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Rationale: Patients with and without cardiovascular diseases have been shown to be at risk of influenza-mediated cardiac complications. Recent clinical reports support the notion of a direct link between laboratory-confirmed influenza virus infections and adverse cardiac events. Objective: Define the molecular mechanisms underlying influenza virus–induced cardiac pathogenesis after resolution of pulmonary infection and the role of necroptosis in this process. Methods and Results: Hearts from wild-type and necroptosis-deficient (MLKL [mixed lineage kinase domain-like protein]-KO) mice were dissected 12 days after initial influenza A virus (IAV) infection when viral titers were undetectable in the lungs. Immunofluorescence microscopy and plaque assays showed presence of viable IAV particles in the myocardium without generation of interferon responses. Global proteome and phosphoproteome analyses using high-resolution accurate mass-based LC-MS/MS and label-free quantitation showed that the global proteome as well as the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts in a strain-independent manner. Necroptosis-deficient mice had increased survival and reduced weight loss post-IAV infection, as well as increased antioxidant and mitochondrial function, indicating partial protection to IAV infection. These findings were confirmed in vitro by pretreatment of human and rat myocytes with antioxidants or necroptosis inhibitors, which blunted oxidative stress and mitochondrial damage after IAV infection. Conclusions: This study provides the first evidence that the cardiac proteome and phosphoproteome are significantly altered post–pulmonary influenza infection. Moreover, viral particles can persist in the heart after lung clearance, altering mitochondrial function and promoting cell death without active replication and interferon responses. Finally, our findings show inhibition of necroptosis or prevention of mitochondrial damage as possible therapeutic interventions to reduce cardiac damage during influenza infections. Graphic Abstract: A graphic abstract is available for this article.

Hazard studies for nanomaterials (NMs) commonly assess whether they activate an inflammatory response. Such assessments often rely on rodents, but alternative models are needed to support the implementation of the 3Rs principles. Zebrafish (Danio rerio) offer a viable alternative for screening NM toxicity by investigating inflammatory responses. Here, we used non-protected life stages of transgenic zebrafish (Tg(mpx:GFP)i114) with fluorescently-labeled neutrophils to assess inflammatory responses to silver (Ag) and zinc oxide (ZnO) NMs using two approaches. Zebrafish were exposed to NMs via water following a tail fin injury, or NMs were microinjected into the otic vesicle. Zebrafish were exposed to NMs at 3 days post-fertilization (dpf) and neutrophil accumulation at the injury or injection site was quantified at 0, 4, 6, 8, 24, and 48 h post-exposure. Zebrafish larvae were also exposed to fMLF, LTB4, CXCL-8, C5a, and LPS to identify a suitable positive control for inflammation induction. Aqueous exposure to Ag and ZnO NMs stimulated an enhanced and sustained neutrophilic inflammatory response in injured zebrafish larvae, with a greater response observed for Ag NMs. Following microinjection, Ag NMs stimulated a time-dependent neutrophil accumulation in the otic vesicle which peaked at 48 h. LTB4 was identified as a positive control for studies investigating inflammatory responses in injured zebrafish following aqueous exposure, and CXCL-8 for microinjection studies that assess responses in the otic vesicle. Our findings support the use of transgenic zebrafish to rapidly screen the pro-inflammatory effects of NMs, with potential for wider application in assessing chemical safety (e.g. pharmaceuticals).

We evaluated the effect of copper oxide nanomaterials (CuO NMs), uncoated and with 3 different surface coatings (carboxylated, pegylated, and ammonia groups), on acute toxicity and accumulation dynamics in Daphnia magna. With the use of biodynamic modelling, biosorption and elimination rate constants were determined for D. magna following waterborne exposure to dissolved Cu and CuO NMs. The relationship between modeled parameters and acute toxicity endpoints was evaluated to investigate whether accumulation dynamics parameters could be used as a predictor of acute toxicity. The Langmuir equation was used to characterize the biosorption dynamics of Cu NMs and Cu chloride, used as dissolved Cu control. Uptake rates showed the following NM rankings: pristine-CuO > NH3 -CuO > aqueous Cu > polyethylene glycol (PEG)-CuO > COOH-CuO. To determine Cu elimination by D. magna, a one-compartment model was used. Different elimination rate constants were estimated for each chemical substance tested. Those that were easily biosorbed were also easily removed from organisms. Biosorption and depuration properties of NMs were correlated with zeta potential values and diameters of NM agglomerates in the suspensions. No link was found between biosorption and toxicity. Waterborne exposures to more difficult-to-biosorb CuO NMs were more likely to induce adverse effects than those that biosorbed easily. It is proposed that some physicochemical properties of NMs in media, including zeta potential and agglomerate diameter, can lead to higher biosorption but do not necessarily affect toxicity. The mode of interaction of the NMs with the organism seems to be complex and to depend on chemical speciation and physicochemical properties of the NMs inside an organism. Moreover, our findings highlight that coating type affects the biosorption dynamics, depuration kinetics, and dissolution rate of NMs in media. Environ Toxicol Chem 2020;39:287-299. © 2019 SETAC.

Organically modified clays can be used as nanofillers in polymer-clay nanocomposites to create bio-based packaging with improved strength and barrier properties. The impact of organic modification on the physico-chemical properties and toxicity of clays has yet to be fully investigated but is essential to ensure their safe use. Two organoclays, named N116_HDTA and N116_TMSA, were prepared using a commercially available sodium bentonite clay and the organic modifiers hexadecyl trimethyl ammonium bromide (HDTA) and octadecyl trimethyl ammonium chloride (TMSA). An in vitro hazard assessment was performed using HaCaT skin cells, C3A liver cells, and J774.1 macrophage-like cells. Organic modification with HDTA and TMSA increased the hazard potential of the organoclays in all cell models, as evidenced by the higher levels of cytotoxicity measured. N116_TMSA caused the greatest loss in viability with IC50 values of 3.2, 3.6 and 6.1 μg/cm2 calculated using J774.1, HaCaT and C3A cell lines, respectively. Cytotoxic effects were dictated by the amount of free or displaced organic modifier present in the exposure suspensions. The parent bentonite clay also caused distinct cytotoxic effects in J774.1 macrophage-like cells with associated TNF-α release. Such information on the hazard profile of organoclays, can feed into risk assessments for these materials.

Natural Layered Silicates (NLS) and Synthetic Layered Silicates (SLS) are a diverse group of clay minerals that have attracted great interest in various branches of industry. However, despite growing demand for this class of material, their impact on human health has not been fully investigated. Therefore, the aim of this study was to evaluate and compare the potential toxic effects of a wide range of commercially available SLS and NLS of varying physicochemical properties (lithium (Li) or fluoride (F) content and size). Mouse BALB/c monocyte macrophage (J774A.1) and human monocyte-derived macrophages (MDMs) were chosen as in vitro models of alveolar macrophages. Montmorillonite, hectorite, Medium (med) F/High Li and Low F/Med Li particles, were cytotoxic to cells and induced potent pro-inflammatory responses. The remaining particles (No F/Very (V)Low Li, No F/Med Li, No F/Low Li, High F/Med Li and High F/Med Li washed) were non- to relatively low- cytotoxic and inflammogenic, in both type of cells. In an acellular condition none of the tested samples increased reactive oxygen species (ROS), while ROS generation was observed following exposure to sublethal concentrations of Med F/High Li, Low F/Med Li, montmorillonite and hectorite samples, in J774A.1 cells. Based on the results obtained in this study the toxic potency of tested samples was not associated with lithium or fluoride content, but appeared to be dependent on particle size, with the platelets of larger dimension and lower surface area being more potent than the smaller platelet particles with higher surface area. In addition, the increased bioactivity of Med F/High Li and Low F/Med Li was associated with endotoxin contamination. Obtained results demonstrated that layered silicate materials have different toxicological profiles and suggest that toxicological properties of a specific layered silicate should be investigated on an individual basis.

