Toxicological impact of organic ultrafine particles (UFPs) in human bronchial epithelial BEAS-2B cells at air-liquid interface.

Air pollution has significant health effects worldwide, and airborne particles play a significant role in these effects. Ultrafine particles (UFPs) have an aerodynamic diameter of 0.1 µm or less, can penetrate deep into the respiratory tree, and are more toxic due to their large specific surface area, which should adsorb organic compounds. The aim of this study is to show the toxicological effects of UFPs with high organic content at low dose on BEAS-2B cells through at air-liquid interface (ALI) exposure using a Vitrocell® technology and a miniCAST (Combustion Aerosol Standard) generator. In conjunction with this approach, chemical analysis of particles and gas phase was performed to evaluate the presence of polycyclic aromatic hydrocarbons (PAHs). Chemical analyses confirmed the presence of PAHs in UFPs. With this experimental setup, exposure of the BEAS-2B cells induced neither cytotoxicity nor mitochondrial dysfunction. However, an increase of oxidative stress was observed, as assessed through Nrf2, NQO1, HO-1, CuZnSOD, MnSOD, and Catalase gene expression, together with significant induction of genes related to xenobiotic metabolism CYP1A1 and CYP1B1. Negative regulation of inflammatory genes expression (IL-6 and IL-8) was present three hours after the exposition to the UFPs. Taken together, this experimental approach, using repeatable conditions, should help to clarify the mechanisms by which organic UFPs induce toxicological effects.

Prevalence and correlates of having sexual and reproductive health priorities met by HIV providers among women living with HIV in a Canadian setting.

OBJECTIVE: To: (1) describe the prevalence of key reproductive health outcomes (e.g., pregnancy, unintended pregnancy; abortion); and (2) examine social-structural correlates, including HIV stigma, of having key sexual and reproductive health (SRH) priorities met by participants' primary HIV provider, among women living with HIV. METHODS: Data were drawn from a longitudinal community-based open cohort (SHAWNA) of women living with HIV. The associations between social-structural factors and two outcomes representing having SRH priorities met by HIV providers ('being comfortable discussing sexual health [SH] and/or getting a Papanicolaou test' and 'being comfortable discussing reproductive health [RH] and/or pregnancy needs') were analyzed using bivariate and multivariable logistic regression models with generalized estimating equations for repeated measures over time. Adjusted odds ratios (AOR) and 95% confidence intervals [95% CIs] are reported. RESULTS: Of 314 participants, 77.1% reported having SH priorities met while 64.7% reported having RH priorities met by their primary HIV provider at baseline. In multivariable analysis, having SH priorities met was inversely associated with: sexual minority identity (AOR: 0.59, 95% CI: 0.37-0.94), gender minority identity (AOR: 0.52, 95% CI: 0.29-0.95) and recent verbal or physical violence related to HIV status (AOR: 0.55, 95% CI: 0.31-0.97) and positively associated with recently accessing women-centred services (Oak Tree Clinic) (AOR: 4.25, 95% CI: 2.20-8.23). Having RH priorities met was inversely associated with: sexual minority identity (AOR: 0.56, 95% CI: 0.40-0.79), gender minority identity (AOR: 0.45, 95% CI: 0.25-0.81) and being born in Canada (AOR: 0.29, 95% CI: 0.15-0.56) and positively associated with recently accessing women-centred services (AOR: 1.81, 95% CI: 1.29-2.53) and a history of pregnancy (AOR: 2.25, 95% CI: 1.47-3.44). CONCLUSION: Our findings suggest that there remain unmet priorities for safe SRH care and practice among women living with HIV, and in particular, for women living with HIV with sexual and/or gender minority identity and those who experience enacted HIV stigma. HIV providers should create safe, non-judgmental environments to facilitate discussions on SRH. These environments should be affirming of all sexual orientations and gender identities, culturally safe, culturally humble and use trauma-informed approaches.

Piloting an evidence-based intervention for HIV prevention among street youth in Eldoret, Kenya.

