Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7.

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 µm, and Nav1.5 is inactive at 10 µm We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

The novel collagen matrikine, endotrophin, is associated with mortality and cardiovascular events in patients with atherosclerosis.

BACKGROUND: Rupture of atherosclerotic plaques is the major cause of acute cardiovascular events. The biomarker PRO-C6 measuring Endotrophin, a matrikine of collagen type VI, may provide valuable information detecting subjects in need of intensified strategies for secondary prevention. OBJECTIVE: In this study, we evaluate endotrophin in human atherosclerotic plaques and circulating levels of PRO-C6 in patients with atherosclerosis, to determine the predictive potential of the biomarker. METHODS: Sections from the stenotic human carotid plaques were stained with the PRO-C6 antibody. PRO-C6 was measured in serum of patients enrolled in the Carotid Plaque Imagining Project (CPIP) (discovery cohort, n = 577) and the innovative medicines initiative surrogate markers for micro- and macrovascular hard end-points for innovative diabetes tools (IMI-SUMMIT, validation cohort, n = 1,378). Median follow-up was 43 months. Kaplan-Meier curves and log-rank tests were performed in the discovery cohort. Cox proportional hazard regression analysis (HR with 95% CI) was used in the discovery cohort and binary logistic regression (OR with 95% CI) in the validation cohort. RESULTS: PRO-C6 was localized in the core and shoulder of the atherosclerotic plaque. In the discovery cohort, PRO-C6 independently predicted future cardiovascular events (HR 1.089 [95% CI 1.019 -1.164], p = 0.01), cardiovascular death (HR 1.118 [95% CI 1.008 -1.241], p = 0.04) and all-cause death (HR 1.087 [95% CI 1.008 -1.172], p = 0.03). In the validation cohort, PRO-C6 predicted future cardiovascular events (OR 1.063 [95% CI 1.011 -1.117], p = 0.017). CONCLUSION: PRO-C6 is present in the atherosclerotic plaque and associated with future cardiovascular events, cardiovascular death and all-cause mortality in two large prospective cohorts.

Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

STUDY QUESTION: What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)? SUMMARY ANSWER: The diagnostic performance of sequencing a gene panel based on genes associated with mouse azoospermia was relatively successful in idiopathic NOA patients and allowed the discovery of two novel genes involved in NOA due to meiotic arrest. WHAT IS KNOWN ALREADY: NOA is a largely heterogeneous clinical entity, which includes different histological pictures. In a large proportion of NOA, the aetiology remains unknown (idiopathic NOA) and yet, unknown genetic factors are likely to play be involved. The mouse is the most broadly used mammalian model for studying human disease because of its usefulness for genetic manipulation and its genetic and physiological similarities to man. Mouse azoospermia models are available in the Mouse Genome Informatics database (MGI: http://www.informatics.jax.org/). STUDY DESIGN, SIZE, DURATION: The first step was to design of a 'mouse azoospermia' gene panel through the consultation of MGI. The second step was NGS analysis of 175 genes in a group of highly selected NOA patients (n = 33). The third step was characterization of the discovered gene defects in human testis tissue, through meiotic studies using surplus testicular biopsy material from the carriers of the RNF212 and STAG3 pathogenic variants. The final step was RNF212 and STAG3 expression analysis in a collection of testis biopsies. PARTICIPANTS/MATERIALS, SETTING, METHODS: From a total of 1300 infertile patients, 33 idiopathic NOA patients were analysed in this study, including 31 unrelated men and 2 brothers from a consanguineous family. The testis histology of the 31 unrelated NOA patients was as follows: 20 Sertoli cell-only syndrome (SCOS), 11 spermatogenic arrest (6 spermatogonial arrest and 5 spermatocytic arrest). The two brothers were affected by spermatocytic arrest. DNA extracted from blood was used for NGS on Illumina NextSeq500 platform. Generated sequence data was filtered for rare and potentially pathogenic variants. Functional studies in surplus testicular tissue from the carriers included the investigation of meiotic entry, XY body formation and metaphases by performing fluorescent immunohistochemical staining and immunocytochemistry. mRNA expression analysis through RT-qPCR of RNF212 and STAG3 was carried out in a collection of testis biopsies with different histology. MAIN RESULTS AND THE ROLE OF CHANCE: Our approach was relatively successful, leading to the genetic diagnosis of one sporadic NOA patient and two NOA brothers. This relatively high diagnostic performance is likely to be related to the stringent patient selection criteria i.e. all known causes of azoospermia were excluded and to the relatively high number of patients with rare testis histology (spermatocytic arrest). All three mutation carriers presented meiotic arrest, leading to the genetic diagnosis of three out of seven cases with this specific testicular phenotype. For the first time, we report biallelic variants in STAG3, in one sporadic patient, and a homozygous RNF212 variant, in the two brothers, as the genetic cause of NOA. Meiotic studies allowed the detection of the functional consequences of the mutations and provided information on the role of STAG3 and RNF212 in human male meiosis. LIMITATIONS, REASONS FOR CAUTION: All genes, with the exception of 5 out of 175, included in the panel cause azoospermia in mice only in the homozygous or hemizygous state. Consequently, apart from the five known dominant genes, heterozygous variants (except compound heterozygosity) in the remaining genes were not taken into consideration as causes of NOA. We identified the genetic cause in approximately half of the patients with spermatocytic arrest. The low number of analysed patients can be considered as a limitation, but it is a very rare testis phenotype. Due to the low frequency of this specific phenotype among infertile men, our finding may be considered of low clinical impact. However, at an individual level, it does have relevance for prognostic purposes prior testicular sperm extraction. WIDER IMPLICATIONS OF THE FINDINGS: Our study represents an additional step towards elucidating the genetic bases of early spermatogenic failure, since we discovered two new genes involved in human male meiotic arrest. We propose the inclusion of RNF212 and STAG3 in a future male infertility diagnostic gene panel. Based on the associated testis phenotype, the identification of pathogenic mutations in these genes also confers a negative predictive value for testicular sperm retrieval. Our meiotic studies provide novel insights into the role of these proteins in human male meiosis. Mutations in STAG3 were first described as a cause of female infertility and ovarian cancer, and Rnf212 knock out in mice leads to male and female infertility. Hence, our results stimulate further research on shared genetic factors causing infertility in both sexes and indicate that genetic counselling should involve not only male but also female relatives of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS (grant number: FIS/FEDER-PI14/01250; PI17/01822) awarded to CK and AR-E, and by the European Commission, Reproductive Biology Early Research Training (REPROTRAIN, EU-FP7-PEOPLE-2011-ITN289880), awarded to CK, WB, and AE-M. The authors have no conflict of interest.

A short-term increase in dietary cholesterol and fat intake affects high-density lipoprotein composition in healthy subjects.

BACKGROUND AND AIMS: High-cholesterol and high-fat diets alter biochemical composition and anti-oxidant properties of high-density lipoproteins (HDL) in animals. Whether this occurs in humans is unknown. Therefore, we examined the effect of a short-term elevation in dietary cholesterol and fat intake on HDL composition in healthy subjects. METHODS AND RESULTS: In a randomized, crossover clinical trial, 14 healthy young volunteers followed a 14-day low-cholesterol/low-fat diet (LChF) and a 14-day isocaloric high-cholesterol/high-fat diet (HChF) in a random order. After each diet, we measured HDL concentrations of hydroxyeicosatetraenoic acids (HETE), hydroxyoctadecadienoic acids (HODE), and haptoglobin, as well as serum amyloid A (SAA) and paroxonase-1 activity (PON-1). HDL concentrations of 15-HETE (+254%, p = 0.002), 5-HETE (+116%, p = 0.004), 13-HODE (+102%, p = 0.049), and SAA levels (+75%, p = 0.007) were significantly higher after the HChF than after the LChF. Furthermore, haptoglobin was marginally increased (+32%, p = 0.091) while PON-1 activity was unaffected (-16%, p = 0.366) by the HChF. CONCLUSION: In healthy subjects, a short-term elevation in dietary cholesterol and fat intake increases HDL lipid hydroperoxide content (15-HETE, 5-HETE, 13-HODE) and SAA levels, which are key features of dysfunctional HDL. This is the first study showing that a physiologic manipulation of dietary cholesterol and fat intake affects HDL lipidome and proteome in healthy subjects independently of weight changes. CLINICAL TRIAL REGISTRATION: NCT02549144.

