Influence of dietary total antioxidant capacity on the association between smoking and hypertension in Brazilian graduates (CUME project).

BACKGROUND AND AIMS: Hypertension (HTN) is a chronic non-communicable disease influenced by non-modifiable risk factors, such as sex and age, as well as modifiable risk factors such as lifestyle, including diet and smoking. Moreover, diet quality among smokers is worse than that of non-smokers, mainly in terms of antioxidant content. Thus, the current study aimed to investigate whether dietary total antioxidant capacity (dTAC) influences the association between smoking and HTN. METHODS AND RESULTS: This cross-sectional study included 4303 graduates (69.35% women) from the Cohort of Minas Gerais Universities (CUME) project. An online food frequency questionnaire was administered to participants, and dTAC was estimated using the ferric reducing antioxidant power method. In the questionnaires, individuals reported smoking status, systolic and diastolic blood pressure values, previous HTN diagnosis, and use of antihypertensive drugs. Logistic regression models were used to estimate the odds ratio and 95% confidence interval between smoking and HTN, stratified by the median dTAC. Current and former smokers had higher dTAC values despite their lower fruit intake. Moreover, coffee was the main contributor to dTAC among them. Smoking was associated with a higher likelihood of HTN, mainly among individuals with a higher dTAC. However, after exclusion of coffee antioxidant capacity, there was an association between only smoking and HTN in individuals with lower dTAC. CONCLUSIONS: The controversial association between higher dTAC and HTN can result from high coffee intake. Higher dTAC without coffee intake may mitigate the association between smoking and HTN in this population.

Synthetic Peptides Derived From Lycosa Erythrognatha Venom: Interaction With Phospholipid Membranes and Activity Against Resistant Bacteria.

Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of µmol L-1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.

Novel components of Tityus serrulatus venom: A transcriptomic approach.

Several research groups have studied the components produced by the venom gland of the scorpion Tityus serrulatus, which has one of the most lethal venoms in the world. Various methodologies have been employed to clarify the complex mechanisms of action of these components, especially neurotoxins and enzymes. Transcriptomes and proteomes have provided important information for pharmacological, biochemical, and immunological research. Next-generation sequencing (NGS) has allowed the description of new transcripts and completion of partial sequence descriptions for peptides, especially those with low expression levels. In the present work, after NGS sequencing, we searched for new putative venom components. We present a total of nine new transcripts with neurotoxic potential (Ts33-41) and describe the sequences of one hyaluronidase (TsHyal_4); three enzymes involved in amidation (peptidyl-glycine alpha-amidating monooxygenase A, peptidyl-alpha-hydroxyglycine alpha-amidating lyase, and peptidylglycine alpha-hydroxylating monooxygenase), which increases the lethal potential of neurotoxins; and also the enzyme Ts_Chitinase1, which may be involved in the venom's digestive action. In addition, we determined the level of transcription of five groups: toxins, metalloproteases, hyaluronidases, chitinases and amidation enzymes, including new components found in this study. Toxins are the predominant group with an expression level of 91.945%, followed by metalloproteases with only 7.790% and other groups representing 0.265%.

Moving Pieces in a Cellular Puzzle: A Cryptic Peptide from the Scorpion Toxin Ts14 Activates AKT and ERK Signaling and Decreases Cardiac Myocyte Contractility via Dephosphorylation of Phospholamban.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Relationship Between Level of Care Dependency and Quality of Life of Family Caregivers of Care-Dependent Patients.

A cross-sectional study was conducted with 139 family caregivers in two community health center areas in the city of Belo Horizonte, located in the southeastern state of Minas Gerais, Brazil. The purpose of the study was to analyze the relationship between level of care dependency and quality of life (QOL) of family caregivers. A socio-demographic questionnaire, the Barthel Index for assessing patients' care dependency level, and the World Health Organization's Quality of Life-BREF (WHOQOL-BREF) for evaluating family caregivers' QOL were used. Patients' care dependency level was not significantly associated with family caregivers' QOL. However, the QOL decreased when family caregivers had chronic disease and were dependent on public health care services, and increased when they received caregiver education and practiced a leisure activity. These factors are modifiable or controllable; therefore, family health nurses have an important role in formulating strategies to support family caregivers, especially those related to health education aimed at health care, health promotion, control, and prevention of diseases.

Dietary Folate Intake Is Negatively Associated with Excess Body Weight in Brazilian Graduates and Postgraduates (CUME Project).