OBJECTIVES: This study presents findings from piloting an adapted evidence-based intervention, Stepping Stones and Creating Futures, to change street-connected young people's HIV knowledge, condom-use self-efficacy, and sexual practices. METHODS: Eighty street-connected young people participated in a pre- and post-test mixed methods design in Eldoret, Kenya. The primary outcome of interest was HIV knowledge. Secondary outcomes included condom-use self-efficacy and sexual practices. Multiple linear regression models for change scores with adjustment for socio-demographic variables were fitted. Qualitative and quantitative findings are presented together, where integration confirms, expands on, or uncovers discordant findings. RESULTS: Participants had a significant increase in HIV knowledge from pre- to post-intervention. The median HIV knowledge score pre-intervention was 11 (IQR 8-13) and post-intervention 14 (IQR 12-16). Attendance was significantly associated with HIV knowledge change scores. Qualitatively participants reported increased HIV and condom-use knowledge and improved condom-use self-efficacy and health-seeking practices. CONCLUSIONS: Our findings support the potential for further testing with a rigorous study design to investigate how best to tailor the intervention, particularly by gender, and increase the overall effectiveness of the program.

Impact of after-treatment devices and biofuels on diesel exhausts genotoxicity in A549 cells exposed at air-liquid interface.

Using an air-liquid interface (ALI) device in dynamic conditions, we evaluated the efficiency of fuel after-treatment strategies (diesel oxidation catalysis, DOC, and diesel particulate filter, DPF, devices) and the impact of 7% and 30% rapeseed methyl esters (RME) blending on oxidative stress and genotoxicity induced in A549 lung cells after 3h exposure to whole Diesel exhausts. Oxidative stress was studied using assays of ROS production, glutathione level, catalase and superoxide-dismutase (SOD) activities. No oxidative stress and no clear differences on cytotoxicity patterns between biodiesel and standard Diesel exhausts were found. A weak but significant genotoxicity (8-oxodGuo adducts) and, for standard Diesel only, a DNA damage response (DDR) as evidenced by ƔH2AX foci, remained after DOC+DPF flowing. All together, these data could contribute to the improvement of the after treatment strategies and to health risk assessment of current diesel exhausts.

A social ecological approach to understanding correlates of lifetime sexual assault among sexual minority women in Toronto, Canada: results from a cross-sectional internet-based survey.

Stigma, discrimination and violence contribute to health disparities among sexual minorities. Lesbian, bisexual and queer (LBQ) women experience sexual violence at similar or higher rates than heterosexual women. Most research with LBQ women, however, has focused on measuring prevalence of sexual violence rather than its association with health outcomes, individual, social and structural factors. We conducted a cross-sectional online survey with LBQ women in Toronto, Canada. Multivariate logistic regression analyses were conducted to assess correlates of lifetime sexual assault (LSA). Almost half (42%) of participants (n = 415) reported experiences of LSA. Participants identifying as queer were more likely to have experienced LSA than those identifying as lesbian. When controlling for socio-demographic characteristics, experiencing LSA was associated with higher rates of depression, sexually transmitted infections (STIs), receiving an STI test, belief that healthcare providers were not comfortable with their LBQ sexual orientation, and sexual stigma (overall, perceived and enacted). A history of sexual violence was associated with lower: self-rated health, overall social support, family social support and self-esteem. This research highlights the salience of a social ecological framework to inform interventions for health promotion among LBQ women and to challenge sexual stigma and sexual violence.

HIV vaccine acceptability and culturally appropriate dissemination among sexually diverse Aboriginal peoples in Canada.

This study explored HIV vaccine acceptability and strategies for culturally appropriate dissemination among sexually diverse Aboriginal peoples in Canada, among those at highest HIV risk. We conducted four focus groups (n=23) with Aboriginal male (1) and female (1) service users, peer educators (1) and service providers (1) in Ontario, Canada. Transcripts were analysed with narrative thematic techniques from grounded theory, using NVivo. Participants' mean age was 37 years; about half (52%) were female, half (48%) Two-spirit or lesbian, gay or bisexual (LGB)-identified, 48% had a high-school education or less and 57% were unemployed. Vaccine uptake was motivated by community survival; however, negative HIV vaccine perceptions, historically based mistrust of government and healthcare institutions, perceived conflict between western and traditional medicine, sexual prejudice and AIDS stigma within and outside of Aboriginal communities, and vaccine cost may present formidable obstacles to HIV vaccine acceptability. Culturally appropriate processes of engagement emerged on individual levels (i.e., respect for self-determination, explanations in Native languages, use of modelling and traditional healing concepts) and community levels (i.e., leadership by Aboriginal HIV advocates and political representatives, identification of gatekeepers, and procuring Elders' endorsements). Building on cultural strengths and acknowledging the history and context of mistrust and social exclusion are fundamental to effective HIV vaccine dissemination.

Genome-wide functions of PML-RARα in acute promyelocytic leukaemia.