Dental pulp regeneration via cell homing.

The typical treatment for irreversibly inflamed/necrotic pulp tissue is root canal treatment. As an alternative approach, regenerative endodontics aims to regenerate dental pulp-like tissues using two possible strategies: cell transplantation and cell homing. The former requires exogenously transplanted stem cells, complex procedures and high costs; the latter employs the host's endogenous cells to achieve tissue repair/regeneration, which is more clinically translatable. This systematic review examines cell homing for dental pulp regeneration, selecting articles on in vitro experiments, in vivo ectopic transplantation models and in situ pulp revascularization. MEDLINE/PubMed and Scopus databases were electronically searched for articles without limits in publication date. Two reviewers independently screened and included papers according to the predefined selection criteria. The electronic searches identified 46 studies. After title, abstract and full-text examination, 10 articles met the inclusion criteria. In vitro data highlighted that multiple cytokines have the capacity to induce migration, proliferation and differentiation of dental pulp stem/progenitor cells. The majority of the in vivo studies obtained regenerated connective pulp-like tissues with neovascularization. In some cases, the samples showed new innervation and new dentine deposition. The in situ pulp revascularization regenerated intracanal pulp-like tissues with neovascularization, innervation and dentine formation. Cell homing strategies for pulp regeneration need further understanding and improvement if they are to become a reliable and effective approach in endodontics. Nevertheless, cell homing currently represents the most clinically viable pathway for dental pulp regeneration.

Testicular sperm extraction after laparoscopic orchiectomy for bilateral postpubertal intra-abdominal cryptorchidism: What chance of sperm retrieval?

Infertility occurs in up to 54% of men with bilateral undescended testes. Orchiectomy is considered the best therapeutic approach, especially when cryptorchidism is diagnosed in adulthood, due to a high risk of malignancy. A 33-year-old man was referred with a clinical presentation of empty scrotum and an ultrasonography and magnetic resonance imaging evaluation of intra-abdominal bilateral cryptorchidism. Follicle-stimulating hormone was 23.20 IU/L, luteinising hormone was 14.10 IU/L, total testosterone was 12.1 nmol/L, and 17-beta-oestradiol was 0.16 nmol/L. Semen analysis showed absolute azoospermia. Tumour marker levels were in the normal range. Testicular volume was 4.0 ml for right testis and 4.6 ml for left testis. The patient underwent a laparoscopy bilateral orchiectomy and subsequently a testicular sperm extraction (TESE), in the purpose to finding mature spermatozoa. The biological examination revealed the presence of immature sperm cells, not efficient for a cryopreservation. The histologic analyses show a pattern of Sertoli cell-only syndrome and maturation arrest. TESE might be a good option for patients with absolute azoospermia and cryptorchidism, especially if bilateral. The procedure, performed after orchiectomy, is safe and does not have any impact on patient's health, although it is important to clarify the very low potential of sperm recovery.

Are hormone measurements and ultrasounds really predictors of sperm retrieval in testicular sperm extraction? A case report and literature review.

