A simple approach to synthetize folic acid decorated magnetite@SiO2 nanostructures for hyperthermia applications.

Superparamagnetic magnetite nanoparticles were synthetized and capped by a SiO2 shell in order to avoid oxidation and aggregation of the iron oxide nanostructures. The inorganic capping was then further decorated by folic acid molecules, by using a very simple procedure exploiting supramolecular interactions among the organic moieties and the inorganic nanoparticles. The supramolecular nanoadduct thanks to folic acid molecules could act as a "Trojan horse" for the cancer cells and due to its superparamagnetic properties could induce local heat generation upon an appropriate magnetic field application. In fact, temperature was increased up to 42 °C when a 18 mT magnetic field was applied to the nanoparticles and the hybrid nanostructures were verified to be selectively internalized by HeLa cells, a human cervical cancer line known to overexpress the folic acid receptor.

Long-term Results of 18 Fat Injections in Unilateral Vocal Fold Paralysis.

OBJECTIVE: The purposes of this study were to assess the long-term efficacy of medialization laryngoplasty via injection of autologous fat in patients with unilateral laryngeal paralysis, and to discuss the results based on the volume of fat injected and the size of the initial glottic gap. STUDY DESIGN: This was a retrospective study with clinical reevaluation of 18 patients treated for unilateral laryngeal paralysis at Liège University Hospital between April 1, 2011, and December 1, 2014. METHOD: The voice examination included subjective scales (GRBAS and the Voice Handicap Index), a report on acoustic and aerodynamic voice parameters, and laryngostroboscopic assessment of glottic closure and mucosal wave (T0: preoperative; T1: immediate postoperative; T2: >1 year). RESULTS: The median follow-up duration was 19 months. We observed a significant improvement in maximum phonation time; mean flow rate; jitter; the Voice Handicap Index; the G, R, B, and A subscales of the GRBAS-I; and glottic closure at T1. These parameters remained stable over time, without any significant difference except for shimmer and glottic closure between T1 and T2. Regardless of the volume of fat injected and the magnitude of the initial glottic gap, we did not see any significant difference from one assessment time to another. CONCLUSION: Medialization laryngoplasty by means of the injection of autologous fat is a safe, effective surgical technique for patients with unilateral laryngeal paralysis regardless of the size of the initial glottic gap. The results are maintained longer than 1 year, and no evidence of significant resorption of the fat was found in our study over a period of 12-58 months. The volume of fat injected does not seem to affect the functional results.

Drastic nickel ion removal from aqueous solution by curcumin-capped Ag nanoparticles.

A completely green synthesis protocol has been adopted to obtain silver nanoaggregates capped by the natural compound (1E, 6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-diene), also known as curcumin. The synthesis has been monitored by infrared, Raman, visible and fluorescence spectroscopies. Characterization confirms that curcumin reduces and caps the nanoparticles, and such a procedure allows its solubility in water and drastically increases curcumin stability. Silver nanoparticles (AgNPs)/curcumin complex has been dispersed in a water solution containing a known nickel ion concentration. After three days, a grey precipitate is observed and nickel concentration in the solution is reduced by about 70%.

Diet choice by goats as effect of milk production level during late lactation.

The diet self-regulation ability of goats during late lactation has been studied with regard to their production level. Two groups of seven Girgentana goats producing 1100 ± 157 g/day (H group) and 613 ± 138 g/day (L group) were housed in individual pens and were given alfalfa pelleted hay (1.5 kg), whole grains of maize (0.5 kg), barley (0.5 kg), faba bean (0.5 kg) and pelleted sunflower cake (0.5 kg) on a daily basis. During a 7-day pre-experimental period, goats received a mixed ration based on the same feeds used during the experimental period (1.5 kg of hay and 0.4 kg of each concentrate). Individual choice of feeds was continuously recorded for 7 days using a 24-h IR video surveillance system equipped with four video cameras. The nutrient intake in both groups was much higher than needed. Goats in the H group ate more (2016.3 v. 1744.3 g dry matter (DM)/day) and selected less hay (26.9% v. 34.6% DM), more high-protein feeds (faba bean and sunflower cake: 14.0% and 15.9% v. 8.8% and 7.9% DM, respectively) and less maize (21.5% v. 25.0% DM), reaching a higher CP concentration in the diet (17.3% v. 15.0% DM) compared with the goats in the L group. During the 24-h trial period, hay was more constantly selected (on average never reaching <20% of the total hourly basis feeding time, apart from the first hour after feed administration) compared with concentrate feeds. This feeding behaviour has probably exercised a 'curative' effect that enabled the goats to continue to take in very high levels of starch and protein, without manifesting any symptom of metabolic disease. Shifting goats from the pre-experimental diet, based on a mixture of the same feeds used during the experimental period, to the free-choice feeding caused more than 20% increase in milk production in both groups. From the results of the intake, we are unable to conclude that the goats can select their diet to meet their requirements, as goats consumed much more than needed. However, when free to choose their diet, the animals improved milk performance, despite the late-lactation stage.