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

One of the most widely used approaches for improving drug permeation across the stratum corneum barrier of the skin is the use of chemical penetration enhancers, such as surfactants. In this study, two anionic surfactants, named C12-OPK and C18-OPK, were synthesized via condensation of itaconic acid and fatty amines, with C12 and C18 alkyl chains, respectively. Assessment of impacts on HaCaT keratinocyte cell viability was used as indicator of their potential to cause skin irritation 24 h post exposure (Alamar Blue assay). The LC50 values of C12-OPK and C18-OPK (144 and 85 mg/L, respectively) were lower than LC50 values of the most used commercial surfactants (e.g. SDS). The effect of different surfactant concentrations (up to ten times the critical micellar concentration, CMC) on hydrocortisone (HC) solubility and permeation through porcine skin was also evaluated. Results showed that drug solubility increased linearly with increasing concentrations of both surfactants, as a consequence of the association between drug and micelles. In vitro permeation results showed that the permeability coefficient increased at surfactant concentrations lower than the CMC. In particular, a higher enhancement effect on drug permeation was obtained with C18-OPK, due to its hydrophobic properties that ensured a more effective HC permeation in comparison to C12-OPK.

To meet the demands of increased global meat consumption, animal production systems will have to become more efficient, or at least maintain the current efficiency utilizing feed ingredients that are not also used for human consumption. Use of growth promoters is a potential option for increasing production animal feed efficiency and increased muscle growth. The objective of this manuscript is to describe the mechanisms by which the growth promoters, beta-adrenergic agonists and growth hormone, mediate their effects, with specific consideration of the aspects which have implications for meat quality.

In the near twenty-year existence of the human and mammalian artificial chromosome field, the technologies for artificial chromosome construction and installation into desired cell types or organisms have evolved with the rest of modern molecular and synthetic biology. Medical, industrial, pharmaceutical, agricultural, and basic research scientists seek the as yet unrealized promise of human and mammalian artificial chromosomes. Existing technologies for both top-down and bottom-up approaches to construct these artificial chromosomes for use in higher eukaryotes are very different but aspire to achieve similar results. New capacity for production of chromosome sized synthetic DNA will likely shift the field towards more bottom-up approaches, but not completely. Similarly, new approaches to install human and mammalian artificial chromosomes in target cells will compete with the microcell mediated cell transfer methods that currently dominate the field.

Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism in vitro using primary human neuron cell cultures and in vivo with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies., Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

Previously, we highlighted induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist. Hence we tested the hypothesis that the ER-stress inhibitor, sodium 4-phenylbutyrate (PBA), would inhibit Clenbuterol-mediated muscle growth and reduce expression of genes that are known indicators of an ISR in mice. Clenbuterol (1mg/kg/day) administered to C57BL6/J mice for 21 days increased body weight (p

Skeletal muscle homeostasis depends on an intricate balance between muscle hypertrophy, atrophy and regeneration. As we age, maintenance of muscle homeostasis is perturbed, resulting in a loss of muscle mass and function, termed sarcopenia. Individuals with sarcopenia exhibit impaired balance, increased falls (leading to subsequent injury) and an overall decline in quality of life. The mechanisms mediating sarcopenia are still not fully understood but clarity in our understanding of the precise pathophysiological changes occurring during skeletal muscle ageing has improved dramatically. Advances in transcriptomics has highlighted significant deregulation in skeletal muscle gene expression with ageing, suggesting epigenetic alterations may play a crucial and potentially causative role in the skeletal muscle ageing process. microRNAs (miRNAs, miRs), novel regulators of gene expression, can modulate many processes in skeletal muscle, including myogenesis, tissue regeneration and cellular programming. Expression of numerous evolutionary conserved miRNAs is disrupted in skeletal muscle with age. Given that a single miRNA can simultaneously affect the functionality of multiple signaling pathways, miRNAs are potent modulators of pathophysiological changes. miRNA-based interventions provide a promising new therapeutic strategy against alterations in muscle homeostasis. The aim of this review is two-fold; firstly to outline the latest understanding of the pathophysiological alterations impacting the deregulation of skeletal muscle mass and function with ageing, and secondly, to highlight the mounting evidence for a role of miRNAs in modulating muscle mass, and the need to explore their specific role in sarcopenia.

The potential toxicity of carbon nanotubes (CNTs) has been compared to pathogenic fibres such as asbestos. It is important to test this hypothesis to ascertain safe methods for CNT production, handling and disposal. In this study aspects reported to contribute to CNT toxicity were assessed: length, aspect ratio, iron content and crystallinity; with responses compared to industrially produced MWCNTs and toxicologically relevant materials such as asbestos. The impacts of these particles on a range of macrophage models in vitro were assessed due to the key role of macrophages in particle clearance and particle/fibre-induced disease. Industrially produced and long MWCNTs were cytotoxic to cells, and were potent in inducing pro-inflammatory and pro-fibrotic immune responses. Short CNTs did not induce any cytotoxicity. Frustrated phagocytosis was most evident in response to long CNTs, as was respiratory burst and reduction in phagocytic ability. Short CNTs, metal content and crystallinity had less or no influence on these endpoints, suggesting that many responses were fibre-length dependent. This study demonstrates that CNTs are potentially pathogenic, as they were routinely found to induce detrimental responses in macrophages greater than those induced by asbestos at the same mass-based dose.

Background The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. Results The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. Conclusions The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

The study aim was to investigate how gold nanoparticles (NPs) of different sizes (20 and 100 nm) influence primary hepatocytes, the hepatocyte cell line C3A and macrophage cytokine responses when dispersed in lung or blood relevant fluids. Gold Au NPs induced cytotoxicity in primary hepatocytes at the highest dose of 66 μg/cm2, this effect was modified by the dispersant, the effect was greater with lung lining fluid (LLF). Release of interleukin (IL)-6, Monocyte chemoattractant protein-1 (MCP-1) and IL-1β was enhanced by the Au NPs and the effects were influenced by the particle size and dispersant. In medium, the smallest particle size was most effective at inducing IL-6 release, while in LLF the largest particles were most effective at inducing IL-6 release. Both 20 nm and 100 nm particles enhanced MCP-1 and IL-1β in the presence of LLF. The Au particles had no cytotoxic effects nor did they stimulate the release of cytokines in the C3A hepatoma cell line. The Au NPs had no significant impact on macrophage viability. Particles induced IL-6 and TNF-α release. LLF and serum reduced the IL-6 response while albumin enhanced the TNF-α response compared to medium dispersed Au NPs. The Au NPs did not impact on MCP-1 release, but this cytokine was enhanced by albumin and serum, while it was depressed by LLF. The macrophage responses were lower than those evoked in primary hepatocytes. In conclusion, when assessing the cytotoxic and pro-inflammatory responses induced by Au NPs, the response is influenced by the dispersant, with different dispersants having different effects in different cell types.

