hour period. On the other hand, efficacy of eplerenone versus enalapril as monotherapy in systemic hypertension was evaluated by Williams et al which demonstrated that eplerenone was as effective as enalapril for monotherapy in patients with stage 1 or 2 hypertension. Efficacy and safety of eplerenone therapy were also evaluated in Japanese patients with hypertension.–12 The report from Saruta et al was a randomized, double-blind, placebo-controlled, parallel-group, dose-ranging study in 193 patients. Eplerenone has been shown to be an efficacious and well-tolerated agent and should be considered as one of the options for the management of patients with essential hypertension. The manuscript by Sato et al reported that the add-on therapy in Japanese patients with essential hypertension had an extremely beneficial antihypertensive effect, when eplerenone was co-administered with an ACE-I or long-acting CCB. Tsutsui et al also reported eplerenone was well tolerated in Japanese patients with heart failure with reduced ejection fraction.Although we compared between 50 and 100 mg of eplerenone and 1–2 mg of trichlormethiazide in this study, Krum et al–15 Sato et al reported a randomized controlled study of finerenone versus eplerenone in Japanese patients with worsening chronic heart failure and diabetes and/or chronic kidney disease. They found that mean changes in serum potassium levels were similar between finerenone and eplerenone. Khosla et al suggested that caution should be paid when using eplerenone for BP control in patients with advanced stage 3 nephropathy with a serum potassium of 4.5 mEq/l. Eschalier et al also recommended that eplerenone, including in patients with eGFR > 30 ml/min/1.73 m2 and potassium <5.0 mmol/l, was both efficacious and safe when carefully monitored in patients with chronic heart failure and reduced ejection fraction. Regarding the efficacy and safety of eplerenone in the management of mild-to-moderate hypertension, systematic review and meta-analysis by Pelliccia et al cautioned the increased potassium levels; however, average incidence of hyperkalemia (i.e. >5.5 mmol/l) in the all previous reports was below 2%. Only 0.1 mmol/l as average potassium increase in eplerenone group was found in our study. When we compared between eplerenone and trichlormethiazide by the analysis of covariance adjusted for age, sex, and BMI, a significant potassium increase (P = .002) was found in eplerenone group.As for safety, some studies are focusing on hyperkalemia.5.2 Therefore, this study examined whether eplerenone, a selective aldosterone blocker, can improve the glucose metabolism such as insulin resistance. There have been some reports concerning the beneficial effect of eplerenone on glucose metabolism or metabolic syndrome. Bender et al suggested in their review that aldosterone and mineralocorticoid receptor (MR) signaling represent an ideal candidate pathway linking early promoters of diabetes, especially obesity, to vascular insulin resistance. McMurray et al also suggested reassuring evidence of the neutral effect of eplerenone on insulin action in hypertensive, nondiabetic patients. However, negative data have been also reported, in which Hwang et al did not support a contributing role for MR in endothelial dysfunction and insulin resistance in older adults with metabolic syndrome. Thus, as Tirosh et al also suggested that the evidence of MR blockade in metabolic syndrome are still scant, although several clinical trials have demonstrated the beneficial effects of MR antagonists on heart failure, hypertension, and diabetic nephropathy.–23 On the other hand, Japanese investigators have reviewed the effects of eplerenone on salt-sensitive hypertension and chronic kidney disease and metabolic syndrome.,25 The present study also did not show a beneficial effect on insulin resistance or metabolic syndrome in the eplerenone group, which might be caused by the insufficient period (6 months) to improve the metabolic-related factors.Our previous study in a general population demonstrated that high levels of plasma aldosterone predicted the development of insulin resistance after 10 years in subjects without insulin resistance at baseline.5.3The strengths of the current study are the use of PROBE design, multicenter trial, in which data were independently and strictly monitored and analyzed by statistics experts. Moreover, our data significantly contribute to scientific progress in a balanced perspective to the proposed important pathophysiological role of MR activation in insulin resistance and in metabolic syndrome although we could not indicate the beneficial effect.5.4First, we have studied a small number of Japanese hypertensives with obesity. Of the enrolled 204 patients, 39 subjects were dropped out. Second, although the readers may be interested in the 24 h, night, and day BP changes, the data of SBP and DBP were obtained only by office measurement. Third, since we did not perform echocardiology to the enrolled patients, eplerenone may have a different efficacy in patients affected by heart failure. Finally, our results might have been influenced by adherence of eplerenone and trichlormethiazide of the patients.6Our data demonstrated, for the first time using PROBE, that the eplerenone therapy was more effective for hypertension with overweight or obesity than the trichlormethiazide therapy, especially in elderly patients.We thank Drs. Bekki H (Bekki Clinic), Uemura S, Ohga M, Matsumoto M , Onitsuka I , Shihara M, Kaneyuki M, , Jinnouchi J, Jinnouchi Y, , Tamai O , Kusaba K, Shimada T , Sugi K, Katsuki S , Wada Y, Eguchi H (Wada Clinic), Tahara N , Kumagai E, Okina N , Ohuchida M, Umei T , Kai H , Yokota T, Yoshida T , Miyamoto T , Kanaya S, Yokoyama S , Nohara Y , Miyake Y , Kuwano K (Kuwano Clinic), Nagata T, Takeshita N , Morita H , Yamamoto K (Yamamoto Clinic), Nakao K, Ohbu K , Nakamura T , Tokuda K (Tokuda Clinic), Maki S , Ishizaki T (Ishizaki Clinic), Hashimoto Y , Nohara M (Nohara Clinic). We also thank Mrs. Makiko Tokuyasu for her secretarial assistance.Data curation: Eita Kumagai.Formal analysis: Tatsuyuki Kakuma, Machiko Kawaguchi.Investigation: Hisashi Adachi.Supervision: Yoshihiro Fukumoto.Writing – original draft: Hisashi Adachi.


Similarly, in study 2, total-cholesterol AUC0–10h in TRL was not different but triacylglycerol AUC0–10h in TRL was greater for 100 g CWE vs. no egg. In both studies, whole egg consumption did not affect plasma total-cholesterol AUC0–10h, while triacylglycerol AUC0–10h was increased. These results suggest that the dietary cholesterol in whole egg was not well absorbed, which may provide mechanistic insight for why it does not acutely influence plasma total-cholesterol concentration and is not associated with longer-term plasma cholesterol control.Whole egg is a food source of dietary cholesterol and inconsistent research findings exist about the effect of dietary cholesterol from whole egg on blood cholesterol concentration. We assessed the effect of co-consuming cooked whole egg (CWE) on dietary cholesterol absorption from two randomized-crossover studies. For study 1, 16 men consumed raw vegetables with no egg, 75 g CWE, or 150 g CWE. For study 2, 17 women consumed cooked vegetables with no egg or 100 g CWE. Triacylglycerol-rich lipoprotein fractions (TRL) were isolated from collected blood. In study 1, total-cholesterol areas under the curve (AUC) Cholesterol’s important functions in the human body include an essential structural component in cell membranes and being a chemical precursor of steroid hormones and bile acids ,2. CholeWhole egg is a well-known food source of dietary cholesterol and contains unsaturated fat, high-quality protein, and varying quantities of vitamins and minerals ,13. DespThe main objective of this research was to assess the effect of consuming cooked whole eggs on cholesterol absorption in adults, measured in TRL fractions. Triacylglycerol concentration within TRL fractions was also measured and used as a control comparison. The results presented in this manuscript were obtained using secondary data from two randomized, cross-over, controlled acute feeding studies designed to assess the impact of consuming eggs on the absorption of fat soluble nutrients from a co-consumed meal ,22.The Purdue University Biomedical Institutional Review Board approved both studies. All subjects signed an informed consent form and received monetary compensation for their participation. Clinical trial profiles for study 1 and study 2 are described at NCT01951313 and NCT02679794, respectively.Sixteen men from the greater Lafayette, IN, region completed the study . InclusiFor this single-blinded, randomized and crossover-design study , all subOn each of the three testing days, subjects consumed a raw mixed-vegetable salad (0 mg dietary cholesterol) including