Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench. [Authors]

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo.

AIMS: High-mobility group box 1 (HMGB1) is a nuclear protein actively secreted by immune cells and passively released by necrotic cells that initiates pro-inflammatory signalling through binding to the receptor for advance glycation end-products. HMGB1 has been established as a key inflammatory mediator during myocardial infarction, but the proximal mechanisms responsible for myocardial HMGB1 expression and release in this setting remain unclear. Here, we investigated the possible involvement of peroxynitrite, a potent cytotoxic oxidant formed during myocardial infarction, on these processes. METHODS AND RESULTS: The ability of peroxynitrite to induce necrosis and HMGB1 release in vitro was evaluated in H9c2 cardiomyoblasts and in primary murine cardiac cells (myocytes and non-myocytes). In vivo, myocardial HMGB1 expression and nitrotyrosine content (a marker of peroxynitrite generation) were determined following myocardial ischaemia and reperfusion in rats, whereas peroxynitrite formation was inhibited by two different peroxynitrite decomposition catalysts: 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III) (FeTPPS) or Mn(III)-tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). In all types of cells studied, peroxynitrite (100 μM) elicited significant necrosis, the loss of intracellular HMGB1, and its passive release into the medium. In vivo, myocardial ischaemia-reperfusion induced significant myocardial necrosis, cardiac nitrotyrosine formation, and marked overexpression of myocardial HMGB1. FeTPPS reduced nitrotyrosine, decreased infarct size, and suppressed HMGB1 overexpression, an effect that was similarly obtained with MnTBAP. CONCLUSION: These findings indicate that peroxynitrite represents a key mediator of HMGB1 overexpression and release by cardiac cells and provide a novel mechanism linking myocardial oxidative/nitrosative stress with post-infarction myocardial inflammation.

Effects of prison work programmes on the employability of ex-prisioners

This paper presents the main results of a study that relates information from the prison system with information for the Spanish Social Security in order to study the employability of the former inmates of prisons in Catalonia, Spain who obtained final release from 1/1/2004 to 31/12/2007. The results show that 43.6% of the ex-prisoners find a job after serving their sentences, but their integration in the labour market tends to be fragile, confirming that it is a very vulnerable group. It was also found that prison work has a favourable effect on employability and that vocational training could be useful for those who have not previously worked and have no education or job skills.

Little effect of seasonal constraints on population genetic structure in eusocial paper wasps.

Climate has long been suggested to affect population genetic structures of eusocial insect societies. For instance, Hamilton [Journal of Theoretical Biology7 (1964) 17] discusses whether temperate and tropical eusocial insects may show differences in population-level genetic structure and viscosity, and how this might relate to differences in the degree of synchrony in their life cycles or modes of nest founding. Despite the importance of Hamilton's 1964 papers, this specific idea has not been tested in actual populations of wasps, probably due to the paucity of studies on tropical species. Here, we compare colony and population genetic structures in two species of primitively eusocial paper wasps with contrasting ecologies: the tropical species Polistes canadensis and the temperate species P. dominulus. Our results provide important clarifications of Hamilton's discussion. Specifically, we show that the genetic structures of the temperate and tropical species were very similar, indicating that seasonality does not greatly affect population viscosity or inbreeding. For both species, the high genetic differentiation between nests suggests strong selection at the nest level to live with relatives, whereas low population viscosity and low genetic differentiation between nest aggregations might reflect balancing selection to disperse, avoiding competition with relatives. Overall, our study suggests no prevalence of seasonal constraints of the life cycle in affecting the population genetic structure of eusocial paper wasps. These conclusions are likely to apply also to other primitively eusocial insects, such as halictine bees. They also highlight how selection for a kin structure that promotes altruism can override potential effects of ecology in eusocial insects.

Comparative Study of Chemoembolization, Loadable Beads: In vitro Drug Release and Physical Properties of DC Bead and Hepasphere, Loaded with Doxorubicin and Irinotecan

Purpose To characterize in vitro the loadability, physical properties, and release of irinotecan and doxorubicin from two commercially available embolization microspheres. Materials and Methods DC Bead (500-700 μm) and Hepasphere (400-600 μm) microspheres were loaded with either doxorubicin or irinotecan solutions. Drug amount was quantified with spectrophotometry, bead elasticity was measured under compression, and bead size and loading homogeneity were assessed with microscopy image analysis. Drug release was measured over 1-week periods in saline by using a pharmacopeia flow-through method. Results Almost complete drug loading was obtained for both microsphere types and drugs. Doxorubicin-loaded DC Beads maintained their spherical shape throughout the release. In contrast, Hepaspheres showed less homogeneous doxorubicin loading and, after release, some fractured microspheres. Incomplete doxorubicin release was observed in saline over 1 week (27% ± 2 for DC beads and 18% ± 7 for Hepaspheres; P = .013). About 75% of this amount was released within 2.2 hours for both beads. For irinotecan, complete release was obtained for both types of beads, in a sustained manner over 2-3 hours for DC Beads, and in a significantly faster manner as a 7-minute burst for Hepaspheres. Conclusions The two drug-eluting microspheres could be efficiently loaded with both drugs. Incomplete doxorubicin release was attributed to strong drug-bead ionic interactions. Weaker interactions were observed with irinotecan, which led to faster drug release.