Recent laboratory successes in the development of genetically engineered mosquitoes for controlling pathogen transmission have fostered the need for standardized procedures for advancing the technical achievements to practical tools. It is incumbent in many cases for the same scientists doing the in-laboratory discovery research to also take on the initial challenges of developing the pathway that will move the technologies to the field. One of these challenges is having a set of criteria for selecting collaborators and sites for efficacy and safety field trials that combine rigorous science with good ethical and legal practices. Specific site-selection criteria were developed in four categories-Scientific, Regulatory, Community Engagement, and Resources-in anticipation of open-field releases of a transgenic mosquito strain designed to suppress populations of the dengue vector mosquito, Aedes aegypti. The criteria are derived from previous published material, discussions, and personal experiences with the expectation of providing guidance to laboratory scientists for addressing the conceptual and operational considerations for identifying partner researchers and countries with whom to collaborate. These criteria are not intended to be prescriptive nor can they be applied to every circumstance where genetic approaches are proposed for deployment. However, we encourage those involved in the discovery phase of research to consider each criterion during project planning activities, and where appropriate, incorporate them into a "go/no-go" decision-making process for further development and testing of the technologies.

The rapid expansion of the incorporation of nano-sized materials in consumer products overlaps with the necessity for high-throughput reliable screening tools for the identification of the potential hazardous properties of the nanomaterials. The ToxTracker assay (mechanism-based reporter assay based on embryonic stem cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress) is one such tool, which could prove useful in the field of particle toxicology allowing for high throughput screening. Here, ToxTracker was utilised to evaluate the potential hazardous properties of two particulates currently used in the food industry (vegetable carbon (E153) and food-grade TiO2 (E171)). Due to the fact that ToxTracker is based on a stem cell format, it is crucial that the data generated is assessed for its suitability and comparability to more conventionally used relevant source of cells - in this case cells from the gastrointestinal tract and the liver. Therefore, the cell reporter findings were compared to data from traditional assays (cytotoxicity, anti-oxidant depletion and DNA damage) and tissue relevant cell types. The data showed E171 to be the most cytotoxic, decreased intracellular glutathione and the most significant with regards to genotoxic effects. The ToxTracker data showed comparability to conventional toxicity and oxidative stress assays; however, some discrepancies were evident between the findings from ToxTracker and the comet assay.

We investigated the effects of silica particles and nanoparticles (NPs) (50 nm and 200 nm) with a neutral and positively charged surface when dispersed in saline, bovine serum albumin (BSA) or lung lining fluid (LLF) 24 h post instillation into the lungs of rats. There was a significant increase in the recruitment of neutrophils in animals instilled with 50 nm plain and aminated NPs compared with 200 nm particles when dispersed in saline or BSA, but not when dispersed in LLF. There was no evidence of toxicity or an increase in the albumin content of the bronchoalveolar lavage fluid. Immunostaining for the transcription factor Nrf2 in BAL cells indicated that there was a significant increase in nuclear colocalisation in animals treated with plain and aminated 50 nm NPs compared with plain and aminated 200 nm particles when dispersed in saline, but no difference was observed between 50 nm and 200 nm aminated particles when dispersed in BSA. There was no difference in nuclear colocalisation with any of the particle types dispersed in LLF.This study suggests that low dose intratracheal exposure to silica nanoparticles can produce an acute inflammatory response and that the dispersion medium may influence the magnitude of this response.

The regulation of muscle fibre transitions has mainly been studied in vivo using conventional histological or immunohistochemical techniques. In order to investigate the molecular regulation of myosin heavy chain (MyHC) isoform expression in cell culture studies, we first characterised the normal transitions in endogenous expression of the MyHC isoforms and the myogenic regulatory factors during differentiation of C2C12 muscle cells. Interestingly, across the time course of differentiation, MyHC mRNA isoforms were expressed in a distinct temporal pattern as two distinct cohorts, one including MyHC I, embryonic and neonatal, the other including MyHC IIa, IIx and IIb. The pattern of expression suggests a transition in MyHC isoforms, from one cohort to another, occurs during muscle cell differentiation and that these transitions occur independent of nerve innervation. To our knowledge, this is the most comprehensive analysis of in vitro MyHC mRNA isoform transitions and provides important information for studying the regulation of transitions in MyHC isoforms in cell culture systems.

The aim of this study was to assess the effects of a series of different surface coated quantum dots (QDs) (organic, carboxylated [COOH] and amino [NH₂] polytethylene glycol [PEG]) on J774.A1 macrophage cell viability and to further determine which part of the QDs cause such toxicity. Cytotoxic examination (MTT assay and LDH release) showed organic QDs to induce significant cytotoxicity up to 48 h, even at a low particle concentration (20 nM), whilst both COOH and NH₂ (PEG) QDs caused reduced cell viability and cell membrane permeability after 24 and 48 h exposure at 80 nM. Subsequent analysis of the elements that constitute the QD core, core/shell and (organic QD) surface coating showed that the surface coating drives QD toxicity. Elemental analysis (ICP-AES) after 48 h, however, also observed a release of Cd from organic QDs. In conclusion, both the specific surface coating and core material can have a significant impact on QD toxicity.

BACKGROUND Phenoxodiol, a synthetic analog of Genistein, is being assessed in several clinical studies against a range of cancer types and was shown to have a good efficacy and safety profile. In this study we tested the effects of Phenoxodiol against prostate cancer cell lines. METHODS Cell-cycle analysis, plasmatic membrane damage, clonogenic assay, comet assay, and Western blot methodologies were employed to assess the effects of Phenoxodiol on prostate cancer cell lines. An in vivo model confirmed the potential therapeutic efficacy of Phenoxodiol when administered orally to tumor bearing mice. RESULTS Phenoxodiol treatment promoted a marked inhibition of proliferation and loss of colony formation in LNCaP cells in a dose- and time-dependent manner. Similar effects were also observed in the metastatic prostate cell lines PC3 and DU145. Activation of poly(ADP ribose) polymerase 1 (PARP-1) clearly indicates the induction of DNA damage by Phenoxodiol. Oral administration of Phenoxodiol induced a considerable growth inhibition of malignant tumors generated by inoculation of LNCaP cells into Balb/c nu/nu athymic mice. CONCLUSIONS These data demonstrated that Phenoxodiol promotes apoptosis, as determined by PARP-1 degradation, via mitochondrial depolarization and G1/S cell-cycle arrest thereby confirming that it is active against androgen-dependent and independent prostate cancer cells. Although a precise target for Phenoxodiol has not been identified, these data contribute to our understanding of the mechanism by which this drug promotes cell death in prostate cancer cells, and warrants the continued clinical development of Phenoxodiol as a therapeutic for the treatment of metastatic prostate cancer. Prostate 70: 1211–1221, 2010. © 2010 Wiley-Liss, Inc.

