Comparison Between Folic Acid and gH625 Peptide-Based Functionalization of Fe3O4 Magnetic Nanoparticles for Enhanced Cell Internalization.

A versatile synthetic route based on magnetic Fe3O4 nanoparticle (MNP) prefunctionalization with a phosphonic acid monolayer has been used to covalently bind the gH625 peptide on the nanoparticle surface. gH625 is a membranotropic peptide capable of easily crossing the membranes of various cells including the typical human blood-brain barrier components. A similar synthetic route was used to prepare another class of MNPs having a functional coating based on PEG, rhodamine, and folic acid, a well-known target molecule, to compare the performance of the two cell-penetrating systems (i.e., gH625 and folic acid). Our results demonstrate that the uptake of gH625-decorated MNPs in immortalized human brain microvascular endothelial cells after 24 h is more evident compared to folic acid-functionalized MNPs as evidenced by confocal laser scanning microscopy. On the other hand, both functionalized systems proved capable of being internalized in a brain tumor cell line (i.e., glioblastoma A-172). These findings indicate that the functionalization of MNPs with gH625 improves their endothelial cell internalization, suggesting a viable strategy in designing functional nanostructures capable of first crossing the BBB and, then, of reaching specific tumor brain cells.

Egg envelopes of Armadillidium vulgare (Latreille, 1804) (Crustacea, Isopoda Oniscidea): Ultrastructure and lectins binding.

The ultrastructural study carried out on (a) oocytes of Armadillidium vulgare during vitellogenesis, (b) mature eggs taken from the ovaries during the parturial moult of the posterior half of the body, and (c) fertilized eggs collected within a few hours of their release into the brood pouch, has clearly demonstrated that before the fertilization the chorion is the only envelope present in the egg of oniscidean isopods. In the mature eggs, the chorion appears as a uniformly electron-dense lamina, about 0.4-0.5 µm thick, which does not show any specialized area. A second envelope, described by other authors as vitelline envelope, is formed above the oolemma only right after fertilization and appears separated from the chorion by a space full of liquid. The ways in which the genesis of this envelope is realized are not yet clear; it could be interpreted rather as a fertilization membrane. The investigations carried out with the aid of a battery of FITC-lectins have highlighted the presence at the chorion surface of unfertilized eggs of various saccharide residues distributed in uniform way. No significant change was observed in the pattern of lectins binding to the chorion of eggs taken from the brood pouch, thus demonstrating how, after the fertilization, no significant rearrangement in the distribution of saccharide residues present on the egg surface occurs in A. vulgare. The ways in which, therefore, the recognition, the binding and the entry of the peculiar sperm of oniscidean isopods into the egg occur, still remain all to be deciphered. Microsc. Res. Tech. 79:792-798, 2016. © 2016 Wiley Periodicals, Inc.

Multifunctional magnetic nanoparticles for enhanced intracellular drug transport.

In this paper we report the synthesis and characterization of biocompatible multi-functional magnetic nanoparticles (MNPs) able to enhance the intracellular transport of N-methylated drugs. The Fe3O4 magnetic core was first functionalized with a mixed monolayer consisting of two different phosphonic acids having terminal acetylenic and amino groups, which provide an active platform for further functionalization with organic molecules. Then, a tetraphosphonate cavitand receptor (Tiiii) bearing an azide moiety and the N-hydroxysuccinimide (NHS) activated forms of poly(ethylene glycol) (PEG), folic acid (FA) and carboxy-X-rhodamine (Rhod) were covalently anchored on alkyne and amine moieties respectively, through 1,3-dipolar cycloaddition and EDC/NHS coupling reactions. The obtained MNPs are biocompatible and possess magnetic, luminescence and recognition properties which make them suitable for multimodal theranostic applications. In particular, combined confocal microscopy and cytotoxicity experiments showed that these multi-functional MNPs are able to recognize a specific drug "in situ" and promote its cellular internalization, thus enhancing its efficiency.

Bioaccumulation of cadmium and lead and its effects on hepatopancreas morphology in three terrestrial isopod crustacean species.

