Surface decoration with gH625-membranotropic peptides as a method to escape the endo-lysosomal compartment and reduce nanoparticle toxicity.

The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.

Fluorescent (rhodamine), folate decorated and doxorubicin charged, PEGylated nanoparticles synthesis.

PEGylated silica nanoparticles, giving very stable aqueous sols, were successfully functionalised with rhodamine, one of the more stable fluorophore; they were also decorated with the targeting agent folic acid (FA) and charged with the well known drug doxorubicin. Rhodamine functionalization required a modification of the synthesis route of the nanoparticles (NP). Functionalization with FA required activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Folate decorated NP were easily charged with doxorubicin. The experimental results proved the successfulness of the functionalization. The bond to the NP does not reduce the therapeutic efficacy of the drug. The calculated encapsulation efficiency (32 %) was only a little lower than the value (47 %) reported for the very popular PEGylated PLGA NP.

Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters.

Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design "cell instructive" materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm(-1)) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribution of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gradient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.

Toxicological properties of nanoparticles of organic compounds (NOC) from flames and vehicle exhausts.

We examined the biological reactivity in vitro of nanoparticles of organic compounds (NOC) with diameters, d = 1-3 nm, a class of combustion-generated particulate relatively unstudied compared to larger more graphitic soot particles because of their small size even though they may contribute significantly to the organic fraction of PM sampled from vehicle exhausts and urban atmospheres. We tested NOC samples collected from 2004 model vehicle emissions and laboratory flames. NOC produced a dose dependent mutagenic response in Salmonella bacteria, suggesting that NOC may add significantly to the overall mutagenicity of vehicle emissions. Incubation with peptides caused agglomeration and precipitate of the otherwise stable NOC suspension, but the chemical and/or physical nature of the NOC-peptide interactions could not be resolved. A significant cytotoxic response was measured above a critical dose of NOC in mouse embryo fibroblasts NIH3T3 cells along with possible evidence of cellular uptake by optical and confocal microscopy. The toxicological assays showed that NOC collected from flames and vehicle exhausts effectively interacted in vitro with both prokaryotic and eukaryotic cells. Differences in mutagenic potencies observed for various Salmonella strains with and without metabolic activation indicate differences in the chemical composition of NOC collected from different vehicles and flames.

Engineered mu-bimodal poly(epsilon-caprolactone) porous scaffold for enhanced hMSC colonization and proliferation.

The use of scaffold-based strategies in the regeneration of biological tissues requires that the design of the microarchitecture of the scaffold satisfy key microstructural and biological requirements. Here, we examined the ability of a porous poly(epsilon-caprolactone) (PCL) scaffold with novel bimodal-micron scale (mu-bimodal) porous architecture to promote and guide the in vitro adhesion, proliferation and three-dimensional (3-D) colonization of human mesenchymal stem cells (hMSCs). The mu-bimodal PCL scaffold was prepared by a combination of gas foaming (GF) and selective polymer extraction (PE) from co-continuous blends. The microarchitectural properties of the scaffold, in particular its morphology, porosity distribution and mechanical compression properties, were analyzed and correlated with the results of the in vitro cell-scaffold interaction study, carried out for 21days under static conditions. Alamar Blue assay, scanning electron microscopy, confocal laser scanning microscopy and histological analyses were performed to assess hMSC adhesion, proliferation and 3-D colonization. The results showed that the combined GF-PE technique allowed the preparation of PCL scaffold with a unique multiscaled and highly interconnected microarchitecture that was characterized by mechanical properties suitable for load-bearing applications. Study of the cell-scaffold interaction also demonstrated the ability of the scaffold to support hMSC adhesion and proliferation, as well as the possibility to promote and guide 3-D cell colonization by appropriately designing the microarchitectural features of the scaffold.

Effects of fibronectin and laminin on structural, mechanical and transport properties of 3D collageneous network.

Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN- and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN- and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.

Platelet function and anesthetics in cardiac surgery: an in vitro and ex vivo study.

UNLABELLED: We studied the effects of the anesthetics commonly used in cardiac surgery on platelet function. Fentanyl, droperidol, succinylcholine, pancuronium, thiopental, and diazepam at therapeutic concentrations were tested for their in vitro effects on the expression of platelet membrane glycoproteins Ib and IIbIIIa (GpIb, GpIIb-IIIa) and of P-selectin in anticoagulated whole blood by flow cytometry. The expression of P-selectin was determined under basal conditions, after the incubation of blood with adenosine diphosphate (ADP) 10 micromol/L, and the stable prostaglandin endoperoxide analog U46619 1 micromol/L. No drug affected the expression of P-selectin in unstimulated and ADP- or U46619-stimulated platelets, with the exception of thiopental, which markedly decreased the U46619-induced expression of P-selectin. Thiopental concentration-dependently inhibited U46619-induced and ADP-induced platelet aggregation, with effects on U46619-induced aggregation at therapeutic concentrations. To assess ex vivo effects, the same platelet markers were also assessed in blood obtained from 10 patients undergoing elective coronary surgery. Compared with basal values, platelet response to U46619 was significantly reduced just after the administration of anesthetic drugs, and the effect persisted for 48 h after surgery. Our study suggests that, at therapeutic concentrations, thiopental inhibits U46619-induced platelet activation both in vitro and ex vivo. The mechanisms responsible of this effect, together with its clinical significance, require further investigation. IMPLICATIONS: Thiopental inhibited prostaglandin-induced platelet activation at therapeutic concentrations both in vitro and ex vivo in cardiac surgical patients whereas adenosine diphosphate-induced activation was affected only at supratherapeutic drug concentrations. Thus, administration of sodium thiopental may contribute to the in vivo impairment of platelet function in patients undergoing elective cardiac surgery.