Azoospermia can be diagnosed in about 10%-15% of the infertile male population. To overcome the problem of failure to produce spermatozoa in the ejaculate in patients with nonobstructive azoospermia (NOA), testicular sperm extraction (TESE) may be performed to find the focal area of spermatogenesis. A 47-year-old man with NOA presented for treatment of secondary couple infertility. The patient underwent a first TESE 7 years earlier with cryopreservation, and an intracytoplasmic sperm injection-embryo transfer ended in a term pregnancy. He reported a history of repeated testicular traumas. At the present time, a complete medical workup was carried out, including clinical history, general and genital physical examination, scrotal and transrectal ultrasounds. Hormone measurements showed follicle-stimulating hormone level of 42.7 IU/L, luteinising hormone of 11.4 IU/L, total testosterone of 2.6 ng/ml and right and left testicular volume, respectively, of 4 and 3.9 ml. He underwent a second TESE, with successful sperm retrieval and cryopreservation. The histological pattern was hypospermatogenesis. In cases of extreme testicular impairment, although in the presence of very high follicle-stimulating hormone value and small testicular volume, estimating poor sperm recovery potential, the integration of clinical and anamnestic data, could help the surgeon to practise the more appropriate method of treatment.

Metformin effects on the heart and the cardiovascular system: A review of experimental and clinical data.

BACKGROUND: Metformin, the eldest and most widely used glucose lowering drug, is likely to be effective also on cardiac and vascular disease prevention. Nonetheless, uncertainty still exists with regard to its effects on the cardiovascular system as a whole and specifically on the myocardium, both at the organ and cellular levels. METHODS: We reviewed the available data on the cardiac and vascular effects of metformin, encompassing both in vitro, either tissue or isolated organ, and in vivo studies in experimental animals and humans, as well as the evidence generated by major clinical trials. RESULTS: At the cellular level metformin's produces both AMP-activated kinase (AMPK) dependent and independent effects. At the systemic level, possibly also through other pathways, this drug improves endothelial function, protects from oxidative stress and inflammation, and from the negative effects of angiotensin II. On the myocardium it attenuates ischemia-reperfusion injury and prevents adverse remodeling induced by humoral and hemodynamic factors. The effects on myocardial cell metabolism and contractile function being not evident at rest or in more advanced stages of cardiac dysfunction, could be relevant during transient ischemia, during an acute increase in workload and in the early stages of diabetic/hypertensive cardiomyopathy as confirmed by few small clinical trials and some observational studies. The overall evidence emerging from both clinical trials and real world registry is in favor of a protective effect of metformin with respect to both coronary events and progression to heart failure. CONCLUSIONS: Given this potential, its efficacy and its safety (and also its low cost) metformin remains the central pillar of the therapy of diabetes.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

Measures of atherosclerotic burden are associated with clinically manifest cardiovascular disease in type 2 diabetes: a European cross-sectional study.

BACKGROUND: There is a need to develop and validate surrogate markers of cardiovascular disease (CVD) in subjects with diabetes. The macrovascular changes associated with diabetes include aggravated atherosclerosis, increased arterial stiffness and endothelial dysfunction. The aim of this study was to determine which of these factors is most strongly associated with clinically manifest cardiovascular events. METHODS: Vascular changes were measured in a cohort of 458 subjects with type 2 diabetes (T2D) and CVD (myocardial infarction, stroke or lower extremity arterial disease), 527 subjects with T2D but without clinically manifest CVD and 515 subjects without T2D and with or without CVD. RESULTS: Carotid intima-media thickness (IMT) and ankle-brachial pressure index were independently associated with the presence of CVD in subjects with T2D, whereas pulse wave velocity and endothelial function provided limited independent additive information. Measurement of IMT in the carotid bulb provided better discrimination of the presence of CVD in subjects with T2D than measurement of IMT in the common carotid artery. The factors most significantly associated with increased carotid IMT in T2D were age, disease duration, systolic blood pressure, impaired renal function and increased arterial stiffness, whereas there were no or weak independent associations with metabolic factors and endothelial dysfunction. CONCLUSIONS: Measures of atherosclerotic burden are associated with clinically manifest CVD in subjects with T2D. In addition, vascular changes that are not directly related to known metabolic risk factors are important in the development of both atherosclerosis and CVD in T2D. A better understanding of the mechanisms involved is crucial for enabling better identification of CVD risk in T2D.