Nanoparticles (NPs) are being used within diverse applications such as medicines, clothing, cosmetics and food. In order to promote the safe development of such nanotechnologies it is essential to assess the potential adverse health consequences associated with human exposure. The liver is recognised as a target site for NP toxicity, due to NP accumulation within this organ subsequent to injection, inhalation or instillation. The uptake of fluorescent polystyrene carboxylated particles (20 nm or 200 nm diameter) by hepatocytes was determined using confocal microscopy; with cells imaged "live" during particle exposure or after exposure within fixed cells. Comparisons between the uptake of polystyrene particles by primary rat hepatocytes, and human hepatocyte cell lines (C3A and HepG2) were made. Uptake of particles by hepatocytes was size, time, and serum dependent. Specifically, the uptake of 200 nm particles was limited, but 20 nm NPs were internalised by all cell types from 10 min onwards. At 10 min, 20 nm NP fluorescence co-localised with the tubulin cytoskeleton staining; after 30 min NP fluorescence compartmentalised into structures located within and/or between cells. The fate of internalised NPs was considered and they were not contained within early endosomes or lysosomes, but within mitochondria of cell lines. NPs accumulated within bile canaliculi to a limited extent, which suggests that NPs can be eliminated within bile. This is in keeping with the finding that gold NPs were eliminated in bile following intravenous injection into rats. The findings were, in the main, comparable between primary rat hepatocytes and the different human hepatocyte cell lines.

SUMMARY The anti-inflammatory properties of parasitic helminths have been largely linked to their excretory–secretory (ES) products. Some studies have noted a lack of TNF-α production and limited recruitment of neutrophils into the lungs after Nippostrongylus brasiliensis infection. We previously reported that instillation of ES from L3 larvae of N. brasiliensis to the lungs could inhibit the recruitment of neutrophils on a background of LPS-induced inflammation. A similar reduction in neutrophil recruitment was observed in this study. This reduction was associated with the significant inhibition in gene transcription of the adhesion molecule, ICAM-1, and the chemokine, MIP-2 in bronchoalveolar lavage (BAL) cells. The LPS-stimulated gene transcription of the pro-inflammatory cytokines TNF-α and IL-1β was also significantly reduced by L3 ES. Inducible nitric oxide synthase (iNOS) is normally elevated in classically activated macrophages, however, in this case gene transcription of iNOS was inhibited by L3 ES and may suggest a phenotype change to anti-inflammatory. The general inhibition of pro-inflammatory mediators observed in this study suggests that infective stage L3 larvae excrete and/or secrete inhibitory products capable of modifying the normally potent LPS inflammatory response.

Increased levels of particulate air pollution (PM10) have been implicated as a causal agent in pulmonary disease exacerbation and increased deaths from respiratory and cardiovascular disorders. The exact mechanism by which PM10 drives toxicity in the lung is still unknown, but studies have focused on inhibition of macrophage function and impaired alveolar clearance mechanisms. To assess the effects of PM10 on pulmonary macrophage clearance mechanisms ex vivo, Wistar rats were instilled with 125 or 250 microg of PM10 collected from the North Kensington, London. Control rats were instilled with sterile saline. The rats were sacrificed after 18 h and a bronchoalveolar lavage (BAL) was performed. Macrophages isolated from the BAL fluid were assessed for ability to migrate towards a positive chemoattractant (ZAS) ex vivo and to perform phagocytosis. Macrophages isolated from the PM10-exposed rats showed inhibition of potential to migrate. Macrophage phagocytic ability ex vivo was also significantly reduced by the presence of PM10 inside the cells. This study indicates that acute PM10 exposure diminishes macrophage motility and phagocytosis in a manner that could prove deleterious to particle clearance from the alveolar region of the lung. Decreased particle clearance promotes inflammation, and hence, warrants further investigation in relation to the effects of chronic PM10 exposure on macrophage clearance mechanisms and establishing the mechanisms behind decreased macrophage migration.

Different particle types cause excessive lung inflammation that is thought to play a role in the various types of pathology they produce. Recently attention has been focused on nanoparticles due to their presence in environmental particulate air pollution, their use and exposure in occupational settings, and their potential use in nanotechnology and novel therapeutics. We have shown previously that the surface area metric drives the overload response. We have instilled a number of low-toxicity dusts of various particle sizes and assessed neutrophil influx into the lung at 18-24 h postinstillation. The extent of inflammation was demonstrated as being a function not of the mass dose instilled but interestingly of the surface area dose instilled. Since low-toxicity nanoparticles present a "special" case of high surface area, they are relatively inflammogenic. We tested whether we could use this approach to model the reactivity of highly toxic dusts. Rats were instilled with either DQ12 quartz or aluminum lactate-treated DQ12 and, as anticipated, the high specific surface toxicity of DQ12 meant that it was much more inflammogenic (63 times more) than the surface area alone would have predicted. By contrast, aluminum lactate-treated DQ12 fell into the line of "low-toxicity" dusts. In addition, as an in vitro testing alternative to that of in vivo testing, interleukin (IL)-8 production in A549 cells exposed to the panel of various particles clearly demonstrated the ability to also identify a relationship between surface area dose and reactivity. These approaches present the possibility of modelling potential toxicity of nanoparticles and nuisance dusts based on the inflammatory response of a given instilled surface area dose.

Increased levels of proinflammatory cytokines are present in bronchoalveolar lavage fluid in various lung diseases. Redox-sensitive transcription factors such as NF-kappaB regulate gene transcription for these cytokines. We therefore studied the effect of a new thiol antioxidant compound, Nacystelyn (NAL), on IL-8 regulation in a human macrophage-derived cell line (THP-1). LPS (10 microg/ml) increased IL-8 release compared with control levels. This LPS activation was inhibited by coincubation with NAL (1 and 5 mM). Pretreatment with cycloheximide or okadaic acid, protein synthesis, and serine/threonine phosphatase inhibitors, respectively, did not modify inhibition of IL-8 release caused by NAL. NF-kappaB and C/EBP DNA binding were increased after LPS treatment compared with control, an effect inhibited by cotreatment with NAL. Activator protein (AP)-1 DNA binding was unaffected. The enhanced neutrophil chemotaxis produced by conditioned media from LPS-treated cells was inhibited when cells were cotreated with NAL. The selectivity of NAL inhibition upon IL-8 expression was studied. LPS-treated THP-1 cells also had higher levels of TNF-alpha, transforming growth factor (TGF)-beta1 and -3, MIP-1alpha and -beta, and RANTES gene expression. However, only LPS-induced IL-8 and TGF-beta1 expressions were inhibited by NAL. An anti-inflammatory effect of NAL was confirmed in vivo as shown by a reduction in LPS-induced neutrophil recruitment to the lungs following instillation of NAL into the lungs. Our studies demonstrate that NAL has anti-inflammatory properties in vitro and in vivo, may therefore have a therapeutic role in lung inflammation, and has the advantage over other antioxidant agents in that it may be administrated by inhalation.

