Earlier studies have shown that skin autofluorescence measured with an AGE reader estimates the accumulation of AGEs in the skin, which increases with ageing and is associated with the metabolic syndrome and type 2 diabetes. In the present study, we examined whether the measurement of skin autofluorescence can predict 4 year risk of incident type 2 diabetes, cardiovascular disease (CVD) and mortality in the general population.1c ≥48 mmol/mol (≥6.5%) at follow-up. Participants were diagnosed as having incident CVD  by self-report. Mortality was ascertained using the Municipal Personal Records Database.For this prospective analysis, we included 72,880 participants of the Dutch Lifelines Cohort Study, who underwent baseline investigations between 2007 and 2013, had validated baseline skin autofluorescence values available and were not known to have diabetes or CVD. Individuals were diagnosed with incident type 2 diabetes by self-report or by a fasting blood glucose ≥7.0 mmol/l or HbAp < 0.001), compared with individuals who survived and remained free of the two diseases. Skin autofluorescence predicted the development of type 2 diabetes, CVD and mortality, independent of several traditional risk factors, such as the metabolic syndrome, glucose and HbA1c.After a median follow-up of 4 years (range 0.5–10 years), 1056 participants (1.4%) had developed type 2 diabetes, 1258 individuals (1.7%) were diagnosed with CVD, while 928 (1.3%) had died. Baseline skin autofluorescence was elevated in participants with incident type 2 diabetes and/or CVD and in those who had died  contains peer-reviewed but unedited supplementary material, which is available to authorised users. The worldwide prevalence of type 2 diabetes is increasing rapidly; it is predicted to be close to 650 million in 2040. Cardiovascular complications are the main drivers of increased morbidity and premature mortality in diabetes –3. SeverIn the last two decades, the role of AGEs in ageing and the pathophysiology of diabetes-related complications has been studied extensively. AGEs are formed in a multistep process by glycation and oxidation of free amino groups of proteins, lipids and nucleic acids. In addition to the classic Maillard reaction, AGEs are formed through the reaction of amino groups with α-dicarbonyls, such as 3-deoxyglucosone, methylglyoxal and glyoxal –10. AGEsThe accumulation of AGEs can be assessed non-invasively by measuring skin autofluorescence (SAF) . This meLong-term prospective studies on the value of SAF to predict development of type 2 diabetes, CVD and mortality in the general population are lacking. However, SAF was associated with increased mortality and cardiovascular events in specific groups and, for example, predicted amputations in individuals with peripheral artery disease , 26.Because of the promise of SAF as a valuable biomarker, the goal of this study was to assess whether SAF was able to predict the development of type 2 diabetes, CVD and mortality in the general population. For this, we performed an extensive prospective follow-up study of individuals participating in the Dutch Lifelines Cohort Study.Participants from the Lifelines Cohort Study, a large population-based study in the northern region of the Netherlands n = 1861) and/or type 1 diabetes (n = 177) and/or type 2 diabetes (n = 2557) or reported a history of gestational diabetes (n = 134) or MODY (n = 4). Furthermore, individuals with new type 1 diabetes (n = 12) and a new history of gestational diabetes (n = 55) at follow-up were excluded, as well as those without documented follow-up (n = 5530). This resulted in 72,880 individuals available for analyses  and 56,086 (77%) participants, respectively.For the present study, we evaluated 82,904 participants of Western European descent between 18 and 90 years of age, who underwent baseline investigations between 2007 and 2013, and for whom validated SAF measurement was available at baseline and prospective follow-up was performed between January 2014 and January 2018. There were no relevant differences in sex distribution, age and glycaemic variables between those with and without SAF measurements. We excluded participants who, at baseline, had clinical CVD . The average of the last three readings was recorded for each BP variable and heart rate.At both baseline and follow-up examination, participants completed a self-administered questionnaire on medical history, past and current diseases, and health behaviour. Medication use was verified at baseline by a certified research assistant and scored using the Anatomical Therapeutic Chemical (ATC) Classification System. Information regarding smoking behaviour  and quantity smoked, as well as coffee consumption (cups/day), was collected from the questionnaires . Weight 2, guarded against surrounding light, with an excitation light source with wavelength between 300 and 420 nm (peak intensity at ~370 nm). Emission light and reflected excitation light from the skin are measured with an internal spectrometer in the range 300–600 nm. SAF was based on the ratio of the average emitted light intensity per nm in the range 420–600 nm and the average reflected light intensity per nm in the range 300–420 nm, multiplied by 100, and is expressed in arbitrary units (AU), taking skin colour into account [z scores) were calculated separately for men and women, based on the total population.SAF was measured non-invasively using an AGE reader , as described previously , 20. The account . More de1c (EDTA-anticoagulated) was analysed using an NGSP-certified turbidimetric inhibition immunoassay on a Cobas Integra 800 CTS analyser . Serum creatinine was measured on a Roche Modular P chemistry analyser  and renal function was calculated as estimated (e)GFR with the formula developed by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) [Blood samples were taken in the fasting state between 08:00 and 10:00 hours and transported to the laboratory facility at room temperature or 4°C, depending on the sample requirements. On the same day, HbACKD-EPI) . Total c1c ≥48 mmol/mol (≥6.5%) at follow-up evaluation. Incident CVD was defined as present when participants reported myocardial infarction, percutaneous transluminal coronary angioplasty (PTCA), stent positioning, coronary artery bypass grafting (CABG), transient ischaemic attack (TIA), cerebrovascular accident (CVA), intermittent claudication or peripheral artery vascular surgery. Vital status was ascertained with the Municipal Personal Records Database (GBA). Data on cause of death were not available. The incidence of type 2 diabetes, CVD and mortality was calculated separately and as a composite outcome for all age-decade groups ..Diagnosis of the metabolic syndrome was established if a participant at baseline satisfied at least three out of five criteria according to the modified guidelines of the National Cholesterol Education Programs Adults Treatment Panel III (NCEP ATPIII criteria): (1) systolic BP ≥130 mmHg and/or diastolic BP ≥85 mmHg and/or use of antihypertensive medication; (2) HDL-cholesterol levels <1.03 mmol/l in men and <1.30 mmol/l in women and/or use of lipid-lowering medication influencing HDL-cholesterol levels; (3) triacylglycerol levels ≥1.70 mmol/l and/or use of triacylglycerol-lowering medication; (4) waist circumference ≥102 cm in men and ≥88 cm in women; (5) fasting glucose level ≥5.6 mmol/l and/or use of blood glucose-lowering medication and/or diagnosis of type 2 diabetes . IncidenU test. The χ2 test was used to analyse categorical variables. Uni- and multivariate logistic regression analyses were performed to examine the association between SAF and the composite outcome of incident type 2 diabetes, CVD and mortality, as well as these outcomes separately, while adjusting for relevant clinical, biochemical and lifestyle risk factors. In our models, we adjusted for the most important determinants of SAF, i.e. age , additionally adjusted for presence of the metabolic syndrome (model 2), glycaemic variables (model 3a/b), confounding non-biochemical factors (model 4) and all relevant factors (model 5). As age is an important factor influencing not only SAF measurements, but also the absolute incidence of events, we calculated the association between SAF and outcome according to four clinically relevant age groups, considered as low, intermediate, high and very high risk: ≤35 years; 36–50 years; ≥51 years; and ≥61 years. Values of p < 0.05 were considered statistically significant.All analyses were conducted using PASW Statistics . Data are presented as mean ± SD, or median and interquartile range (IQR) when not normally distributed. Means were compared between groups with ANOVA. When variables were not normally distributed, medians were compared using the non-parametric Mann–Whitney 1c ≥48 mmol/mol (≥6.5%) in 268, and either elevated blood glucose or HbA1c in 531 participants.The incidence of all outcomes is shown in Table p < 0.001), and had a higher baseline BMI, fasting glucose and HbA1c  had developed CVD at follow-up . As expected, mortality increased with age  in those participants who developed an event  compared with those who remained free from these events.ESM Table p = 1.5 × 10−307). This association remained significant after adjusting for age (model 1), age and the metabolic syndrome (model 2) and also after adjusting for age, fasting glucose  or HbA1c . Additional regression models revealed that the association also remained significant when adjusted for sex, waist circumference and current smoking (model 4), as well as all for other variables including systolic BP, plasma lipids, eGFR and coffee consumption . In the composite multivariate model 5, age, glucose, waist circumference, current smoking, systolic blood pressure and triacylglycerol were most strongly associated with the composite outcome (Table Table p = 4.1 × 10−181). This association remained highly significant after adjusting for age (model 1), presence of the metabolic syndrome (model 2), glycaemic variables (model 3a/b) and other possible confounding, non-biochemical factors (model 4). Model 5 showed that male sex, waist, systolic BP, cholesterol and current smoking, in addition to SAF and age, were independently associated with mortality. Similarly, univariate regression analyses revealed SAF to be strongly associated with both incident type 2 diabetes and incident CVD separately , SAF still was significant, and age, glucose, waist circumference, male sex and triacylglycerol were the strongest factors associated with incident type 2 diabetes, and age, waist circumference, systolic BP and current smoking were the strongest factors associated with incident CVD.Additionally, we assessed the relationship between SAF and the three individual outcomes separately Table . In a unz score tertile was associated with an almost twofold increased risk of mortality compared with the other tertiles.As the time of death of all participants was recorded, we were able to show the effect of SAF on time from baseline to death. As can be seen in ESM Fig. Finally, as age is an important factor influencing SAF measurements, but also the absolute incidence of outcome events Fig. , we calc1c.This prospective study within the general population demonstrates that SAF is significantly associated with new-onset type 2 diabetes, CVD and mortality during a median follow-up of 4 years. SAF predicted these combined outcomes independently of several conventional risk factors, including age, sex, waist circumference, the metabolic syndrome, smoking status, fasting glucose and/or HbA1c were used to define type 2 diabetes at follow-up, which may have caused overestimation of their predictive values. SAF also significantly predicted mortality alone, even after correction for all relevant risk factors, such as age, sex, waist circumference and smoking. Finally, SAF was most strongly predictive in participants aged 36 and above, probably because of the low incidence of events in the lowest age group


N6-(2-(2-Furanyl-2-oxoethyl))-l-lysine (furosine) is a deteriorative reaction product that is produced during heat treatment and storage of milk. This compound affects the quality of commercial dairy products. Accurate determination of furosine is necessary as it may serve as a measure of the degree of protein degradation in dairy products.In this article, two HPLC based methods (1. a novel ion-pairing reagent 2. a strong cation exchange column) are proposed to quantify furosine. These methods were optimized and validated for their application to analyze fluid milk and dried milk powder.•Two methods that can be used for routine milk quality control, including heat damage and adulteration, were developed.•Compared to previous methods, the modified procedures herein using aromatic sulfonic acids  provide less expensive and more sensitive determinations.•The identification and quantification of the furosine chromatographic signal was successfully achieved during analysis of commercial and spiked samples. Furosine is a compound formed in the early stages of the Maillard reaction  ,2; it isMiscellaneous methods for the determination of furosine in dairy include capillary zone electrophoresis  as well The most common methods for the determination of furosine are based on high- performance liquid chromatography (HPLC). For example, a previous report utilizing HPLC with an acetate buffer was applied to assay furosine in a variety of dairy products  . In thisPreviously, sodium heptane sulfonate has been used as pairing agent by several research groups to aid the measurement of furosine in commercial milk , whippinp-toluenesulfonic acid (TsOH) which interacts simultaneously both with the furosine and the C8 column. Alternatively, we used a strong cation exchange column  for furosine determination. These modifications were optimized and validated, resulting in two reproducible and accurate approaches for the determination of furosine.Herein we report modifications of method ISO 18,329:2004(E). IDF: 193:2004(E). We substituted acetic acid and potassium chloride in the mobile phase for a solution of ®), and methanol  were acquired from Merck Millipore . Ultra-High Pure Nitrogen was purchased from Praxair Technology Inc. . Ultrapure water  was obtained using an A10 Milli-Q Advantage system and an Elix 35 system .Furosine analytical standard was attained from Polypeptide Laboratories . Hydrochloric acid , TsOH  and sodium 1-heptane sulfonate  were purchased from Sigma-Aldrich . HPLC grade acetonitrile  equipped with a degasser (DGU-20A5), quaternary pump (LC-20AT), an autosampler (SIL-20 A HT), a system controller (CBM-20 A), a column oven (CTO-20 A), a photodiode array detector (SPD-M20AV) was used during all analysis. Chromatographic data management was performed using LC Solutions (Version. 5.2).™, LC-8, 58297, SUPELCO, St. Louis, MO, USA) and a solvent system that included a 5 mmol TsOH or HSA L−1 solution  and ACN (B). Gradient elution was accomplished as follows: 100% A, 0–10 min; 50% A, 10–20 min; 50% A, 20–22 min and 100% A, 22–27 min. Solvent flow and column compartment temperature, detector wavelength and sample injection volume were kept constant during elution at 1.0 mL min−1, 30 °C, 280 nm, and 20 μL, respectively . A 0.2 mol L of 5 μL F, a flow−1 aqueous solution is added. Immediately thereafter, the vial is capped with a septum, tan PTFE/silicone  and nitrogen is bubbled for 1 min (to purge any oxygen), into the solution through a needle. The resulting mixture was heated adiabatically for 23 h at 110 degrees Celsius. The resulting hydrolysate was filtered through a qualitative filter paper, grade 4  by gravity. Subsequently, 0.5 mL of the hydrolysate was filtered through a SPE cartridge previously conditioned with 5 mL MeOH and 10 mL water .Sample treatment was performed according to the ISO 18,329 IDF 193 reference method. Briefly, to a fluid milk subsample of 2 mL, in a 40 mL glass vial , 6 mL of an HCl 10.6 mol L−1 solution. The eluate was pressure-filtered through a 0.20 μm filter  and recovered in an HPLC 2 mL vial for injection (Shimadzu Prominence). As recommended in the reference ISO 18,329 IDF 193 method, the remaining of the hydrochloric acid hydrolysate is used for protein determination according to method AOAC OMASM 991.20 Nitrogen  in Milk. Kjeldahl Method.Dropwise elution from the SPE cartridge was performed using a 3 mL HCl 3 mol L−1 HCl solutions, poor accuracy  and reproducibility are obtained. However, when the same sample (0.2 g) is first suspended into a volume of 2 mL of water, using an Ultra-Turrax®  at 8 500 rpm, before spiking and dilution with the HCl solution, the resulting recoveries improve drastically  are commonly used during reverse phase chromatography to improve peak symmetry. We believe that in our HPLC method, the use of aromatic sulfonic acids such as TsOH serves two purposes: a) aromatic sulfonate anions exhibit different surfactant properties than other commonly used agents  , b) the in situ within the column; on the solid phase. In contrast with other methods, no organic solvent or ion pair agent is needed during the separation on a SCX column and, as such, this method is less expensive and might be considered as green chemistry. Another advantage resides in that the column uses isocratic mode during separation, this prevents baseline drift during phase shifting  and as the stationary phase in several cation exchange columns . Considering the existence of the latter column, we postulated that it may well retain furosine by a mechanism similar to TsOH ion-pairing, but occuring −1  fold acuter than its HSA counterpart and, in turn, SCX is 2.6 times more sensitive than the former . In the case of the peak symmetry, the signal obtained during SCX has the least tailing factor of the three methods compared; possibly because it involves the direct interaction of the stationary phase with the analyte. Meanwhile, ion-pair separation relies on multiple interactions occurring at once .Moreover, an additional assay was performed using HSA sodium salt instead of TsOH to compare the proposed method with an already established approach . We founU, p < 0.05, Repeatability was attained for four types of milk samples,  values for RSD obtained ranged from 1.31 to 4.06%. No significant differences in variability among milk samples  (d), as Pearson test show variables with a positive association  However, slight variations among laboratories may be caused by i. methodological differences, ii. storage temperature during transport  , Fig. 1Deratures ) and iiiThese methods aim to establish an accurate and reliable technique which will allow assessing basal furosine concentrations in commercial milk C over thN6-carboxymethyllysine) [It is important to notice that furosine alone may not be sufficient to achieve such goal. It is true that multiple or prolonged thermic treatment will increase furosine concentrations. Still, to our knowledge, no legislation has been established. For example, some countries even permit the commercial preparation and sale of recombined milk , but itllysine) . Noteworp < 0.05.Calibration curves parameters , coefficients of determination, limits of detection, and standard errors were computed as a linear fit model using SAS JMP 13 . A Mann-Whitney test was used to evaluate the hypothesis that variability among milk samples was equal. A One-way ANOVA and a Tukey post hoc test were used to compare differences among method results for a group of samples tested (the same samples were tested using 3 different methods as a way to assess robustness). Additionally, a Pearson product-moment correlation test was used to compare results obtained from a third-party method (MET-058) and our data (TsOH and SCX). For all tests, significant results were considered if Furosine is calculated as follows:If the result is to be reported in mass , milk density must be accounted for.To express as a protein base:Protein is determined as directed by AOAC 991.20 using 6.38 as a nitrogen-to-protein conversion factor.


