Early left ventricular abnormality/dysfunction in obese children affected by NAFLD.

BACKGROUND AND AIMS: Although it is generally accepted that non alcoholic fatty liver disease (NAFLD) is linked to increased risk of cardiovascular disease, the presence of abnormalities in cardiac function among NAFLD children is limited and controversial. Aim of the study was to detect cardiac abnormalities/dysfunction in a paediatric population of NAFLD. METHODS AND RESULTS: Anthropometric, laboratory, cardiovascular fitness, 24 h blood pressure monitoring and Doppler echocardiography parameters were obtained in 50 untreated children (37 males; mean age 12.2 + 2.5) with biopsy-proven NAFLD. Abnormalities in both cardiac function and geometry could be identified in the whole study population: prevalence of about 35% in left ventricular hypertrophy, 14% of concentric remodelling and 16% of left atrial dilatation. Furthermore children with NAFLD (NAS score <5) showed lower cardiac alterations compared to NASH patients (NAS score >5). After adjusting for age, sex and BMI, a positive correlation was found only between LV mass and NAS score (p < 0.001). CONCLUSION: Our results suggest that cardiac dysfunction can be detectable early in NAFLD children and this is not linked to cardiovascular and metabolic alteration, other than to liver damage. Although as a preliminary stage, we can speculate a possible direct relationship between liver and heart steatosis, already occurring during childhood.

Galectin-3 expression in thyroid fine needle cytology (t-FNAC) uncertain cases: validation of molecular markers and technology innovation.

Thyroid cancer is not very common, accounting for 1-2% of all cancers, with a population incidence of about 0.004%. Currently, the ability to discriminate between follicular adenoma and carcinoma represents the major challenge in preclinical diagnosis of thyroid proliferative lesions. Better discrimination between the two would help avoid unnecessary thyroidectomy and save valuable resources. Over the years, galectin-3 (Gal-3) has been proposed as a diagnostic marker with varied success. In this paper, we used Environmental Scanning Electron Microscopy Immunogold Labelling (ESEM-IGL) to investigate the expression of Gal-3 on Thin-Prep fine needle aspiration cytology (FNAC). We optimized the ESEM-IGL method on thyroid cell lines (RO-82 and FTC-133) comparing our membrane Gal-3 labeling data with Western blot. We evaluated 183 thyroid FNAC from Italian patients with a uncertain pre-surgical diagnosis. ESEM-IGL method marker sensitivity is 71.2%, while specificity is 53.3% and diagnostic efficacy is 61.2%. Our results confirmed that Gal-3 expression is associated with situations of hypertrophy and/or cellular hyperproliferation, pathophysiological situations common both to adenomas and to thyroid carcinomas. The innovation of thyroid FNAC Thin-Prep ESEM-IGL shows the levels of Gal-3 immunolabeling clearly, even through the individual cells of a thyroid nodule. However, Gal-3 alone, as a molecular marker of thyroid cancer, can still have a limited application in pre-surgery diagnosis.

Immunogold labelling in environmental scanning electron microscopy: applicative features for complementary cytological interpretation.

We have combined environmental scanning electron microscopy (ESEM) and immunogold labelling (IGL) for the analysis of cell morphology and surface protein detection on human fine needle aspiration, which is processed in thin uniform monolayer (a single layer of cells) on a glass slide by Thin Prep technology. Among scanning electron microscopy techniques, we choose the environmental modality (ESEM) because it allows a slight manipulation of biological samples and an operational time comparable with cytological techniques. Moreover, the Thin Prep technology confirmed a reproducible cell monolayer on glass smear, minimizing problems for the determination of appropriate amount of material per slide. The first experimental data in ESEM-IGL on biological samples with fine needle aspiration Thin Prep, in human thyroid nodules, showed that cells retained their morphology and provided a clear IGL. The optimization of conditions (i.e. vacuum pressure, temperature and relative humidity) confirmed the possibility to observe an immunolabelled biological sample and morphological signal, joined with compositional informations, due to peculiar characteristics of gaseous secondary electron detector in ESEM. The ESEM-IGL and fine needle aspiration Thin Prep could be used in combination for the interpretation of cell morphology and cell surface immunolabelling. Our paper suggests this use as a powerful diagnostic tool in a pre-surgical evaluations, opening a new applicative window for electron microscopy.