Ultrafine (Uf) particles are a component of particulate air pollution suggested to be responsible for the health effects associated with elevations of this pollutant. We have previously suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals. Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease. The effect of Uf particles on rat alveolar macrophages and human blood monocytes was investigated with reference to the roles of calcium and reactive oxygen species (ROS). TNF-alpha protein release, intracellular calcium concentration, TNF-alpha mRNA expression, and transcription factor activation were studied as end points after treatment of rat alveolar macrophages or peripheral blood monocytes. The calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, the calmodulin inhibitor W-7, and the antioxidants Trolox and Nacystelin (NAL) were included in combination with Uf particles. Verapamil reduced intracellular calcium concentration in rat alveolar macrophages on stimulation with Uf particles. This effect was also apparent with transcription factor AP-1 activation. All antagonists and antioxidants reduced Uf-stimulated nuclear localization of the p50 and p65 subunits of NF-kappaB in human monocytes. Verapamil, BAPTA-AM, and NAL reduced Uf-stimulated TNF-alpha protein release, whereas only verapamil reduced Uf-stimulated mRNA expression in rat alveolar macrophages. In human monocytes, verapamil, Trolox, BAPTA-AM, and W-7 reduced Uf-stimulated TNF-alpha protein release. These findings suggest that Uf particles may exert proinflammatory effects by modulating intracellular calcium concentrations, activation of transcription factors, and cytokine production through a ROS-mediated mechanism.

Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10).Donaldson K, Stone V, Borm PJ, Jimenez LA, Gilmour PS, Schins RP, Knaapen AM, Rahman I, Faux SP, Brown DM, MacNee W.Colt/ELEGI Laboratories, Centre for Inflammation Research, The University of Edinburgh Medical School, Edinburgh EH8 9AG, Scotland, UK. ken.donaldson@ed.ac.ukThis review focuses on the potential role that oxidative stress plays in the adverse effects of PM(10). The central hypothesis is that the ability of PM(10) to cause oxidative stress underlies the association between increased exposure to PM(10) and both exacerbations of lung disease and lung cancer. Pulmonary inflammation may also underlie the cardiovascular effects seen following increased PM(10), although the mechanisms of the cardiovascular effects of PM(10) are not well understood. PM(10) is a complex mix of various particle types and several of the components of PM(10) are likely to be involved in the induction of oxidative stress. The most likely of these are transition metals, ultrafine particle surfaces, and organic compounds. In support of this hypothesis, oxidative stress arising from PM(10) has been shown to activate a number of redox-responsive signaling pathways in lung target cells. These pathways are involved in expression of genes that play a role in responses relevant to inflammation and pathological change, including MAPKs, NF-kappaB, AP-1, and histone acetylation. Oxidative stress from particles is also likely to play an important role in the carcinogenic effects associated with PM(10) and hydroxyl radicals from PM(10) cause DNA damage in vitro.

PM10 contains an ultrafine component, which is generally derived from combustion processes. This ultrafine fraction may be a factor in the increases in exacerbations of respiratory disease and deaths from cardiorespiratory causes associated with transient increases in levels of PM10. By using four different ultrafine particles (carbon black, cobalt, nickel, and titanium dioxide), we set out to determine the attributes of the ultrafine particle (surface area, chemical composition, particle number, or surface reactivity) that contribute most to its toxicity and proinflammatory effects both in vivo and in vitro. Instillation of 125 micro g ultrafine carbon black (UFCB) and ultrafine cobalt (UFCo) particles induced a significant influx of neutrophils at both 4 and 18 h postinstillation. Accompanying the influx of neutrophils was an increase in macrophage inflammatory protein-2 (MIP-2) (at 4 h) and an increase in gamma-glutamyl transpeptidase (at 18 h) in bronchoalveolar lavage fluid (BAL). Ultrafine nickel (UFNi) did not induce a significant increase in neutrophil influx until 18 h postinstillation. The increase in neutrophils induced by UFNi at this timepoint was comparable to that induced by UFCo and UFCB. UFTi did not induce a significant increase in neutrophils following instillation into the rat lung. The levels of MIP-2 observed at 4 h and neutrophil influx at 18 h induced by the particle samples were consistent with the pattern of surface free radical generation (as measured by the plasmid scission assay) whereby UFCo, UFCB, and UFNi all cause significant increases in inflammatory markers, as well as inducing a significant depletion of supercoiled plasmid DNA, indicative of hydroxyl radical generation. A role for free radicals and reactive oxygen species (ROS) in mediating ultrafine inflammation is further strengthened by the ability of the antioxidants N-acetylcysteine (NAC) and glutathione monoethyl ester (GSHme) to block the particle induced release of tumour necrosis factor-alpha (TNF-alpha) from alveolar macrophages in vitro. The ultrafine particles in PM10 may cause adverse effects via oxidative stress, and this could have implications for susceptible individuals. Susceptible individuals, such as those with COPD or asthma, already exhibit preexisting oxidative stress and hence are in a primed state for further oxidative stress induced by occupational or environmental particles.

In 1997, an IARC Working Group classified quartz (crystalline silica) as a Group 1 lung carcinogen, but only in some industries, i.e., the quartz hazard is a variable entity. The reactivity of the quartz surface may underlie its ability to cause inflammation, and treatments that ameliorate this reactivity will reduce the quartz hazard. In this study we treated quartz (Q) with aluminium lactate (AL), a procedure that is reported to decrease the quartz hazard, and explored the effect this had on the highly reactive quartz surface and on proinflammatory events in rat lungs. Aluminium lactate-treated quartz showed a reduced surface reactivity as measured by electron spin resonance and the hemolysis assay. Eighteen hours after instillation of Q into the rat lung, there was massive inflammation as indicated by the number of neutrophils in the bronchoalveolar lavage (BAL). In addition, Q induced an increase in BAL macrophage inflammatory protein-2 (MIP-2) while ALQ had no significant effect compared to control. Epithelial damage, as indicated by BAL protein and gamma glutamyl transpeptidase, also increased with Q but not with ALQ. Furthermore, Q induced an increase in MIP-2 mRNA by BAL cells while ALQ had no effect compared to controls. There was an increase in nuclear binding of the transcription nuclear factor kappaB (NF-kappaB) in the Q-exposed BAL cells and again no effect on nuclear NF-kappaB binding in BAL cells from ALQ-exposed rats. In conclusion, treatment of the quartz surface with aluminium lactate reduced the reactivity of the particles both in terms of hydroxyl radical generation and in terms of the induction of molecular signaling events leading to inflammation.

L-nucleosides selectively enter malaria infected erythrocytes and have the unique ability to be metabolised by the malarial adenosine deaminase. This has allowed us to design novel L-nucleosides as potential anti-malarials.

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

Ultrafine carbon black (ufCB) 14 nm in diameter and fine carbon black (CB) 260 nm in diameter were instilled intratracheally in rats at mass of 125 microg, and the bronchoalveolar lavage (BAL) profile at 6 h was assessed. UfCB generated a 50% neutrophil alveolitis 6 h after intratracheal instillation compared to CB, which showed similar activity to the phosphate-buffered saline (PBS) vehicle control. UfCB instillation also produced a marked increase in lactate dehydrogenase (LDH) levels in BAL fluid, which was associated with increased epithelial permeability measured as total protein. In contrast, CB had much less of an effect in increasing BAL protein. Although both particle types caused a decrease in glutathione (GSH) in lung tissue compared to control, the greatest depletion was seen in ufCB-treated animals. To investigate time response, bronchoalveolar lavage was carried out at 6 h, 24 h, and 7 days after a single 125-microg instillation of ufCB. Neutrophil influx was relatively persistent and was still maintained 7 days later. Tumor necrosis factor (TNF) production by BAL leukocytes increased gradually postinstillation, whereas NO production became significantly higher at 24 h after instillation and remained at raised levels up to 7 days. Higher doses of CB caused more inflammation than the ufCB. Thus, in the instillation model, a localized dose of particle over a certain level causes the particle mass to dominate the response, rather than the surface area. In contrast to the effect of CB, which showed a dose-related increasing inflammatory response, ufCB at the highest dose caused less of a neutrophil influx than at the lower dose. Six hours after intratracheal instillation, the threshold dose for neutrophil influx occurred at 50 microg. Calculation of surface area of particles instilled suggested that this was likely to be an overload-inducing dose of particles, as gauged from recent experiments with inhaled particles. In summary, this study provides evidence in a rat instillation model that ufCB has greater ability than CB to produce lung inflammation and oxidant stress at a relatively low dose of 125 microg. At high doses, however, BAL is not a reliable indicator of pulmonary response, since the overall response seems to scale to mass or volume of instilled particulate without an influence of surface area.

