Nanoporous silica nanoparticles functionalized with a fluorescent turn-on spirorhodamineamide as pH indicators.

We prepared water soluble, biocompatible fluorescent turn-on pH nanosensors and characterized their behavior as a function of changes in pH. The response relies on a halochromic reaction of a spirorhodamineamide derived from the bright and highly chemically and photo-stable rhodamine 6G, encapsulated in core/nanoporous shell silica nanoparticles. The fluorescent sensors displayed a fast response in the pH range of intracellular compartments. The encapsulation conferred solubility in aqueous environments and biocompatibility. We assessed the two main properties of the sensor, namely the useful pH range and the kinetics of the response, and compared them to those of the free probe. We found that such properties are strongly dependent on the functionalization and position in the silica matrix relative to the core/shell structure. Finally, we demonstrated the cellular uptake of the nanosensors, and their localization in lysosomes of living cells, by fluorescence confocal microscopy.

Selective electrocatalytic hydrogenation using palladium nanoparticles electrochemically formed in layer-by-layer multilayer films.

Sequential adsorption of PdCl4(2-) within weak polyelectrolyte layer-by-layer (LbL) self-assembled multilayer films with further electrochemical reduction to yield Pd(0) nanoparticles (Pd-NPs) has been demonstrated. The electrocatalytic hydrogenation (ECH) of model molecules such as acetophenone and benzophenone on Pd-NPs of different sizes (6 to 35 nm) and bulk Pd crystal surface in hydroalcoholic acid solution has been investigated. Distribution of reaction products (secondary alcohols and alkanes) and faradaic yield was systematically investigated. While the polyelectrolyte multilayers act as nanoreactors by confining PdCl4(2-) ions and preventing the formation of large crystals, their presence also alters the hydrogenation reaction and therefore heat treated surfaces showed only the effect of nanocrystal size on the reaction selectivity and faradaic yield.

Delayed graft dislocation after thoracic aortic endovascular repair.

BACKGROUND: Endograft dislocation in thoracic aorta has not been widely studied. OBJECTIVES: The purpose of this study is to analyze the incidence and predisposing factor in a single centre experience after 117 procedures. METHOD: Between November 2000 and December 2011, all consecutive patients undergoing endovascular repair for descending thoracic or thoraco-abdominal aortic disease were identified. Follow-up imaging protocol included triple-phase CT-angiography at 1, 4, and 12 months, and annually thereafter. Migration was defined as proximal/ distal movements >10 mm relative to anatomical landmarks or any movement leading to symptoms or reintervention. RESULT: We identified 117 patients. Mean follow-up was 32 months (range, 1-144). Overall, five (4.3%) patients with thoracic EG dislocation were identified. Dislocation was classified as collapse/infolding in 3 cases and migration in 2. Mean delay of the dislocation was 12.7 months. Only one patient developed symptoms and required an additional endograft. In the group of dislocated endografts, mean age (53 ± 20 vs. 68 ± 15, P = .032) and the diameter of the aortic lesion were lower (4.1 cm ± 1.6 vs. 5.6 cm ± 1.8, P = .069), and the proximal landing zone at "zones 2 and 3" were more frequently used (5 vs. 65, P = 0.81). All but one patient with collapse/infolding are still alive and doing well at a mean follow-up of 80 months. CONCLUSION: Dislocation is an infrequent complication, but not so rare. Young age, small aortic diameter , and proximal sealing at the distal arch were the most important data associated with this complication.

Neural Stem Cells from Shank3-ko Mouse Model Autism Spectrum Disorders.

Autism spectrum disorders (ASD) comprise a complex of neurodevelopmental disorders caused by a variety of genetic defects and characterized by alterations in social communication and repetitive behavior. Since the mechanisms leading to early neuronal degeneration remain elusive, we chose to examine the properties of NSCs isolated from an animal model of ASD in order to evaluate whether their neurogenic potential may recapitulate the early phases of neurogenesis in the brain of ASD patients. Mutations of the gene coding for the Shank3 protein play a key role in the impairment of brain development and synaptogenesis in ASD patients. Experiments here reported show that NSCs derived from the subventricular zone (SVZ) of adult Shank3Δ11-/- (Shank3-ko) mice retain self-renewal capacity in vitro, but differentiate earlier than wild-type (wt) cells, displaying an evident endosomal/lysosomal and ubiquitin aggregation in astroglial cells together with mitochondrial impairment and inflammasome activation, suggesting that glial degeneration likely contributes to neuronal damage in ASD. These in vitro observations obtained in our disease model are consistent with data in vivo obtained in ASD patients and suggest that Shank3 deficit could affect the late phases of neurogenesis and/or the survival of mature cells rather than NSC self-renewal. This evidence supports Shank3-ko NSCs as a reliable in vitro disease model and suggests the rescue of glial cells as a therapeutic strategy to prevent neuronal degeneration in ASD.