This study was designed to compare cadmium (Cd) and lead (Pb) bioaccumulation in three species of oniscidean isopods - Armadillidium granulatum Brandt, Armadillidium vulgare (Latreille) and Porcellio laevis Latreille which were exposed for three weeks to a contaminated diet, and to determine the morphological and ultrastructural changes in hepatopancreas. Metal accumulation, determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), was linearly associated with the exposed concentration and was a function of the metal and the species tested. All three species accumulated lower levels of Pb than Cd. A. vulgare accumulated the largest concentration of Pb, especially at the higher doses, whereas P. laevis showed the greatest Cd accumulation, and the highest Cd concentration was lethal for all exposed species. The highest concentrations of Pb and Cd induced significant changes both in the general morphology of tubules and in the ultrastructural organization of epithelial cells in hepatopancreas. Some Pb/Cd induced alterations include: brush border disorganization; reduction of the basal labyrinth formed by the plasma membrane; condensation of some cytoplasm areas and of chromatin; rough endoplasmic reticulum and mitochondrial alterations; increase of secondary lysosomes and of type B granules in S cells. Some of the ultrastructural changes observed overlap with those induced by prolonged starvation, whereas others can be useful biomarkers of heavy metal toxicity. This study has confirmed that in terrestrial isopods, the accumulation of the different metals occurs in a species-specific manner; therefore ecological monitoring and assessment studies should consider each species individually. The research has confirmed that in the terrestrial isopods the accumulation of the different metals occurs in a species-specific way; therefore each species should first be evaluated in view of its employ in biomonitoring programs.

Dispersion of a Nanoliter Bolus in Microfluidic Co-Flow.

Microfluidic systems enable reactions and assays on the scale of nanoliters. However, at this scale nonuniformities in sample delivery become significant. To determine the fundamental minimum sample volume required for a particular device, a detailed understanding of mass transport is required. Co-flowing laminar streams are widely used in many devices, but typically only in the steady-state. Because establishing the co-flow steady-state consumes excess sample volume and time, there is a benefit to operating devices in the transient state, which predominates as the volume of the co-flow reactor decreases. Analysis of the co-flow transient has been neglected thus far. In this work we describe the fabrication of a pneumatically controlled microfluidic injector constructed to inject a discrete 50nL bolus into one side of a two-stream co-flow reactor. Using dye for image analysis, injections were performed at a range of flow rates from 0.5-10µL/min, and for comparison we collected the co-flow steady-state data for this range. The results of the image analysis were also compared against theory and simulations for device validation. For evaluation, we established a metric that indicates how well the mass distribution in the bolus injection approximates steady-state co-flow. Using such analysis, transient-state injections can approximate steady-state conditions within predefined errors, allowing straight forward measurements to be performed with reduced reagent consumption.

Functionalization of PEGylated Fe3O4 magnetic nanoparticles with tetraphosphonate cavitand for biomedical application.

In this contribution, Fe3O4 magnetic nanoparticles (MNPs) have been functionalized with a tetraphosphonate cavitand receptor (Tiiii), capable of complexing N-monomethylated species with high selectivity, and polyethylene glycol (PEG) via click-chemistry. The grafting process is based on MNP pre-functionalization with a bifunctional phosphonic linker, 10-undecynylphosphonic acid, anchored on an iron surface through the phosphonic group. The Tiiii cavitand and the PEG modified with azide moieties have then been bonded to the resulting alkyne-functionalized MNPs through a "click" reaction. Each reaction step has been monitored by using X-ray photoelectron and FTIR spectroscopies. PEG and Tiiii functionalized MNPs have been able to load N-methyl ammonium salts such as the antitumor drug procarbazine hydrochloride and the neurotransmitter epinephrine hydrochloride and release them as free bases. In addition, the introduction of PEG moieties promoted biocompatibility of functionalized MNPs, thus allowing their use in biological environments.

Durability of sustained response shown in paediatric patients with chronic hepatitis C who were treated with interferon alfa-2b plus ribavirin.

Long-term studies in adults indicate that sustained virologic response (SVR) after combination treatment for chronic hepatitis C (CHC) predicts long-term clearance. Although peginterferon plus ribavirin is now standard care for children with CHC, long-term follow-up studies are not yet available. This study evaluated durability of virologic response over 5 years in children previously treated with interferon alfa-2b plus ribavirin (IFN/R). Ninety-seven of 147 children with CHC, who were treated with IFN/R and completed the 6-month follow-up in two previous clinical trials, participated in this long-term follow-up study. All were assessed annually for up to 5 years; patients with SVR were assessed for durability of virologic response. Children with SVR (n = 56) and those with detectable hepatitis C virus (HCV) RNA 24-week post-treatment (n = 41) were followed for a median of 284 weeks. Overall, 70% (68/97) of patients completed the 5-year follow-up. One patient with genotype 1a CHC had SVR and relapsed at year 1 of follow-up with the same genotype. Kaplan-Meier estimate for sustained response at 5 years was 98% (95% CI: 95%, 100%). Six patients with low-positive HCV RNA levels (n = 4) or missing HCV RNA at the 24-week follow-up visit (n = 2) in the initial treatment studies had virologic response during this long-term follow-up study. Linear growth rate was impaired during treatment with rapid increases in the immediate 6 months post-treatment. Mean height percentile at the end of the 5-year follow-up was slightly less than the mean pretreatment height percentile. Five patients experienced serious adverse events; none related to study drug exposure. SVR after IFN/R predicts long-term clearance of HCV in paediatric patients; growth normalized in the majority of children during the long-term follow-up. Similar long-term results could be expected after peginterferon alfa-2b plus ribavirin treatment.