Exposure to asbestos is associated with several lung diseases. The carcinogenic action of asbestos is not fully understood but oxidative stress is considered to play a role. Iron on the surface can lead to Fenton chemistry and the Haber Weiss reaction producing free radicals such as the hydroxyl radical, which is likely to be important. Little is known of the pathogenic action of man- made fibres. This study involved the use of a panel of man-made fibres, some of which were shown to be pathogenic and others shown to be non-pathogenic in recent animal studies. A short term assay measuring Fe 3+ release from the fibres over a 20 hour time period, and also a longer study of 12 weeks, found that pathogenic and non-pathogenic fibres could not be differentiated according to Fe 3+ release only. Iron release from native fibres was compared with that from surfactant- coated fibres, and in all cases surfactant coated fibres released more Fe 3+ inferring that in vivo lung lining fluid coats native fibres and therefore affects the fibre surface chemistry and hence reactivity.© 1998 Published by Elsevier Science Ltd on behalf of British Occupational Hygiene Society.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria. Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria. This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia. Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A. NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment. As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate. A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source. Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii. The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production. Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals. Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport. Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

Policy Platform A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico Janine M. Ramsey 1 , J. Guillermo Bond 1 , Maria Elena Macotela 1 , Luca Facchinelli 2,3 , Laura Valerio 2,4 , David M. Brown 5 , Thomas W. Scott 2 , Anthony A. James 5,6 * 1 Centro Regional de Investigacio´n en Salud Pu´blica, Instituto Nacional de Salud Pu´blica, Tapachula, Chiapas, Me´xico, 2 Department of Entomology, University of California, Davis, California, United States of America, 3 Department of Experimental Medicine, Functional Genomics Center, University of Perugia, Perugia, Italy, 4 Pasteur Institute–Cenci Bolognetti Foundation, University of Rome Sapienza, Rome, Italy, 5 Department of Microbiology and Molecular Genetics, University of California, Irvine, California, United States of America, 6 Department of Molecular Biology and Biochemistry, University of California, Irvine, California, United States of America Introduction Sustainable and effective control of dengue is hampered due to a number of factors, including the lack of evidence- based, locally relevant interventions; insuf- ficient information regarding key compo- nents of virus transmission and vector ecology; failure to implement precise and efficient surveillance systems; inefficient healthcare systems; ineffective health pro- motion and outreach resulting in lack of community dialogue and participation; and a paucity of efficient diagnostic strategies and clinical attention [1]. In- creased research efforts in response to the complexity of this problem have focused on the development of novel technologies that would enhance existing tools for vector-borne disease prevention [2–4]. Genetic strategies to reduce or replace mosquito populations and thereby inter- rupt transmission of dengue viruses are among the new approaches being consid- ered [5–7]. Many of these approaches take advantage of molecular genetic tools to engineer traits that cause lethal pheno- types or confer resistance to the pathogen in the mosquito. Genetic strategies are being advanced through a series of overlapping domains that inform the decision making on feasibility, safety, efficacy, and acceptabil- ity. Although the need to focus on science- based regulation using a risk-assessment framework is gaining support [8], there has been a relative lack of attention on broader community regulations that are explicitly or indirectly required to bring a genetics-based product to the field [3,9– 11]. An evidence-based approach would facilitate the integration, efficacy, and acceptability of policy for an intervention strategy. We addressed the regulatory challenges associated with testing a strain of Aedes aegypti engineered to result in population suppression in contained field trials in southwestern Mexico [12]. This large research effort (designated hereafter as the ‘‘Project’’) combined elements of scientific and social discovery and devel- opment as the basis for moving a new technology from the laboratory to the field. Unlike the rollout of other public- health products such as drugs, vaccines, and insecticides, no pipelines exist to move candidate genetically modified mosquitoes (GMMs) from the laboratory through safety and efficacy trials to field deploy- ment. This lack of a preexisting structure made it necessary for the scientists in the Project to play critical, unbiased roles in formulating the product development pathway. The challenge offered a unique opportunity for potential end users and beneficiaries of the technology to be involved from the beginning in product discovery and development. This ap- proach ensures that requirements for safety and efficacy are included as design features engineered into the modified mosquito strains [13]. It is incumbent on the researchers to identify gaps and assist in development of regulatory norms that should be applied to the products they create. These norms include not only statutory regulations but also a broader regulatory environment that addresses the needs and concerns of all communities in which the product will be applied. We describe here the regulatory and social structures used for obtaining approvals in Mexico. This review of our approach is intended to stimulate analysis and dia- logue that will help refine regulatory practices of genetic-based strategies for vector-borne disease control. Regulatory Domains for the Discovery and Development of Genetically Modified Mosquitoes An initial challenge of the Project was identifying relevant communities [14]. No consensus existed as to what comprises a relevant community to engage for a GMMs research project, nor were there any widely accepted methods for identify- ing their members. We adopted a defini- tion (modified from [15]) in which the community consists of all those individuals who share the identified risks and/or will benefit from the outcome of the proposed research project. In this context, the community coalesces as a result of the project and evolves continuously as it progresses through conceptualization, dis- cussion, and implementation [16]. The community is formed ultimately by those individuals, groups, organizations, and agencies that have legitimate interest in the research, and therefore they must be engaged in an effective and timely man- ner. Successfully moving a novel technology from the laboratory to practical application Citation: Ramsey JM, Bond JG, Macotela ME, Facchinelli L, Valerio L, et al. (2014) A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico. PLoS Negl Trop Dis 8(3): e2623. doi:10.1371/journal.pntd.0002623 Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America Published March 13, 2014 Copyright: s 2014 Ramsey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research and Policy for Infectious Disease Dynamics program of the Science and Technology Directory, Department of Homeland Security; Fogarty International Center, National Institutes of Health; Pasteur Institute – Cenci Bolognetti Foundation; Foundation for the National Institutes of Health through the Grand Challenges in Global Health (GCGH) initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare no conflict of interest. * E-mail: aajames@uci.edu PLOS Neglected Tropical Diseases | www.plosntds.org March 2014 | Volume 8 | Issue 3 | e2623

