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Regular endurance exercise training is an effective intervention for the maintenance of metabolic health and the prevention of many age-associated chronic diseases. Several metabolic and inflammatory factors are involved in the health-promoting effects of exercise training, but regulatory mechanisms remain poorly understood. Cellular senescence-a state of irreversible growth arrest-is considered a basic mechanism of aging. Senescent cells accumulate over time and promote a variety of age-related pathologies from neurodegenerative disorders to cancer. Whether long-term intensive exercise training affect the accumulation of age-associated cellular senescence is still unclear. Here, we show that the classical senescence markers p16 and IL-6 were markedly higher in the colon mucosa of middle-aged and older overweight adults than in young sedentary individuals, but this upregulation was significantly blunted in age-matched endurance runners. Interestingly, we observe a linear correlation between the level of p16 and the triglycerides to HDL ratio, a marker of colon adenoma risk and cardiometabolic dysfunction. Our data suggest that chronic high-volume high-intensity endurance exercise can play a role in preventing the accumulation of senescent cells in cancer-prone tissues like colon mucosa with age. Future studies are warranted to elucidate if other tissues are also affected, and what are the molecular and cellular mechanisms that mediate the senopreventative effects of different forms of exercise training., (© 2023. The Author(s).)

Objective: Whether blood eosinophils (bEOS) in chronic obstructive pulmonary disease (COPD) are associated with disease progression is a topic of debate. We aimed to evaluate whether the differential white blood cell (WBC) count, symptoms and treatment may predict lung function decline and exacerbations in COPD patients., Methods: We retrospectively examined stable COPD patients with a minimum follow-up of 3 years at our outpatients' clinic. We collected information about lung volumes (FEV1, FVC), the total and differential WBC count, acute exacerbations of COPD (number in the 12 months before the beginning of the study=AE-COPD-B, and during the follow-up=AE-COPD-F), smoking status and treatment. FEV1 decline and AE-COPD-F were described by using a generalized linear model and a 2-level random intercept negative binomial regression, respectively. The models included eosinophil and neutrophil counts as potential predictors and were adjusted by sex, age, smoking status, AE-COPD-B, treatment with bronchodilators and inhaled corticosteroids (ICS)., Results: Sixty-eight patients were considered, 36 bEOS- (<170 cells/μL, the median value) and 32 bEOS+ (≥170 cells/μL). ∆FEV1 was higher in bEOS+ than bEOS- (34.86 mL/yr vs 4.49 mL/yr, p=0.029). After adjusting for potential confounders, the eosinophil count was positively (β=19.4; CI 95% 2.8, 36.1; p=0.022) and ICS negatively (β=-57.7; CI 95% -91.5,-23.9; p=0.001) associated with lung function decline. bEOS were not found to be associated with the number of AE-COPD-F., Conclusion: In stable COPD patients, a higher level of blood eosinophils (albeit in the normal range) predicts a greater FEV1 decline, while ICS are associated with a slower progression of airflow obstruction.

Cardiac hypertrophy and renal damage associated with hypertension are independent predictors of morbidity and mortality. In a model of hypertensive heart disease and renal damage, we tested the actions of continuous administration of Vastiras, a novel compound derived from the linear fragment of ANP (atrial natriuretic peptide), namely pro-ANP 31-67 , on blood pressure and associated renal and cardiac function and remodeling. Of note, this peptide, unlike the ring structured forms, does not bind to the classic natriuretic peptide receptors. Dahl/Salt-Sensitive rats fed a 4% NaCl diet for 6 weeks developed hypertension, cardiac hypertrophy, and renal damage. Four weeks of treatment with 50 to 100 ng/kg per day of Vastiras exhibited positive effects on renal function, independent of blood pressure regulation. Treated rats had increased urine excretion, natriuresis, and enhanced glomerular filtration rate. Importantly, these favorable renal effects were accompanied by improved cardiac structure and function, including attenuated cardiac hypertrophy, as indicated by decreased heart weight to body weight ratio, relative wall thickness, and left atrial diameter, as well as reduced fibrosis and normalized ratio of the diastolic mitral inflow E wave to A wave. A renal subtherapeutic dose of Vastiras (25 ng/kg per day) induced similar protective effects on the heart. At the cellular level, cardiomyocyte size and t-tubule density were preserved in Vastiras-treated compared with untreated animals. In conclusion, these data demonstrate the cardiorenal protective actions of chronic supplementation of a first-in-class compound, Vastiras, in a preclinical model of maladaptive cardiac hypertrophy and renal damage induced by hypertension.