tomatoes, shredded carrots, baby spinach, lettuce, and Chinese wolfberry with no egg, with 75 g cooked whole egg (CWE), or with 150 g CWE. All salads were also served with 3 g canola oil. A served raw mixed-vegetable salad with 3 g canola oil provided 92 kcal, 3 g protein, 12 g carbohydrate, 4 g fat, and 0 mg of cholesterol. The no egg, 75 g CWE, and 150 g CWE included 0 mg, 280 mg, and 560 mg of total cholesterol, respectively. Cooked whole eggs were prepared uniformly from large eggs based on the American Egg Board’s recommendation  and portSeventeen women from the greater Lafayette, IN, region completed the study . InclusiFor this investigator-blinded, randomized, crossover-design study SAS 9.2 software  was used to randomize the trial orders and all subjects completed the two trials. Prior to each testing day, subjects consumed a prescribed low carotenoid and low-vitamin D diet for 7-day. On each of the two testing days, a catheter was placed into an antecubital vein and after a baseline blood sample was collected, subjects consumed a test meal. After the test meal was consumed, blood samples were collected hourly for 10 h and lunch was consumed after blood was collected at hour 5. Three-week dietary washout periods were scheduled between the 2 prescribed diet and testing periods since menstrual cycle phase may affect lipid and lipoprotein metabolism ,24.On each of the two testing days, subjects consumed sautéed vegetables (0 mg dietary cholesterol) including carrots, spinach, pepper, tomato salsa, and vitamin D enriched Portobello mushroom and 3 g canola oil with no egg or with 100 g CWE. The served sautéed vegetables with 3 g canola oil provided 92 kcal, 3 g protein, 12 g carbohydrate, 4 g fat, and 0 mg of cholesterol. The no egg and 100 g CWE included 0 mg and 373 mg of total dietary cholesterol, respectively. The low cholesterol lunch consumed at hour 5 provided 443 kcal, 15 g protein, 88 g carbohydrates, 3 g fat, and 14 mg of cholesterol. A registered dietitian developed all menus, as described above.g at 4 °C for 15 min. Serum tubes were sent to MidAmerica Clinical Laboratories  and lipid-lipoprotein profiles  cholesterol) were measured using photometric assays  and low density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation [Fasting state bloods were collected into serum separator tubes and tubes were held at room temperature for 30 min and centrifuged at 4000× equation .g, 15 min, 4 °C) to obtain plasma and aliquots of plasma were stored at −80 °C until thawed for analyses. Ten mL of fresh plasma were also processed to isolate the TRL fractions as previously reported [On each testing day, bloods were collected into EDTA tubes and portions of collected blood samples were centrifuged . Postprandial total cholesterol and triacylglycerol concentrations within the TRL fractions and plasma were baseline corrected by subtracting fasting concentrations from each time point. The 0–10 h positive incremental areas under the curve (AUC) of total cholesterol and triacylglycerol concentrations in TRL fractions were then calculated.Since the assessment of whole egg consumption on cholesterol absorption is a secondary objective of both studies, subject sample size estimates were not done based on this outcome of interest. Retrospectively, we conducted effect-size calculations before implementing the data analysis. For these within-subject, crossover-designed studies, an a priori power calculation was completed for two dependent means  to detect a difference equal to 1 SD between treatments . The effect size was one and the total number of participants needed for each study was estimated to be 13, which is less than the 16 subjects tested for Study 1 and 17 subjects tested for Study 2.0–10h of total cholesterol and triacylglycerol in TRLs and plasma. All of the analyses were performed using SAS 9.2  and data are presented as least-squares means (lsmeans) ± standard error (SE) of the lsmean unless otherwise noted. Statistical significance was accepted at p < 0.05 (2-tailed).Age and BMI adjusted repeated-measures ANOVA with post hoc Tukey’s test was performed to determine differences of baseline corrected total cholesterol and triacylglycerol concentrations at each time point. Age and BMI adjusted one-factor ANOVA with post hoc Tukey’s test was also applied to assess the differences in baseline-corrected positive incremental AUC2, respectively. Blood lipid and lipoprotein concentrations were 99 ± 14 (triacylglycerol), 171 ± 8 , 52 ± 3 (HDL cholesterol), and 100 ± 7 (LDL cholesterol) mg/dL.The mean ± SE age and BMI of the 16 men were 24 ± 1 year and 24 ± 1 kg/m2. Blood lipid and lipoprotein concentrations were as follows: triacylglycerol, 90 ± 12; total cholesterol, 183 ± 11; HDL cholesterol, 56 ± 3; and LDL cholesterol, 109 ± 10 mg/dL.Among the 17 women, mean ± SE age was 45 ± 4 year and BMI was 25 ± 2 kg/m0–10h within TRL fractions also were not different among 150 g CWE vs. 75 g CWE vs. no egg    . In cont = 0.10) . The tri 0.0006) .0–10h within TRL fractions were not different between 100 g CWE vs. no egg    . At hour = 0.30)  and the  0.0001) .0–10h were not different among 150 g CWE vs. 75 g CWE vs. no egg    . From ho = 0.83)  and the  0.0099) .0–10h were not different between 100 g CWE vs. no egg    . From ho = 0.66)  and the  0.0281) .Historically, consumers were discouraged from consuming whole egg due to the relatively high content of dietary cholesterol in egg yolk. Yet, previous studies showed no relation between the whole egg intake and blood cholesterol concentration ,27,28. SWhile fasting state lipid and lipoprotein concentrations mainly reflect body homeostasis, their post-prandial responses reflect the capacity to handle an acute dietary fat and cholesterol load . ExperimWhile results from both study 1 and study 2 indicate that consuming cholesterol-rich whole eggs did not acutely increase cholesterol absorption or plasma total cholesterol concentrations, higher triacylglycerol concentrations within the TRL fractions and plasma were observed. These results are consistent with research showing that higher dietary fat intake (0 g vs. 45 g) caused greater triacylglycerol within TRL fractions and plasma , and thaStrengths of this research include using data from two investigator-blinded, randomized, crossover, diet-controlled studies and assessing cholesterol and triacylglycerol absorption based on results from TRL fractions versus plasma. Although assessments of cholesterol and triacylglycerol responses in TRL fractions were secondary measurements, retrospective power calculations support both studies having adequate sample size. It is important to note that these results come from acute feeding trials and postprandial changes in total cholesterol content in TRL fractions includes both dietary and biliary cholesterol. Estimates indicate that biliary cholesterol contributes about 75–85% of intestinal cholesterol content . The expIn conclusion, results from these two randomized controlled acute feeding studies indicate that dietary cholesterol contained in whole egg is not well absorbed and does not increase plasma total cholesterol concentration. These findings provide a mechanism to help explain why dietary cholesterol intake may not affect long-term plasma total cholesterol control.


Mean total 24 h NTX excreted in MBPM was −10% (p = 0.035) lower than CON. Urinary PYD and DPD were unaffected by treatment. This study demonstrates the acute effects of bedtime ingestion of a calcium-fortified, milk-based protein matrix on bone remodeling.The diurnal rhythm of bone remodeling suggests nocturnal dietary intervention to be most effective. This study investigated the effect of bedtime ingestion of a calcium-fortified, milk-derived protein matrix (MBPM) or maltodextrin (CON) on acute (0–4 h) blood and 24-h urinary change in biomarkers of bone remodeling in postmenopausal women with osteopenia. In CON, participants received 804 ± 52 mg calcium, 8.2 ± 3.2 µg vitamin D and 1.3 ± 0.2 g/kg BM protein per day. MBPM increased calcium intake to 1679 ± 196 mg, vitamin D to 9.2 ± 3.1 µg and protein to 1.6 ± 0.2 g/kg BM. Serum C-terminal cross-linked telopeptide of type I collagen (CTX) and procollagen type 1 amino-terminal propeptide (P1NP), and urinary N-telopeptide cross-links of type I collagen (NTX), pyridinoline (PYD) and deoxypyridinoline (DPD) was measured. Analyzed by AUC and compared to CON, a −32% lower CTX ( Low bone mineral density  and a diminution in bone quality (bone microarchitecture) are attributes of risk of fracture in people with osteopenia . Bone arBone turnover markers (BTM) of bone resorption  and formation  inform of change in bone metabolism and bone health earlier, and with greater sensitivity than change in bone mineral density (BMD) . Defined1–42) and glucagon-like peptide-1 (GLP-17–36) acting