The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly.

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.

Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation.

The distribution of the synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on synaptic vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to synaptic vesicles at rest. The value of this ratio was intermediate in synaptic vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with synaptic vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release.

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the variations organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

A cell-free system to study regulation of focal adhesions and of the connected actin cytoskeleton.

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of beta1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of beta1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated beta1 receptors show that the cytoplasmic portion of beta1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified alpha-actinin colocalizes and redistributes with beta1 receptors on ventral plasma membranes depleted of actin, implicating binding of alpha-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

Human platelets express the synaptic markers VGLUT1 and 2 and release glutamate following aggregation.

Vesicular glutamate transporters (VGLUTs) are involved in storing glutamate for secretion at the level of glutamatergic axon terminals, and for this reason they have been extensively used as markers to identify glutamate-releasing cells. Platelets have been considered as a suitable model for studying glutamatergic dysfunction because they perform glutamate uptake and express both external transporters, and NMDA-like receptors. Here, we show that platelets express the pre-synaptic markers VGLUT1 and VGLUT2 and release glutamate following aggregation, implying a possible contributory role in the pathophysiology of stroke, migraine, and other excitotoxic disorders.

Assessing the effects of biomechanical overload on dairy parlor workers' wrist: Definition of a study approach and preliminary results.

BACKGROUND: Dairy milking is a demanding work task that has been associated with hand and wrist musculoskeletal disorders. Clinical approaches to identify the early effects of musculoskeletal disorders among dairy parlor workers' wrist have not been well defined. OBJECTIVES: The purpose of this pilot study was to develop a study protocol that would assist in the identification and quantification of hand and wrist disorders among dairy workers that perform tasks in the dairy parlor. Additionally, such a study protocol was needed to perform relatively rapid assessments of the wrist/hand on large samples of dairy workers. METHODS: Fourteen dairy parlor workers were assessed for i) upper limb symptoms and work history through questionnaire, ii) a physical examination of the upper limb and in particular wrists and iii) wrist ultrasonography. An additional 21 unexposed paired participants (the control group) also participated in the data collection. RESULTS: The study results identified two ultrasound acoustic windows characterized by the highest predictive value for alteration of the wrist's structure. Study results indicated an impairment of the distal median nerve in structure and mobility and impairment of the distal tendon of the muscle extensor carpi ulnaris that included dislocation and frank damage. CONCLUSIONS: This study identified at least two acoustic windows that should be assessed with ultrasound studies on larger groups and in prospective periodical health surveillance of dairy workers. The study confirmed the wrist was at risk for biomechanical stress among workers conducting milking tasks in the dairy parlors.

Staple Line Reinforcement During Laparoscopic Sleeve Gastrectomy: Absorbable Monofilament, Barbed Suture, Fibrin Glue, or Nothing? Results of a Prospective Randomized Study.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is associated with serious complications, such as staple line (SL) leaks and bleeding. In order to prevent the occurrence of these complications, surgeons have advocated the need to strengthen the staple line. The aim of this randomized controlled study was to compare the efficacy of three different ways of strengthening of the SL in LSG in preventing surgical post-operative complications. METHODS: Between April 2012 and December 2014, 600 patients (pts) scheduled for LSG were prospectively randomized into groups without SL reinforcement (group A) or with SL reinforcement including fibrin glue coverage (group B), or oversewn SL with imbricating absorbable (Monocryl™; group C) or barbed (V lock®) running suture (group D). Primary endpoints were post-operative leaks, bleeding, and stenosis, while secondary outcomes consisted of the time to perform the staple line reinforcement (SLR) and total operative time. RESULTS: Mean SLR operative time was lower for group B (3.4 ± 1.3 min) compared with that for groups C (26.8 ± 8.5 min) and D (21.1 ± 8.4 min) (p < 0.0001). Mean total operative time was 100.7 ± 16.4 min (group A), 104.4 ± 22.1 min (group B), 126.2 ± 18.9 min (group C), and 124.6 ± 22.8 (group D) (p < 0.0001). Post-operative leaks, bleeding, and stenosis were recorded in 14 pts (2.3 %), 5 pts (0.8 %), and 7 pts (1.1 %), respectively, without statistical difference between the groups. CONCLUSION: Our study suggests that SLR during LSG, with an imbricating or non-imbricating running suture or with fibrin glue, is an unrewarding surgical act with the sole effect of prolonging the operative time.