Citrus bergamia Risso et Poiteau), a fruit mostly produced in the Ionian coastal areas of Southern Italy , is a valuable source of health-promoting constituents with, among other, antioxidant properties. In order to investigate the potential antiaging effects of this Mediterranean natural antioxidant source, bergamot juices of three different cultivars  were herein characterized by the mean of high-performance liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry. Then, juices were investigated for the evaluation of total polyphenolic and flavonoid contents, cell-free model antioxidant activities, and in vitro antiaging properties on two different cellular models of induced myocardial senescence. The best performing juice was also assessed in vivo. The phytochemical profiles confirmed that juices were rich in flavonoids, both flavone and flavanone glycosides. In addition, two limonoid glycosides were also identified in all cultivars. Each cultivar showed different phenolic and flavonoid contents. In tube results showed the juice robust antioxidant activities that correlate with their phenolic and flavonoid contents. Moreover, for the first time, the ability of juice to counteract the chemical-induced senescence was here demonstrated in both cellular models. Lastly, the in vivo data obtained from mouse hearts evidenced an increase in transcription of genes involved in antiaging and antioxidant responses. The overall results suggest that bergamot juice exerts antioxidant and antisenescence effects, making it useful for nutraceutical purposes.Aging is one of the main risk factor for the onset of cardiovascular diseases; one of the possible explanations could be linked to the age-associated overproduction of free radicals. This increase of oxidative stress can be overcome with a high intake of food antioxidants. In this context, a number of studies have been addressed to assess the antiaging potential of natural antioxidant compounds. Recently, it has been shown that the juice of bergamot ( The pathogenesis of many age-associated diseases can be due to an increase of oxidative stress; indeed, the overproduction of free radicals could impair protein and fatty acid functions and could lead to DNA damage, predisposing to age-related disorders . In partCitrus fruits have gained a high attention for their content of flavonoids and other beneficial components. In particular, bergamot (Citrus bergamia Risso et Poiteau), a fruit mostly produced in the Ionian coastal areas of Southern Italy , has been demonstrated to be a valuable source of health-promoting molecules that contribute to its antioxidant, anti-inflammatory, and cholesterol reduction capacities .–37.35–37e °C see  for 30 sin vitro antisenescence activity data are presented as the means ± standard errors of the means (SEM) of triplicate samples and are representative of three different experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni's corrected t-test for post hoc pair-wise comparisons. p < 0.05 was considered statistically significant.The nonlinear multipurpose curve-fitting program GraphPad Prism  was used for data analysis and graphic presentations. The cell-free model antioxidant activity data were expressed as mean ± standard deviation of three independent experiments. Pearson coefficient was used to determine the correlation among polyphenol and flavonoid contents and antioxidant activity. The C. bergamia juices obtained from the different cultivars are displayed in In the present study, the HPLC-PDA profiles recorded at 325 nm of the three 1–14 were detected in all cultivars and were identified comparing their HPLC elution orders, ESI-MS/MS, and UV data (Compounds  UV data  with tho1) −). The base peak ion at m/z 595 can be attributed to eriocitrin or neoeriocitrin, two flavanone O-glycoside isomers previously reported in C. bergamia juice −) and 579 ([M−H−144−162]−), leading to suppose that compound 7 can be a naringin or narirutin ([M−H]− at m/z 579) with a hydroxy-3-methylglutaryl moiety and a hexose residue linked to the molecule.Full spectrum of compound ia juice . A very The quantification of the main flavonoids detected in all sample juices was determined on the naringin basis by HPLC-PDA recorded at 325 nm. Results obtained from quantitative analysis are listed in C. bergamia fruits were then investigated. Each cultivar showed different phenolic and flavonoid contents  have not been previously reported for measuring the activity of bergamot juice samples. Last, the RACI was also calculated.In order to characterize the total antioxidant capacity of bergamot juices , four di2•− is a radical oxygen species normally produced inside the body, but it is known to be very harmful to cellular components as a precursor of a more reactive oxygen species, for example, the hydroxyl radical. The activity of ascorbic acid (IC50 = 4.34 ± 0.39 mg/mL), used as reference, was compared with the samples by IC50 values , followed by “fantastico” (IC50 = 1.13 ± 0.11 mg/mL). Both these juices demonstrated to have a stronger superoxide radical scavenging activity than ascorbic acid. Juice obtained from “femminello” exhibited an activity (IC50 = 4.77 ± 0.58) similar to ascorbic acid. The scavenging activities were in accordance with previous investigation [O0 values . Juice otigation .FRAP test evaluates the ability of plant extracts to reduce ferric ions. In our study, the FRAP assay revealed that “fantastico” cultivar had the highest reducing power (9.83 ± 0.54 mg TE/g), followed by “femminello” and “castagnaro”  .β-carotene/linoleic acid system was also investigated by a BCB test. The oxidation of linoleic acid generates peroxyl free radicals, which will then oxidize the highly unsaturated β-carotene. The presence of antioxidants minimizes the oxidation of β-carotene. All samples showed moderate β-carotene bleaching inhibition activity; in fact, results ranged from 22.42 ± 1.15 to 31.60 ± 0.50 % and the highest value was found in cultivar “castagnaro” , followed by “castagnaro” (−0.09), and “femminello” (−0.45).C. bergamia cultivar juices using two radical scavenging assays . The highest antioxidant capacity has been found in “castagnaro” juice compared to “fantastico” and then “femminello”. On the contrary, comparing the radical scavenging activity of the three bergamot cultivars using just DPPH assay, the previous paper [Recently, Sicari and Pellicanò  have invus paper  has estaThese discrepancies among literature and in respect to present results may be ascribed to the fruit phenolic content that depends on many factors, such as the degree of maturity at harvest, genetic differences, and environmental conditions . NotablyThen, in order to verify if there was a correlation between juice amount of polyphenols and antioxidant properties, Pearson analyses were conducted using averaged values of each variable, as reported in r = 0.89), suggesting a consistent relationship between polyphenolic and flavonoid content and the ferric reducing antioxidant power.The highest positive correlation was observed between FRAP and TPC and TFC , as previously reported [β-galactosidase  was monitored as a marker of cellular senescence. For chemical insults, in order to establish the effective dose of DOX or H2O2 able to induce cellular senescence, H9c2 cells were challenged with increasing concentrations of DOX (ranging from 0.01 to 0.1 μM) or H2O2 (ranging from 5 to 100 μM), diluted in water, for 3 h. Subsequently, the cells were cultured with fresh complete medium for 3 days, and then the presence of sa-β-Gal was examined. A concentration-dependent increase of sa-β-gal was observed following the H9c2 cell treatment with DOX . Conversely, only 60 μM H2O2 was effective to induce the staining of sa-β-Gal significantly (p < 0.01). A higher dose of H2O2 (100 μM) did not further increase the senescence marker, probably due to the cytotoxic effect exerted by such a high concentration of H2O2. Together, these results allowed us to establish 0.05 μM DOX and 60 μM H2O2 as the effective drug concentrations to be used for the following H9c2 cell experiments.H9c2 cell senescence was induced by cell exposure to exogenous oxidative insults  and the pivotal senescence hallmark, the sa-β-Gal staining, was assessed after 3 days. 2O2-induced (light gray bars) and DOX-induced (dark gray bars) senescence model.Therefore, with the aim to explore the protective potential of bergamot juice against cellular senescence, the DOX- or Hβ-Gal staining cells are in light blue. As reported in 2O2 or DOX evidenced a significant sa-β-Gal staining in respect of control cells . Blood total cholesterol, HDL, LDL, triglyceride, glycaemia, and glycated haemoglobin values were superimposable with the corresponding values measured in young-adult animals, suggesting that these animals did not have cardiometabolic diseases. Indeed, lipid and glycemic parameters measured in BFJ and control groups were not significantly different .Nevertheless, the real-time PCR analyses, performed on hearts of the old mice fed with FBJ or vehicle for three months, showed statistically significant increases in mRNA levels of three regulator genes, SIRT1, NRF2, and FOXO3, which are largely involved in antiaging and antioxidant responses .The silent information regulator 2 family proteins (SIRT), also called “sirtuins,” have been demonstrated to coordinate metabolic responses to changes in nutritional availability and maintain physiological homeostasis. Brain-specific Sirt1-overexpressing (BRASTO) transgenic mice have shown significant life span extension and delay in the aging process compared to control mice . The nucIn order to further demonstrate the involvement of these pathways in the antioxidant effects of the active compounds contained in bergamot juice, two additional genes, NAD(P)H dehydrogenase quinine 1 (NQO1) and heme oxygenase 1 (HO-1), routinely assessed as markers of NRF2 activity, have been investigated. These two NRF2 target genes are markers of a robust antioxidant response . As repoThen, considering that oxidative stress has been pointed out as relevant cause of both in heart aging and in the development of several cardiac diseases, an approach with BFJ could guarantee an improvement of these targets  typically altered and at the basis of cardiac dysfunction and of the consequent tissue damage .Therefore, all together, these data indicated that the bergamot juice exerts beneficial health effects counteracting senescence in myocardial H9c2 cells and increases the antiaging and antioxidant defenses in mouse hearts, opening new perspectives on the potential pharmaceutical application of this natural source.Citrus genus displayed different pharmacological activities; in particular, a placebo-controlled, double-blind study demonstrated that limonin glucoside may be useful in the prevention and/or treatment of different chronic inflammatory diseases, such as cancer, diabetes, and cardiovascular diseases [in vitro and in vivo.Natural polyphenols have gained considerable attention as potential agents for prevention and treatment of oxidative stress-related diseases, such as aging and cardiovascular diseases , 51, anddiseases . The resdiseases , 39, 41.These findings supported the nutraceutical properties of the bergamot fruit, encouraging the use of its pulp as health-promoting resource.