Non-equilibrium turbulence scalings and self-similarity in turbulent planar jets.

We study the self-similarity and dissipation scalings of a turbulent planar jet and the theoretically implied mean flow scalings. Unlike turbulent wakes where such studies have already been carried out (Dairay et al. 2015 J. Fluid Mech. 781, 166-198. (doi:10.1017/jfm.2015.493); Obligado et al. 2016 Phys. Rev. Fluids 1, 044409. (doi:10.1103/PhysRevFluids.1.044409)), this is a boundary-free turbulent shear flow where the local Reynolds number increases with distance from inlet. The Townsend-George theory revised by (Dairay et al. 2015 J. Fluid Mech. 781, 166-198. (doi:10.1017/jfm.2015.493)) is applied to turbulent planar jets. Only a few profiles need to be self-similar in this theory. The self-similarity of mean flow, turbulence dissipation, turbulent kinetic energy and Reynolds stress profiles is supported by our experimental results from 18 to at least 54 nozzle sizes, the furthermost location investigated in this work. Furthermore, the non-equilibrium dissipation scaling found in turbulent wakes, decaying grid-generated turbulence, various instances of periodic turbulence and turbulent boundary layers (Dairay et al. 2015 J. Fluid Mech. 781, 166-198. (doi:10.1017/jfm.2015.493); Vassilicos 2015 Annu. Rev. Fluid Mech. 95, 114. (doi:10.1146/annurev-fluid-010814-014637); Goto & Vassilicos 2015 Phys. Lett. A 3790, 1144-1148. (doi:10.1016/j.physleta.2015.02.025); Nedic et al. 2017 Phys. Rev. Fluids 2, 032601. (doi:10.1103/PhysRevFluids.2.032601)) is also observed in the present turbulent planar jet and in the turbulent planar jet of (Antonia et al. 1980 Phys. Fluids 23, 863055. (doi:10.1063/1.863055)). Given these observations, the theory implies new mean flow and jet width scalings which are found to be consistent with our data and the data of (Antonia et al. 1980 Phys. Fluids 23, 863055. (doi:10.1063/1.863055)). In particular, it implies a hitherto unknown entrainment behaviour: the ratio of characteristic cross-stream to centreline streamwise mean flow velocities decays as the -1/3 power of streamwise distance in the region, where the non-equilibrium dissipation scaling holds.

Vitronectin absorbed on nanoparticles mediate cell viability/proliferation and uptake by 3T3 Swiss albino mouse fibroblasts: in vitro study.

We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5 mg/mL results for NPs. However, NPs in the range 0.01-0.30 mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1 µg/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation.

Energy expenditure and insulin sensitivity evaluation in obese children affected by hepatosteatosis.

OBJECTIVES: The aim of our study was to evaluate the physical and sedentary activities and energy expenditure (EE) in a group of children affected by non-alcoholic fatty liver disease (NAFLD), compared with normal and obese subjects, using a physical activity questionnaire (PAQ) and a SenseWear armband (SWA). METHODS: Forty NAFLD (10 females), 41 lean (NRM; 11 females) and 30 obese (OB; 10 females), age- and pubertal stage-matched, children were included. RESULTS: Sedentary activity (PAQ) was similar in NAFLD and NRM but less in OB, while SWA showed that NAFLD spent less time in physical activity and more in sedentary activities compared with NRM, but not with OB. Insulin sensitivity index result is related to active EE (cal kg(-1) d(-1) ) in NAFLD, while homeostatic model assessment index result was negatively related to total EE in OB. CONCLUSIONS: Regular physical activity must be encouraged in all obese children affected by NAFLD or not, and SWA might be a possible valid tool for evaluating actual EE.

Impaired energy expenditure despite normal cardiovascular capacity in children with type 1 diabetes.