We studied asbestos, vitreous fiber (MMVF10), and refractory ceramic fiber (RCF1) from the Thermal Insulation Manufacturers' Association fiber repository regarding the following: free radical damage to plasmid DNA, iron release, ability to deplete glutathione (GSH), and activate redox-sensitive transcription factors in macrophages. Asbestos had much more free radical activity than any of the man-made vitreous fibers. More Fe3+ was released than Fe2+ and more of both was released at pH 4.5 than at pH 7.2. Release of iron from the different fibers was generally not a good correlate of ability to cause free radical injury to the plasmid DNA. All fiber types caused some degree of oxidative stress, as revealed by depletion of intracellular GSH. Amosite asbestos upregulated nuclear binding of activator protein 1 transcription factor to a greater level than MMVF10 and RCF1; long-fiber amosite was the only fiber to enhance activation of the transcription factor nuclear factor kappa B (NF kappa B). The use of cysteine methyl ester and buthionine sulfoximine to modulate GSH suggested that GSH homeostasis was important in leading to activation of transcription factors. We conclude that the intrinsic free radical activity is the major determinant of transcription factor activation and therefore gene expression in alveolar macrophages. Although this was not related to iron release or ability to deplete macrophage GSH at 4 hr, GSH does play a role in activation of NF kappa B. Images Figure 1. Figure 5. A Figure 5. B Figure 6. A Figure 6. B

A wide range of fiber types was tested in two in vitro assays: toxicity to A549 epithelial cells, as detachment from substrate, and the production of the proinflammatory cytokine tumor necrosis factor (TNF) by rat alveolar macrophages. Three of the fibers were also studied in vivo, using short-term inhalation followed by a) bronchoalveolar lavage to assess the inflammatory response and b) measurement of cell proliferation in terminal bronchioles and alveolar ducts, using incorporation of bromodeoxyuridine (BrdU). The amount of TNF produced by macrophages in vitro depended on the fiber type, with the man-made vitreous fibers, and refractory ceramic fibers being least stimulatory and silicon carbide (SiC) whiskers providing the greatest stimulation. In the epithelial detachment assay there were dose-dependent differences in the toxicity of the various fibers, with long amosite being the most toxic. However, there was no clear relationship to known chronic pathogenicity. Fibers studied by short-term inhalation produced some inflammation, but there was no clear discrimination between the responses to code 100/475 glass fibers and the more pathogenic amosite and SiC. However, measurements of BrdU uptake into lung cells showed that amosite and SiC produced a greater reaction than code 100/475, which itself caused no more proliferation than that seen in untreated lungs. These results mirror the pathogenicity ranking of the fibers in long-term experiments. In conclusion, the only test to show potential as a predictive measure of pathogenicity was that of cell proliferation in lungs after brief inhalation exposure (BrdU assay). We believe that this assay should be validated with a wider range of fibers, doses, and time points.

Engineered nanoparticles are increasingly used in medical applications and day-to-day consumer products, leading to concerns about the potential environmental and human health impacts. Silver nanoparticles are particularly prevalent because of their use as anti-bacterial agents in many commonly available products. Nanoparticles (NPs) are believed to accumulate, often preferentially, in the liver. This study therefore investigates the effect of a silver NP (20 nm) on the liver, and in particular, the role of Kupffer cells (KCs; resident liver macrophages) in the overall inflammatory response in the organ. Cytokine expression in the normal liver was measured in terms of IL2, IL4, TNF-α, IFN-γ and IL10 released from the organ with significant up-regulation of TNF-α and IL10 being observed. For livers in which the KC population was specifically targeted and destroyed this cytokine increase was significantly decreased in comparison to the normal tissue. IL10 was secreted at approximately three times the concentration of TNF-α in all the test cases. The high levels of IL10 released from the normal tissue in comparison to the KC depleted livers suggest that the cytokine may help to protect against a pro-inflammatory response to these Ag NPs. This may indicate a potentially important role for KCs in the anti-inflammatory response and suggests that tolerance to the Ag NPs is favoured over a fully activated immune response. In addition, albumin production was measured as an indicator of hepatic function. It was noted that the liver function was unaffected by the Ag NPs.

The potential hazard to humans exposed to nanomaterials such as silica and iron oxide was investigated using an in vitro macrophage cell culture system. Amorphous silica and iron oxide particles and nanomaterials (NMs) were dispersed in cell culture medium supplemented with either bovine serum albumin (BSA), lung lining fluid (LLF) or serum, in order to mimic the body fluids encountered during different routes of exposure in the body. End points investigated included macrophage viability and cytokine production. Silica NMs and particles (50 and 200 nm, respectively) were unmodified (plain) or aminated (NH2 ). Iron oxide NMs and particles, Fe3 O4 45 nm and Fe2 O3 280 nm were also used in this study. Silica particles and NMs induced a dose-dependent increase in cytotoxicity as measured by lactate dehydrogenase (LDH) release. Serum enhanced silica-induced interleukin (IL)-6, IL-10, IL-1β and MCP-1 release, whereas albumin partially inhibited MCP-1 release. Aminated silica, 50 nm was more potent than the 200-nm particles at inducing monocyte chemoattractant protein-1 (MCP-1) production when dispersed in medium or LLF, suggesting a size specific effect for these particles and this cytokine. Iron oxide particles were relatively inert compared with the silica particles and NMs; however, serum and albumin did affect cytokine release in some treatments. In conclusion, the data suggests that serum, compared with medium, BSA and LLF is very potent at enhancing macrophage responses to silica and iron oxide particles and NMs. Size was only influential in LLF for a limited number of parameters, whereas surface chemistry was not of consequence in this in vitro macrophage system.

Recombinant platelet-derived growth factor (rPDGF-BB) stimulates migration and proliferation of fibroblasts and smooth muscle cells and induces the rapid development of granulation tissue. This study investigated the use of rPDGF-BB and skin grafting to heal avascular ulcers using a rabbit model. We further investigated the effect of a hyaluronic acid carrier and a vascular pedicle on rPDGF-induced tissue generation in this model.Large avascular ulcers were created on both ears of New Zealand white rabbits. One ulcer was treated with topical rPDGF-BB, the other with control buffer. After 5 or 7 days, the amount of granulation tissue migration from the wound margin was measured, and the wounds were skin grafted. In another group of ulcers, rPDGF-BB treatment was combined with a hyaluronic acid disk or an intact central axial ear artery and vein.Whereas control wounds generated 1.00 +/- 0.27 mm and 1.75 +/- 0.25 mm of granulated tissue migration from the wound margin by days 5 and 7, respectively, rPDGF-BB treatment increased the amount of granulation tissue migration to 1.88 +/- 0.23 mm and 3.00 +/- 0.86 mm by days 5 and 7 after treatment, respectively (P0.05 at both time points). Hyaluronic acid disks stimulated 2.50 +/- 0.22 mm of granulation tissue migration by day 7 in controls; when rPDGF-BB was added to the carrier, the migration increased to 4.50 +/- 0.29 mm from the wound margin (P0.05). Granulation tissue migration was further increased with an intact vascular pedicle: 5.75 +/- 0.48 mm in controls versus 7.75 +/- 0.25 mm with rPDGF-BB treatment (P0.01). Skin grafts applied to the treated ulcers failed to survive in all groups except those with intact vascular pedicles. CONCLUSIONS. This study demonstrates that rPDGF-BB can increase the amount of granulation tissue generated over an avascular wound. This tissue generation is enhanced by both a hyaluronic acid carrier and a vascular pedicle. A vascular pedicle was required for skin graft survival. This study supports the role of hyaluronic acid in rPDGF-BB induced tissue generation and the requirement of a direct blood supply for functional cytokine-induced tissue generation.