Background: The research was conducted in the frame of a population-based, case control study, called Genes Environment Interaction in Respiratory Disease., Objective: To assess the association between protein intake and physical performance in a general population sample., Design: Researchers investigated the association between the participants' dietary information and their physical performance using the 6-min walking test and the distance walked in metres (6MWD) as main outcome measure. Information on dietary intake was collected using the validated European Investigation into Cancer and Nutrition food frequency questionnaires (FFQs). Then, daily intake of energy and macronutrients was estimated by means of the NAF software (nutritional analysis of FFQ). Linear regression models were used to evaluate the associations between vegetable, animal and total protein intakes and the 6MWD. The models were adjusted for socio-demographic features, total fats and available carbohydrate intakes., Results: The participants were 223 subjects (57% females) aged between 23 and 68 years. Their mean vegetable and animal proteins intake for gram/kg of body weight/day were, respectively, 0.4 and 0.7. After adjusting for all the potential confounders, there was a significant increase of 20.0 (95% CI 0.8; 39.2) m in the distance walked for an increase in 10 g/day of vegetable proteins and non-significant variations of -1.8 (95% CI -9.3; 5.7) m for an increase in 10 g/day of animal proteins and of 0.5 (95% CI -6.8; 7.7) for an increase in 10 g/day of total proteins., Discussion and Conclusions: Our result suggests a positive role of vegetable proteins on physical performance. Whether this result is related to the high protein intake itself or may be a consequence of the other properties of plant-based foods deserves further investigation., Competing Interests: The authors declare no potential conflicts of interest., (© 2019 Diana Gazzani et al.)

Background: Fat intake has been associated with respiratory diseases, with conflicting results., Objective: We studied the association between asthma and rhinitis with dietary fats, and their food sources in an Italian population., Methods: Clinical and nutritional information was collected for 871 subjects (aged 20-84) from the population-based multi-case-control study Genes Environment Interaction in Respiratory Diseases (GEIRD): 145 with current asthma (CA), 77 with past asthma (PA), 305 with rhinitis and 344 controls. Food intake was collected using the EPIC (European Investigation into Cancer and Nutrition) Food Frequency Questionnaire. The associations between fats and respiratory diseases were estimated by multinomial models. Fats and their dietary sources were analysed both as continuous variables and as quartiles., Results: Monounsaturated fatty acids and oleic acid were associated with a reduced risk of CA in both continuous (RRR = 0.68, 95%CI: 0.48; 0.96; RRR = 0.69; 95%CI: 0.49; 0.97, per 10 g, respectively) and per-quartile analyses (p for trend = 0.028 and 0.024, respectively). Olive oil was associated with a decreased risk of CA (RRR = 0.80; 95%CI: 0.65; 0.98 per 10 g). An increased risk of rhinitis was associated with moderate total fat and SFA intake., Conclusions: High dietary intakes of oleic acid and of olive oil are associated with a lower risk of asthma but not of rhinitis., (© 2019 John Wiley & Sons Ltd.)

Heparin is a complex mixture of heterogeneous sulfated polysaccharidic chains. Its physico-chemical characterization is based on the contribution of several methods, but advantages of the use of complementary techniques have not been fully investigated yet. Strong-Anion-Exchange HPLC after enzymatic digestion and quantitative bidimensional 1 H- 13 C NMR (HSQC) are the most used methods for the determination of heparin structure, providing the composition of its building blocks. The SAX-HPLC method is based on a complete enzymatic digestion of the sample with a mixture of heparinases I, II and III, followed by the separation of the resulting di- and oligo-saccharides by liquid chromatography. The NMR-HSQC analysis is performed on the intact sample and provides the percentage of mono- and di-saccharides by integration of diagnostic peaks. Since, for both methods, accuracy cannot be proved with the standard procedures, it is interesting to compare these techniques, highlighting their capabilities and drawbacks. In the present work, more than 30 batches of porcine mucosa heparin, from 8 manufacturers, have been analyzed with the two methods, and the corresponding results are discussed, based on similarities and differences of the outcomes. The critical comparison of both common and complementary information from the two methods can be used to identify which structural features are best evaluated by each method, and to verify from the concordance of the results the accuracy of the two methods, providing a powerful tool for the regular characterization of single, commercial preparations of Heparin.