Folate, vitamin B6, and vitamin B12 intake can be important regulators for obesity development. Thus, we investigated the possible association between the intake of these vitamins and the excess body weight or obesity prevalence in the participants of the Cohort of Universities in Minas Gerais (CUME project). This study analyzed cross-sectional data of 2695 graduates and postgraduates from universities in the state of Minas Gerais (801 men, 1894 women, ages 36.2 ± 9.4). The first step consisted of collecting data online, and the second step consisted of blood collecting in the subsample living in the city of Viçosa and its region (Minas Gerais). Excess body weight and obesity prevalence were 38.1% and 10.1%, respectively. Inadequate intake of folate, B6, and B12 were 12, 6.3, and 11.1%, respectively. Beans/lentils and French bread presented the highest contribution to folate intake (23.45% and 10.01%, respectively). Those individuals in the third tertile for folate intake (≥511.12 µg/d) had a lower excess body weight [prevalence ratio (PR): 0.79, confidence interval (CI): 0.71⁻0.8] and obesity prevalence (PR: 0.60, CI: 0.45⁻0.78). These associations were maintained when the sample was categorized by sex. In addition, serum folate was positively associated with dietary folate (p for trend = 0.032) and negatively associated with serum homocysteine (p for trend = 0.003) in the subsample. Dietary folate intake was negatively associated with excess body weight and obesity in CUME participants, indicating the relevance of this vitamin dietary assessment.

The synthetic peptide LyeTxI-b derived from Lycosa erythrognatha spider venom is cytotoxic to U-87 MG glioblastoma cells.

Antimicrobial peptides present a broad spectrum of therapeutic applications, including their use as anticancer peptides. These peptides have as target microbial, normal, and cancerous cells. The oncological properties of these peptides may occur by membranolytic mechanisms or non-membranolytics. In this work, we demonstrate for the first time the cytotoxic effects of the cationic alpha-helical antimicrobial peptide LyeTx I-b on glioblastoma lineage U87-MG. The anticancer property of this peptide was associated with a membranolytic mechanism. Loss of membrane integrity occurred after incubation with the peptide for 15 min, as shown by trypan blue uptake, reduction of calcein-AM conversion, and LDH release. Morphological studies using scanning electron microscopy demonstrated disruption of the plasma membrane from cells treated with LyeTx I-b, including the formation of holes or pores. Transmission electron microscopy analyses showed swollen nuclei with mild DNA condensation, cell volume increase with an electron-lucent cytoplasm and organelle vacuolization, but without the rupture of nuclear or plasmatic membranes. Morphometric analyses revealed a high percentage of cells in necroptosis stages, followed by necrosis and apoptosis at lower levels. Necrostatin-1, a known inhibitor of necroptosis, partially protected the cells from the toxicity of the peptide in a concentration-dependent manner. Imaging flow cytometry confirmed that 59% of the cells underwent necroptosis after 3-h incubation with the peptide. It is noteworthy that LyeTx I-b showed only mild cytotoxicity against normal fibroblasts of human and monkey cell lines and low hemolytic activity in human erythrocytes. All data together point out the anticancer potential of this peptide.

LyeTxI-b, a Synthetic Peptide Derived From Lycosa erythrognatha Spider Venom, Shows Potent Antibiotic Activity in Vitro and in Vivo.

The antimicrobial peptide LyeTxI isolated from the venom of the spider Lycosa erythrognatha is a potential model to develop new antibiotics against bacteria and fungi. In this work, we studied a peptide derived from LyeTxI, named LyeTxI-b, and characterized its structural profile and its in vitro and in vivo antimicrobial activities. Compared to LyeTxI, LyeTxI-b has an acetylated N-terminal and a deletion of a His residue, as structural modifications. The secondary structure of LyeTxI-b is a well-defined helical segment, from the second amino acid to the amidated C-terminal, with no clear partition between hydrophobic and hydrophilic faces. Moreover, LyeTxI-b shows a potent antimicrobial activity against Gram-positive and Gram-negative planktonic bacteria, being 10-fold more active than the native peptide against Escherichia coli. LyeTxI-b was also active in an in vivo model of septic arthritis, reducing the number of bacteria load, the migration of immune cells, the level of IL-1ß cytokine and CXCL1 chemokine, as well as preventing cartilage damage. Our results show that LyeTxI-b is a potential therapeutic model for the development of new antibiotics against Gram-positive and Gram-negative bacteria.

Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host.

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.

Ts14 from Tityus serrulatus boosts angiogenesis and attenuates inflammation and collagen deposition in sponge-induced granulation tissue in mice.