It has been reported that excretory-secretory (ES) material from the parasitic nematode Nippostrongylus brasiliensis has potential modulatory effects on the host's immune system. We observed that intratracheal instillation of ES from the L3 stage of the parasite reduced neutrophil numbers in LPS-induced inflammation as assessed by bronchoalveolar lavage.

Background Transgenic mosquito strains are being developed to contribute to the control of dengue and malaria transmission. One approach uses genetic manipulation to confer conditional, female-specific dominant lethality phenotypes. Engineering of a female-specific flightless phenotype provides a sexing mechanism essential for male-only mosquito, release approaches that result in population suppression of target vector species. Methods An approach that uses a female-specific gene promoter and antibiotic-repressible lethal factor to produce a sex-specific flightless phenotype was adapted to the human malaria vector, Anopheles stephensi. Transposon- and site-specific recombination-mediated technologies were used to generate a number of transgenic An. stephensi lines that when combined through mating produced the phenotype of flight-inhibited females and flight-capable males. Results The data shown here demonstrate the successful engineering of a female-specific flightless phenotype in a malaria vector. The flightless phenotype was repressible by the addition of tetracycline to the larval diet. This conditional phenotype allows the rearing of the strains under routine laboratory conditions. The minimal level of tetracycline that rescues the flightless phenotype is higher than that found as an environmental contaminant in circumstances where there is intensive use of antibiotics. Conclusions These studies support the further development of flightless female technology for applications in malaria control programmes that target the vectors.

The delivery of large DNA vectors (>100 000 bp) remains a limiting step in the engineering of mammalian cells and the development of human artificial chromosomes (HACs). Yeast is commonly used to assemble genetic constructs in the megabase size range, and has previously been used to transfer constructs directly into cultured cells. We improved this method to efficiently deliver large (1.1 Mb) synthetic yeast centromeric plasmids (YCps) to cultured cell lines at rates similar to that of 12 kb YCps. Synchronizing cells in mitosis improved the delivery efficiency by 10-fold and a statistical design of experiments approach was employed to boost the vector delivery rate by nearly 300-fold from 1/250 000 to 1/840 cells, and subsequently optimize the delivery process for multiple mammalian, avian, and insect cell lines. We adapted this method to rapidly deliver a 152 kb herpes simplex virus 1 genome cloned in yeast into mammalian cells to produce infectious virus.

Increased airspace epithelial permeability is an early event in lung inflammation and injury. In this study, we have developed a rat model to study the mechanisms of the epithelial permeability to 125iodine-labeled bovine serum albumin (125I-BSA), instilled intratracheally during acute lung inflammation. Epithelial permeability was measured as the percentage of instilled 125I-BSA appearing in the blood. The increase in epithelial permeability induced by intratracheal instillation of heat-killed Corynebacterium parvum produced a peak influx of neutrophils into the bronchoalveolar space at 16 h, which occurred after the peak increase in epithelial permeability (8 h). The increased epithelial permeability induced by C. parvum did not appear to be protease- or oxidant-mediated. Depletion of peripheral blood neutrophils was achieved by an intravenous injection of anti-neutrophil polyclonal antibody. The consequent profound reduction in neutrophil and macrophage influx into the airspaces 8 h after instillation of C. parvum reduced the epithelial permeability to control values. Bronchoalveolar lavage (BAL) leukocytes from rats 8 h, but not 16 h, after treatment with C. parvum caused a modest increase in epithelial permeability when re-instilled intratracheally into control rat lungs. Separation of the leukocytes before re-instillation indicated that macrophages rather than neutrophils were predominantly responsible for the increased epithelial permeability. The presence of dramatically increased levels of tumor necrosis factor (TNF) in BAL 8 h in contrast to a slight increase in BAL 16 h after C. parvum, the release of TNF from 8 h macrophages, the increased epithelial permeability induced by TNF in epithelial monolayers in vitro, and the inhibition of C. parvum-induced epithelial permeability by TNF antibody support the premise that TNF is a major player in the increased epithelial permeability that occurs during C. parvum-induced acute alveolitis.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

Non-denaturing polyacrylamide gels were used to analyse superoxide dismutase (SOD), catalase, peroxidase, NADH oxidase and NADH peroxidase in the microaerophilic protozoan parasite Giardia duodenalis. A cytosolic H2O-producing NADH oxidase and membrane-associated NADH peroxidase were readily detected from G. duodenalis. In all Giardia strains investigated the NADH oxidase was present in high levels (1.2-2 U (mg protein)-1). Using the same technique, NADH oxidase activity was also detected in the microaerophilic protozoan parasites Tritrichomonas foetus, Trichomonas vaginalis and Entamoeba histolytica and in the bacterium Escherichia coli. The conventional enzymes of oxidative stress management (superoxide dismutase, catalase and peroxidase) were not detected in particulate or cytosolic extracts from recent and established strains of Giardia assayed in situ. Spectrophotometric assays also yielded negative results. The same methodology readily detected one or more of these enzyme activities in T. foetus, T. vaginalis and E. coli. Superoxide dismutase activity was not detected in lines of Giardia resistant to high levels of metronidazole or furazolidone. Furthermore, the agents 1,10 phenanthroline, diamide, MnCl2 and KNO3, which induce SOD in anaerobically cultured E. coli, did not induce SOD in Giardia. 1,10 phenanthroline has also been shown to induce iron-containing (Fe-) SOD in Entamoeba. Neither peroxidase nor catalase activities were detected in a peroxide-resistant line of Giardia. Viable trophozoites from parent lines were able to decompose H2O2 at a significant rate. It appears that the conventional SOD, catalase and peroxidase utilised in aerobic metabolism have been substituted in Giardia by NADH oxidase and NADH peroxidase, similar to anaerobic bacteria. The O2-scavenging NADH oxidase explains the previously observed futile 'respiration' in Giardia.

We studied renal autoregulation in nondiabetic and streptozotocin diabetic Munich-Wistar rats 6 weeks after diabetes induction. We had previously shown that diabetic rats had greater preservation of renal blood flow (RBF) at reduced renal perfusion pressures (RPP) than control rats and speculated that this could be due to higher protein intake in the diabetic rats. Because of hyperphagia, the diabetic rats in the present study on a 24% protein diet (D-24) had 70% or greater increase in dietary protein intake compared to controls on the same diet (C-24). Diabetic rats on a 14% protein diet (D-14) had a dietary protein intake similar to C-24 and less than that of D-24 animals. Baseline glomerular filtration rate (GFR) and renal blood flow were lower and renal vascular resistance (RVR) was higher in the D-14 compared to the C-24 or D-24 rats. Nonetheless, at markedly reduced RPP both diabetic groups had better sustained RBFs and lower RVRs than the controls. Thus, increased dietary protein intake cannot explain the autoregulatory abnormalities in diabetes. Bilateral carotid artery ligation increased systemic blood pressure similarly in the C-24, D-24, and D-14 rats. All three groups responded with increased RVR to carotid artery ligation. Following carotid artery ligation, differences were no longer seen between diabetic and control rats for RBF at reduced RPPs. These studies indicate that diabetic rats are capable of generating increased RVR, ruling out impaired vascular constrictive capacity as an explanation of these hemodynamic abnormalities. It is hypothesized that carotid artery ligation resulted in sympathic stimulation of intrarenal renin release which overcame the decreased glomerular angiotensin II receptor density in diabetic rats, thus normalizing the renal vascular responses to hypoperfusion.