Carotenoid consumption is related to lower lipid oxidation and DNA damage in middle-aged men.

The present cross-sectional study assessed the potential relationships of carotenoid intake with lipid and oxidative stress markers in middle-aged men. A total of 296 apparently healthy middle-aged men (mean age 50.5 (SD 5.0) years, BMI 25.8 (SD 3.5) kg/m(2)) were recruited to participate in the study. Dietary intake, anthropometry, blood pressure, lifestyle features, blood and urine biomarkers were assessed using validated procedures. The lipid markers included NEFA, Castelli index, and TAG:HDL ratio; oxidative stress markers included urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-iso-PGF2α and plasma oxidised-LDL (ox-LDL). We observed a significant inverse association (P < 0.05) between NEFA concentrations and consumption of lutein plus zeaxanthin, ß-carotene, α-carotene and total carotenoid, while Castelli index was negatively associated with daily intake of lycopene, ß-carotene and total carotenoids. Regarding oxidative stress biomarkers, urinary 8-OHdG and ox-LDL concentrations were also inversely associated (P < 0.05) with consumption of lycopene, lutein plus zeaxanthin, ß-carotene, α-carotene and total carotenoids, regardless of confounding variables. Moreover, there was a negative association of urinary 8-iso-PGF2α concentration with dietary lutein plus zeaxanthin (ß - 0.135, 95% CI - 0.268, - 0.001), ß-carotene (ß - 0.156, 95% CI - 0.277, - 0.034) and with the sum of all carotenoids (ß - 0.189, 95% CI - 0.333, - 0.046). In conclusion, total daily carotenoid intake based on five investigated carotenoid types (ß-cryptoxanthin, lycopene, lutein plus zeaxanthin, ß-carotene and α-carotene) was inversely associated with relevant lipid and oxidative stress markers in middle-aged men, with emphasis on ß-carotene that was negatively associated with five of the six lipid and oxidative stress markers evaluated in the present study.

Mutations in voltage-gated potassium channel KCNC3 cause degenerative and developmental central nervous system phenotypes.

Potassium channel mutations have been described in episodic neurological diseases. We report that K+ channel mutations cause disease phenotypes with neurodevelopmental and neurodegenerative features. In a Filipino adult-onset ataxia pedigree, the causative gene maps to 19q13, overlapping the SCA13 disease locus described in a French pedigree with childhood-onset ataxia and cognitive delay. This region contains KCNC3 (also known as Kv3.3), encoding a voltage-gated Shaw channel with enriched cerebellar expression. Sequencing revealed two missense mutations, both of which alter KCNC3 function in Xenopus laevis expression systems. KCNC3(R420H), located in the voltage-sensing domain, had no channel activity when expressed alone and had a dominant-negative effect when co-expressed with the wild-type channel. KCNC3(F448L) shifted the activation curve in the negative direction and slowed channel closing. Thus, KCNC3(R420H) and KCNC3(F448L) are expected to change the output characteristics of fast-spiking cerebellar neurons, in which KCNC channels confer capacity for high-frequency firing. Our results establish a role for KCNC3 in phenotypes ranging from developmental disorders to adult-onset neurodegeneration and suggest voltage-gated K+ channels as candidates for additional neurodegenerative diseases.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (µ-TRTX-Hh2a).

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

Characterization of the anisotropic mechanical behaviour of colonic tissues: experimental activity and constitutive formulation.