via an entero-osseous axis [per diem. Few nutritional studies of bone metabolism time supplemental intervention to act at the nocturnal peak of bone remodeling [Bone remodeling is sensitive to modulation by feeding and nutrient specific intake. The response of bone remodeling to food ingestion is linked putatively to the secretion of enterogastric hormones glucose-dependent insulinotropic peptide . Participants were informed of the purpose of the study and all known risks before providing written, informed consent. The study was conducted in accordance with the ethical standards outlined in the most recent version of the Declaration of Helsinki and registered with clinicaltrials.gov identifier NCT03337971.n = 1; medication with Gabapentin™: n = 1). One participant was unable to attend during the intervention periodThe remaining 16 volunteers progressed to the RCT and randomly assigned to receive either a milk-based protein supplement (MBPM) or an isoenergetic, maltodextrin control (CON) over two consecutive days  Study database by invitation. ULBC subjects had preassigned alphanumeric ID codes that continued to the present study. Subject volunteers were free-living, lactose tolerant, and devoid of treatment for osteoporosis or drugs affecting calcium absorption. Dual energy X-ray absorptiometric  measurement of AP spine (L1–L4) and dual femur BMD , conducted and analyzed in accordance with the International Society for Clinical Densitometry , confirmive days . SupplemA block-randomized, within-subject crossover design examining acute (0–4 h) and 24 h change in biomarkers of bone turnover in healthy, postmenopausal women with osteopenia following ingestion of MBPM or CON was conducted. Estimated from a least significant change in bone resorption  statistical power (1-β) of 80% and two-sided α of 0.05 would be achieved by a sample size of 16 participants.™ Dietary Analysis Software (Nutritics© software version 4.0 for Ireland). Dietary intake was compared to reference intakes (DRIs) developed by the Food and Nutrition Board (FNB) at the Institute of Medicine (IOM) of The National Academies [Following instruction from a qualified dietitian, participants recorded their dietary intake for seven consecutive days  using a weighed food and fluid log. Participants reported food type, brand name, quantity, cooking method, type, time, and location of meal preparation. Subsequent coding and analysis was completed using Nutriticscademies .Based on the 7-day dietary intake data a 48 h dietary standardization, representative of the individual’s reported habitual dietary patterns of the research population, compliant with dietary recommendations (IOM DRIs) for energy, protein, and calcium, reflective of participants’ food preferences and normalized to body mass was prescribed for each participant by a registered dietitian. Each participant received the same meal plan on each of day of the intervention. Individuals’ standardized diet was supplemented with CON or MBPM. At bedtime, participants received a supplemental intake of MBPM or CON. The nutrient composition of MBPM and CON  compriseParticipants were resident at the University of Limerick during the consecutive 2-day, 2-night trial . Particig at 4 °C for 5 min and frozen at −80 °C until analysis. Sixteen of the 192 blood samples drawn were hemolyzed and not analyzed. This reduced the full analysis from 3 participants. Active plasma GLP-1(7-36) and total GIP (1-42) was measured using the MSD® metabolic assay kits  based on sandwich ELISA and according to the manufacturer’s instructions. The inter-assay CVs were 7.3 and 16.1 for active GLP-1 and total GIP, respectively. Plasma CTX and serum P1NP by 2-site immunometric assay using electrochemiluminescent detection . Plasma parathyroid hormone (PTH) measured using the human bone magnetic bead panel-1 kit  employing Luminex™ technology. The interassay CV was 5.3% for CTX, 4.5% for P1NP and <10% for PTH. 24 h urine collection commenced at 17:00 h on day 1, following full void, to 17:00 h of the 2nd day. An aliquot of urine was frozen at −20 °C until batch analysis for urinary free pyridinoline (fPYD) and deoxypyridinoline (fDPD) by LC-MS/MS [® C501, Roche, Burgess Hill, UK). Urinary assays were undertaken by the Bioanalytical Facility, University of East Anglia, Norwich, UK.Serum and plasma were separated by centrifugation at 10,000× t-test. Cohen’s effect size was calculated using the standard formula (t-score/√n). Statistical significance was set at p ≤ 0.05.Shapiro–Wilk test was applied to check for normality of the data that are presented as the mean ± SD unless stated otherwise. Temporal change in serially sampled data was analyzed by repeated measures ANOVA, the level of significance for post-hoc tests subject to Bonferroni correction. Difference between mean change attributed to CON and MBPM was analyzed by paired The baseline characteristics of the subject population are representative of postmenopausal women age 55 to 70 y . SubjectReported mean daily intake, from a 7-day food and fluid log  demonstrFeeding induced a differential response for GIP compared to GLP-1. The temporal change in GIP was to increase by 40% from 109 ± 32 pmol/L to a peak of 152 ± 37 pmol/L 60 min postingestion in CON and by 50% from 99 ± 30 pmol/L to a peak of 150 ± 39 pmol/L 60 min postingestion in MBPM. Thereafter, GIP decreased to 60 ± 17 pmol/L residing 45% lower than prefeeding 4 h postingestion. Analyzed by AUC, no difference in overall response between CON and MBPM was observed a.In contrast, GLP-1 decreased in a linear manner with time postfeeding in both trials. After 4h GLP-1 decreased by 38% from 65 ± 16 pmol/L prefeeding to 40 ± 11 pmol/L after 4 h postingestion in CON and by 30% from 72 ± 16 pmol/L to 51 ± 12 pmol/L 4 h postingestion in MBPM. No difference in overall response between CON and MBPB was observed b.p = 0.002, d = 1.08)  a.p = 0.011, d =0.83)  b.p = 0.52, d = 0.2)  c.p = 0.89), providing confidence that total urine collection was attained.Participants’ urine output varied from −750 to 1100 mL, resulting in a 4.1 ± 23% mean difference in total 24-h urine volumes between days. However, total 24-h urinary creatinine excretion was not significantly different between days , A large interindividual variation in 24 h urinary BTM excretion was observed . Mean ton = 9) of participants actually achieving this by food intake alone. A difference of 11% was observed in dietary calcium intake between our study sample and NANS data (MDI = 995 ± 573 mg/day) [The reported baseline dietary intakes of the present cohort of Irish women with osteopenia were comparable to results from the Irish National Adult Nutrition Survey (NANS) . The mea mg/day) . The habp = 0.12, d = 0.46) compared to CON. Though moderate in magnitude, the outcome of the nocturnal feed was a transient reduction in the nocturnal peak of bone remodeling.The principal outcome of this block randomized, within subject crossover trial in postmenopausal women with osteopenia was to effect a positive change in bone remodeling by nocturnal feeding. Timed to coincide with peak rate of bone remodeling MBPM effected an acute (4 h) reduction in the serum bone resorption marker CTX, a corresponding increase in the bone formation marker P1NP and a moderately greater reduction in the P1NP/CTX ratio  and 24 h urinary NTX were significantly correlated , whereas DPD  and PYD  were not. Earlier studies demonstrate the value of urinary cross-links to monitor the circadian rhythm of bone resorption [Exhibiting higher diurnal amplitude, the overall influence on circadian, 24-h bone resorption is informed by the measurement of 24 h urinary N-telopeptide cross-links of type I collagen (NTX), and associated cross-links pyridinoline (PYD) and deoxypyridinoline (DPD). A dilution of the acute (0–4 h), transient change in BTM response to nocturnal feeding within a 24 h cycle was expected, but only 24 h urinary NTX reflected the circulatory changes in BTM in the acute phase postfeeding. Furthermore, the associated response of CTX  . EvidencIn summary, the reduction in AUC of CTX (−30% compared to CON) confirms our initial hypothesis that a dairy-based protein supplement fortified with calcium (MBPM) fed at bedtime has a potent effect on nocturnal rates of bone resorption in healthy osteopenic postmenopausal women. The synergistic, pluripotent quality of a milk-based protein matrix and timing of ingestion to the nocturnal, peak rate of bone remodeling transiently depressed bone turnover. We conclude that a late-evening supplement of calcium-fortified milk protein affects a beneficial decrease in the homeostatic rate of bone remodeling in persons at risk of degenerative bone disease. A 24-w longitudinal study  is currently in progress to assess the efficacy of outcome in bone turnover and BMD in this population.


Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg , Gln , Glu (acidic), and Ala  through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid  maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid  impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile FeMoco citrate) . FeMoco residues . Homocitresidues .Major research progress has been made, particularly in regard to nitrogenase structure and the reduction of non-biological substrates . However2 production is obligately coupled with N2 fixation. N2 replaces H2 when it binds to reduced FeMoco, and the N2/H2 molar ratio is 1:1. In fact, H2 production is correlated with N2 partial pressure, and always exceeds N2 fixation amount , glutamine , glutamic , and alanine  separately. Comparison at cytological and enzymatic levels of characteristics of wild-type (WT) and mutant strains clarified the role of α-Lys424 in substrates reduction of nitrogenase.Here, we extend the 2014 study by investigating the function of α-Lys424 was analyzed and simulated by PyMOL 2.0.0 software program.MoFe protein crystal structure data were downloaded from RCSB Protein Data Bank . Amino acid structure of α-LysnifD gene fragment was cloned into the replication vector, pUC18, and the target bases were mutated by PCR. Suicide vector pGPCm containing mutational fragment was constructed and transferred into K. oxytoca M5al by bacterial conjugation and fused into K. oxytoca M5al genome by recombination. DNA sequencing was used to confirm the target mutation and avoid polar effect. Detailed methods were described in our previous work , kanamycin , chloramphenicol .e medium . SupplemKlebsiella oxytoca M5al and its mutant strains were activated on plates and grown overnight in nitrogenous liquid medium at 30°C. Cells were collected, centrifuged, and re-suspended in 60 mL nitrogen-free medium under N2 atmosphere, with initial OD600 ∼0.25. Every 4 h, OD600, acetylene and proton reduction activity were detected. OD600 was measured by a UV–visible spectrophotometer , acetylene and proton reduction activity was measured in a10-mL serum with a sealed stopper according to a previous report (2H4) and H2 were, respectively, analyzed by gas chromatography with flame ionization detector (GC-FID) and thermal conductivity detector (GC-TCD) .s report . Ethylen600 was adjusted to 0.4, and serum bottles were filled with Ar, then 10 mL Ar was removed, and 10 mL 15N2 gas was injected to induce nitrogenase expression and 15N2 fixation (negative control: no 15N2 injection). Cells were incubated at 30°C in water bath for12 h, and bacterial sludge was harvested and freeze-dried. 15N2 content (mg/g) was measured by Shenzhen Huake Isotope Testing Technology Co., Ltd .Cells were cultured in nitrogenous medium for 12 h, centrifuged, washed, and suspended in 120 mL nitrogen-free medium. ODgroES (coding chaperone), and target genes were nifA (coding positive regulator), nifL (coding negative regulator), and nifD (coding α-subunit of MoFe protein). Primers used for qPCR are shown in −ΔΔCt method, as reported by Samples were collected at three points during diazotrophic growth: the original (5 h), exponential (9 h), and maximal (13 h) activity points. Total RNA was extracted and revers-transcribed. Quantitative real-time PCR (qPCR) was performed using Light Cycler 480 with SYBR Green I Master kit . Positive control gene was K. oxytoca M5al were used as probes for Western blotting. Integrated optical density (IOD) of MoFe protein was measured using Image-Pro Plus 6.0 software program .Samples at exponential phase were suspended in lysis buffer and disrupted by ultrasonic. Supernatants were adjusted to equal total protein content, and proteins were separated by Tricine-SDS-PAGE. Antibodies against MoFe protein of 2 in bubbled solution. A major improvement over the previously described method was that 50% sucrose of separation gel was removed to reduce mutational MoFe protein diffusion in separation gel. Purified protein was measured by Tricine-SDS-PAGE and stained by Coomassie Blue, and the protein purity was assessed using BandScan 5.0 software program .WT or mutant strains were cultured, respectively, in a 42-L autofermenter . When nitrogen source for the first phase was depleted, mutant strains should be added with Arg⋅HCl (1 g/L) to induce nitrogenase expression . Acetylein vitro, and it was probably > 2:1 , 20 mM sodium dithionite, 40 mM creatine phosphate, and 20 U creatine phosphokinase. MoFe protein quantity was fixed as 20 μg and Fe protein content was increased gradually. The reaction was initiated by addition of 0.9 mL C2H2, and the C2H4 production was measured by GC-FID after mixing in a water bath for 30 min at 30°C.Molar ratio of Fe protein to MoFe protein was affected by the flow solution ly > 2:1 . It was 2H2 atmosphere with the appropriate ratio of MoFe and Fe protein that determined by the previous protein ratio calibration. The C2H4 production was measured by GC-FID. Proton reduction activity was assessed under 100% Ar atmosphere and measured by GC-TCD.The buffer used for nitrogenase activity detection was the same of that used in the calibration assay above. Acetylene reduction activity of nitrogenase was assessed under 90% Ar/10% C2H2 was set to a series  on the basis of preliminary experiments. C2H2 reduction activity was detected, and kinetic parameters were calculated according to Lineweaver-Burk plot . The data were analyzed using software program CDNN 2.1 .Characteristic three-dimensional structures of proteins  are asymmetric, resulting in an unequal propagation speeds of L and R polarized light beams, and consequent circular dichroism (CD). α-Lys4+, 6Fe2+, and Fe3+ of FeMoco give rise to paramagnetic signal. Electron paramagnetic resonance (EPR) signal intensity reflects the FeMoco content when protein concentrations are the same. MoFe protein was diluted to 3 mg/mL and reduced by sufficient sodium dithionite, degassed with high purity dinitrogen, and quantity 150 μL was placed in EPR tubes in the anaerobic glove box . Sample tubes were refrigerated sequentially in ice/water mixture (0°C) for 30 s, in drikold/ethanol mixture (−80°C) for 30 s, and finally in liquid nitrogen (−196°C). Low-temperature continuous-wave EPR spectra were recorded on an EPR spectrometer  equipped with an Oxford-900 liquid helium cryostat (temperature range 4–8 K)  .2, and 10 mM MgATP were placed in one reservoir syringe. Fe and MoFe protein were placed in the other reservoir syringe. When proteins come into contact with the buffer, [4Fe-4S] cluster of Fe protein valence state will change and the absorption value at 430 nm will increase. Absorbance increase was monitored by stopped-flow spectrometer . Experiments were repeated five times and the second to fourth were selected to analyze the results. Observed rate constant, kobs, for [4Fe-4S]1+→[4Fe-4S]2+ (F1+→F2+) step was carried out by Prism 6.0 .Electron transfer from Fe to MoFe protein is a necessary condition for nitrogenase reduction substrates. The difference of electron transfer rates between WT and mutant nitrogenase helps to determine whether the mutation site affects electron transfer ; 80 mM M424 and related elements were simulated by PyMOL software. The distance between NZ (ζ-N atom) of α-Lys424 and O3 of homocitrate was evaluated as 3.9 Å  or Gln (5.8 Å). Glu and Ala had no N atom on the branched chain; their OE1 (ε-O1 atom) and CB (β-C atom) were, respectively, 5.4 and 6.6 Å away from O3 of homocitrate, respectively.Structures of α-Lysas 3.9 Å . Mutatioimulated . The disnifV) deleted mutant which is used as a control strain; citrate is a substitute in culture. Therefore, we also provided the simulated structure of homocitrate and citrate in 2–) in difference.UNF837 is a homocitrate synthetase gene   . Relativ2⋅OD600−1⋅mL−1⋅h−1). 424R and 424Q had similar activity values compared with WT; while 424E, 424A, and UNF837 were lower than that of WT . 424A lost the ability of diazotrophic growth and acetylene reduction, but retained a very low proton reduction activity, presumably due to the action of hydrogenase in cells.Hydrogen emission is a characteristic of nitrogenase, and high hydrogen producing strains have great potential application value. Proton reduction activity of each cell was tested under 100% Ar and measured with GC-TCD. The results are shown in 15N2 incorporation assay  for WT was 19.2 mg/g. Corresponding values for 424R, 424Q, 424E, and UNF837 were all lower: 14.0, 10.0, 4.5, and 1.8 (mg/g); respectively, 73, 52, 24, and 13% of WT value ; it seemed that replacement by Arg had the least effect on basic properties of nitrogenase.Cytological properties of the four mutants differed from those of WT. Nitrogenase activity was altered by the replacement of a single residue  . Three nif genes were selected to measure. The products of nifA and nifL, respectively, activate and inhibit nif gene cluster transcription as a function of ammonium salt concentration in medium. nifD encodes α-subunit of MoFe protein, and it is the site of mutations. qPCR analysis showed that, at each of the three phases, all three nif genes transcription level in the mutant strains was 0.7–1.5 folds of WT  was measured by Western blotting, and content was analyzed by Image Pro Plus . IOD var4+ was depleted after 8 h culture. Maximal acetylene reduction activity values for WT, 424R, 424Q, and 424E were, respectively, 946, 1051, 588, and 279 (nmol C2H4⋅OD600−1⋅mL−1⋅h−1)  was added to induce nitrogenase expression when the initial NHL−1⋅h−1) , consistThe crushing of cells, collection of supernatant by centrifugation, and purification of MoFe and Fe proteins were performed under anaerobic conditions as established in our previous studies . MoFe anIt was important to ensure nitrogen protection during purification process. Purified proteins were tested by Tricine-SDS-PAGE. As shown in in vivo was 2:1. However, in vivo and in vitro environments were different and it was necessary to determine the in vitro molar ratio prior to analysis