Del ciberespai al paper

El món editorial més tradicional tendeix cada vegada més a apostar per medis electrònics com a base per a la difusió del seu contingut o com a complement de l’edició en paper. És pràcticament un pas natural que han fet o estant fent la majoriade publicacions en paper.El creixement d’Internet, tant en nombre d’usuaris com en inversió publicitària, juntament amb la poca necessitat d’inversió per a la creació de revistes online, ha propiciat el sorgiment de revistes exclusivament virtuals que han esdevingut les mésimportants d’Internet, per sobre de publicacions virtuals de revistes amb edició en paper.Però tot i que Internet es el futur, el medi en paper és i serà un mercat molt important a mitjà termini. El mercat publicitari es paga força millor que en les pàgines web, on els models s’esgoten molt ràpidament i s’ha d’innovar constantment per tal de mantenir el click-rate dels usuaris. L’atenció que l’usuari presta a la publicitat en paper és molt més alta que en les pàgines web i menys molesta per l’usuari.El nostre treball es centrarà en fer el pas contrari a l’habitual, és a dir, en convertir una revista exclusivament virtual en una revista que publiqui també en paper. Analitzarem els possibles beneficis d’aquest pas així com els costos que suposaria adaptar un medi virtual al paper.El repte estarà en investigar quin és el model de revista idoni per completar el pas del medi virtual al paper, aconseguint maximitzar els beneficis.

Nevera, mp3, mòbil... quin és el seu paper actual?

Des dels seus inicis, l’ésser humà ha tingut una sèrie de necessitats. Ha requerit d’aliment per sobreviure, d’un sostre on refugiar-se, de roba per vestirse, etc. Conforme han passat els anys, ha anat generant tècniques per a millorar la seva estància al planeta, des del descobriment del foc per escalfarse fins al desenvolupament de màquines complexes. És per això, que ha anat introduint invents a la seva vida que han cobert les necessitats sorgides al llarg del temps.El treball que desenvolupem a continuació es centra en un seguit d’aparells que han sorgit al llarg del segle XX i que han revolucionat la nostra vida. Concretament, els set aparells escollits com a objecte del nostre estudi són: la nevera, la rentadora, l’automòbil, la televisió, el reproductor d’àudio portàtil, el telèfon mòbil i l’ordinador.L’aparició d’electrodomèstics, com la nevera o la rentadora, no només ens ha permès dedicar menys esforç a les tasques domèstiques, sinó també estalviarnos temps a l’hora de realitzar-les. Aquest fet, que ara potser ni tan sols considerem, va ser del tot determinant a l’època que van sorgir, ja que va permetre alliberar mà d’obra domèstica promovent la inserció al mercat laboral d’un sector de la població com el del sexe femení.Per la seva banda, la introducció de l’automòbil també ha estat un gran avenç. Aquest, ha revolucionat la manera de desplaçar-nos, proporcionant-nos una autonomia fins aleshores impensable.Aquest mateix patró segueixen la resta d’altres aparells escollits; tots van provocar un canvi profund a la societat amb la seva aparició i, encara avui, segueixen mantenint un paper destacat al nostre dia a dia. La importància que tenen a les nostres vides ha estat la motivació que ens ha conduït a dur a terme un treball que els tingués com a protagonistes.El nostre treball consta de dues parts:A la primera, de caràcter teòric, ens hem proposat estudiar la història d’aquests aparells, és a dir, hem volgut donar resposta a una sèrie de preguntes com: quan van aparèixer, quins van ser els seus creadors i quines transformacions han anat experimentant al llarg del temps, entre d’altres.A la segona part, més pràctica, hem realitzat un treball de camp (les característiques del qual expliquem detalladament a l’apartat següent) amb l’objectiu d’analitzar la rellevància que tenen aquests aparells a la societat actual. És a dir, hem volgut saber quins aparells, dels escollits, són els més valorats per la població avui dia i quins són els que menys. Pensem que l’ús que els enquestats fan d’aquests béns serà un criteri clau a l’hora de puntuarlos, encara que no l’únic. Hem realitzat aquest anàlisi diferenciant els individus per franges d’edat i sexes. A més, també hem treballat altres qüestions comserien, entre d’altres, si el seu ús s’entén com una necessitat o va encaminat cap a l’oci; quant temps triguen els usuaris a renovar-los, quin és el motiu, etc. En relació amb aquesta segona part, hem formulat un seguit d’hipòtesis amb l’objectiu de contrastar-les posteriorment amb els resultats de les enquestes.Les hipòtesis són:- Avui dia, els individus donen més importància a aquells aparells que són més moderns, (entenent com a tals, aquells que han sorgit més recentment i estan lligats a les noves tecnologies i la innovació contínua), com poden ser l’ordinador i el telèfon mòbil, que els electrodomèstics més tradicionals com serien la nevera o la rentadora. Tot això, a més, considerant que pel que fa als primers, entenem que part del seu ús està destinat a l’oci; mentre que pels segons el seu ús està motivat per una necessitat, com seria l’alimentació en el cas de la nevera.- Pel que fa a les franges d’edat, les valoracions mostraran diferències importants en els diversos aparells. Entre la franja més jove (de 16 a 25 anys) i la de més edat (més de 60 anys), les valoracions seran més extremes, gairebé oposades.- Pel que fa als dos sexes, les valoracions que donin als aparells seran molt semblants.Hem considerat que per a dur a terme un bon anàlisi, el més adequat és presentar la informació de cada aparell per separat. És per això que cada bé constitueix un apartat independent que consta d’una primera part referent a la seva història, i una segona referent als resultats obtinguts de les enquestes.A continuació, hem procedit a ajuntar els resultats extrets dels diferents aparells i a presentant-los de forma conjunta per a comparar-los. Així, hem pogut contrastar les nostres hipòtesis inicials i hem raonat possibles causes que justificarien dels resultats obtinguts.