The restriction of grazing duration does not compromise lamb meat colour and oxidative stability.

Over 72 days, 33 lambs were fed: concentrates in stall (S), grass at pasture for 8 hours (8 h), or grass at pasture for 4 hours in the afternoon (4h-PM). The 4h-PM treatment did not affect the carcass yield compared to the 8h treatment. Meat colour development after blooming was unaffected by the treatments. The 4 h-PM treatment increased the proportion of polyunsaturated fatty acids (PUFA; P<0.0005) and of the highly peroxidizable fatty acids (HP-PUFA; P<0.001) in meat compared to the 8h treatment. The S treatment increased lipid oxidation (higher TBARS values) and impaired colour stability (higher H* values) of meat over storage compared to the 8h and 4 h-PM treatments (P<0.0005 and P=0.003, respectively). No difference in meat oxidative stability was found between the 8h and the 4h-PM treatments. In conclusion, growing lambs can tolerate a restriction of grazing duration without detrimental effects on performances and meat oxidative stability.

Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytes.

PURPOSE: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. METHODS: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. RESULTS: RHIgM22 co-localized with integrin ß3 but not other integrin ß-chains in OLs. Downstream of integrin ß3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin αvß3 and PDGFαR. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. CONCLUSIONS: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation.

Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein.

The clearance of apoptotic cells by phagocytes is a fundamental process during tissue remodeling and resolution of inflammation. In turn, the phagocytosis of apoptotic cells generates signals that suppress pro-inflammatory activation of macrophages. These events occur during the resolution phase of inflammation and therefore the malfunctioning of this process may lead to inflammation-related tissue damage. Here, we demonstrate that the calcium-binding protein S100A9, normally abundant in the cytoplasm of neutrophils and also released by apoptotic neutrophils, is involved in the suppression of macrophages after the uptake of apoptotic neutrophils. Both, spontaneous and induced production of inflammatory species (nitric oxide, hydrogen peroxide and TNF-alpha) as well as the phagocytic activity were inhibited when macrophages were in presence of apoptotic neutrophils, conditioned medium from neutrophil cultures or a peptide corresponding to the C-terminal region of S100A9 protein. On the other hand, macrophages kept in the conditioned medium of neutrophils that was previously depleted of S100A9 were shown to resume the activated status. Finally, we demonstrate that the calcium-binding property of S100A9 might play a role in the suppression process, since the stimulation of intracellular calcium release with ionomycin significantly reversed the effects of the uptake of apoptotic neutrophils in macrophages. In conclusion, we propose that S100A9 is a novel component of the regulatory mechanisms of inflammation, acting side-by-side with other suppressor factors generated upon ingestion of apoptotic cells.

The C-terminus of murine S100A9 inhibits spreading and phagocytic activity of adherent peritoneal cells.

OBJECTIVE AND DESIGN: In the present study, the effect of a synthetic peptide (H(92)-G(102)) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on adherent peritoneal cell function. MATERIALS AND METHODS: For in vitro assays, peritoneal cells were obtained from the abdominal cavity of mice and incubated, with the different concentrations of mS100A9p, for 1 h, and then their spreading and phagocytosis activities were evaluated. For ex-vivo assays, cells obtained from animals treated for 1 h with the peptide were submitted to the mannose-receptor phagocytosis assay. Shorter homologue peptides to the C-terminus of mS100A9p were also evaluated on in vitro phagocytosis assays of Candida albicans particles. RESULTS: mS100A9p reduced both the spreading index and phagocytic activity, in vitro and ex-vivo, independent of the receptor evaluated. The homologue peptide corresponding to the H(92)-E(97) region of mS100A9p, the zinc-binding motif, was responsible for such an effect. CONCLUSION: These results suggest a modulator effect of the C-terminus of S100A9 protein on the function of adherent peritoneal cells.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion.

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

Understanding pre- and post-hysterectomy levels of negative affect: a stress moderation model approach.