BACKGROUND: Benefit of fitness on children with type 1 diabetes mellitus (T1DM) is still debated. AIM: To evaluate the influence of physical activity on metabolic balance and exercise tolerance in prepubertal children affected by T1DM. METHODS: We analyzed 35 pre-/peripubertal T1DM children and 31 matched controls using an activity monitor (SenseWear Armbad) and physical activity questionnaire (PAQ) to assess energy expenditure (EE), total and active, sedentary and physical activities (h/day and Mets = metabolic equivalents). The maximal cardiopulmonary exercise test (CPET) was also performed. RESULTS: Total physical activities and total and active EE (>3 Mets) resulted higher in controls than in T1DM patients and self-reported perception of physical and sedentary activities was altered in T1DM children as well in controls and were different from the measured data. No differences were found in CPET parameters with the exception of a higher maximal blood pressure in T1DM children. In multivariate analysis HbA1c negatively correlated with VO(2). CONCLUSION: Prepubertal T1DM children seem to have a lower level of physical activity and EE and a probable altered feeling of physical and sedentary activities. On the other hand, T1DM children do not show any alteration of cardiovascular performance, although glycemic control (HbA1c) may play a role in cardiovascular performance.

Impression cytology with scanning electron microscopy: a new method in the study of conjunctival microvilli.

PURPOSE: Recent studies used impression cytology with scanning electron microscopy (SEM) to study the conjunctival surface of bovine eyes and normal human eyes. The purpose of this study was to evaluate the use impression cytology and SEM (ICSEM) in patients affected by tear film abnormalities. METHODS: Forty-five patients were divided into three groups according to mild, moderate or severe subjective sensation of dry eye. Fifteen asymptomatic subjects served as control group. In all patients the tear film was evaluated with break-up time (BUT), Schirmer's, and Ferning test, whereas conjunctival epithelium was evaluated with impression cytology and optic microscopy (ICOM), and ICSEM. The Spearman rank correlation test was used to compare the outcome of these examinations with the subjective sensation of dry eye in each group, and to identify correlations among the five tests. RESULTS: ICSEM findings highly correlated with subjective dry eye sensation (Spearman correlation coefficient, 796; P<0.01). ICSEM revealed incipient epithelial damage (reduction or absence of microvilli) before the appearance of alterations of nucleus and cytoplasm of epithelial cells revealed by optic microscopy. The number of microvilli was correlated with the degree of tear film abnormalities and subjective sensation of dry eye (Spearman correlation coefficient, 796; P<0.01). CONCLUSION: ICSEM was very effective in detecting the reduction in the number of microvilli. Therefore, it could represent an effective method to detect alterations in the conjunctival epithelium resulting from tear film damage even before the epithelial damage occurs and is detected by optic microscopy.

Scanning electron microscopy of the tunica albuginea of the corpora cavernosa in normal and impotent subjects.

The tunica albuginea (TA) of the penis is thought to play a major role in the erection mechanism. It functions by compressing the subalbugineae venulae, which promotes the slower venous flow during erection, and provides a fibrous frame to give an inextensible support for the vessels and nerves. It acts as the inextensible enclosing structure which contains the erectile tissue and gives the erect penis its shape. The functions of the TA result from its structure, consisting for the most part of collagenic and elastic fibers. This study investigated, with the aid of scanning electron microscopy (SEM), the microarchitecture of the TA and the spatial relation of its fibers in ten impotent patients and in six control subjects with normal erectile function. The arrangement of elastic fibers in the TA seems to account for their function, which is to prevent the overstretching of collagenic fibers during maximum intracavernous pressure. In impotent patients, a reduction in the elastic fibers in the TA appears to produce disorders in the arrangement of the collagenic fibers. These alterations in the architecture of the TA in impotent patients can give rise top erection disorders.

Potential allelochemicals fromRuta graveolens L. and their action on radish seeds.

An aqueous extract ofRuta graveolens L. (250 g/liter) was tested for its allelopathic activity in vitro on radish germination and radicle growth in light and darkness. It caused a delay in the onset and a decrease in the rate of germination (40%) in the light. The photoinhibition of germination was accompanied by an inhibition of water uptake into the seed. Furthermore, the inhibition of radicle growth was slightly higher in the light than in darkness. Three potential allelochemicals, biologically active in the light, were isolated from the extract: 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and 4-hydroxy-coumarin at concentrations of 10(-4) M, 2×10(-4) M, and 0.4 ×10(-5) M respectively. At a concentration of 2×10(-4) M, 5-MOP was the most potent inhibitor, decreasing radish germination to 32% and radicle growth to 17% with respect to control. Microscopic observations of radish seeds treated with 5-MOP suggest that this substance changes the swelling of the seed coat and aleurone layer, which precedes radicle protrusion.