Background and Objective: Health-related quality of life (HRQL) in respiratory diseases has been generally investigated in clinical settings, focusing on a single disorder. In this study on a general population sample, we assessed the relationship between HRQL and several respiratory diseases studied simultaneously (COPD, current (CA) and past (PA) asthma, allergic (AR) and non-allergic (NAR) rhinitis and chronic bronchitis (CB)., Methods: Controls (n = 328) and cases of NAR (n = 95), AR (n = 163), CB (n = 48), CA (n = 224), PA (n = 126) and COPD (n = 28) were recruited in the centre of Verona in the frame of the Italian multi-case control GEIRD (Gene Environment Interactions in Respiratory Diseases) study; HRQL was measured through the SF-36 questionnaire. The relationships between HRQL (in terms of Physical (PCS) and Mental Component Scores (MCS)), respiratory diseases, and covariates were evaluated., Results: With respect to controls, the adjusted PCS median score was worse in subjects suffering from current asthma (- 1.7; 95%CI:-2.8;-0.6), CB (- 3.8; 95%CI:-5.7;-1.9), and COPD (- 5.6; 95%CI:-8.1;-3.1). MCS was worse in current asthmatics (- 2.2; 95%CI:-4.1;-0.3), CB (- 5.5; 95%CI:-8.7;-2.2), and COPD cases (- 4.6; 95%CI:-8.8;-0.5) as well., Conclusions: To our knowledge, this is the first study in the general population that analyzed HRQL performing a simultaneous comparison of HRLQ in several respiratory disorders. We found that subjects suffering from COPD, CA, and CB had the poorest HRQL. Clinicians should carefully consider the possible impact of respiratory disorders as CB and not only that of CA and COPD.

The positive association between overweight, obesity, and cardiovascular and all-cause mortality is well established, even though this relation is typically U shaped with an increased risk also in low-weight subjects. However, being overweight or obese has been associated with a better prognosis in subjects suffering from chronic diseases, id est the "obesity paradox". In both community-dwelling and hospitalized patients with COPD, several studies have reported a significant protective effect of obesity on all-cause mortality, indicating that also in obstructive pulmonary diseases, an obesity paradox may be present. Interestingly, the "paradox" is more evident for subjects with severe bronchial obstruction (i.e., a lower FEV1), while in mild-moderate conditions, the weight-related mortality shows a behavior similar to that observed in the general population. Several factors may confound the relation between COPD, obesity and mortality. The lower FEV1 found in obese people may be linked to a restrictive defect rather than to an obstructive one. Due to the modified chest wall mechanical properties-related to increased fat mass-obese COPD patients may present, respect to their lean counterpart, a lower lung hyperinflation which is associated with higher mortality. The traditional classification of COPD attributes to obese "blue bloaters" a low-grade emphysema in opposition to lean "pink puffers"; the fact that emphysema extent is related to mortality may bias the relationship between weight and survival. It is also to underline that the majority of the studies, consider BMI rather than body composition (a better predictor of mortality) when studying the intriguing relation between weight, COPD, and mortality. Reverse bias has also to be taken into account, hypothesizing that an unintentional weight loss may be the deleterious factor related to mortality, rather than considering obesity a protective one. Further prospective studies are needed to shed light on the complexity of this emerging issue., Level of Evidence: Level V: Narrative Review.