Agrin controls synaptic differentiation in hippocampal neurons.

Agrin controls the formation of the neuromuscular junction. Whether it regulates the differentiation of other types of synapses remains unclear. Therefore, we have studied the role of agrin in cultured hippocampal neurons. Synaptogenesis was severely compromised when agrin expression or function was suppressed by antisense oligonucleotides and specific antibodies. The effects of antisense oligonucleotides were found to be highly specific because they were reversed by adding recombinant agrin and could not be detected in cultures from agrin-deficient animals. Interestingly, the few synapses formed in reduced agrin conditions displayed diminished vesicular turnover, despite a normal appearance at the EM level. Thus, our results demonstrate the necessity of agrin for synaptogenesis in hippocampal neurons.

Expression of muscle calsequestrin in epithelial HeLa cells: distribution and functional role.

Calsequestrin is a Ca2+ binding protein expressed by a few cell types (mostly muscle fibers). In these cells the distribution of the protein is within the endoplasmic/sarcoplasmic reticulum, however, not uniformly throughout but at discrete sites of the lumen. In order to investigate the mechanisms of this unusual intracellular distribution together with the possible functions of the protein, we have studied stable transfected clones of epithelial HeLa cells. Treatment of these cells with butyric acid induced a rapid (24 h) and massive (approx. 10-fold) increase of the transfected protein, whereas the other lumenal and membrane proteins of the endoplasmic reticulum were either modified slightly or unchanged. When butyric acid treatment was interrupted the calsequestrin levels returned rapidly (within 24 h) to the pre-treatment level. Such a rapid turnover was due in part to secretion, sustained by both spontaneous and Ca(2+)-dependent release of calsequestrin to the extracellular medium. From the physiological point of view, the transfected cells exhibited only moderate increases of the Ca2+ release responses triggered by either ATP (a ligand addressed to the P2u receptor and working through IP3 generation) or thapsigargin (a blocker of the endoplasmic reticulum Ca2+ ATPase), with no further increase after butyric acid induction of calsequestrin. This result appears to correlate with the occurrence of only small amounts of calsequestrin within the endoplasmic reticulum lumen of all transfected cells. The bulk of calsequestrin, in contrast, was found sequestered within large vacuoles distributed both near the cell surface and, after butyric acid treatment, also in the deep cytoplasm. These vacuoles (possibly a lysosomal subcompartment) appear to contain no Ca2+ as no difference in 45Ca release from transfected cells was observed without or with butyric acid pretreatment when exposed to ionomycin, alone or combined with monensin. We conclude that HeLa cells possess no adequate mechanisms to keep calsequestrin in its physiologically relevant location, the endoplasmic reticulum. In the transfected cell the protein seems therefore to be diverted (possibly by default) to vacuoles destined to be rapidly eliminated by the cell.

Lipid-drug interaction: a structural analysis of pindolol effects on model membranes.

The ternary system constituted by distearoylphosphatidylcholine, pindolol (a vasodilator drug) and water has been investigated by using X-ray diffraction and calorimetric techniques. The structural modifications induced by the drug have been determined and a possible interaction model has been derived. In particular, the pindolol content-temperature dependent phase diagram shows the occurrence of two new phases: the first is an interdigitated gel, and the second is a lamellar structure presenting an unusual mixed disordered-ordered conformation of the hydrocarbon chains (L alpha beta). The comparative analysis of electron density profiles relative to the L alpha beta phase, reveals significant modifications in the paraffinic region of the lipid layer. In agreement with thermodynamic results, the structural data suggest that the drug induces a stiffening and a tightening of the hydrocarbon chains. Moreover, the hydrophilic properties of the membrane (particularly in P beta, and L alpha beta phases) present an evident dependence with the drug concentration.

Reflex cardiovascular control in congestive heart failure.

This article reviews evidence that the reflex control of the cardiovascular system provided by negative feedback mechanisms is impaired in congestive heart failure (CHF). The impairment involves vagal and sympathetic modulation of the heart exerted by arterial baroreceptors. It also affects baroreceptor control of blood pressure and peripheral vascular resistance, as well as the cardiopulmonary receptor's ability to modulate sympathetic activity. The degree of such impairment is most marked in severe CHF but is also apparent, to a minor degree, in mild heart failure. Reflex impairment is due to a reduction in the receptor signal, but other factor under investigation are probably also involved. Digoxin and other pharmacologic treatments of CHF improve reflex function, thereby facilitating a reduction in the elevated sympathetic activity and a stepping up of the reduced vagal activity typical of CHF. This may be relevant to a patient's prognosis.