Anaplasma spp. are gram-negative obligate intracellular bacteria of remarkable importance in both human and veterinary health. Six Anaplasma species have currently been classified in the genus Anaplasma and are known as causative agents of tick-borne diseases1. The impacts of the diseases caused by Anaplasma on the productivity and health of animals have been recognized for over a century, and it is still a significant threat to livestock industries2. A zoonotic role has recently been uncovered for Anaplasma phagocytophilum, resulting in a rising interest in these organisms3. Anaplasma phagocytophilum has been considered the main cause of human anaplasmosis worldwide in recent years. Recently, a potential novel tick-transmitted Anaplasma species was identified in asymptomatic goats in China, and it has been recognized as a cause of human infection; the name “Anaplasma capra” was proposed provisionally4. Sequence and phylogenetic analyses of “Anaplasma capra” based on different gene loci revealed that this organism was distinct from all known Anaplasma species4. Although “A. capra” has not yet been formally recognized, human cases of “A. capra” infection have been recorded in northeastern China, and subsequent reports have also shown that “A. capra” seems to be widely distributed in China7, suggesting this agent may pose a substantial public health concern and may be responsible for anaplasmosis cases of unknown cause.A. phagocytophilum has been identified in a great number of wild animal species2. In previous reports, specific DNA of “A. capra” has been detected in domestic and some species of wild animals9, and “A. capra”-like bacteria were identified in ticks10, indicating “A. capra” might have a broad host range and genetic diversity.Wild animals are important reservoirs for tick-borne pathogens. The zoonotic Budorcas taxicolor) (five) and forest musk deer (Moschus berezovskii) (one); the second-grade nationally protected Himalayan goral  (three); and two nonendangered protected species, Reeves’s muntjac (Muntiacus reevesi) (three), and wild boar (one). Blood or liver samples were collected individually from animals, frozen at −20 °C, and transported in a freezer pack to a laboratory immediately. The total DNA was extracted using a QIAamp DNA Mini Kit according to the protocol. This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.This study was conducted in wild animals from Tangjiahe National Nature Reserve, which is a national protected area located in Sichuan Province, China  and negative controls (double-distilled water) were included in each assay. “Anaplasma capra” was detected in six wild animals, including three takins, two Reeves’s muntjacs, and one forest musk deer , gltA(594 bp), and msp4(656 bp) gene were purified using the AxyPrep DNA gel extraction kit , cloned  and sequenced . Sequence analysis revealed that the 16S rRNA sequences (MH188286) obtained in this study were 100% identical to each other and to those of “A. capra” isolates identified from sheep (MF066918), H. qinghaiensis ticks (KX673825) and Japanese serows (AB509223); and they were 99.8% identical to those of the isolates identified from humans  and H. longicornis ticks (KP314237). Phylogenetic analysis based on the 16S rRNA gene demonstrated that the isolates were clustered within the clade inside the“A. capra” but distinct from other, well-defined Anaplasma species  with 2–6 bp nucleotide substitutions were obtained and showed 98.4–100% identity to those of “A. capra” isolates from sheep (MF071308 and MF071309) and H. qinghaiensis ticks (KX685885 and KX685886), but they were 87.2–88.3% identical to the isolate HLJ-14 from human (KM206274). Moreover, the msp4sequences of these isolates showed 88.4% identity to “A. capra” isolate HLJ-14 (KM206277). Phylogenetic analysis of both gltA and msp4 gene sequences revealed that the “A. capra” strains were clustered independently from Anaplasma species identified previously and formed two distinct subclades with high bootstrap values. The isolates identified in this study were closely related to “A. capra” isolates from H. qinghaiensis and sheep but clearly distinct from human isolate HLJ-14 , roe deer (Capreolus capreolus), white-tailed deer(Odocoileus virginianus), mouflon (Ovis musimon), and chamois (Rupicapra rupicapra)11. In the present study, the novel tick-transmitted zoonotic “A. capra” was first identified in free-ranging wild animals  from a national nature reserve, where contact between wild and domestic animals rarely occurs. These results suggest that “A. capra” is endemic in this protected area and that those wild animals can act as competent sylvatic reservoirs for this organism.A. capra” was first described in cattle from Japan in 200112, and the agent was mistakenly assumed to be A. centrale   and free-living serows  from Japan8, and in cattle (MG910977–9) from Malaysia9. Those findings together with the results obtained in this study suggested that additional tick species and/or domestic and wild animals might be involved in the transmission and maintenance of “A. capra”, which warrants further investigation.The 16S rRNA gene sequence of “07) Fig. 13. More A. capra” isolates obtained from ticks, domestic, and wild animals as well as humans exhibit high sequence similarities to each other (99.8–100%) and cluster in a separate clade within the genus Anaplasma, but they are clearly distinct from the other members of Anaplasma, indicating the novelty of this causative agent. Obviously, these isolates of “A. capra” were a single species according to the classification criteria of bacteria13 (>99% 16S rRNA gene sequence similarity). However, they were classified into two different groups with low sequence similarity and a divergent phylogenetic relationship based on the gltA and msp4 genes (87.2–88.3% for gltA and 88.4% for msp4), indicating two genotypes of “A. capra” in ticks and reservoir hosts. The “A. capra” genotype 1 identified in I. persulcatus, H. longicornis, sheep, goat, and human significantly differs from the genotype 2 identified in H. qinghaiensis, takin, Reeves’s muntjac and forest musk deer, suggesting a high degree of genetic diversity and host tropisms of “A. capra”, as has been documented in A. phagocytophilum2. However, it is unclear whether these two genotypes of “A. capra” have pathogenicity variation for animals and human, which should be clarified in the future.Sequence and phylogenetic analysis based on the 16S rRNA gene demonstrated that “A. phagocytophilum was recognized as the causative agent of human granulocytic anaplasmosis, the cases have constantly increased in many countries14. However, numerous anaplasmosis cases of undetermined cause remain, indicating additional Anaplasma species are responsible for human anaplasmosis15. However, clinical cases of anaplasmosis might be generally neglected because the symptoms of the illness are notoriously nonspecific, making it likely to be confused with other illnesses14. The identification of the novel tick-transmitted zoonotic “A. capra” calls for clinicians to be aware of the possible infection from this causative agent in patients suspected of anaplasmosis or other tick-borne diseases, especially in areas where anaplasmosis can occur.In the past three decades, an increasing number of novel tick-associated microbes with zoonotic potential continue to be discovered, representing a considerable impact on public health worldwide. Since A. capra” infection was first documented in three species of free-ranging wild ruminants from a national nature reserve of China, and a novel genotype of “A. capra” was identified that clearly differed from the genotype identified from human. Further studies should be conducted to fully elucidate the host range, vector ticks, pathogenicity, and geographic distribution of this organism.In summary, “Supplementary Figure S1Supplementary Table S1


In lumbar spinal stenosis (LSS), at most times, several levels are impaired and selecting the correct level remains a common problem for surgeons, as surgery remains invasive, and extended laminectomy may lead to secondary surgical complications. Therefore, helping to select the correct level may be useful for surgeons. The use of diffuse tensor imaging (DTI) and paraspinal mapping (PM) in addition to conventional magnetic resonance imaging (MRI) may be helpful . However, with decompression levels determined by conventional magnetic resonance imaging (MRI) increasing, whether the benefits of reducing decompression level of conventional MRI + (DTI or PM) will be more obvious is unknown.Reduced surgical levels that were different between levels determined by conventional MRI + (DTI or PM) and conventional MRI + neurogenic examination (NE) between groups were compared. Treatment outcome measures were performed at 2 weeks, 3 months, 6 months, and 12 months postoperatively.p = 0.085; two levels/four levels, p = 0.039; three levels/ four levels, p = 0.506, respectively).The reduced levels of three groups showed no statistically significant differences between each other except for two levels and four levels (two levels/three levels, With surgical levels determined by conventional MRI increasing, the benefits of DTI and PM will be uncertainly more obvious. The term lumbar spinal stenosis (LSS) is commonly used to describe patients with symptoms related to anatomical reduction in lumbar spinal canal. Among older individuals, LSS is a highly disabling condition  and is tThe challenge for anatomically based determination is that, while necessary for the diagnosis of LSS, it is not sufficient to determine the severity of symptoms which lead a patient to seek treatment . The extThe use of diffuse tensor imaging (DTI) and paraspinal mapping (PM) techniques can reduce the decompression levels of lumbar spinal stenosis than conventional MRI + NE . HoweverSymptomatic patients of aged 40–90 years with degenerative lumbar spinal stenosis detected on conventional MRI or radiography from October 2015 to October 2017 were enrolled in this study. Since stenosis defining features can be seen on conventional MRI before and more clearer than changes consistent with stenosis can be detected on radiography, patients with degenerative lumbar spinal stenosis on conventional MRI were eligible. The inclusion criteria were as follows: patients had neuro claudication including both lower back pain and one leg pain that was consistent with a lumbar spinal stenosis and had persisted for at least 6 months despite pharmacologic treatment, physical therapy, or limitation of activity. Leg pain was defined as the pain below the buttocks . NE was In terms of the decompression levels determined by conventional MRI, the patients were divided into three groups: 1, two levels; 2, three levels; and 3, four levels.All patients underwent conventional MRI and symptoms, signs, X-ray, DTI, and PM examinations and went for decompression surgeries (laminectomies or laminotomies) with surgical levels determined by conventional MRI + (PM or DTI). All surgeons were trained and performed at least for 50 lumbar spinal decompression surgeries annually. PM and DTI were described as below.Typically, the total MiniPM score was used to indicate the extent of paraspinal denervation. In this study, the MiniPM score at each nerve root, a summary of six scores  were shown to be associated with the level of the radiculopathy used  3 actual voxel size; 0.50 × 0.50 × 4.00 mm3 calculated voxel size and sagittal T2-weighted fast spin-echo  sequences were obtained using a 4/0.4-mm section thickness/gap; 176 × 290 matrix; 0.91 × 1.00 × 4.00-mm3 actual voxel size and 0.50 × 0.50 × 4.00 mm3 calculated voxel size.A 3.0-T conventional MRI scanner  was used in this study. Sagittal T1-weighted fast spin-echo sequences were obtained using a 453/8.0 ms for TR/TE, 4/0.4-mm section thickness/gap; 176 × 290 matrix; 0.91 × 1.00 × 4.00-mm2 was considered normal, 76 to 100 mm2 was considered to be moderately stenotic, and ≤ 76 mm2 was classified as severely stenotic. The nerve root compromise in lateral recess was graded as follows: grade 0, no contact of the disc with the nerve root; grade 1, contact without deviation; grade 2, nerve root deviation; and grade 3, nerve root compression. A nerve root compression was considered to be present when the root was deformed , be3 voxel size was very larger than that in the previous study (1.1 × 1.6 × 3 mm), and therefore, the spatial resolution was unlikely to account for the difference [Our 3.13 × 2.54 × 3.0 mmfference , maybe bfference , the FA The reduced surgical levels showed statistically significant difference between two levels/four levels, but it was not significant between two levels/three levels and three levels/four levels, which showed that, with the surgical levels determined by conventional MRI increasing, the benefits of reducing the decompression level of DTI and PM would be uncertainly more obvious.All postoperative ODI, VAS-BP, and VAS-LP scores at 2 weeks after surgeries were measured to be with improvement than preoperative ODI, VAS-BP, and VAS-LP scores with statistically significance, respectively, and the averages of ODI, VAS-BP, and VAS-LP scores were greatly reduced, indicating that surgical treatment achieved good results. In the postoperative follow-up, there were 4 patients with lumbar compression fractures (because of osteoporosis), 2 patients with femoral neck fractures, which all had nothing to do with the lumbar spinal stenosis. The postoperative VAS-BP and VAS-LP scores and ODI scores in some cases had some fluctuation, but the averages were toward improvement timely, and none of the patients’ symptoms recurred or/and exacerbated or/and required a repeat surgery.We acknowledged that our study had some limitations. One was that a small number of subjects were investigated and a limited follow-up was performed. The further studies were needed to investigate whether our findings remain valid in a larger population and a longer follow-up. Another, we could not repeat the DTI and PM after surgeries because of the spinal instrumentation artifacts, such as those from pedicle screw systems and surgical scar, respectively.This study suggested that with the surgical levels determined by conventional MRI increasing, the benefits of DTI and PM will be uncertainly more obvious.