PML-RAR (retinoic acid receptor)α is the hallmark protein of acute promyelocytic leukaemia, a highly malignant subtype of acute myeloid leukaemia that accounts for approximately 10% of all AML cases. Recently, several studies have been set out to obtain a comprehensive genome-wide view of the molecular actions of this chimeric protein. In this review, we highlight the new insights that arose from these studies, in particular focussing on newly identified PML-RARα target genes, its interplay with RXR and deregulation of epigenetic modifications.

Meta-analysis of health and demographic correlates of stigma towards people living with HIV.

HIV-related stigma may negatively impact the health, quality of life, social support and well-being of people living with HIV (PLHIV). Previous studies have used diverse samples and a multitude of measurement instruments to examine demographic and health correlates of HIV-related stigma, highlighting the importance of synthesizing findings across different studies to gain a better understanding of these associations. This study examined the relationships between HIV-related stigma and a range of demographic, social, physical and health characteristics. A meta-analysis was conducted to assess the overall strength and direction of these relationships. Twenty-four studies of PLHIV, conducted in North America and published in peer-reviewed journals between January of 2000 and November of 2007, were examined and their findings integrated. The heterogeneity of reported results was also assessed and examined. Our review revealed substantial variability in the ways researchers measure participants' HIV-related stigma as well as their physical, emotional and mental health. In spite of this variability, high stigma level was consistently and significantly associated with low social support (r = -0.369, p<0.0005), poor physical health (r = -0.324, p<0.0005), poor mental health (r = -0.402, p<0.0005), age (-0.066, p<0.05) and income (-0.172, p<0.005). These correlations were of a medium size, which would be recognized by the individual in daily life. Health and mental health professionals working with individuals and families impacted by HIV could benefit from an enhanced understanding of correlates of HIV-related stigma, which will inform assessments, interventions and treatment plans. The association between HIV-related stigma and physical health has potential implications for treatment, care and support for people at different stages of HIV infection. AIDS Service Organizations are also encouraged to integrate findings into HIV stigma interventions and social support programs. Additionally, HIV-related stigma scales should be developed and validated, so that future studies using them are able to report findings that are operationally and conceptually consistent.

Single-pair FRET microscopy reveals mononucleosome dynamics.

We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% betaME. Replacing the triplet quencher betaME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10's of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.

Bacterial induction of pleural mesothelial monolayer barrier dysfunction.

Pneumonia remains one of the most common infectious causes of mortality. Patients with pneumonia develop parapneumonic effusions with a high neutrophil count as well as high protein concentrations. We hypothesized that pulmonary parenchymal bacterial infection causes a permeability change in the pleural mesothelium by inducing the production of vascular endothelial growth factor (VEGF). Complicated parapneumonic pleural effusions (empyema) have a 19-fold higher VEGF level than pleural fluids secondary to congestive heart failure and a 4-fold higher level than pleural fluids secondary to uncomplicated parapneumonic effusions. We also analyzed the influence of live Staphylococcus aureus on mesothelial barrier function using a model of confluent mesothelial monolayers. There was a significant drop in electrical resistance across S. aureus-infected pleural mesothelial cell (PMC) monolayers. Recombinant VEGF also decreases PMC electrical resistance. Neutralizing antibodies to VEGF significantly inhibited the drop in PMC electrical resistance caused by S. aureus. S. aureus infection also caused a significant increase in protein leak across confluent mesothelial monolayers. Our results suggest that bacterial pathogens induce VEGF release in mesothelial cells and alter mesothelial permeability, leading to protein exudation in empyema.

The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling.

Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.

Recruitment of chromatin remodeling machines.

The assembly of eukaryotic DNA into folded nucleosomal arrays has drastic consequences for many nuclear processes that require access to the DNA sequence, including RNA transcription, DNA replication, recombination, and repair. Two types of highly conserved chromatin remodeling enzymes have been implicated as regulators of the repressive nature of chromatin structure: ATP-dependent remodeling complexes and nuclear histone acetyltransferases (HATs). Recent studies indicate that both types of enzymes can be recruited to chromosomal loci through either physical interactions with transcriptional activators or via the global accessibility of chromatin during S phase of the cell cycle. Here we review these recent observations and discuss the implications for gene-specific regulation by chromatin remodeling machines.

SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state.

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.

Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates. We also perform assays with arrays reconstituted with hyperacetylated or trypsinized histones and isolated histone (H3/H4)(2) tetramers. The results define three groups of the ATP-dependent family of remodeling enzymes. In addition we investigate the ability of an acidic activator to recruit remodeling complexes to nucleosomal arrays. We propose that ATP-dependent chromatin remodeling enzymes share a common reaction mechanism and that a key distinction between complexes is in their mode of regulation or recruitment.