We have previously described a 25mer anti-hypertensive peptide, previously named TsHpt-I (Tityus serrulatus Hypotensin-I), now Ts14, as an agonist of B2 kinin receptor. Bradykinin is known to play physiological roles in angiogenic, inflammatory, and fibrogenic processes, mostly mediated by B2 receptor. Therefore, we investigated whether Ts14 could modulate key events (neovascularization, inflammatory cell recruitment, and extracellular matrix deposition) of the fibrovascular tissue, induced by polyether-polyurethane sponge implants in mice. Sponges were implanted in the dorsum of 7-week-old C57Bl/6 male mice that received daily intrasponge treatment with Ts14 (27.25µg/sponge/day in 10µL PBS) or vehicle (10µL PBS/sponge/day) and were assessed on day 7 after surgery. Hemoglobin content, blood flow (laser Doppler perfusion imaging), and VEGF levels in the implants, used as indices of vascularization, indicated that Ts14 enhanced angiogenesis in implants relative to the PBS-treated group. Interestingly, Ts14 reduced TNF-α levels and neutrophil infiltration, although stimulated macrophage infiltration into implants, as determined by myeloperoxidase (MPO) and N-acetyl-ß-d-glucosaminidase (NAG) enzyme activities, respectively. Regarding the fibrogenic component (soluble collagen content and Sirius-red histological staining), we observed that Ts14 inhibited collagen deposition in the implants. Overall, our results suggest that Ts14 exerts proangiogenic, anti-inflammatory, and anti-fibrogenic activities. These effects may indicate a therapeutical potential of this peptide in conditions where angiogenesis, inflammation, and fibrogenesis contribute to disease progression and chronicity.

Structural and Functional Elucidation of Peptide Ts11 Shows Evidence of a Novel Subfamily of Scorpion Venom Toxins.

To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies of KTxs (called α, ß, γ, κ, δ, and λ-KTxs). Here we report on a previously described orphan toxin from Tityus serrulatus venom, named Ts11. We carried out an in-depth structure-function analysis combining 3D structure elucidation of Ts11 and electrophysiological characterization of the toxin. The Ts11 structure is highlighted by an Inhibitor Cystine Knot (ICK) type scaffold, completely devoid of the classical secondary structure elements (α-helix and/or ß-strand). This has, to the best of our knowledge, never been described before for scorpion toxins and therefore represents a novel, 6th type of structural fold for these scorpion peptides. On the basis of their preferred interaction with voltage-gated K channels, as compared to all the other targets tested, it can be postulated that Ts11 is the first member of a new subfamily, designated as ε-KTx.

Identification of metal-binding to proteins in seed samples using RF-HPLC-UV, GFAAS and MALDI-TOF-MS.

An extraction procedure using Tris-HCl buffer solution was employed in order to extract water-soluble proteins from seed samples of oat, wheat and soybean. Initially, the total protein concentration was determined by the Bradford method in each solution, after the extraction procedure. The soybean sample showed a higher concentration of total protein compared to the others. The protein extracts obtained were separated by reverse-phase chromatography (RP-HPLC-UV). The protein fractions were collected and analyzed by graphite furnace atomic absorption spectrometry (GFAAS) and matrix-assisted laser desorption/ionization (MALDI-TOF-MS) for determination of Cu, Fe, Mn and Zn and identification of proteins, respectively. The combination of techniques such as RP-HPLC-UV, GFAAS and MALDI-TOF-MS allowed the identification of several proteins bound to metals present in the seed samples.

Potassium channel blockers from the venom of the Brazilian scorpion Tityus serrulatus ().

Potassium (K(+)) channels are trans-membrane proteins, which play a key role in cellular excitability and signal transduction pathways. Scorpion toxins blocking the ion-conducting pore from the external side have been invaluable probes to elucidate the structural, functional, and physio-pathological characteristics of these ion channels. This review will focus on the interaction between K(+) channels and their peptide blockers isolated from the venom of the scorpion Tityus serrulatus, which is considered as the most dangerous scorpion in Brazil, in particular in Minas-Gerais State, where many casualties are described each year. The primary mechanisms of action of these K(+) blockers will be discussed in correlation with their structure, very often non-canonical compared to those of other well known K(+) channels blockers purified from other scorpion venoms. Also, special attention will be brought to the most recent data obtained by proteomic and transcriptomic analyses on Tityus serrulatus venoms and venom glands.

δ-Ctenitoxin-Pn1a, a Peptide from Phoneutria nigriventer Spider Venom, Shows Antinociceptive Effect Involving Opioid and Cannabinoid Systems, in Rats.