Obesity, metabolic syndrome, and hyperleptinemia are associated with aging and age-associated diseases including prostate cancer. One experimental approach to inhibit tumor growth is to reduce dietary protein intake and hence levels of circulating amino acids. Dietary protein restriction (PR) increases insulin sensitivity and suppresses prostate cancer cell tumor growth in animal models, providing a rationale for clinical trials. We sought to demonstrate that biomarkers derived from plasma extracellular vesicles (EVs) reflect systemic leptin and insulin signaling and respond to dietary interventions. We studied plasma samples from men with prostate cancer awaiting prostatectomy who participated in a randomized trial of one month of PR or control diet. We found increased levels of leptin receptor in the PR group in total plasma EVs and in a subpopulation of plasma EVs expressing the neuronal marker L1CAM. Protein restriction also shifted the phosphorylation status of the insulin receptor signal transducer protein IRS1 in L1CAM+ EVs in a manner suggestive of improved insulin sensitivity. Dietary PR modifies indicators of leptin and insulin signaling in circulating EVs. These findings are consistent with improved insulin and leptin sensitivity in response to PR and open a new window for following physiologic responses to dietary interventions in humans., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Protein-restricted (PR), high-carbohydrate diets improve metabolic health in rodents, yet the precise dietary components that are responsible for these effects have not been identified. Furthermore, the applicability of these studies to humans is unclear. Here, we demonstrate in a randomized controlled trial that a moderate PR diet also improves markers of metabolic health in humans. Intriguingly, we find that feeding mice a diet specifically reduced in branched-chain amino acids (BCAAs) is sufficient to improve glucose tolerance and body composition equivalently to a PR diet via metabolically distinct pathways. Our results highlight a critical role for dietary quality at the level of amino acids in the maintenance of metabolic health and suggest that diets specifically reduced in BCAAs, or pharmacological interventions in this pathway, may offer a translatable way to achieve many of the metabolic benefits of a PR diet., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Calorie restriction (CR) retards aging, acts as a hormetic intervention, and increases serum corticosterone and HSP70 expression in rodents. However, less is known regarding the effects of CR on these factors in humans. Serum cortisol and molecular chaperones and autophagic proteins were measured in the skeletal muscle of subjects on CR diets for 3-15 years and in control volunteers. Serum cortisol was higher in the CR group than in age-matched sedentary and endurance athlete groups (15.6 ± 4.6 ng/dl versus 12.3 ± 3.9 ng/dl and 11.2 ± 2.7 ng/dl, respectively; p ≤ 0.001). HSP70, Grp78, beclin-1, and LC3 mRNA and/or protein levels were higher in the skeletal muscle of the CR group compared to controls. Our data indicate that CR in humans is associated with sustained rises in serum cortisol, reduced inflammation, and increases in key molecular chaperones and autophagic mediators involved in cellular protein quality control and removal of dysfunctional proteins and organelles., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Pharmaceutical iron sucrose is an iron (III) replacement for the treatment of iron deficiency anemia in patients with chronic kidney disease. The drug product (injection) is a colloidal solution of ferric hydroxide in complex with sucrose, containing 20 mg/mL elemental iron; according to United States pharmacopoeia (USP), the limit of iron (II) is 0.4% w/v. A selective CE method for the simultaneous determination of iron (III) and its potential impurity iron (II), was developed by applying a dual precapillary complexation. In particular, 1,10-phenanthroline and 1,2-diaminocyclohexanetetraacetic acid were used for complexation of iron (II) and iron (III), respectively. Sample preparation was optimized to achieve mineralization of pharmaceuticals using HCl 6 M, by avoiding perturbation of the oxidation status of both iron species. Simple CZE conditions, involving a 60 mM (pH 9.3) tetraborate buffer at the constant voltage of 25 KV and 25°C, allowed fast separation of iron (II) and iron (III) complexes that were detected at 265 nm. Sensitivity for iron (II) determination was found to be 4.80 μM (LOQ) corresponding to 0.15% w/w with respect to the total iron test level. The method was validated by following International Conference on Harmonization guidelines for specificity, linearity, precision, accuracy, and robustness and it was applied to real pharmaceutical samples. The obtained results suggested that the method can be a useful alternative to the official USP and British pharmacopoeia polarographic method., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reduced dietary protein intake and intermittent fasting (IF) are both linked to healthy longevity in rodents, and are effective in inhibiting cancer growth. The molecular mechanisms underlying the beneficial effects of chronic protein restriction (PR) and IF are unclear, but may be mediated in part by a down-regulation of the IGF/mTOR pathway. In this study we compared the effects of PR and IF on tumor growth in a xenograft mouse model of breast cancer. We also investigated the effects of PR and IF on the mechanistic Target Of Rapamycin (mTOR) pathway, inhibition of which extends lifespan in model organisms including mice. The mTOR protein kinase is found in two distinct complexes, of which mTOR complex 1 (mTORC1) is responsive to acute treatment with amino acids in cell culture and in vivo. We found that both PR and IF inhibit tumor growth and mTORC1 phosphorylation in tumor xenografts. In somatic tissues, we found that PR, but not IF, selectively inhibits the activity of the amino acid sensitive mTORC1, while the activity of the second mTOR complex, mTORC2, was relatively unaffected by PR. In contrast, IF resulted in increased S6 phosphorylation in multiple metabolic tissues. Our work represents the first finding that PR may reduce mTORC1 activity in tumors and multiple somatic tissues, and suggest that PR may represent a highly translatable option for the treatment not only of cancer, but also other age-related diseases.