of nitrogenase properties. It should be noted that to avoid differences in the reaction process arising from different sources of Fe protein, all reactions were conducted using WT-Fe protein, and MoFe protein quantity was fixed as 20 μg. MoFe protein of the mutant strains and WT showed similar trends; acetylene reduction activity rose gradually as Fe protein content increased, and did not rise further after Fe protein content reached 80 μg. The result is shown in The molar ratio of Fe to MoFe protein 2H2, was 1493 nmol C2H4⋅mg−1⋅min−1 for WT. Values for 424R, 424Q, and 424E were 1559, 967, and 479 (nmol C2H4⋅mg−1⋅min−1), which were 104, 65, and 32% of WT, respectively   .2 production accompanies N2 reduction. The detection was determined under 100% Ar atmosphere. The results showed that the activity was very similar for WT and 424R: 1162 and 1124 (nmol H2⋅mg−1⋅min−1), respectively (2⋅mg−1⋅min−1), which were 50 and 20% of WT value.Proton reduction activity is one of fundamental properties of nitrogenase. Usually, Hectively . In cont424 is located in the α-helix (α423-431) of MoFe protein. To evaluate effect of mutation on the α-helix, secondary structure of MoFe protein was examined by CD spectroscopy. The results indicated that each of the three substituted MoFe proteins displayed the same characteristic α-helix peaks  as MoFe protein of WT. α-helix and β-sheet contents of all nitrogenases were in the range 47.2–47.4 and 14.3–15.0%  and complex (1994 amino acids), it is not surprising that replacement of a single residue did not greatly alter its integral structure. This is what we expected, that it is meaningful to judge the role of target amino acid according to the result of property analysis.α-Lys.3–15.0% . These r.3–15.0% . These i2 production of nitrogenase is generally correlated with reduction of FeMoco content in MoFe protein . Amino acid substitution in MoFe protein did not result in new EPR signal. g factor and line width were also unchanged. Our findings indicated that mutations of α-Lys424 did not alter FeMoco structure but did affect insertion of FeMoco in MoFe protein. Reduction of FeMoco content resulted in decreased proton reduction activity.FeMoco is the active center of nitrogenase, and its Mothe same . When g 1+→F2+). F2+ will be re-reduced to F1+ (F2+→F1+), and this process involves the dissociation of Fe and MoFe protein. As the process of re-reduction is complicated, we only consider the process F1+→F2+, and its observed rate constant, kobs, is calculated.When reduced Fe protein transfers an electron to resting state MoFe protein, the valence state of [4Fe-4S] cluster will go up from +1 to +2 . But absorbance of WT, 424R, 424Q, and 424E were 0.044, 0.035, 0.023, and 0.015. The depressed absorbance values of mutations demonstrated a decreased amount of electron transfer.As shown in 424 is located near homocitrate long arm. Homocitrate serves as a terminus of FeMoco. nifV− mutation, in which homocitrate was replaced by citrate resulted in significant decreases of acetylene, proton, and nitrogen reduction activity, indicating an important role of homocitrate in nitrogenase activity , and activity of 424Q is therefore lower than that of 424R. Gln and Glu are generally similar in structure, but the groups on Glu’s R-group have no N atom, and O atom forms a weaker hydrogen bond. The change of nitrogenase activity for 424E is more moderate than that of 424Q.FeMoco is situated deep below MoFe protein and surrounded by amino acid residues, supported by α-subunit mainly. The residues play varying roles in catalytic properties and orientation. α-Lysactivity . Nitrogeactivity . Fe2 andthe view . α-Lys19on sites . α-Lys42nifV− single mutant. They were not only replaced the homocitrate in MoFe protein, but also disrupted the microenvironment of active sites due to the replacement of amino acids. That was why they lost enzymatic properties more thoroughly.The four amino acids were also substituted in UNF837 strain. As shown in Our findings supplemented the structure/function relationship in nitrogenase. The amino acids surrounding FeMoco are precisely selected by evolution in nitrogen-fixing microorganisms.2 production is inconsistent, and percentages of cytological production are higher than those of enzymatic, indicating that there are other enzymes in K. oxytoca, for example hydrogenase, that can reduce the proton to H2. Enzymatic H2 production reflected ability of nitrogenase reduction proton. And the process is affected by numerous factors. For example, it is inhibited by N2 in WT and by carbon monoxide (CO) when the α-Gln190 residue is replaced by Lys , and this short distance results in a strong hydrogen bonding according to 424 and α-Ile423 jointly maintain the location of homocitrate in MoFe protein.The acid-base property of amino acid at α-424 site obviously affects the content of FeMoco in MoFe protein. Alkaline Lys and Arg do not affect FeMoco content, while neutral Gln or acidic Glu decrease FeMoco content to varying degrees. As 190 and α-His194 are involved in electron transfer from P-cluster to S2B of FeMoco. In this study, acetylene and proton reduction activities of mutants were correlated with the FeMoco content of MoFe proteins, but nitrogen fixation activity of the mutants reduced to a greater degree than acetylene reduction activity, presumably because of increased potential around FeMoco. Results of stopped-flow also provided indirect evidence for FeMoco potential increase, because for 424R the number of electrons FeMoco received decreased more obviously than WT when their electron transfer rate and FeMoco contents were the same. The lower the potential of a metal cluster, the stronger its reducibility. Amino acids interact with metal clusters and affect their potential  of homocitrate (2, because regardless of where the binding occurs, high potential of FeMoco will inhibit the nitrogen fixation activity.Electron transfer between the Fe and MoFe proteins is essential for nitrogenase activity . Amino aotential . α-Lys42ocitrate . DynamicE. coli, yeast, chloroplast, etc. (Researchers have been attempting for many decades to construct an efficient “nitrogen fixation plant”, but the dream has not yet been achieved. The major obstacle is the low activity observed when nitrogenase is expressed in st, etc. . The low424 to coordinate insertion of FeMoco to MoFe protein, in combination with certain other amino acids; and demonstrated its function in maintaining low potential of the microenvironment to guarantee efficient catalytic activity of nitrogenase.In conclusion, the results of the present study supplemented experimental evidence of alkaline α-LysJL, YL, and WJ conceived the project and designed the experiments. LS, PL, QG, WJ, and CZ performed the experiments and data analysis. JL, CZ, and AB provided useful suggestions and discussion. LS and YL wrote the manuscript. All authors read and approved the finalized 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.


Lactobacillus fermentum MBC2 was investigated through in vitro and in vivo approaches. Caenorhabditis elegans was used as an in vivo model to analyze pro-longevity and anti-aging effects. L. fermentum MBC2 showed a high gut colonization capability compared to E. coli OP50 (OP50) or L.rhamnosus GG (LGG). Moreover, analysis of pumping rate, lipofuscin accumulation, and body bending showed anti-aging effects in L. fermentum MBC2-fed worms. Studies on PEPT-1 mutants demonstrated that pept-1 gene was involved in the anti-aging processes mediated by this bacterial strain through DAF-16, whereas the oxidative stress protection was PEPT-1 independent. Moreover, analysis of acid tolerance, bile tolerance, and antibiotic susceptibility were evaluated. L. fermentum MBC2 exerted beneficial effects on nematode lifespan, influencing energy metabolism and oxidative stress resistance, resulted in being tolerant to acidic pH and able to adhere to Caco-2 cells. Overall, these findings provide new insight for application of this strain in the food industry as a newly isolated functional starter. Furthermore, these results will also shed light on C. elegans molecular players involved in host-microbe interactions.Lactic acid bacteria (LAB) are involved in several food fermentations and many of them provide strain-specific health benefits. Herein, the probiotic potential of the foodborne strain  Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) microorganisms mainly involved in various food fermentations ,2.Lactobacillus genus are commonly used as probiotics, and accepted as safe by the US Food and Drug Administration and the European Food Safety Authority. Among them, L. fermentum is a heterofermentative species belonging to the Firmicutes phylum  III), pept-1(lg601) and CF1038 daf-16(mu86) mutants used in experiments were propagated on nematode growth medium (NGM) modified to be peptone-free (mNGM) [L. fermentum. Afterward, 25 μL of each overnight culture, resuspended in M9 buffer, corresponding to 10 mg of bacterial cells, was spread on 3.5 cm diameter mNGM plates. Heat-killed lactobacilli cells were prepared as follows: bacteria were cultured overnight and resuspended in M9 medium as described above; cells were then incubated at 65 °C for 90 min and deposited onto mNGM agar plates. Heat-killed cells were also plated on MRS agar in parallel to ensure that no viable cells remained. OP50 cells were treated in the same manner.The wild-type e (mNGM) . Worms wSynchronized worms obtained as above were incubated at 16 °C on mNGM plates seeded with bacteria, allowing embryos laying. The number of progeny and body length analysis were recorded as described by Reference .For each experiment, 10 animals at L4 stage and at 5 days of adulthood were washed and lysed according to Reference . Whole wAbout 10 worms of each condition were analysed at the stage of 13 days adult, as described in Reference .Nematodes at L4 stage, grown as indicated above, were stained as described by Reference .