SRC64 regulates the localization of a Tec-family kinase required for Drosophila ring canal growth.

Mutation of the Src64 gene of Drosophila results in ovarian ring canal defects and reduced female fertility. We used a dosage-sensitive modifier screen to search for downstream components of the SRC64 signaling pathway. We show that mutations affecting Tec29, an essential gene encoding a member of the Tec family of protein tyrosine kinases, dominantly enhance the Src64 ring canal phenotype. Loss of Tec29 function in the female germline results in a phenotype strikingly similar to that caused by the loss of Src64 function. In each case, the ring canals are reduced in size and phosphotyrosine content. We further demonstrate that TEC29 localizes to the ring canal, and this subcellular localization requires Src64 function. These data suggest that TEC29 is a downstream target of SRC64, and that regulating TEC29 localization during ring canal growth may be a crucial SRC64 function.

Src64 is required for ovarian ring canal morphogenesis during Drosophila oogenesis.

The Src family of protein tyrosine kinases have been implicated as important regulators of cellular proliferation, differentiation and function. In order to understand further the role of Src family kinases, we have generated loss-of-function mutations in Src64, one of two Src family kinases known in Drosophila melanogaster. Animals with reduced Src64 function develop normally and are fully viable. However, Src64 female flies have reduced fertility, which is associated with the incomplete transfer of cytoplasm from nurse cells to the developing oocyte. Analysis of Src64 egg chambers showed defects in the ring canals that interconnect the oocyte and its 15 associated nurse cells. Src64 ring canals fail to accumulate the high levels of tyrosine phosphorylation that are normally present. Despite the reduced tyrosine phosphorylation, known ring canal components such as filamentous actin, a ring canal-specific product of the hu-li tai shao gene, and the kelch protein localize properly. However, Src64 ring canals are reduced in size and frequently degenerate. These results indicate that Src64 is required for the proper growth and stability of the ovarian ring canals.

Efficacy and financial benefit of an anesthesiologist-directed university preadmission evaluation center.

STUDY OBJECTIVE: To study the effectiveness of an anesthesiologist-directed preadmission evaluation center (PEC) in our institution. DESIGN: I: Preoperative test costs were measured on two sets of patients undergoing same-day surgery. II: Rate of cancellation was measured on all patients undergoing same-day surgery in a subsequent one-year time period. SETTING: The PEC, short procedure unit, and same-day admission unit of a university hospital. PATIENTS: I: 3,062 male and female patients undergoing same-day surgery between January 1, 1992, and August 31, 1992. II: 9,454 male and female patients undergoing same-day surgery between July 1, 1993, and June 30, 1994. INTERVENTIONS: Age, ASA physical status, type of surgery performed, and tests ordered were recorded in two groups of same-day surgical patients. Group S had testing primarily ordered by surgeons, augmented by the anesthesiologists in the PEC. Group A had testing primarily ordered by the anesthesiologists in the PEC, but surgeons could still order tests they felt necessary. On the day of surgery, the attending anesthesiologist recorded any additional testing that was required or would have altered intraoperative management. In a follow-up study, cancellations of same-day surgical patients were recorded for a one-year period. MEASUREMENTS AND MAIN RESULTS: I: With the exception of complete blood counts with differentials, significantly fewer tests were ordered in Group A than Group S. These changes produced an average cost savings of $20.89 per patient. There were no recorded cancellations or apparent alterations in intraoperative management attributable to inadequate testing. II: Of the 9,454 same-day procedures from 7/1/93 to 6/31/94, 66 were cancelled on the day of the procedure. None of the patients seen in the PEC were cancelled due to causes possibly preventable by a PEC, unlike the cases of 4 patients who had not been evaluated in teh PEC and were cancelled. CONCLUSION: A PEC, in which the anesthesiologist primarily orders preoperative tests and approves patients' readiness for surgery, is both an efficient and cost-effective system.

Change of the cytochrome oxidase level during exponential growth in Neurospora crassa.

In Neurospora cells growing in various media, the specific activity of cytochrome oxidase increases very markedly during early exponential growth, reaching a maximum after 4-5 duplication times, then it slowly declines.