The aim was to investigate the biomechanical behaviour of colonic tissues by a coupled experimental and numerical approach. The wall of the colon is composed of different tissue layers. Within each layer, different fibre families are distributed according to specific spatial orientations, which lead to a strongly anisotropic configuration. Accounting for the complex histology of the tissues, mechanical tests must be planned and designed to evaluate the behaviour of the colonic wall in different directions. Uni-axial tensile tests were performed on tissue specimens from 15 fresh pig colons, accounting for six different loading directions (five specimens for each loading direction). The next step of the investigation was to define an appropriate constitutive framework and develop a procedure for identification of the constitutive parameters. A specific hyperelastic formulation was developed that accounted for the multilayered conformation of the colonic wall and the fibre-reinforced configuration of the tissues. The parameters were identified by inverse analyses of the mechanical tests. The comparison of model results with experimental data, together with the evaluation of satisfaction of material thermomechanics principles, confirmed the reliability of the analysis developed. This work forms the basis for more comprehensive activities that aim to provide computational tools for the interpretation of surgical procedures that involve the gastrointestinal tract, considering the specific biomedical devices adopted.

The ideal blood pressure target to prevent cardiovascular disease in type 2 diabetes: a neutral viewpoint.

Type 2 diabetes mellitus (T2DM) and essential hypertension are often associated, and retrospective data analyses suggest an association between lower blood pressure (BP) values and lower cardiovascular (CV) risk in patients with T2DM. However, the most recent intervention trials fail to demonstrate a further CV risk reduction, for BP levels <130/80 mm Hg, when compared to levels <140/90 mm Hg. Moreover, a J-shaped, rather than a linear, relationship of BP reduction with incident CV events has been strongly suggested. We here debate the main available evidences for and against the concept of 'the lower the better', in the light of the main intervention trials and meta-analyses, with a particular emphasis on the targets to be pursued in elderly patients. Finally, the most recent guidelines of the scientific societies are critically discussed.

Impact of mild to moderate reductions of glomerular filtration rate on coronary artery disease severity.

BACKGROUND & AIMS: The bases of the link between reduced glomerular filtration rate (GFR) and coronary artery disease (CAD) are complex and to some extent still unclear. We performed this observational, single referral center, cohort study to evaluate whether mild to moderate GFR reduction is associated with more severe CAD and/or with a worse cardiac prognosis independently of proteinuria, diabetes and traditional risk factors. METHODS AND RESULTS: In 1752 consecutive non-diabetic patients without proteinuria or moderate/severe kidney disease undergoing a clinically driven coronary angiography, coronary arteries lesions, myocardial function and hypertrophy and 10-yrs incidence of cardiac events and death were evaluated in relation to classes of estimated GFR defined according the lowest eGFR value (105+, 90+, 75+, 60+, 45+). A reduced eGFR was independently associated with hypertension, myocardial hypertrophy and stress induced ischemia, while the excess coronary lesions and the worse myocardial systolic function were both largely explained by age and cardiovascular risk factors. When compared to subjects 75+, both the risk of cardiac death (1.67[1.10-2.57] and 3.06[1.85-5.10]) and non-fatal myocardial infarction (2.58[1.12-6.49] and 2.73[1.31-6.41]) adjusted for age and comorbidities were higher in eGFR 60+ and 45+ patients. CONCLUSIONS: A mild-moderate reduction of eGFR is closely associated to higher rates of stress-induced ischemia, myocardial hypertrophy and higher risk of fatal and non-fatal cardiac events. The associations of reduced eGFR with coronary atherosclerosis and myocardial systolic dysfunction are both largely explained by age and traditional risk factors.

HDL lipid composition is profoundly altered in patients with type 2 diabetes and atherosclerotic vascular disease.

BACKGROUND AND AIMS: We have previously shown that the anti-inflammatory and anti-oxidant functions of HDL are impaired in T2D patients. In this study, we examined whether HDL from T2D patients contains elevated levels of oxidized fatty acids and whether those levels correlate with cardiovascular disease (CVD). METHODS AND RESULTS: HETEs and HODEs on HDL were determined by LC-MS/MS in 40 non-diabetic controls (ND), 40 T2D without CVD (D⁺CVD⁻) and 38 T2D with known history of CVD (D⁺CVD⁺). HDL oxidant index was evaluated by a cell-free assay using dichlorofluorescein. Twenty-six randomly selected subjects from the three groups underwent coronary calcium score evaluation (CAC). Major cardiovascular risk factors were similar among the groups. HETEs and HODEs content were significantly increased in HDL from D⁺CVD⁺ when compared to D⁺CVD⁻ and ND patients. HDL oxidant index was not different among the three groups; however, it was significantly higher in patients with CAC score >100 when compared to patients with CAC score <100. CONCLUSION: Patients with D⁺CVD⁻ and D⁺CVD⁺ are characterized by a severe, graded enrichment of oxidized fatty acids on HDL. In the present study, a loss of HDL function (as estimated by the HDL oxidant index) is observed only in patients with more advanced atherosclerosis.