Osteointegration of bioactive glass-coated zirconia in healthy bone: an in vivo evaluation.

Osteointegration of yttria stabilised tetragonal zirconia (YSTZ), either coated with bioactive glass named RKKP bioglaze (RKKP) or uncoated, was evaluated in an animal model. RKKP-coated and uncoated (controls) YSTZ cylinders were implanted in the distal femoral epiphyses of 14 Sprague Dawley rats under general anaesthesia. At the experimental times of 30 and 60 days after sacrifice, histomorphometry and SEM microanalysis were performed on methylmethacrylate-embedded undecalcified sections to determine the osteointegration rate. At 30 days, a significantly higher affinity index was demonstrated in vivo by histomorphometric evaluation in RKKP-coated versus uncoated YSTZ implants p < 0.05); at 60 days, the coated implants behaved better than controls (affinity index of + 32%), but the difference observed lay within the statistical uncertainty. SEM analysis demonstrated better bone adhesion to the material in RKKP-coated YSTZ at both 30 and 60 days. These findings suggest that YSTZ coated with the bioactive glass named RKKP enhances osteointegration of ceramics.

Thermodynamic and structural effects of propranolol on DPPC liposomes.

Some thermodynamic and structural aspects of propranolol-DPPC liposomes interaction were investigated by DSC and X-ray diffraction: the lamellar arrangement of the lipid matrix remains intact even at high concentrations of the drug (until 1:1 drug/lipid molar ratio). However, the bilayer thickness increases significantly and the chains become perpendicular to the lamellar planes, for increasing drug content. At still higher propranolol concentrations a hexagonal phase occurs followed by a lamellar phase, in which the liposomes are destroyed. Moreover, the presence of propranolol has been found to impart fluidity to the lipid matrix.

The freezing and thawing of water in poultry meat and vegetables irradiated by electrons at doses of 0.1-4 kGy.

Poultry meat and some vegetables, irradiated by 5 MeV electrons (0.1-4 kGy), were analysed by differential scanning calorimetry, from 24 h after irradiation. The temperature and enthalpy transitions of the water contained in the irradiated samples were measured and compared with those of unirradiated samples. We analysed 18 meat and 10 vegetable samples for each irradiation dose together with a similar number of unirradiated controls. The mean supercooling temperatures of water in the irradiated poultry meat samples and in some vegetables are significantly lower than those of controls. Moreover, the freezing enthalpies of the irradiated poultry breast are significantly lower than those of controls, while they are unchanged in the other cases. The mean ice melting temperatures and enthalpies are similar for all samples. The amount of the lowering of the water-ice transition depends on the nature of the sample and is highest in poultry breast and lowest in vegetables.

Osteointegration of bioactive glass-coated and uncoated zirconia in osteopenic bone: an in vivo experimental study.

In elderly and osteoporotic patients an age-related loss of osteoinductivity could be the biological cause of implant failure regardless of the high quality of the implanted device. yttria stabilized tetragonal zirconia (YSTZ), either coated with the bioactive glass named RKKP bioglaze (RKKP) or uncoated, was implanted in the distal femurs of sham-operated and ovariectomized female rats. Animals were sacrificed at 30 and 60 days. Histomorphometry and microhardness tests were performed to assess osteointegration rate as well as bone quality around the implants. Significant decreases (p < 0.0005) in trabecular bone volume, BV/TV (41%), trabecular bone surface BS/TV (33%), trabecular thickness Tb.Th (20%), and trabecular number Tb.N (32%), together with a significant increase in trabecular separation Tb.Sp (184%), were found for the osteopenic rats compared with the sham-operated rats. At both experimental times the RKKP coating ensured a better osteointegration rate with higher AI values than the uncoated YSTZ, even when osteopenic rats were used (48% at 30 days and 12% at 60 days). No differences were observed at the bone-biomaterial interfaces for either material when comparing sham-operated with osteopenic rats. The present results demonstrate that the RKKP bioactive glass used as a coating ensures a high osteointegration rate even in osteoporotic bone, which is already visible from postoperative day 30 and is still apparent on day 60.

Casting immobilized pH gradients into cylindrical polyacrylamide gels.

A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 microliter each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5 degrees to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, followed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90 degrees and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradients is ensured by using stabilizing density gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).