M = 28.282) of the United States, F = 105.560, p < 0.001; obesity prevalence was highest in the Southcentral (32.300%) and Southeast (32.200%) regions, x2(9731) = 10,850, p < 0.001; and FE was most prevalent in the Mid-Atlantic region (67.3%), x2(9882) = 11,291, p < 0.001.The results of this study closely mirror results found in studies of the general population. A better understanding of the geographic distribution of these outcomes could guide the targeting of sub-populations for public health programs. In particular, Team Red, White & Blue community growth and other fitness based public health programs could be expanded to reach more veterans.The purpose of the present study was to examine regional differences in average self-reported BMI, obesity prevalence, and frequent exercise (FE) among members of Team Red, White, and Blue (Team RWB) – a military veteran service organization founded to increase physical activity in veterans. A total of 10,015 military veterans participated in a needs assessment conducted by Team RWB between December 2014 and August 2016. Multivariate regression analysis with bootstrapped coefficients revealed that: BMI was highest in the Midwest region ( Althoug22.1Team RWB is a 501(c) organization focused on enriching veterans' lives through physical fitness and social activities to address reintegration challenges and offset physical health declines and weight gain following military to civilian transition . Since in = 10,015).Team RWB staff collected baseline assessments of new members in 2014. Team RWB staff/researchers developed an instrument for the assessment. The instrument was reviewed by subject-matter experts and pilot-tested for completion time and psychometric properties. The final version of the online survey was offered to all new Team RWB members with a t-shirt incentive. Between December 2014–August 2016, 37,229 active-duty service-members, National Guard, reserve personnel, or veterans joined Team RWB. During this time-frame, 19,218 military-connected participants (52%) completed the survey. In this study, only veterans were analyzed (2.22 ∗ 703]) , and frequent exercise (FE). BMI was calculated with self-reported height and weight (BMI = [(weight(lbs) / height(in)) ∗ 703]) . Three r ∗ 703]) . FE was 2.3The primary independent variable was Team RWB region. Team RWB stratifies states into seven regions : Northwe2.4To isolate the effect of region on BMI, OB, and FE, and in keeping with other similar studies of veterans , severalOne item assessed anxious mood: “In the past month, I have felt nervous, anxious, or on edge.” One item assessed depressed mood: “In the past month, I have felt down, depressed, or hopeless.” For these constructs, a five-point response scale was offered: strongly disagree to strongly agree. “Strongly agree” and “agree” responses were grouped together (Code = 1) and all other responses were grouped together (Code = 0). Survey completion year was also controlled for in the present study to rule out historical effects.Daily tobacco use was measured with the following question: “How would you characterize your use of tobacco products?” The following response options were available: never, once a month or less, once a week or less, weekly, daily, and frequently during the day. “Daily” or “frequently during the day” responses were grouped together (Code = 1) and all other responses were grouped together (Code = 0). Daily alcohol use was measured with the following question: “How would you rate your consumption of alcohol?” The following response options were available: never, once a month or less, once a week or less, occasional drinks during the week, every day, and more than a couple drinks every day. Responses of “every day” or “more than a couple drinks every day” were grouped together (Code = 1) and all other responses were grouped together (Code = 0).2.5d), OB (Cohen's h), and FE (Cohen's h) based on significant results of the regression models.Descriptive statistics for BMI, OB and FE were calculated according to the independent and control variables. Maps were created in QGIS  in order3Average BMI was highest in the Midwestern region , Fig. 1,F = 105.560, p < 0.001. Veterans in the Northeastern, Southeastern, Midwestern, and Southcentral regions had significantly higher BMIs than veterans in the Pacific region. Cohen's d was calculated for the aforementioned statistically significant regional differences in order to determine the practical difference in means: Midwest vs. Pacific, d = 0.139; Southeast vs. Pacific, d = 0.128; Southcentral vs. Pacific, d = 0.127; and Northeast vs. Pacific, d = 0.084. Covariates associated with higher BMI included being male, having VA health care benefits, exercising infrequently, reporting symptoms consistent with depressed mood, not using alcohol/cigarettes daily, having served in the Gulf/Vietnam/Korean war eras, and being disabled.The overall BMI model  was statx2(9731) = 10,850, p < 0.001. All regions except the Northwest had significantly higher proportions of OB than the Pacific region. Cohen's h was calculated for the aforementioned statistically significant regional differences to determine the practical difference in proportions: Southcentral vs. Pacific, Cohen's h = 0.146; Southeast vs. Pacific, Cohen's h = 0.144; Midwest vs. Pacific, Cohen's h = 0.133; Northeast vs. Pacific, Cohen's h = 0.089; Mid-Atlantic vs. Pacific, Cohen's h = 0.061. Covariates associated with greater likelihood of OB included being male, having VA healthcare benefits, infrequent exercise, having depressed mood, not using alcohol/tobacco daily, having served in the Gulf/Vietnam/Korean war era, and having a disability.The overall OB model  was statx2(9882) = 11,291, p < 0.001. The Pacific, Southeast, and Mid-Atlantic regions had higher proportions of FE than the Northeast region. Cohen's h was calculated for the aforementioned statistically significant regional differences to determine the practical difference in proportions: Mid-Atlantic vs. Northeast, Cohen's h = 0.084; Pacific vs. Northeast, Cohen's h = 0.078; Southeast vs. Northeast, Cohen's h = 0.046. Covariates associated with greater likelihood of FE included being male, not having VA health care benefits, having a lower BMI, not reporting symptoms consistent with anxious/depressed mood, not using tobacco daily, being employed, and not having a disability.The overall FE model  was stat4Results of this study showed that veteran BMI, obesity prevalence, and engagement in FE varied by U.S. region, although effect sizes for differences were small and veterans in all regions demonstrated an average BMI within the overweight BMI category. Outcomes of multivariate regression models showed that high BMI and high obesity prevalence were concentrated in the Midwestern and Southern regions, which corresponds to studies of the general population . Furthermore, results showed that FE was most prevalent in the Mid-Atlantic region, but ranged from 63.3% to 67.0% in other regions.Several covariates in the present study exhibited significant associations with our outcomes. Specifically, depressed/anxious mood, risky health behaviors , and a service-connected disability were associated with higher BMI, greater likelihood of obesity, and reduced likelihood of FE. These results correspond to other studies of the veteran population .When considering the findings of this report, several limitations must be acknowledged. Secondary analysis of survey data, while providing a large sample of new Team RWB respondents, limited the scope of questions asked. Data were self-reported, which could be problematic due to respondent recall or reluctance to truthfully answer sensitive questions . Further, regarding this point, it is possible that what constitutes FE could vary from person to person. Some research has shown that differences in self-report and objective measures of physical activity and medical conditions differ among military veterans .The items assessing anxious/depressed mood were broad . As a re5This study is the first to nationally evaluate regional differences in veteran BMI/obesity. It provides important information for the military-connected community. Team RWB was designed to implement physical activities to improve health outcomes in transitioning service members. Their national presence and ability to create engaging physical activities for veterans addresses a critical need. In geographic locations where chapters do not exist, other public health programs aimed at reducing risk for obesity and increasing exercise frequency among the general population could be tailored to target military veterans. Additionally, information about obesity risk in specific Team RWB regions is useful to the organization. Programming to promote supported physical activity programs , promote mental well-being, and decrease negative health behaviors around smoking and drinking are especially warranted.


Zooplankton and epibenthic crustaceans, as the fundamental consumers, exhibit δ13C and δ15N values ranging from −21.3 to −19.8‰ and +5.1 to +7.5‰, respectively, and can utilize the vent POM for 38–53% of their diets. The vent-obligate crab Xenograpsus testudinatus shows a large variation in δ13C (from −18.8 to −13.9‰) and δ15N values (from 1.1 to 9.8‰), although an omnivorous trophic level (2.5) is identified for it using δ15N values of amino acids, and it can utilize the vent POM for 6–87% of its diet. The consistently low (< 10.0‰) and overlapping δ15N values for most of the analyzed macroinvertebrates suggest extensive ingestion of chemosynthetic production complementing the photosynthetic production, a weak prey–predator relationship and low trophic complexity possibly imposed by the extreme environmental contexts of shallow-water hydrothermal ecosystems.Shallow-water hydrothermal vent ecosystems are distinct from the deep-sea counterparts, because they are in receipt of sustenance from both chemosynthetic and photosynthetic production and have a lack of symbiosis. The trophic linkage and energy flow in these ecosystems, however remain elusive, which allows us poor understanding of the whole spectrum of biological components distributed across such environmental gradients. In this study, a thorough isotopic survey was conducted on various biological specimens and suspended particulates collected along four transects across the venting features of a shallow-water hydrothermal field off Kueishan Island, Taiwan. The isotope data combined with a Bayesian-based mixing model indicate that the vent-associated particulate organic matter (vent POM), as primary contribution of chemoautotrophic populations, has a high δ Unlike the vast majority of marine ecosystems sustained by photosynthetic primary production, benthic ecosystems associated with deep-sea hydrothermal vents are primarily driven by chemoautotrophic production that harvests metabolic energy from the oxidation of abundant reducing compounds , dominates over other benthic community members [The ecosystem near Kueishan Island  off northeastern Taiwan provides a model field for studying energy flow in shallow-water hydrothermal vents. It is unique and rare in that the vent-obligate crab,  members . In addi members . The benX. testudinatus, which is thought to occupy the highest trophic level in the KST hydrothermal ecosystem [15N values of the primary producers. This isotopic survey identified potential food sources and consumers across both vertical and horizontal scales and established a general model of energy flow and benthic−pelagic coupling in shallow-water vent ecosystems.This study aims to investigate a thorough spectrum of food source utilization, trophic structure, and energy flow in the KST ecosystem. Particulate organic matter (POM), fauna, and macroalgae were collected across the venting features and analyzed for their bulk carbon and nitrogen isotopic compositions to quantitatively distinguish the respective contributions of chemosynthetic and photosynthetic production . To bettcosystem , the nitca. 