Investigation of the mechanical behaviour of the foot skin.

BACKGROUND/PURPOSE: The aim of this work was to provide computational tools for the characterization of the actual mechanical behaviour of foot skin, accounting for results from experimental testing and histological investigation. Such results show the typical features of skin mechanics, such as anisotropic configuration, almost incompressible behaviour, material and geometrical non linearity. The anisotropic behaviour is mainly determined by the distribution of collagen fibres along specific directions, usually identified as cleavage lines. METHODS: To evaluate the biomechanical response of foot skin, a refined numerical model of the foot is developed. The overall mechanical behaviour of the skin is interpreted by a fibre-reinforced hyperelastic constitutive model and the orientation of the cleavage lines is implemented by a specific procedure. Numerical analyses that interpret typical loading conditions of the foot are performed. The influence of fibres orientation and distribution on skin mechanics is outlined also by a comparison with results using an isotropic scheme. RESULTS: A specific constitutive formulation is provided to characterize the mechanical behaviour of foot skin. The formulation is applied within a numerical model of the foot to investigate the skin functionality during typical foot movements. Numerical analyses developed accounting for the actual anisotropic configuration of the skin show lower maximum principal stress fields than results from isotropic analyses. CONCLUSION: The developed computational models provide reliable tools for the investigation of foot tissues functionality. Furthermore, the comparison between numerical results from anisotropic and isotropic models shows the optimal configuration of foot skin.

Systemic inhibition of nitric oxide synthesis in non-diabetic individuals produces a significant deterioration in glucose tolerance by increasing insulin clearance and inhibiting insulin secretion.

AIMS/HYPOTHESIS: Endogenous NO inhibits insulin release in isolated beta cells and insulin-degrading enzyme activity in hepatocytes, while NO release from endothelial cells has been suggested to enhance insulin action. We assessed the overall effect of systemic inhibition of endogenous NO synthesis on glucose homeostasis in humans. METHODS: Twenty-four non-diabetic volunteers underwent two hyperglycaemic (+7 mmol/l) clamps with either saline or L-NG-nitroarginine methyl ester (L-NAME, at rates of 2.5, 5, 10 and 20 µg min⁻¹ kg⁻¹) infusion. Another five volunteers underwent an OGTT with either saline or L-NAME (20 µg min⁻¹ kg⁻¹) infusion. Blood pressure and heart rate were measured to monitor NO blockade; during the OGTT, endothelial function was assessed by peripheral arterial tonometry and insulin secretion by C-peptide deconvolution and insulin secretion modelling. RESULTS: Compared with saline, L-NAME at the highest dose raised mean blood pressure (+20 ± 2 mmHg), depressed heart rate (-12 ± 2 bpm) and increased insulin clearance (+50%). First-phase insulin secretion was impaired, but insulin sensitivity (M/I index) was unchanged. During the OGTT, L-NAME raised 2 h plasma glucose by 1.8 mmol/l (p < 0.01), doubled insulin clearance and impaired beta cell glucose sensitivity while depressing endothelial function. CONCLUSIONS/INTERPRETATION: In humans, systemic NO blockade titrated to increase blood pressure and induce endothelial dysfunction does not affect insulin action but significantly impairs glucose tolerance by increasing plasma insulin clearance and depressing insulin secretion, namely first-phase and beta cell glucose sensitivity.