Before and after hysterectomy, 60 women completed self-report questionnaires. Measures of personality (NEO-Five Factor Inventory, NEO-FFI), coping (Coping Inventory for Stressful Situations, CISS), and procedure appraisal were completed pre-operatively. Measures of depression and anxiety were completed pre- and post-operatively. Pre-op, 34% of women reported depression at clinical levels, and 29% reported clinical anxiety. The prevalence of depression fell to 8% 3-months post-op although clinical levels of anxiety persisted post-op in 22% of women. Regression analyses revealed that the principal risk factors for post-op negative affect were pre-op levels of depression and concerns about hysterectomy outcome. In assessing proposed models of post-hysterectomy outcome, structural equational modelling revealed the key position of neuroticism and extraversion, which were both directly and indirectly related to pre- and post-operative depression and anxiety. The mediating variables in this model included coping dispositions and procedure appraisal. It is concluded that the variables contained within stress moderation models provide a useful framework for understanding the processes that may lead to elevated levels of negative affect both before and after hysterectomy. Such an approach may prove beneficial for other surgical-outcome studies.

VIN 3: a clinicopathologic review.

A retrospective review of the management of vulvar intraepithelial neoplasia 3 (VIN 3) over a 16-year period from 1981 to 1997 was conducted. Complete information was available for analysis on 101 patients. The mean age was 53.9 years (range 14-102 years). The mean duration of follow-up was 36 months (range 2-184 months). Fifty-eight percent of patients presented with pruritus. The disease was multifocal in 51% and unifocal in 49% of cases and the left labium majus was the most frequently affected site (27%). Co-existent or previous genital disease was identified in 39% of patients and 8% had a history of invasive gynecological cancer. Histologic evidence of human papillomavirus (HPV) infection was found in 31% of patients. Wide local excision was the most frequently used treatment modality (78%). Thirty-eight percent of patients required at least one further treatment for recurrent disease. Smoking, multifocality, HPV effect, and positive surgical margins were not found to be significant predictors of recurrence. There were three (3%) cases of progression to invasive squamous cell carcinoma of the vulva, one at 6, 7, and 7 years after initial treatment.

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases.

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Deformation-induced lipid trafficking in alveolar epithelial cells.

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.

Membrane traffic in sphingolipid storage diseases.

In this review, we summarize our studies of membrane lipid transport in sphingolipid storage disease (SLSD) fibroblasts. We recently showed that several fluorescent SL analogs were internalized from the plasma membrane predominantly to the Golgi complex of normal cells, while in ten different SLSD cell types, these lipids accumulated in endosomes and lysosomes (The Lancet 1999;354: 901-905). Additional studies showed that cholesterol homeostasis is perturbed in multiple SLSDs secondary to SL accumulation and that mistargeting of SL analogs was regulated by cholesterol (Nature Cell Biol 1999;1: 386-388). Based on these findings, we hypothesize that endogenous sphingolipids, which accumulate in SLSD cells due to primary defects in lipid catabolism, result in an altered intracellular distribution of cholesterol, and that this alteration in membrane composition then results in defective sorting and transport of SLs. The importance of SL/cholesterol interactions and potential mechanisms underlying the regulation of lipid transport and targeting are also discussed. These studies suggest a new paradigm for regulation of membrane lipid traffic along the endocytic pathway and could have important implications for future studies of protein trafficking as well as lipid transport. This work may also lead to important future clinical developments (e.g. screening tests for SLSD, new methodology for screening drugs which abrogate lipid storage, and possible therapeutic approaches to SLSD).

Broad screening test for sphingolipid-storage diseases.

BACKGROUND: Lipid-storage diseases are collectively important because they cause substantial morbidity and mortality, and because they may present as dementia, major psychiatric illness, developmental delay, or cerebral palsy. At present, no single assay can be used as an initial general screen for lipid-storage diseases. METHODS: We used a fluorescent analogue of lactosylceramide, called N-[5-(5,7-dimethylborondipyrromethenedifluoride)-1-pentanoyl]D- lactosylsphingosine (BODIPY-LacCer), the emission of which changes from green to red wavelengths with increasing concentrations in membranes, to examine the intracellular distribution of the lipid within living cells. FINDINGS: During a brief pulse-chase experiment, the fluorescent lipid accumulated in the lysosomes of fibroblasts from patients with Fabry's disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs and Sandhoff forms), metachromatic leucodystrophy, mucolipidosis type IV, Niemann-Pick disease (types A, B, and C), and sphingolipid-activator-protein-precursor (prosaposin) deficiency. In control cells, the lipid was mainly confined to the Golgi complex. In a masked study, replicate samples of 25 of 26 unique cell lines representing ten different lipid-storage diseases, and 18 of 20 unique cell lines representing controls were correctly identified; the sensitivity was 96.2% (95% CI 80.4-99.9) and the specificity 90.0% (68.3-98.8). INTERPRETATION: This method may be useful as an initial general screen for lipid-storage diseases, and, with modification, could be used for large-scale automated screening of drugs to abrogate lysosomal storage in various lipidoses. The unexpected accumulation of BODIPY-LacCer in several biochemically distinct diseases raises important questions about common mechanisms of cellular dysfunction in these disorders.