15 km off the northeastern coast of Taiwan and aligned with the southwestern extension of the Okinawa Trough. It is composed of andesitic rocks formed during the subduction of the Philippine Sea plate underneath the Eurasian continent and represents the most westward extension of volcanic activity within the Okinawa Trough [2-rich, extremely acidic (the lowest recorded pH in the world of 1.52 and 2.77 on average [2) of vent-obligate crabs, e.g., X. testudinatus, have been found to inhabit areas near the chimneys and rock crevices where gas bubbles emerge [KST is located a Trough  Fig 1)..ca. 15 k average ) and hot average ). Helium average . An extrs emerge .ca. 10 m) with continuous discharge of whitish fluids and sulfur deposited at the venting peripheral was chosen as the center for our sampling campaign conducted in August and September of 2015. Sampling was conducted along four transects (100 m in length with the exception of the shoreward NW transect) extending from the vent center to the peripheral region  in order to collect samples with sufficient biological diversity and to uncover the effects of venting on trophic structure. The horizontal intervals for sampling benthos and seawater were 10 m and 20–50 m, respectively. The vent-influenced area is usually milky blue in color and its range exhibits great spatiotemporal variation depending on the tides and currents.No specific permission was required for the sampling as the location is not private-owned or protected under any regulation, and this study did not involve endangered or protected species. One of the vents  were collected from the venting orifice using a titanium pipe connected with a Teflon pipe and a pre-vacuumed sterile bottle. An extra venting fluid sample was collected in a close but different vent in October 2016 to verify the composition of vent fluids. The collected fluids were filtered using pre-combusted  glass fiber filters (pore size of 0.7 μm) to collect POM for isotopic analysis. Zooplankton was collected by obliquely towing a 200-μm mesh plankton net at the sea surface in both vent-influenced and non-vent locations. The sampled zooplankton were then separated into five size categories  for further isotopic analyses using stainless steel sieves. For small epibenthic crustaceans, a light trap equipped with a 5-L plastic bottle and a light source was deployed overnight on the seabed of both venting and non-vent areas. Benthic macrofauna, including the predominant vent crab (X. testudinatus) and sea anemones (Anthopleura sp.) species and other coexisting species including corals (Tubastraea sp.) and sea snails , were collected along the transects by scuba divers. Red (Gelidiopsis sp.) and green (Cladophora catenata) macroalgae were plucked off the hard substrate. Species identification was mainly based on a previous survey [Various samples, including suspended POM , zooplankton, benthic fauna, and macroalgae were retrieved from different compartments of the ecosystem to provide a full spectrum of biological diversity for trophic reconstruction. Seawater (4 to 8 L) was collected from the sea bottom by scuba divers and from the sea surface by towing a bucket at each sampling site. Venting fluids , therefore, the effect of acid treatment on the isotopic composition was considered negligible. To obtain sufficient sample amounts for isotopic analysis, several individuals of zooplankton and epibenthic crustaceans, or muscle tissue from more than five individuals of sea snail A. misera at the same sites were pooled. However, the bottom POM from the NW transect was not sufficient for reliable nitrogen isotope determination and, therefore, is not reported.Soft tissues were extracted from sea anemones and corals and muscle tissues were extracted from the crab’s chelae and the sea snail’s foot. All the tissue and POM samples were dried at 60°C for a minimum of 24 h and ground into powder for further analyses. A portion of the powders was subject to acid treatment (1 N HCl) to remove any carbonate minerals in the tissues or POM. The acid treatment did not yield appreciable changes in the δ13C or 15N, R is 13C/12C or 15N/14N, and the standards for δ13C and δ15N are Vienna Pee Dee Belemnite and atmospheric N2 gas, respectively. The U.S. Geological Survey standard #40 , was used as the reference standard. The analytical error (1σ) derived from multiple analyses of the reference standards for both δ13C and δ15N is better than 0.2‰.Carbon and nitrogen isotope ratios were determined using an elemental analyzer (Thermo Flash EA) connected with a Thermo Finnigan MAT253 isotope ratio mass spectrometer at Institute of Oceanography, National Taiwan University. The analyzed results were reported in δ notation:X. testudinatus, compound-specific nitrogen isotope analysis of amino acids was conducted for two individuals from the same site (SW0) with completely distinct δ13C (−13.9‰ versus −18.1‰) and δ15N values (+1.1‰ versus +9.0‰) to accurately estimate its trophic level (TL). The amino acid nitrogen isotope analysis was carried out at the Department of Biogeochemistry of the Japan Agency for Marine-Earth Science and Technology. The preparation of crab tissues for the amino acid isotope analysis mainly followed the procedures described by Chikaraishi et al. [To precisely quantify the trophic position of the crab i et al. . In briei et al. . The nit13C and △δ15N), which exhibit great variability among taxonomic classes and feeding guilds [13C and δ15N were −0.41 ± 1.14‰ and +2.52 ± 2.50‰ for herbivores, and +0.91 ± 1.04‰ and +3.23 ± 0.41‰ for carnivores, respectively [13C and δ15N values and described in the literature, e.g., for E. contractus [X. testudinatus [An isotopic mixing model based on the Bayesian framework was used to quantify the contributions of potential energy sources to vent consumers by applying the open source Stable Isotope Analysis in R (SIAR) package . The SIAg guilds ,25, playg guilds ; thus, wectively . Decisioectively . Therefontractus  and X. tudinatus ,19. For X. testudinatus was generally considered to occupy the top trophic rank in this ecosystem [15N values of the primary producers [15N values of amino acids which exhibit a high degree of trophic enrichment  with those showing little change in the δ15N values with trophic transfer , the trophic level of one organism can be estimated based on the following equation [x and y represent trophic and source amino acids, respectively, βx/y stands for the inherent isotopic difference between amino acids x and y in primary producers, and Δx and Δy indicate the 15N-enrichment factors for each trophic level for amino acids x and y, respectively. Among various amino acids, glutamic acid and phenylalanine have been widely accepted as the most useful pair to derive the precise estimation of trophic levels [While cosystem , two indroducers ,29. By cc levels .X. testudinatus constituted the majority of the faunal abundance, followed by the sea anemones Anthopleura sp. and sea snails A. misera. More than 150 and 130 individuals of X. testudinatus and Anthopleura sp. were collected, respectively. Both species were distributed along the survey transect from the vent center to peripheral sites (100 m from the vent). The abundance of vent crabs decreased beyond 50 m from the vent, whereas sea anemones became more dominant at the periphery. Coral colonies (Tubastraea sp.) were mainly distributed within 50 to 100 m from the vent center, except for one colony observed at a site 10 m from the vent center in the NW transect. The sea snails were generally present with patches of green macroalgae (C. catenata) with A. misera being more abundant than E. contractus. The coverage of green and red macroalgae (Gelidiopsis sp.) was generally observed beyond 30 m from the vent center.During our visual survey through scuba diving, the vent crab 13C and δ15N values of POM associated with the vent fluid were −18.2 ± 1.1‰ and −1.7 ± 0.4‰, respectively, and appear to be the most 15N-depleted end-member having a heavier carbon isotope composition among all potential food sources  among sampling sites  ranged between −32.9 and −22.2‰ (the most 13C-depleted carbon source), whereas the δ15N values ranged between +1.7 and +4.0‰. The δ13C and δ15N values of green macroalgae (C. catenata) ranged from −22.8‰ to −17.3‰ and from +3.7‰ to +5.7‰, respectively. Both red and green macroalgae exhibited extremely large δ13C variations across sampling sites  were higher than those for large sized zooplankton from the same area. Zooplankton collected from venting areas possessed slightly but significantly higher mean δ13C values than those from non-venting areas .The δ13C and δ15N values ranging from −20.0 to −19.8‰ and +5.6 to +6.5‰, respectively. Their δ13C values were slightly higher than those for zooplankton  exhibited slightly larger isotopic variations than corals with the δ13C and δ15N values varying from −20.7 to −18.7‰ and from +7.9 to +9.7‰, respectively. Corals and sea anemones, which are classified as carnivorous suspension feeders, exhibited comparable δ15N values yet obviously more