Heparin, a highly sulfated polydispersed glycosaminoglycan (GAG), is the most widespread clinical anticoagulant; it binds antithrombin III (AT), a member of serine proteinases superfamily, accelerating its antagonist effect on blood coagulation. The binding interaction with AT is an important aspect in characterization of physicochemical properties of GAGs. With the aim at profiling several clinical and experimental heparin batches from different sources (porcine, bovine and ovine mucosa), a quantitative investigation of the binding heparin-AT, was undertaken by means of Affinity Capillary Electrophoresis (ACE). In dynamic-equilibrium ACE, the electrophoretic mobility of the receptor (AT), analyzed in a BGE containing the ligand (the considered GAG), is correlated to ligand concentration and binding constant. In particular, a 20mM sodium phosphate, pH 7.4 buffer (the BGE) was chosen as the neat medium and the experiments were carried out in a highly hydrophilic poly(vinyl alcohol) coated capillary (effective length 8.5 cm). The applied sample, consisting in the receptor AT (0.30 μM) and phenylacetic acid (PAA; 10.0 μM) used as a reference compound, was electrophoresed at each of the studied concentration levels of the ligand (heparin samples, 0.30-10.0 × 10(-7)M; heparan sulfate, 0.35-8.0 × 10(-5)M) supplemented to the BGE. The migration time ratio of PAA to AT was assumed as the chemical response to be correlated to the ligand concentration and the binding constant estimation was based on the application of a nonlinear regression method (rectangular hyperbola). Under these conditions, a number of heparin samples were analyzed and their binding constants (Kd) were found within 14.2 and 56.1 nM (SD ≤ ± 2.0; n=3; coefficient of determination r(2) ≥ 0.96). The good correlation of Kd values to the in-vitro activity (anti-factor Xa and anti-factor IIa), confirmed that the affinity for the target AT is an important feature of heparin samples and could be included among their physico-chemical characteristics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5cmx50mum i.d.) at 40 degrees C and -30kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, DeltaUA-->GalNAc-4S Na(2) and DeltaUA-->GalNAc-6S Na(2) were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector., (Copyright 2010 Elsevier B.V. All rights reserved.)

During the past 25 years, heparin extraction and purification processes have changed. The results of these changes are reflected by the continuous increase in potency of the International Standard for heparin. This increase is due not only to a higher purity, but also to a number of changes in the physico-chemical characteristics of heparin. For long time, all these changes have been disregarded as non-critical by regulatory authorities. Heparin marketing authorisation was reviewed only two years ago and Pharmacopoeia monographs were reviewed just for the addition of new tests, mainly aimed at tackling the oversulfated chondroitin sulfate (OSCS) crisis. Currently, heparin monographs are again under revision. Such changes, different for each manufacturer, have caused a further increase in the heterogeneity of individual batches of heparin. This review aims at showing that chemical, physical and biological characteristics of heparin (such as disaccharide composition, amount of low sulfated and high sulfated sequences, molecular weight profiles [MW], activities, structural artifacts, fingerprints and glycosaminoglycans impurities) are all process-dependent and may significantly vary when different processes are used to minimise the content of dermatan sulfate. The wide heterogeneity of the physico-chemical characteristics of currently marketed heparin and the lack of suitable and shareable reference standards for the identification/quantification of process-related impurities caused, and are still causing, heated debates among scientific institutions, companies and authorities.

Chemical and physical characteristics, building blocks, constitutive disaccharides, sulfation degree, and biological activities of heparins (UFHs) and of low molecular weight heparins (LMWHs) obtained by different depolymerization processes are examined comparatively in terms of structure characteristics, content of 1,6-anhydro rings, and other fingerprints. The heterogeneity of different LMWHs depends on different manufacturing processes and on particular specifications of pharmacopoeias. The reported examples prove that the variability among samples of LMWHs manufactured by the same process is quite limited. Most of the variability is derived from the parent UFH. In contrast, fingerprint groups and residues are specific to the depolymerization process and their extent can be roughly controlled through the process parameters.

Modified heparin disaccharides were obtained by the alkaline treatment of a solution containing the disulfated heparin disaccharide DeltaHexA-alpha-(1-->4)-D-GlcNSO(3),6SO(3). Their structures were characterized by one- and two-dimensional NMR spectroscopy: DeltaHexA-alpha-(1-->4)-1,6-anhydro-GlcNSO(3), DeltaHexA-alpha-(1-->4)-1,6-anhydro-ManNSO(3) and DeltaHexA-alpha-(1-->4)-ManNSO(3),6OSO(3). NMR spectroscopy, in combination with HPLC, provided the composition of the mixture. Characteristic NMR signals of the disaccharides were identified, even at low levels, in a high field of (1)H-(13)C correlation NMR spectra (HSQC) of a low molecular weight heparin (LMWH) obtained by beta-elimination (alkaline hydrolysis) of heparin benzyl ester, providing a more complete structural profile of this class of compounds.

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.