−1 sodium azide. Lipofuscin autofluorescence was detected by fluorescence microscopy .The autofluorescence of intestinal lipofuscin was measured as an index of senescence at day 13 of adulthood. Randomly selected worms from the plates with bacterial lawn were washed three times with M9 buffer. Worms were then placed onto a 3% agar pad containing 20 mmol L−1) on 3% agarose pad on a glass slide and the fluorescence was viewed under a Zeiss Axiovert 25 microscope. The experiments were repeated three times and 15 worms per group were used in each experiment.Synchronized transgenic worms were transferred daily on mNGM plates containing different bacteria and incubated at 16 °C, as described above. At the stage of one or 13 days adult the worms were anesthetized with sodium azide  according to Reference [−1 H2DCF-DA  in methanol was added to the wells. H2DCF-DA is a membrane-permeable non-fluorescent probe, which can enter the cells of the worm and it is intracellularly converted to H2DCFs. This probe can be oxidized by ROS to yield the fluorescent dye DCF. The changes of fluorescence in worms indicate the accumulation of ROS at different time points . The measurement was performed using a multiplate reader  at excitation/emission wavelengths of 485 and 520 nm.ROS formation in eference  with somL. fermentum MBC2 strain was evaluated as described in Reference [Tolerance to gastrointestinal conditions of eference .L. fermentum MBC2 or LGG (OD600 = 1.3) were spread onto MRS agar plates, in which the antibiotic discs were placed. The plates were then incubated under anaerobic conditions for 24 h at 37 °C. The zones of inhibition were measured from the center of the disc and recorded.The antibiotic discs used in the susceptibility test were purchased from Biolab Zrt. . Each disc of 6 mm diameter contained the following antibiotic at the amount reported in parenthesis: amikacin (30 µg), aztreonam (30 µg), vancomycin (30 µg), streptomycin (25 µg), erythromycin (15 µg), tetracycline (30 µg), cefalotin (30 µg), gentamicin (10 µg), cefotaxime (30 µg), chloramphenicol (30 µg), clindamycin (30 µg), penicillin G (10 µg), ampicillin (10 µg), tobramycin (10 µg), cefuroxime (30 µg), oxacillin (1 µg), fosfomycin (50 µg), rifampicin (30 µg), carbenicillin (100 µg), and mezlocillin (75 µg). 100 μL of overnight cultures of L. fermentum MBC2 and LGG were diluted 1:100 in MRS broth and grown for 5 h to the exponential growth phase. After monitoring the OD600, appropriate amounts of bacterial cells were harvested by centrifugation at 5000× g for 10 min, resuspended in antibiotic- and serum-free DMEM and added to cell monolayers at a concentration of 1 × 108 or 1 × 109 CFU/well. Co-cultures of bacteria and Caco-2 cells were incubated at 37 °C for 1.5 h. Non-adhering bacteria were then removed by 5 washes with Hanks’ Balanced Salt solution  and cell monolayers were lysed with 1% Triton-X-100, according to Reference [The human intestinal Caco-2/TC7 cell line was provided by Monique Rousset  and routinely maintained as described in Reference . For theeference . AdherinC. elegans, the statistical significance was determined by Student’s t test or one-way ANOVA analysis coupled with a Bonferroni post test . For in vitro experiments, statistical significance was evaluated by one-way ANOVA, followed by post-hoc Tukey honestly significant difference (HSD) test. Statistical univariate analysis was performed with the “Statistica” software package . Differences with p values < 0.05 were considered significant and were indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.All experiments were performed at least in triplicate. Data are presented as mean ± SD. Prior to analysis, normal distribution and homogeneity of variance of all variables were assumed with Shapiro–Wilk and Levene’s tests, respectively. For in vivo experiments in L. fermentum MBC2 was previously isolated as a predominant member of the MBC microbiota, which was shown to protect mice from obesity-associated inflammation [ammation .C. elegans physiology, the survival rate was first examined. The median lifespan of wild type nematodes, fed L. fermentum MBC2 from embryo hatching, resulted significantly extended as compared to the LGG and OP50-fed controls , characterised by low pH (2.5), exerted a mild reduction of bacterial counts with respect to the initial time point, suggesting that this strain was able to endure acidic environments. However, subsequent treatment of the surviving bacterial cells in simulated pancreatic juice (SPJ) containing bile salts and pancreatin, severely affected bacterial survival, as revealed by the absence of L. fermentum MBC2 colonies recovered after 2 h of incubation  and to detect the presence of the corresponding resistance genes. It must be pointed out that the possibility of horizontal gene transfer to pathogens within the host gut should be carefully verified, to exclude associated risks for health. On the other hand, the presence of intrinsic antibiotic resistance could not represent a safety issue in itself, in light of the possible application of resistant probiotic strains for restoring the normal gut microbiota balance following antibiotic treatment [The ithelium . Adhesioithelium . An impoithelium . Overallreatment .L. fermentum MBC2 isolated from a foodborne microbiota influenced nematode physiology. In particular, the strain positively impacted on C. elegans longevity and influenced energy metabolism as well as oxidative stress resistance. The molecular players involved resulted in being dependent on the PEPT-1 transporter and on the transcriptional factor DAF-16, highlighting their possible involvement in host-microbe interactions. Moreover, L. fermentum MBC2 resulted in being tolerant to acidic pH and able to adhere to intestinal Caco-2 cells. Taken together, these findings provide new insight for a possible application of this strain in the food industry as a novel functional starter.In the present work, administration of


Streptococcus pneumoniae colonizes the upper airways of children and the elderly. Colonization progresses to persistent carriage when S. pneumoniae forms biofilms, a feature required for the development of pneumococcal disease. Nasopharyngeal biofilms are structured with a matrix that includes extracellular DNA (eDNA), which is sourced from the same pneumococci and other bacteria. This eDNA also allows pneumococci to acquire new traits, including antibiotic resistance genes. In this study, we investigated the efficacy of lactoferrin (LF), at physiological concentrations found in secretions with bactericidal activity , in eradicating pneumococcal biofilms from human respiratory cells. The efficacy of synthetic LF-derived peptides was also assessed. We first demonstrated that LF inhibited colonization of S. pneumoniae on human respiratory cells without affecting the viability of planktonic bacteria. LF-derived peptides were, however, bactericidal for planktonic pneumococci but they did not affect viability of pre-formed biofilms. In contrast, LF (40 and 80 μM) eradicated pneumococcal biofilms that had been pre-formed on abiotic surfaces  and on human pharyngeal cells, as investigated by viable counts and confocal microscopy. LF also eradicated biofilms formed by S. pneumoniae strains with resistance to multiple antibiotics. We investigated whether treatment with LF would affect the biofilm structure by analyzing eDNA. Surprisingly, in pneumococcal biofilms treated with LF, the eDNA was absent in comparison to the untreated control (∼10 μg/ml) or those treated with LF-derived peptides. EMSA assays showed that LF binds S. pneumoniae DNA and a time-course study of DNA decay demonstrated that the DNA is degraded when bound by LF. This LF-associated DNase activity inhibited acquisition of antibiotic resistance genes in both in vitro transformation assays and in a life-like bioreactor system. In conclusion, we demonstrated that LF eradicates pneumococcal-colonizing biofilms at a concentration safe for humans and identified a LF-associated DNAse activity that inhibited the acquisition of resistance. Streptococcus pneumoniae is a Gram positive bacterium that resides in the children nasopharynx principally. These bacteria reside asymptomatically in the host, but may become invasive, resulting in pneumococcal disease  and the elderly (>65 years old). However, it can lead to a variety of other pathologies, such as otitis media, meningitis, or septicemia  plays a critical role via comC and the luxS system  and tears (25 μM) and LF-derivative peptides against pneumococcal biofilms. The current study demonstrated a remarkable antimicrobial activity of bLF against pneumococci colonizing human respiratory cells, including antimicrobial activity against antibiotic resistant strains at a concentration safe for humans of <80 μM. We also described a non-previously recognized DNAse activity of bLF.Streptococcus pneumoniae strains utilized in this study are shown in Bovine LF (bLF), approximately 20% iron saturated, was purchased from Abial Biotech (Spain). LPS contamination and iron concentration were previously evaluated . Synthet2 atmosphere until about 85–100% confluency for future use in experiments.HEp-2 (ATCC CCL-23) human laryngeal cells, A549 (ATCC CC-L185) human lung cells, and Detroit 562 (ATCC CCL-138) human nasopharyngeal cells were used in this study, the cells were thawed and resuspended in aseptic conditions in a T-25 culture flask  in EMEM (Detroit and HEp-2) and F-12K (A-549) media supplemented with 10% fetal bovine serum, antibiotics 1% . They were incubated at 37°C in a 5% COS. pneumoniae grown on blood agar plates (BAP) was used to prepare a bacterial suspension in THY broth. This suspension was incubated at 37°C, 5% CO2 until the culture reached an OD600 of ∼0.2 (early log phase). Glycerol was then added to give a final 10% (v/v) and stored at −80°C until used.Inoculums were prepared as previously described . An over4 CFU/ml of S. pneumoniae strain D39 was inoculated in 24-well plates containing A549 cells (1 × 104 confluence) and treated with the following: 40 μM bLF, 10 μM LFcin17-30, LFampin265-284, or LFchimera, and 20 μM erythromycin for 4 or 6 h in F-12K medium without antibiotics and fetal bovine serum, then it was incubated at 37°C in 5% CO2. Supernatants were taken to determinate viability of planktonic bacteria by serial dilution and CFU/ml and the density of pneumococcal biofilm was quantified as described earlier (7.5 × 10 earlier . Briefly4 CFU/ml of S. pneumoniae (strains with or without antibiotic resistance) were inoculated (1) onto tissue culture-treated, polystyrene, 24-well plates (Corning) and (2) HEp-2 cells (ATCC CCL-23) with a confluency of 1 × 104 of cells per well that were subsequently fixed with 2% paraformaldehyde (Sigma) during 15 min. To form biofilms, samples were incubated during 4 or 8 h, respectively at 37°C in a 5% CO2 atmosphere. After that, planktonic cells were removed and fresh THY or cell culture medium was added. Except in the biofilm control wells, 20, 40, or 80 μM bLF, 10 μM LFamp265-285, LFcin17-30 or LFchimera were added and incubated for 6 or 12 h at 37°C with 5% CO2. Finally, biofilms were washed and prepared for quantification as was previously mentioned.