depleted δ13C values (−19.9 to −20.3‰) than scavenger/detritivores. No clear spatial trend in the isotopic compositions was observed for most of the macroinvertebrate species, except for the vent crabs, whose isotopic compositions show a clear negative correlation   and seawater POM (47–48%) . A totalplankton , constitme crabs  suggest 4 ± 20%) , with th13C values similar to epibenthic crustaceans (mean Δδ13C < 0.1‰) and zooplankton (mean Δδ13C = 1.1‰). Their computed dietary composition was highly consistent across all transects, with equal ingestion of zooplankton and epibenthic crustaceans , and their diet compositions were not significantly different among coral colonies , corpses of epibenthic crustaeans (35 ± 4% for E. contractus and 40 ± 3% for A. misera), and zooplankton (21 ± 2% for E. contractus and 27 ± 4% for A. misera)  . Both E.15N value (−1.2‰) but a mid-ranged δ13C value (−21.4‰) [13C and δ15N values).Our data revealed three major groups of isotopically distinct food sources in the KST vent field: vent POM, seawater POM, and green benthic macroalgae . The dis(−21.4‰) , which s13C and depleted in 15N . This distinct isotopic composition is rarely reported and is comparable with those speculated for free-living chemosynthetic bacteria in deep-sea hydrothermal vents [13C values of autotrophic organisms are strongly dependent on (1) isotopic fractionation during biosynthetic processes, and (2) the isotopic composition of inorganic carbon sources [2 and tends to yield significantly less carbon isotopic fractionation (Δδ13Ccell ranged from −3.3 to −13.3‰) than the Calvin cycle (light-independent reaction of photosynthesis) and the reductive acetyl-CoA pathway [13C-enriched organic carbon. In the bottom sediments of the KST vent area and crab tissues, 16S ribosomal RNA gene amplicon pyrosequencing demonstrated the predominance of Epsilon-Proteobacteria (Sulfurovum and Sulfurimonas) [13C in vent POM could also be derived from dissolved inorganic carbon (DIC) with a higher δ13C value. However, the carbon isotopic compositions of CO2 gas released from venting fluids in the KST vent field, ranging from −8.2 to −5.5‰ [13C-enriched DIC for chemoautotrophs in the KST venting fluids. The distinct high δ13C value of vent POM obtained in this study has likely originated from the chemoautotrophic microbial populations using the rTCA cycle as the major biosynthetic pathway and exploiting vent DIC as the primary carbon source.Among these food sources, the vent POM, which is primarily composed of chemoautotrophic microbial biomass ,33, was al vents ,34,35. T sources ,36. In t sources ,38. The  pathway ,39, thusrimonas) –42, mostto −5.5‰ , seems t15N-depleted. Bacterial mats in hydrothermal vent fields (sulfide oxidizing bacteria and other unidentified forms) have been widely and consistently characterized by low δ15N values (ca. −9.6 to +1.6‰) [15N ≈ +4 to +8‰ [15N-depleted isotope composition can likely be attributed to the pronounced bacterial NH4+ assimilation by autotrophic microorganisms [2 fixation or NO3- assimilation  ,19,44,454 to +8‰ ). Such a 0 to 5‰ ,48). In 13C values between −24.0 and −19.0‰ and δ15N values between +4.0 and +8.0‰ [13C values between −23.0 and −19.9‰ and δ15N values between +3.7 and +4.3‰ for POM from the outer shelf of the East China Sea [13C values between −24.8 and −21.1‰ and δ15N values between +3.0 and +4.5‰ for POM in sediment trap and sediments from the Okinawa Trough [13C-enriched and 15N-depleted isotopic signature was barely observed in the seawater POM, suggesting restricted influence of the vent fluid on the surrounding water mass. However, the high spatial variations in the δ13C value of POM at four 20-m sites (The isotopic compositions of seawater POM obtained in this study resembled those for marine planktonic phototrophs (δnd +8.0‰ ). These hina Sea  and δ13Ca Trough , 51). Th-m sites  suggest 13C value (> 5.0‰). Their δ13C values still fell in an extremely large range reported for worldwide marine macroalgae (−3.0 to −35‰) [in situ temperature, and taxonomy [Both green and red macroalgae exhibited large variations in δto −35‰) ,53. Suchtaxonomy .13C values between most benthic invertebrates and seawater POM was observed  and passive sinking of detrital materials [In shallow-water vents, chemosynthesis takes place not only in the bottom water layer but also near the sea surface, and even spreads over the surface layer due to the warmth and buoyancy of hydrothermal fluids ,10. Althaterials , thus enaterials , lateral13C values between suspension feeders and scavenger/detritivores reached ca. 3‰, yet the differences in the δ15N value were less than 1‰. Such discordance between different isotopes has also been observed for deep sea counterparts [Our study revealed distinct carbon isotope compositions among consumers affiliated with different feeding guilds. The disparity in δterparts ,19,57, rterparts .Tubastraea sp. and Anthopleura sp., were close to those for zooplankton and epibenthic crustaceans, whereas the difference in their nitrogen isotopic values approached 3‰. The isotopic pattern suggests a close prey–predator relationship between suspension feeders and these fundamental species. The mixing model further demonstrated almost equal dietary proportions from zooplankton and epibenthic crustaceans across all the sampling sites for both corals and sea anemones  associaic level )  and chemosynthetic primary production (TL = 1). Although the green macroalgae sampled from some sites also exhibited high δ13C values, a previous study revealed common yet very limited use of the green algae Ulvophyceae by X. testudinatus, based on its 18S ribosomal DNA libraries of stomach and gut content [15N values for a portion of the crab individuals were even lower than those of the macroalgae verified the limited contribution of macroalgae to the crab’s diet.Using vent POM as an end member, our modeling computation indicated that the vent POM could contribute 9−67% of crab’s diet on average for different sites . In partediments . Aside fediments . Our est content . The facA. misera and E. contractus are common species distributed along the shallow coastal areas of Taiwan. In comparison with the vent crabs, A. misera and E. contractus had similar isotopic compositions  [15N values of the vent crabs X. testudinatus reached below ca. 2‰. Even the carnivorous sea anemones Anthopleura sp., which possessed the highest δ15N values among all species (mean δ15N = 9.2 ± 0.3‰), were characterized by δ15N values lower than its congeneric species, the algal symbiont-host Anthopleura xanthogrammica inhabiting Pacific intertidal areas [15N for consumers  in the KST vent system further supports the extensive consumption of chemosynthetic production as a complement to a photosynthesis-based diet in shallow-water hydrothermal vent ecosystems.Vent-associated species have been reported to possess distinctly lower δ species ,4,34,65.sibogae) , the δ15al areas . The con13C and δ15N values distinct from that of zooplankton, epibenthic crustaceans, and vent POM, the dietary compositions of the vent crabs should be reliable. Slightly greater uncertainty may arise in the diet determination for corals and sea anemones due to the relatively small difference in δ13C values between the consumers and food sources.The stable isotope mixing model based on the Bayesian framework provided a quantitative insight into both chemosynthetic and photosynthetic contributions in a shallow-water hydrothermal food web. However, such a mixing model has been demonstrated to be sensitive to discrimination factors ,27. Vari13C value greater than that of the heaviest crab individual might be needed to account for the crab’s diet. With this potential diet source, the contribution of hypothetical vent POM to the diet of various consumers would decrease accordingly. To quantitatively constrain this source composition, we first consider a scenario where the trophic position of the crab with the heaviest carbon isotopic composition is 2.5 and the other food sources for this crab were a mixture of zooplankton and epibenthic crustaceans. The simple mass balance generates δ13C and δ15N values of −8.0‰ and −9.9. for the hypothetical vent POM, respectively. Using these compositions, the SIAR simulation yielded that 15–22% of the diet for zooplankton and small epibenthic crustaceans, and 48% of the diet for the heaviest crab are derived from the hypothetical vent POM. Alternatively, if the peculiar crab with the greatest δ13C value relies completely on the hypothetical vent POM (trophic position is 2), the δ13C and δ15N values for the hypothetical vent POM would be −13.5‰ and −1.4‰, respectively. Using these compositions, the SIAR simulation yielded that 28–40% of the diet for zooplankton and small epibenthic crustaceans and 79% of the diet for the heaviest crab are derived from the hypothetical vent POM. Regardless of which scenario, the vent POM still contributes significantly to the diet of a fraction of crab individuals and other primary consumers. Apparently, an accurate estimation of the vent POM contribution relies on a fully evaluation of the isotopic composition of vent POM.Since several vent crab individuals exhibited carbon isotope composition even heavier than that of the measured vent POM, a hypothetical vent POM with a δIn deep-sea hydrothermal systems, the transfer of energy from plume-associated chemosynthetic production to higher trophic levels, particularly to the pelagic populations, was rarely observed due to the lack of sampling at a fine depth resolution  and biom15N values , suggesionships . Under tionships , 72. Theionships ,73.via a thorough isotopic survey of potential food sources and vent-related species with knowledge-based numerical modeling of mixed diets. Our results indicated that the chemosynthetic and photosynthetic productions contributed nearly equally to the food web. Both zooplankton and epibenthic crustaceans acted as an important trophic mediator transferring basic food sources to the upper trophic levels. While vent-obligate crabs are omnivorous, acquiring energy from all available food sources, few of them exhibited the potential to proliferate exclusively on chemoautotrophic energy. Given that hydrothermal vents are characterized by highly temporal variations in habitat conditions [This study provides the first quantitative evaluation of chemosynthetic and photosynthetic contributions to the shallow-water hydrothermal vent food web nditions  and isotnditions ,4, we reS1 Table(DOCX)Click here for additional data file.