Presence of F-actin in sperm head of Armadillidium peraccae (Isopoda, Oniscidea).

Sperm of Armadillidium peraccae have been examined with cytochemical and immunocytochemical methods for fluorescence and electron microscopic visualization of cytoskeleton components. Sperm incubation in an antibody anti-ß-tubulin shows only the presence of two centrioles located in the cytoplasmic region above the nucleus; no other microtubules are present in the sperm head. Instead, fluorescence microscopy of sperm incubated in FITC-phalloidin allowed to detect the presence of a large amount of F-actin in the apical region of the sperm head. The incubation of ultrathin sections of sperm embedded in Lowicryl K4M with a phalloidin-gold complex allowed a more precise localization of F-actin in the amorphous part of the acrosome and in the cytoplasmic region between acrosome and nucleus; F-actin is also present in the thin cytoplasmic layer between plasma membrane and nuclear envelope at the apical portion of the nucleus. Although the sperm was always found completely devoid of motility, the discovery of the presence of an actin cytoskeleton leads us to hypothesize a possible acquisition of motility by the sperm at the time of its interaction with the female gamete. Such a hypothesis is supported by what is known for ostracods whose aflagellate sperm implement a type of amoeboid movement only at the time of their interaction with the female gamete.

How do biomaterials affect the biological activities and responses of cells? An in vitro study.

AIM: As part of regenerative bone surgery, according to the principles of tissue engineering and GBR, the use of biomaterials aims to restore bone deficiencies by restoring both functionality and original morphology of the bone tissue. Besides being biocompatible, biofunctional and reabsorbable, the ideal scaffolding should possess an osteoinductive geometry, which depends on many physical-chemical characteristics and, in particular, on a three-dimensional morphology and the placement of molecules, which would determine pore size and interconnection between them. The purpose of this study was, therefore, to carry out an analysis with a scanning electron microscope, in order to evaluate the effect of the diameter size of the scaffold pores on the bio-molecular interaction between osteoblast-like MG63 cells and four biomaterials with different pore sizes: polylactic-co-glycolic acid, deproteinized bovine bone, equine bone, demineralized bone matrix (DBM). METHODS: Through the observations made with SEM and X-ray microanalysis, it is possible to infer how the morphology, the proliferative ability, the modality of adhesion and the differentiation of MG63 cells are influenced in different ways by the porous structure of the various biomaterials used as a substrate for growth. RESULTS: From among all of the biomaterials examined, DBM represented the best substrate for growth; in fact, there would be a more intense and precocious adhesion of MG-63 cells. CONCLUSION: The strong osteoconductive effect observed in human DBM, seems to be related to the presence of the organic component, while the osteoinductive effect would be mainly attributed to the presence of BMPs.

Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft.

Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.

Burkholderia cepacia complex in cystic fibrosis and non-cystic fibrosis patients: identification of a cluster of epidemic lineages.

This study was performed in order to compare Burkholderia cepacia complex strains from cystic fibrosis (CF) and non-CF patients at the genomovar, genetic and epidemiological levels. A total of 92 B. cepacia respiratory tract isolates were obtained from patients attending the following CF centres: Catania and Palermo, Sicily; Gualdo Tadino, Central Italy, and Milan, Northern Italy. A total of 23 B. cepacia isolates were obtained from blood, surgical wound, and intravenous catheter sources of patients without CF, hospitalized in Catania and Varese, Northern Italy. Genomovar status identification, clonality and genetic relatedness determination, antibiotic susceptibility pattern determination and electron microscopy were performed. Transmission of infection was shown in both CF and non-CF patients by identifying clonality of responsible strains. In total 13 clones were involved in cross-transmission episodes. No outbreak was described involving both CF and non-CF patients. The present study indicates the existence of a distinct cluster of strains responsible for epidemics in CF and non-CF patients, based on their genetic relatedness, distinct from strains associated with no or negligible transmissibility. This result suggests that transmissibility is not only associated with a specific genomovar in CF patients, but also with a group of genetically related lineages in CF and non-CF patients. A key role is shown for both segregation measures and careful surveillance of infection, based on selective culture, molecular identification and epidemiological characterization of individual isolates.