Teichoic acids are not required for Streptococcus pneumoniae and Staphylococcus aureus cell walls to trigger the release of tumor necrosis factor by peripheral blood monocytes.

In gram-negative bacteria, the outer membrane lipopolysaccharide is the main component triggering cytokine release from peripheral blood mononuclear cells (PBMCs). In gram-positive bacteria, purified walls also induce cytokine release, but stimulation requires 100 times more material. Gram-positive walls are complex megamolecules reassembling distinct structures. Only some of them might be inflammatory, whereas others are not. Teichoic acids (TA) are an important portion (> or =50%) of gram-positive walls. TA directly interact with C3b of complement and the cellular receptor for platelet-activating factor. However, their contribution to wall-induced cytokine-release by PBMCs has not been studied in much detail. In contrast, their membrane-bound lipoteichoic acids (LTA) counterparts were shown to trigger inflammation and synergize with peptidoglycan (PGN) for releasing nitric oxide (NO). This raised the question as to whether TA are also inflammatory. We determined the release of tumor necrosis factor (TNF) by PBMCs exposed to a variety of TA-rich and TA-free wall fragments from Streptococcus pneumoniae and Staphylococcus aureus. TA-rich walls from both organisms induced measurable TNF release at concentrations of 1 microg/ml. Removal of wall-attached TA did not alter this activity. Moreover, purified pneumococcal and staphylococcal TA did not trigger TNF release at concentrations as high as > or =100 microg/ml. In contrast, purified LTA triggered TNF release at 1 microg/ml. PGN-stem peptide oligomers lacking TA or amino-sugars were highly active and triggered TNF release at concentrations as low as 0.01 microg/ml (P. A. Majcherczyk, H. Langen, et al., J. Biol. Chem. 274:12537-12543,1999). Thus, although TA is an important part of gram-positive walls, it did not participate to the TNF-releasing activity of PGN.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits.

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

Solving two production scheduling problems with sequence-dependent set-up times

In today’s competitive markets, the importance of goodscheduling strategies in manufacturing companies lead to theneed of developing efficient methods to solve complexscheduling problems.In this paper, we studied two production scheduling problemswith sequence-dependent setups times. The setup times areone of the most common complications in scheduling problems,and are usually associated with cleaning operations andchanging tools and shapes in machines.The first problem considered is a single-machine schedulingwith release dates, sequence-dependent setup times anddelivery times. The performance measure is the maximumlateness.The second problem is a job-shop scheduling problem withsequence-dependent setup times where the objective is tominimize the makespan.We present several priority dispatching rules for bothproblems, followed by a study of their performance. Finally,conclusions and directions of future research are presented.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.