∼7 × 10S. pneumoniae, biofilms destruction assays were done. S. pneumoniae TIGR4 (∼7 × 104 CFU/ml) were inoculated onto Detroit 562 cells (ATCC CCL-138) grown to 85–100% confluency. To form biofilms, bacterial suspensions were incubated for 4 h at 37°C in a 5% CO2 atmosphere. After that, planktonic cells were removed and DMEM medium was added. Once the 4 h-biofilm was grown, 40 and 80 μM bLF was added and was incubated for 6 h at 37°C with 5% CO2. Following incubation, supernatants were discarded and biofilms were washed and fixed with 2% paraformaldehyde for 15 min at room temperature (RT). Biofilms were then washed two times with 1X PBS and cells were blocked with 2% BSA for 1 h at RT. For identification of the mammalian cells, sialic acid was stained with 2.5 μg/ml Wheat Germ agglutinin (WGA) conjugated with Alexa flour  for 15 min at RT (S. pneumoniae (40 μg/ml) for 1 h at RT  was inoculated onto (1) tissue culture-treated polystyrene 24-well plates  or (2) onto 1 × 104 HEp-2 cells (ATCC CCL-23) that had been fixed with 2% paraformaldehyde  and extensively washed to remove the PFA. Inoculated plates, or infected cells, were incubated during 4 h or 8 h, at 37°C with 5% CO2 to form biofilms. Treatment with bLF (40 and 80 μM) or bLFpeptides (10 μM) was subsequently added as was indicated before. The supernatants were then collected and centrifuged for 10 min at 12,000 × g in a refrigerated centrifuge  and filtered by using a 0.2 μM syringe filter. They were subsequently mixed with a 0.5 volume of ethanol and vortexed for 10 s. The DNA from the supernatants was then purified by using the QIAamp DNA minikit  following the manufacturer’s instructions. To quantify amounts of extracellular DNA, quantitative PCR (qPCR) assay, targeting the lytA gene was utilized , 300 nM of each primer and 4 ng/μl of DNA template. Reactions were run in duplicate using a CFX96 Real-Time PCR Detection System  with the following conditions: 1 cycle at 55°C for 3 min, 1 cycle at 95°C for 2 min, 40 cycles of 95°C for 15 s, 55°C for 1 min, and 72°C for 1 min. Melting curves were generated utilizing a cycle of 95°C for 1 min, 65°C for 1 min, and 80 cycles starting at 65°C with 0.5°C increments. For quantification purposes, standards containing 1 × 103, 1 × 102, 1 × 101, 1 × 100, 1 × 10–1, 5 × 10–2, or 1 × 10–3 pg of S. pneumoniae DNA were run in parallel to generate a standard curve and amounts of eDNA were calculated by using the software Bio-Rad CFX manager .utilized . ReactioStreptococcus pneumoniae D39 (∼7 × 104 CFU/ml) was inoculated into THY medium and incubated for 2.5 h at 37°C with 5% CO2. The suspension was centrifuged for 10 min at 12,000 × g in a refrigerated centrifuged. DNA was purified by using the Wizard Genomic DNA Purification Kit  following the manufacturer’s instructions. To analyze the interactions between bLF and DNA, both bLF and albumin proteins were used and evaluated with the Electrophoretic Mobility-Shift Assay (EMSA) kit  following the manufacturer’s instructions. To visualize the DNA-protein interaction, the samples were separated in a 2% agarose gel and dyed with gel red . Gels were imaged using the Kodak model E1 logia 100 imaging system.DNA purified as above was incubated with 40 μM bLF or 40 μM albumin for 80 min at 37°C. To measure DNA decay, DNA concentrations were monitored every 10 min using the Nanodrop 2000 spectrophotometer .S. pneumoniae, we performed the classic transformation assay. The experiment was carried out in S. pneumoniae D39 competent cells using standard procedures. To induce transformation and test the effect of bLF on S. pneumoniae’s ability to transform, a positive control consisting of competent cells, 100 ng of competence-stimulating peptide 1 (CSP1 [EMRLSKFFRDFILQRKK]), 100 ng/mL of DNA purified from strain SPJV22 encoding resistance to Str (200 μg/ml), in complete transformation media was mixed. To test bLF activity against the transformation process, 40 and 80 μM bLF were added to the same reaction as that of the positive control. The negative control lacked CSP1 to prevent transformation. Every sample had a final volume of 200 μl  + Str (200 μg/ml) BAP. These plates were incubated at 37°C with 5% CO2 overnight. To calculate rF, bacteria growth in obtained from BAP with str and tet was divided between bacteria growth in Tet. Recombination frequency was calculated by dividing the number of Tet + Str-resistant recombinants by the total population obtained on blood agar plates without antibiotics.To evaluate the effect of bLF on recombination frequency (rF), we followed the methodology established by t-test using the SigmaPlot software version 12.0 .All experiments were performed in triplicate and analyzed by ANOVA test and student S. pneumoniae strain D39 and treated with bLF, or LF-derived peptides. As a control, some A549-infected cells were treated with erythromycin. Infected and treated human lung cells were incubated for 4 or 6 h after which the viability of planktonic cells or colonizing biofilms was evaluated by dilution and platting. Experiments demonstrated that the density on planktonic pneumococci was not affected by the treatment with bLF while LF-derivative peptides, and erythromycin, eradicated planktonic bacteria  challenged with 20, 40, and 80 μM bLF. In all experiments, a statistically significant reduction of eDNA was observed in biofilms treated with bLF (3-fold reduced) after the treatment with 40 μM of bLF, eDNA of naturally resistant pneumococcal strains was 10-fold reduced, but not completely degraded, indicating that those strains might have evolved a mechanism to partially inactivate the DNAse activity of bLF.Degradation of eDNA was also evaluated in biofilms formed by antibiotic resistance strains  in this life-like environment. In bioreactor chambers containing human pharyngeal cells and inoculated with strains D39obtained . Bioreacin vitro models to mimic two important biological events leading to persistence of pneumococcus in the human airways , to evaluate the effect of physiologically relevant concentrations of LF and LF-derivative peptides on biofilm persistence. We first demonstrated that bLF, and LF-derivative peptides, inhibited initial colonization events on human respiratory cells. The mechanism(s) that blocked colonization was different for bLF compared to that of LF-derivative peptides. For the latter, inhibition of colonization was caused by the antibacterial activity of LF-derivative peptides against planktonic pneumococci. The mechanism utilized by bLF to inhibit pneumococcal colonization was different since bLF presented low antibacterial activity against planktonic bacteria. In line with our experiments, an effect for LF against colonization by other organisms such as Pseudomonas aeruginosa, Streptococcus mutans, and Candida albicans has been demonstrated  was similar to the concentrator of LF found in colostrum (100 μM) and tears (25 μM) and therefore it is safe for humans. Both colostrum and tears protect the human host from bacterial infections, in part because of the antimicrobial activity of LF.P. aeruginosa biofilms and reduced 1 log reduction in viability, but in their experiments a combination LF and xylitol reduced ∼3 unit/logarithmic  and therefore supports a potential therapeutic and/or prophylactic application for LF.It has been reported that LF has anti-biofilm properties when mixed with other molecules. For example, Ammons and colleagues (2009) demonstrated that LF penetrates arithmic . Sheffiebiofilms  whereas nstrated . These sS. pneumoniae biofilms? There are a number of possibilities. For example, given that LF chelates iron and although the metabolism in biofilms decreases these quiescent bacteria still may require iron to maintain its structure and/or vital activities. Another possibility is its capacity to interact with negative components of bacteria such as LPS  has ∼72% aminoacids homology with bLF , it is likely that hLF is also able to degrade DNA. We in the field have speculated for a number of years now that the future of therapeutics would include targeting important components of the pneumococcal transformation machinery including the competence stimulating peptide (CSP) and eDNA (Our studies identified an activity of bLF that had not previously been recognized, DNAse activity . The amiand eDNA . WhereasAll datasets generated for this study are included in the manuscript/supplementary files.UA-Z, JV, and NL-S conceived and designed the study. UA-Z, KN, JB, CL-S, and BA collected the data. UA-Z, AC-R, JV, and NL-S analyzed and interpreted the data. UA-Z and BA drafted the manuscript. AC-R, JV, and NL-S critically revised the manuscript. UA-Z, AC-R, KN, JB, CL-S, BA, JV, and NL-S approved the final version of the 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.