PnTx4(6-1), henceforth renamed δ-Ctenitoxin-Pn1a (δ-CNTX-Pn1a), a peptide from Phoneutria nigriventer spider venom, initially described as an insect toxin, binds to site 3 of sodium channels in nerve cord synaptosomes and slows down sodium current inactivation in isolated axons in cockroaches (Periplaneta americana). δ-CNTX-Pn1a does not cause any apparent toxicity to mice, when intracerebroventricularly injected (30 µg). In this study, we evaluated the antinociceptive effect of δ-CNTX-Pn1a in three animal pain models and investigated its mechanism of action in acute pain. In the inflammatory pain model, induced by carrageenan, δ-CNTX-Pn1a restored the nociceptive threshold of rats, when intraplantarly injected, 2 h and 30 min after carrageenan administration. Concerning the neuropathic pain model, δ-CNTX-Pn1a, when intrathecally administered, reversed the hyperalgesia evoked by sciatic nerve constriction. In the acute pain model, induced by prostaglandin E2, intrathecal administration of δ-CNTX-Pn1a caused a dose-dependent antinociceptive effect. Using antagonists of the receptors, we showed that the antinociceptive effect of δ-CNTX-Pn1a involves both the cannabinoid system, through CB1 receptors, and the opioid system, through µ and δ receptors. Our data show, for the first time, that δ-Ctenitoxin-Pn1a is able to induce antinociception in inflammatory, neuropathic and acute pain models.

Snacking between main meals is associated with a higher risk of metabolic syndrome in a Mediterranean cohort: the SUN Project (Seguimiento Universidad de Navarra).

OBJECTIVE: To evaluate the association of snacking between main meals with the risk of developing metabolic syndrome. DESIGN: A dynamic prospective cohort study (the SUN Project; Seguimiento Universidad de Navarra). Snack consumption was evaluated using the question: 'Do you have the habit of snacking between main meals?' Metabolic syndrome was defined according to the updated harmonizing criteria. We estimated multivariable-adjusted relative risks (RR) of metabolic syndrome and their 95 % confidence intervals using Poisson regression models. An exploratory factor analysis was also used to identify patterns of snacking. SETTING: University of Navarra, Spain. SUBJECTS: The study included 6851 university graduates, initially free of metabolic syndrome, and followed-up them for a median of 8·3 years. RESULTS: Among our participants, 34·6% reported usual snacking between main meals. The cumulative incidence of metabolic syndrome was 5·1 % (9·5% among men and 2·8% among women). Snacking between main meals was significantly associated with higher risk for developing metabolic syndrome after multivariable adjustment (RR=1·44; 95%CI 1·18, 1·77). Higher adherence to an 'unhealthy snacking pattern' was also independently associated with increased incidence of metabolic syndrome (fourth quartile of adherence compared with non-snacking: RR=1·68; 95% CI 1·23, 2·29; P for trend <0·001). CONCLUSIONS: Our findings suggest that avoidance of snacking between main meals can be included among the preventive approaches to reduce the risk of metabolic syndrome development, especially when snacks contain foods of poor nutritional quality.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

ß/δ-PrIT1, a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria reidyi (F.O. Pickard-Cambridge, 1897).

A potent insecticidal toxin, ß/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. ß/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-ß/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes ß/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, ß/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, ß/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for ß/δ-PrIT1, leading to two different pharmacological responses. ß/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.

Association between yogurt consumption and the risk of metabolic syndrome over 6 years in the SUN study.

BACKGROUND: The role of yogurt consumption in the development of metabolic syndrome (MetS) is not fully understood and the available epidemiologic evidence is scarce. The aim of our study was to assess the association between total, whole-fat, or low-fat yogurt consumption and the risk of developing MetS. METHODS: Yogurt consumption was assessed at baseline through a 136-item validated FFQ. MetS was defined following the harmonized definition for MetS according to the AHA and the IDF criteria. Logistic regression models were used. RESULTS: During the first 6-y of follow-up of the SUN cohort, 306 incident cases of MetS were identified. Frequent consumption [≥875 g/week (≥7 servings/week) versus ≤ 250 g/week (2 servings/week)] of total, whole-fat and low-fat yogurt consumption showed non-significant inverse associations with MetS [OR = 0.84 (95% CI: 0.60-1.18); 0.98 (95% CI: 0.68-1.41); and 0.63 (95% CI: 0.39-1.02) respectively]. Only one component of the MetS, central adiposity, was inversely associated with total and whole-fat yogurt consumption [OR = 0.85 (95% CI: 0.74-0.98) and 0.85 (95% CI: 0.73-0.99) respectively]. In the joint assessment of exposure to total yogurt consumption and fruit consumption, those in the highest category of total yogurt consumption, and having a high fruit consumption (above the median ≥264.5 g/day) exhibited a significantly lower risk of developing MetS [OR = 0.61 (95% CI: 0.38-0.99)] compared with those in the lowest category of total yogurt consumption and had fruit consumption below the study median. CONCLUSION: No significant association between yogurt consumption and MetS was apparent. Only one component out of the 5 MetS criteria, central adiposity, was inversely associated with high yogurt consumption. The combination of high consumption of both yogurt and fruit was inversely associated with the development of MetS.