Inhibition of radish germination and root growth by coumarin and phenylpropanoids.

Thirteen natural and synthetic phenylpropanoids as well as coumarin (2×10(4)M) were tested for their biological activity on radish germination and subsequent root growth in light and darkness. Coumarin was the most potent inhibitor. With some exceptions, phenylpropanoids with a carboxylic group in the side chain inhibited root growth. Coumarin was formed spontaneously by photooxidation of 2-hydroxycinnamic acid. Microscopic observations of root treated with coumarin suggest that this substance inhibits the elongation of cells of the differentiating zone of the root.

Allelic association of gene markers on chromosome 11q in Italian families with atopy.

UNLABELLED: In our study, the genetic linkage of the Fcepsilon RIbeta gene with atopy in 77 affected sibling pairs recruited from an Italian panel of 201 subjects has been examined. Atopy was defined by the presence of a positive skin prick test to one or more common aeroallergens, a positive RAST test to one or more common aeroallergens and an elevated circulating total IgE. Genotype analysis was performed by PCR amplification of Fcepsilon RIbeta CA and CI11-319 CA microsatellites. All the family members were also tested for the Ilepsilon 181 mutation with the ARMS method and for Leu181/Leu183 polymorphism. Seventy-two point five percent (72.5%) of the affected sibling pairs shared their maternal allele and 27.5% did not. Therefore, an increased maternal allele sharing was observed: chi2 = 8.10, p < 0.01. Comparing paternal versus maternal allele sharing, a significant difference was observed for the C1II-319 CA marker (chi2 = 4.32, p < 0.05). Atopy phenotype with positive skin prick test, RASTs, and high total serum IgE also showed greater sharing of maternal than paternal alleles in affected sibling pairs. Of the 201 subjects studied, 17 (8.4%) were positive for Leu181. Ten of these were children and seven (70%) had inherited the variant maternally. The seven children had maternally inherited Leu181/Leu183 and were atopic. Within this sample the maternal inheritance of Fcepsilon RIbeta Leu181/Leu183 was associated with an increased risk of IgE responses to common allergens, raised eosinophil counts and increased skin prick test reactions. Therefore, the variant identified a genetic risk factor for atopy. CONCLUSION: The central role of Fcepsilon RIbeta in atopy and the linkage data presented here point to the possibility that genetic variation in Fcepsilon RIbeta or its controlling element may cause differences in the extent of IgE responses between atopic and non-atopic subjects. A search for such mutations or polymorphisms will need to take into account some carriers of atopy among the normal population and genetic heterogeneity among atopic individuals.

Accumulation, localisation, and toxic effects of cadmium in the liverwort Lunularia cruciata.

Accumulation, tissue and intracellular localisation, and toxic effects of cadmium were investigated in the liverwort Lunularia cruciata. The results of analyses carried out by atomic absorption spectrometry on single plants showed that the cadmium accumulation was dose- and time-dependent. Cadmium localisation was assessed by X-ray scanning electron microscopy microanalysis in gemmalings and in the different tissues of the thallus and by X-ray transmission electron microscopy microanalysis at the cellular level. The metal preferentially accumulated in the hyaline parenchyma and at the base of the gemma cups. Inside the cell, cadmium accumulated in the vacuoles and the cell wall. Metal accumulation was accompanied by a concomitant increase in sulphur content within the vacuoles of stressed cells. Gel-permeation chromatography showed that most of the cadmium was associated with a low-molecular-mass fraction eluting at a ratio of elution volume to void volume corresponding to that of phytochelatins. The excess of sulphur deposited in the vacuoles may well have been caused by the stress-induced synthesis of phytochelatins. At the ultrastructural level, sublethal concentrations of cadmium caused alterations of the fine structure of the cells, inducing marked alterations of the chloroplast structure. Cadmium also induced a dose-dependent inhibition of apical thallus growth and gemma germination.

Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus).

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.