Vestibular Migraine (VM) and Meniere's Disease (MD) are episodic vestibular syndromes defined by a set of associated symptoms such as tinnitus, hearing loss or migraine features during the attacks. Both conditions may show symptom overlap and there is no biological marker to distinguish them. Two subgroups of MD patients have been reported, according to their IL-1β profile. Therefore, considering the clinical similarity between VM and MD, we aimed to investigate the cytokine profile of MD and VM as a means to distinguish these patients. We have also carried out gene expression microarrays and measured the levels of 14 cytokines and 11 chemokines in 129 MD patients, 82 VM patients, and 66 healthy controls. Gene expression profile in peripheral blood mononuclear cells (PBMC) showed significant differences in MD patients with high and low basal levels of IL- 1β and VM patients. MD patients with high basal levels of IL- 1β (MDH) had overall higher levels of cytokines/chemokines when compared to the other subsets. CCL4 levels were significantly different between MDH, MD with low basal levels of IL- 1β (MDL), VM and controls. Logistic regression identified IL- 1β, CCL3, CCL22, and CXCL1 levels as capable of differentiating VM patients from MD patients (area under the curve = 0.995), suggesting a high diagnostic value in patients with symptoms overlap. Meniere's Disease (MD) is a syndrome characterized by attacks of recurrent vertigo associated with ipsilateral cochlear symptoms, such as fluctuating sensorineural hearing loss (SNHL), tinnitus or aural pressure , 2.Vestibular Migraine (VM) is defined by the occurrence of episodic vestibular symptoms and a history of migraine, with a temporal association in at least 50% of the attacks . VM has It has been described that 8.4% of MD patients have headache compatible with migraine, during vertigo attacks . The majVM patients have a favorable response to anti-migraine drugs, which supports an underlying migraine mechanism. However, this evidence is insufficient as it is based on uncontrolled clinical case series, thus the apparent efficacy may be due to confounding factors, such as placebo response, spontaneous improvement and multiple drug effect . MoreoveRecently, the Meniere's disease Consortium identified five clinical subgroups in patients with unilateral and bilateral MD: Group 1 was defined by SNHL starting first in one ear and involving the second ear in the next months or years; Group 2 characterized by simultaneous hearing loss in both ears since the onset of the disease; Group 3 clustered familial MD; Group 4 associated with migraine in all cases and Group 5 was found in patients with an autoimmune disease in addition to MD , 11. AddConsidering that the cytokine profile may allow to subgroup MD patients and the clinical similarity of MD and VM, we propose to investigate if the pro-inflammatory signature of these diseases may allow to distinguish these patients.This study included a total of 129 patients with definite MD, 82 patients with VM, and 66 healthy controls. Patients were diagnosed according to the diagnostic criteria of the Barany Society for MD . The exp6 cells/mL in 6-well plates. PBMC were incubated during 16 h at 37°C in 7% CO2. After the incubation, PBMC were centrifuged, RNA was harvested, and supernatants were collected and stored at −80°C.Peripheral blood was diluted 1:1 with 1 × PBS and disposed carefully onto the corresponding 25:15 volume of Lymphosep, Lymphocyte Separation Media . Samples were centrifuged for 20 min at 2,000 rpm to separate blood content. PBMC were collected and washed with 1 × PBS and cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum  and plated at 1.25 × 10RNA was isolated using the High Pure RNA Isolation Kit  following the manufacturer's protocol. RNA concentration was measured on Nanodrop . RNA quality was checked using Agilent 2100 Bioanalyzer .Expression levels were measured using the HumanHT-12 v4 Expression BeadChip  with 500 ng of total RNA and processed with the high-resolution scanner iScan .The number of biological replicates for each condition was: 5 healthy controls, 4 MD patients with low cytokine basal levels, 3 MD patients with high cytokine basal levels and 6 VM patients.p-value 0.05 cut-offs were used.Expression data analysis, normalization, differential expression analysis and Principal Component Analysis (PCA) was carried out as previously described by Frejo et al. . For paihttp://www.ingenuity.com/products/ipa) software, using the differentially expressed genes (DEG) with an adjusted p-value cut-off of 0.02.Core analysis was performed using Ingenuity Pathways Analysis , and 11 chemokines  were measured using the commercially available Multiplex Bead-Based Kits . The measurements were done in accordance with the kit-specific protocols provided by Millipore, using a Luminex 200  and read with Luminex x PONENT 3.1 software (Luminex Corp.). The minimum detection limit for the assays can be found in T-test and Krustal-Wallis H test. Qualitative variables were compared using Pearson Chi-square Test and Krustal-Wallis H test. The level of significance considered was p-value < 0.05.A descriptive analysis was conducted using SPSS software v.22  for the clinical data and displayed as mean ± standard error for the mean (SEM). Quantitative variables were compared using Mann-Whitney U test, Student's unpaired Representative heatmaps of the cytokine levels were done using the gplots package for R.p-values, until we obtained a model for which all cytokines were significant. The coefficient of determination R2, which summarizes the proportion of variance in the dependent variable associated with the predictor variables, was estimated by using Nagelkerke's R2. A receiver operating characteristic (ROC) curve was generated to determine the ability to predict VM based on a model composed of cytokine production. Area under the curves (AUC) was calculated for the ROC curves.We identified cytokines with differential production between MD and VM using Mann-Whitney test. Next, we performed logistic regressions, removing step-by-step the variables which had higher p = 1.20 × 10−5) and had an earlier onset of the disease (p = 4.41 × 10−4) than MD patients. As expected, MD patients had worse hearing (p = 5.32 × 10−22), higher number of vertigo attacks (p = 2.87 × 10−3) and were more functionally affected by the disease (p = 1.44 × 10−11), when compared to VM patients. A significant difference was found in the prevalence autoimmune disease between VM and MD, namely MDL had the highest history of autoimmune disease (p = 9.77 × 10−3). On the other hand, VM patients suffered more from headaches (p = 4.43 × 10−16), and more specifically migraine episodes (p = 1.89 × 10−26) than MD patients. We also observed a higher number of drop attacks in patients with MDH .We compared gene expression profiles of PBMCs from 6 VM patients, 7 MD patients [4 with low basal levels of IL-1β and 3 with high basal levels of IL-1β ] and 5 hp-value = 0.05, the samples were clustered into 4 distinct groups by Principal Component Analysis (PCA), which corresponded to our 3 patient groups and healthy controls, as seen in Using 1,894 genes, with 2 fold-change (FC) and an adjusted p-value < 0.02 and FC>2; CACNA2D2 . The core analysis with IPA software gave us a list of 25 networks. The top ranked network had a score of 37 and 30 focus molecules  high cytokine production in all groups, (b) cytokines which are higher in MDH than in the other groups and (c) cytokines that are higher in patients than in controls, as observed in Also, we can observe that the cytokine levels of VM patients are very similar to MDL patients and that controls always have lower cytokine levels than the patients.p > 0.1)  . On the p = [2.12 × 10−4−3.01 × 10−2]) and CXCL10 (p = [2.18 × 10−4-2.10 × 10−2]), for which VM patients hold the highest levels.Overall, MDH patients have higher levels of all cytokines, except for CCL22 (p = [6.30 × 10−5-1.04 × 10−2]). Moreover, CXCL10 allows to distinguish most groups p = [2.18 × 10−4-3.84 × 10−2]), however it does not discriminate MDH from MDL (p = 0.354).We observed that CCL4 allows to distinguish between all groups of patients and between patients and controls , CXCL1 , and IL-8  are significantly higher in PBMC from VM patients than in MDL patients.Despite the high similarity of cytokine levels of VM patients and MDL patients, the levels of IL-10  and have approximately a decade earlier onset than MD patients, (p = 1.20 × 10−5) and MD patients have more severe hearing loss (p = 5.32 × 10−22). Nevertheless, there is a symptom overlap which could be especially difficult to discriminate in earlier stages of the disease. Interestingly, MD type 4 patients, which are sporadic MD patients that suffer from migraine have been described to have a significantly earlier onset than the remaining MD patients  has an essential role in the recruitment and activation of neutrophils, mediating its function through CXCR2 signaling on neutrophils and by binding to glycosaminoglycans (GAG) on endothelial and epithelial cells and the extracellular matrix . CXCL1 iCCL3 or MIP1- α  is a macrophage secreted chemokine with inflammatory and chemokinetic properties, with CCR1, CCR4 and CCR5 binding. Recently, ES fibroblast cell lines, derived from MD patients have been established by Yamada et al.  and theyCCL22 or MDC (Macrophage-derived chemokine) is a chemoattractant for monocytes, dendritic cells and natural killer cells and it binds to CCR4. IL-1β is a potent inflammatory mediator and is associated with various cellular mechanisms, such as cell activation, proliferation, and apoptosis .The expression of CXCL1, CCL3, CCL22, and IL- 1β has been observed in mouse cochlear tissue , and CXCConsidering that we observed a cytokine increase in PBMCs extracted from peripheral blood, this could indicate that patients with MD and VM have a systemic proinflammatory response, which could explain some patients response to anti-inflammatory drugs , 27. On Some studies have quantified cytokine levels in serum from migraine patients during the attack and non-attack periods. Fidan et al.  observedAutoinflammatory diseases are caused by a hyperactive inflammatory response, that leads to an immune deregulation, driven by IL-1, type I interferon, and NFκB . These dIn this study we were able to find a method that could allow a more accurate differential diagnosis between VM and MD. Since the cohort of patients for which we could measure all cytokines was limited (VM = 16 and MD = 48), these results should be replicated in a larger cohort of patients with VM, MD, and Migraine, also considering clinical variables to better characterize MD endophenotypes.Patients with MD or VM have a different pro-inflammatory signature. A cytokine panel including IL- 1β, CCL3, CCL22, and CXCL1 could be used as biological markers for the differential diagnosis of VM and MD.GSE109558: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109558.The datasets analyzed for this study can be found in the Gene Expression Omnibus with the accession number This study was carried out in accordance with the recommendations of the Declaration of Helsinki with written informed consent from all subjects. The protocol number PI17/1644 was approved by the Granada Ethical Review Board on the 29/01/18.MF and LF performed experimental work including, PBMC isolation and culture, RNA extraction and cytokine measurement. MF, LF, AG-M performed statistical and bioinformatic analysis. EM-S, MR-I, JA-D, AS-V, SS-P, AB-C, JE-S, PP-C, MM-M, IA and JL-E recruited patients and obtained informed consent from all individuals. JL-E designed the study. JL-E supervised all experiments and MF and JL-E drafted the manuscript. All authors revised and approved the final version of the manuscript.Cytokine/chemokine panel for the differential diagnosis of episodic vestibular syndrome. Patent P201930255, March 20, 2019, owned by the Servicio Andaluz de Salud . 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.