With the growing number of young-older adults (baby-boomers), there is an increasing demand for assessment tools specific for this population, which are able to detect subtle balance and mobility deficits. Various balance and mobility tests already exist, but suffer from ceiling effects in higher functioning older adults. A reliable and valid challenging balance and mobility test is critical to determine a young-older adult’s balance and mobility performance and to timely initiate preventive interventions. The aim was to evaluate the concurrent validity, inter- and intrarater reliability, internal consistency, and ceiling effects of a challenging balance and mobility scale, the Community Balance and Mobility Scale (CBM), in young-older adults aged 60 to 70 years.ρ). Inter- and intrarater reliability were calculated as Intra-class-correlations (ICC), and internal consistency by Cronbach alpha and item-total correlations (ρ). Ceiling effects were determined by obtaining the percentage of participants reaching the highest possible score.Fifty-one participants aged 66.4 ± 2.7 years  were assessed with the CBM. The Fullerton Advanced Balance scale (FAB), 3-Meter Tandem Walk (3MTW), 8-level balance scale, Timed-Up-and-Go (TUG), and 7-m habitual gait speed were used to estimate concurrent validity, examined by Spearman correlation coefficient , 3MTW errors , 3MTW time , the 8-level balance scale , the TUG , and 7-m habitual gait speed . Inter- , intrarater reliability  were excellent, and internal consistency  was good to satisfactory. The CBM did not show ceiling effects in contrast to other scales.The CBM significantly correlated with the FAB .Trial number:  Balance ability generally starts to decline in the third decade of life , with anRegular physical activity (PA) is important to maintain independence and prevent functional decline. Current guidelines for older adults aged ≥65 years recommend at least 150 min of moderate intensity or 75 min of vigorous intensity aerobic training per week . PersonsIn order to promote early balance and mobility interventions, adequate assessment strategies are needed to identify subtle balance and mobility deficits in relatively active, high-functioning young-older adults. To date, most balance and mobility assessment tools have been developed to quantify deficits in frail older adults aged ≥70 years –16. CurrCurrent systematic reviews focusing on mobility in older adults conclude that tests such as the Timed Up and Go (TUG) test, the Dynamic Gait Index (DGI), or the Performance Oriented Mobility Assessment also suffer from ceiling effects when applied in independently living, higher functioning older adults , 17. TheIn summary, several studies have shown that balance and mobility measures developed for older, frailer adults show ceiling effects when applied in high-functioning older adults , 20, 23.In this context, the applicability of the Community Balance and Mobility Scale (CBM) has recently generated significant interest in clinical practice for assessing balance and mobility deficits in community-dwelling older adults, either healthy (mean age 70.3 years ) or withρ = 0.87), good correlations with the Timed Up and Go test (ρ = − 0.69) and self-selected gait speed (ρ = − 0.65) [2,k = 0.95; 95% CI = 0.88–0.98) and intrarater reliability  [Recently, the CBM has been validated in a sample of independently living, community-dwelling older adults aged ≥65 years (mean age 73 ± 7), showing excellent correlations with the BBS ( − 0.65) . Reliabi93–0.98) . Moreove93–0.98) .While these findings suggest that the CBM has added value in the assessment of community-dwelling older adults, the measurement properties in the specific population of young-older adults aged 60–70 years are yet to be evaluated. Young-older adults are an extremely heterogeneous population, where some older adults have substantial balance and mobility deficits while others have only minor deterioration in balance performances . The CBMIn this study, we aimed to examine the concurrent validity and reliability of the CBM in community-dwelling healthy young-older adults (60 to 70 years). The evaluation was performed as preparatory part of the European Commission funded project PreventIT (Horizon 2020 grant no 689238), which aims to develop a lifestyle-integrated training intervention to prevent functional decline in young-older adults.The first aim of the present study was to examine the concurrent validity of the CBM by comparing its scores to other established balance and mobility measures thought to have related theoretical constructs. We expected a positive association with the Fullerton Advanced Balance Scale  as this We used a cross-sectional study design for evaluating the concurrent validity and potential ceiling effects of the CBM. The inter- and intra-reliability was also obtained based on video-recordings of the assessments (described below). The data collection was embedded into the PreventIT project (phase 1). PreventIT is a three-year project aiming at developing a lifestyle-integrated training intervention for young-older adults aged 60 to 70 years. Phase 1 of the PreventIT project included pilot studies at the sites involved in the project . The pilot studies aimed to test the measurement properties of balance and mobility instruments in young-older adults. Another purpose of the PreventIT pilot studies was to test the feasibility of the lifestyle-integrated training intervention using questionnaires and focus groups. This feasibility testing occurred after the cross-sectional study for validating the CBM and did not influence this study.For the purpose of evaluating the measurement properties of the CBM in the specific population of young-older adults, we included 51 community-dwelling young-older adults. Inclusion criteria for this study were: community-dwelling older adults aged between 60 and 70 years, able to walk independently, and no cognitive impairment  scale is designed to identify balance deficits , 37 and The 8-level balance scale is an extended version of the SPPB  that incn = 31).The three meter tandem walk (3MTW) test is a modified version of the FAB , measuriThe Timed-Up-and-Go (TUG) test is a valid test evaluating basic functional mobility of older adults . The tesGait speed measurement was derived from the InChianti gait assessment . ParticiThe CBM scale evaluates high-level balance and mobility on 13 items, with six items performed with both the right and left side of the body, resulting in a total of 19 tasks, scored from zero (“unable to perform”) to five (“performs independently”) and is suggested to represent underlying functional skills required in the community . The tasData collection took place in movement laboratories at four test sites: (1) Germany , (2) Germany (Heidelberg University), (3) Norway (Norwegian University of Science and Technology), and (4) the Netherlands (Vrije Universiteit Amsterdam). All tests were conducted in a single assessment lasting about 1.5–2 h. All participants wore their own low-heeled shoes and were allowed sufficient rest periods at any given time. Trained research staff conducted the assessments.The CBM testing sessions were videotaped with a digital camera (Sony HDR-CX240E) in full HD, which also recorded the sound, an important feature for the subsequent rating . Camera height was fixed at 1 m and specific camera positions and angles for each task were predetermined in order to standardize the video recording. The videotaped assessments were scored by two experienced examiners to evaluate interrater reliability. Both raters had on average five years’ experience in assessing balance and mobility using different scales. They received a standardized manual on how to perform the CBM and carried out over 10 assessments. One rater was an exercise scientist (MW), the other a physical therapist (KG). Both raters scored each item independently, being allowed to watch the videos twice, and each of them was blinded to the rating of the other assessor. To determine intrarater reliability, videotaped performance on the CBM was assessed by the same rater a second time three weeks after the first rating.ρ) since the results of the 8-level balance scale (p < .001), errors during 3MTW (p < .001), and gait speed test (p < .05) were not normally distributed according to the Kolmogorov-Smirnov test. Correlation coefficients of ρ < 0.25 were considered as small; 0.25–0.50 as moderate; 0.50–0.75 as good; and > 0.75 as excellent [Concurrent validity between the CBM and the other balance and mobility tests was assessed using the Spearman’s rank correlation coefficient . T-test was used since the results of the CBM were normally distributed.2,k) and intrarater  reliability [ρ). Internal consistency with an α > 0.9 was considered as excellent, > 0.8–0.9 as good, > 0.7–0.8 as acceptable, > 0.6–0.7 as questionable, > 0.5–0.6 as poor, and ≤ 0.5 as unacceptable [Intraclass Correlation Coefficients (ICC) were utilized for total score interrater .Statistical analysis was performed using IBM SPSS Statistics Version 24.0 .N = 31–51). For the TUG and gait speed test, the first five participants were not assessed. For the participants in Heidelberg (n = 16), 3MTW performance was rated only by errors, but not by time. Because time was unavailable, these participants were excluded from statistical analysis on the 3MTW test, resulting in a subsample of 31 participants for which information on time and errors was available.A total of 51 participants aged 66.4 ± 2.7 years  were tested. Participant characteristics are summarized in Table ρ = 0.75; 95% CI = 0.59; 0.85, p < .001).Figure ρ = − 0.61; 95% CI = − 0.83; − 0.33, p < .001). Moderate correlations were found between CBM and gait speed , TUG , 8-level balance scale , and 3MTW time  (Table p = .09).Good correlations were found between CBM and 3MTW errors  and ICC3,k evaluating intrarater reliability at 1.00 (95% CI = 0.99–1.00).Inter- and intrarater reliability coefficients were excellent with ICCp < 0.001). For intrarater reliability, kappa values for 10 of the 19 items were above 0.80 (very good agreement), the other nine were between 0.61 and 0.80 (good agreement). For interrater reliability, two items were above 0.80, ten between 0.61 and 0.80, five between 0.41 and 0.60 (moderate agreement). Two items showed low kappa value of 0.31 and 0.34 respectively [Kappa values for individual item reliability are summarized in Table ectively .Table 3IInternal consistency was evaluated, with a Cronbach’s alpha of 0.88, indicating good internal consistency.Item-total correlations ranged from 0.81 (“Hopping forward left”) to 0.28 . The five items which most strongly correlated with the CBM total score were “Hopping forward left/right”, “Unilateral stance left”, “Forward to backward walking”, and “Lateral foot scooting left” Table .Table 4IThe participants’ scores are presented in Table This study is the first to analyze the measurement properties of the CBM in a sample of young-older adults aged 60 to 70 years. As hypothesized, a good correlation with the FAB was found, indicating strong construct validity of the CBM in the target population of young-older adults. Furthermore, moderate to good correlations with other measures suggest that the CBM measures mobility performance (TUG), dynamic steady-state balance control  and static steady-state balance control . This is in line with previous studies estimating the measurement properties of the CBM in older adults  or thoseρ = − 0.42), which was lower than expected and lower than reported in a previous study which validated the CBM in older adults [ρ = − 0.69) [A good correlation was found between the CBM and FAB, showing that both measure a similar construct. Both scales assess performance of more challenging balance tasks, including static, dynamic, proactive, and reactive balance control , 30, 32.r adults . The lowr adults , 33, 38. − 0.69) . The poo − 0.69) .ρ = − 0.61). The 3MTW errors classify a subject based on errors made during a challenging dynamic balance task, which is similar to the classification scheme of the CBM which may explain the good correlation. For 3MTW time, the correlation was lower (ρ = − 0.35) as compared to 3MTW errors. This suggests that the quality of task execution (3MTW errors) is more strongly linked to CBM performance as compared to the time of task execution (3MTW time).The CBM showed good correlation with 3MTW errors (ρ = 0.46). This suggests that a simple assessment of gait speed, commonly applied in older adults aged ≥70 years [Habitual gait speed, a less challenging measure of dynamic balance, showed a moderate correlation with the CBM . The 8-level balance scale is a measure of static steady-state balance control whereas the CBM primarily evaluates dynamic aspects of balance during complex mobility tasks. In line with the present findings, previous studies have reported moderate associations between static and dynamic steady-state balance control, suggesting that both aspects of balance control are partly interrelated, but represent distinct aspects of balance control  [As expected, a moderate correlation was found between the CBM and the 8-level balance scale (.08–0.39  or one-l05–0.23) .An excellent inter- and intrarater reliability of the CBM total score was found, exceeding the recommended standards of 0.90 to 0.95 for clinical assessments . For theThe Cronbach’s alpha as a measure for internal consistency was 0.88. Although it does not exceed the value of 0.90 suggesting redundancies among items , furtherA limitation of this study is that the sample consists of participants from three countries. While beneficial, cross-national research has limitations. It might be that variation in the performance across the countries could have occurred, despite standardized operating procedures.p = .09). A larger sample is needed to evaluate the validity for discriminating fallers from non-fallers.Additionally, females were overrepresented in our sample (75%) as compared to the general population aged ≥60 years (56% ). HoweveThis cross-sectional study did not allow the determination of responsiveness. Further studies are needed to evaluate the responsiveness of the CBM in the target population.https://clinicaltrials.gov/ct2/show/NCT03065088). The CBM may help to better understand the mechanisms of early balance and mobility decline in young-older adults and inform the development of treatments and intervention programmes aimed at improving early deterioration in balance and mobility, which is in line with the recently updated guidelines for early implementation of neuromotor exercise training in public health approaches [This study provides evidence that the CBM is a suitable tool for the assessment of challenging balance and mobility performances in healthy, young-older adults. The CBM tasks represent meaningful everyday performances which are specifically required to ambulate safely within an everyday environment. With trained assessors, the scale is easily administered, requires little equipment, and most importantly, is valid and reliable in the studied target population. Based on the present results, the CBM has been selected as an end point within the EU project PreventIT and is currently used within a randomized controlled trial evaluating a lifestyle-integrated training intervention for preventing functional decline in healthy, young-older adults (registered online; proaches .