Bovine seminal RNase induces apoptosis in normal proliferating lymphocytes.

Bovine seminal ribonuclease is a member of the RISBAses (ribonucleases with special biological actions) family. It exerts specific anti-tumor, embryotoxic, aspermatogenic and immunosuppressive activities. The cytotoxic effect of bovine seminal ribonuclease on tumor cells is accompanied by the induction of apoptosis. We provide ultrastructural and flow cytometry evidence of apoptotic death following bovine seminal ribonuclease treatment, in normal cells and phytohemagglutinin-stimulated lymphocytes. Transmission and scanning electron microscopy, which were fully supported by flow cytometry data, showed typical features of apoptosis, such as decreased cell size, chromatin condensation, fragmentation in micronuclei, and the presence of apoptotic bodies.

Liver disease in polyglandular autoimmune disease type one: clinicopathologic study of three patients and review of the literature.

We report the findings from liver biopsies of three patients with polyglandular autoimmune disease type 1. The livers in two patients had histologic features of chronic hepatitis that eventuated in bridging fibrosis and cirrhosis over a period of several years. Nonspecific lobular and portal tract inflammation was present in the liver biopsy of the third patient. Although these patients did not have liver-specific autoantibodies, the liver disease in polyglandular autoimmune disease type 1 possibly represents an expression of autoimmune hepatitis.

Cholestasis in infancy.

It is imperative that serum bilirubin fractionation be performed in any infant whose jaundice is prolonged to identify the infant who has cholestasis. Conjugated hyperbilirubinemia always is pathologic, and a well-organized and expedient diagnostic evaluation should be undertaken to identify those conditions that are treatable medically or surgically if they are recognized early. The complications of prolonged cholestasis need to be recognized and the appropriate medical therapy instituted to allow patients to maximize their growth potential, avoid the problems of nutrient deficiencies, and maintain a reasonable quality of life. Hepatic transplantation now offers the opportunity for long-term survival for infants whose liver disorders previously were fatal.

Rectal mucosal major basic protein in infants with dietary protein-induced colitis.

Dietary protein-induced colitis is a frequent cause of rectal bleeding in infants. The exact pathogenic mechanism is unknown but the disorder has been thought to be due to an allergic response. Rectal mucosal edema and eosinophilia are typically found but there are no specific markers currently available. Because eosinophil degranulation, as evidenced by the release of major basic protein, has been implicated in hypersensitivity disorders, we aimed to assess major basic protein deposition as a marker of dietary protein-induced colitis occurring in young infants. Suction rectal biopsies from five infants aged 1 to 7 months with findings consistent with dietary protein-induced colitis were compared histologically with five age matched controls who underwent rectal biopsies to rule out Hirschsprung's disease. An established indirect immunofluorescent staining method was used to identify tissue major basic protein. Comparable rectal deposition of major basic protein was found for the controls and colitic patients. Mucosal eosinophilia but not mast cell content was more prominent in the colitic patients (P < .05) than in the controls. Some of the colitic infants had elevated serum IgE levels (1 of 5), positive RAST for milk (2 of 5), and peripheral blood eosinophilia (1 of 5). Our findings do not support the concept that dietary protein-induced colitis of infancy is due solely to an immediate hypersensitivity response. The results also indicate that major basic protein is probably not a marker or likely primary mediator of this disorder.

Multicenter trial of d-alpha-tocopheryl polyethylene glycol 1000 succinate for treatment of vitamin E deficiency in children with chronic cholestasis.

BACKGROUND: Malabsorption and deficiency of vitamin E causing neurological degeneration are common consequences of chronic childhood cholestatic liver disease. The objective of this study was to determine the long-term efficacy and safety of d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) in correcting vitamin E deficiency in children with chronic cholestasis who were unresponsive to other forms of oral vitamin E. METHODS: Sixty vitamin E-deficient children with chronic cholestasis unresponsive to 70-212 IU.kg-1.day-1 of oral vitamin E were entered into a trial at eight centers in the United States. After initial evaluation, treatment was started with 25 IU.kg-1.day-1 of TPGS. Vitamin E status, neurological function quantitated by a specific scoring system, and clinical and biochemical parameters were monitored during therapy. RESULTS: All children responded to TPGS with normalization of vitamin E status. Neurological function, which had deteriorated before entry in the trial, improved in 25 patients, stabilized in 27, and worsened in only 2 after a mean of 2.5 years of therapy. No adverse effects were observed. CONCLUSIONS: TPGS (20-25 IU.kg-1.day-1) appears to be a safe and effective form of vitamin E for reversing or preventing vitamin E deficiency during chronic childhood cholestasis.