Recruitment of the SWI/SNF chromatin remodeling complex by transcriptional activators.

SWI/SNF is a chromatin remodeling complex that facilitates expression of a number of yeast genes. Here we demonstrate that SWI/SNF can be recruited from yeast nuclear extracts by a transcriptional activator. Recruitment is dependent on an activation domain but not on promoter sequences, TBP, or RNA polymerase II holoenzyme. We also show that acidic activation domains can target SWI/SNF remodeling activity. These results demonstrate that SWI/SNF activity can be targeted by gene-specific activators and that this recruitment can occur independently of Pol II holoenzyme.

The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes.

SWI/SNF and RSC are large, distinct multi-subunit complexes that use the energy of ATP hydrolysis to disrupt nucleosome structure, facilitating the binding of transcription factors or restriction enzymes to nucleosomes [Cote, J., Quinn, J., Workman, J. L., and Peterson, C. L. (1994) Science 265, 53-60 (1); Lorch, Y., Cairns, B. R., Zhang, M., and Kornberg, R. D. (1998) Cell 94, 29-34 (2)]. Here we have used a quantitative assay to measure the activities of these ATP-dependent chromatin remodeling complexes using nucleosomal arrays reconstituted with hypoacetylated, hyperacetylated, or partially trypsinized histones. This assay is based on measuring the kinetics of restriction enzyme digestion of a site located within the central nucleosome of a positioned 11-mer array [Logie, C., and Peterson, C. L. (1997) EMBO J. 16, 6772-6782 (3)]. We find that the DNA-stimulated ATPase activities of SWI/SNF and RSC are not altered by the absence of the histone N-termini. Furthermore, ATP-dependent nucleosome remodeling is also equivalent on all three substrate arrays under reaction conditions where the concentrations of nucleosomal array and either SWI/SNF or RSC are equivalent. However, SWI/SNF and RSC cannot catalytically remodel multiple nucleosomal arrays in the absence of the histone termini, and this catalytic activity of SWI/SNF is decreased by histone hyperacetylation. These results indicate that the histone termini are important for SWI/SNF and RSC function; and, furthermore, our data defines a step in the remodeling cycle where the core histone termini exert their influence. This step appears to be after remodeling, but prior to intermolecular transfer of the remodelers to new arrays.

Positive and negative discrimination of estrogen receptor agonists and antagonists using site-specific DNA recombinase fusion proteins.

Activation of the estrogen receptor (ER) by hormone involves at least two steps. First, hormone binding initially relieves repression, a property imposed on ER in cis by its ligand-binding domain (EBD). Subsequently, the derepressed ER binds specific genomic sites and regulates transcription. In addition to the natural hormone, ER binds a broad range of ligands that evoke a spectrum of responses ranging from full ER activation by agonists to partial activation and inhibition by partial or complete antagonists. How these different ligands evoke different ER responses remains unclear. To address this issue, we have developed a nontranscriptional assay for ER ligand responsiveness based on Flp recombinase/human EBD protein chimeras. These fusion proteins transduce the transient event of ligand binding into a permanent DNA change in a human cell line system. A fusion protein including ER D, E, and F domains was activated by all the ER ligands tested, demonstrating that both agonists and antagonists serve to relieve initial repression, and that differences between them lie downstream in the activation pathway. Mutant variants of the Flp-ER protein that distinguish between agonists and antagonists, and a mutant EBD that selectively lost the ability to respond to 17beta,-estradiol but not to other ligands, were also identified. Thus, agonists and antagonists can be functionally distinguished in a nontranscriptional assay.

FLP recombinase/estrogen receptor fusion proteins require the receptor D domain for responsiveness to antagonists, but not agonists.

The ligand-binding domains of steroid receptors convey ligand-dependent regulation to certain proteins to which they are fused. Here we characterize fusion proteins between a site-specific recombinase, FLP, and steroid receptor ligand-binding domains. These proteins convert ligand binding into DNA recombination. Thus, ligand binding is directly coupled to an enzyme activity that is easily measured by DNA rearrangements or heritable genetic changes in marker gene expression, as opposed to the multiple events leading to transcription. Recombination by a FLP-estrogen receptor (FLP-EBD) fusion is activated by all tested estrogens, whether agonists or antagonists, indicating that all induce EBD release from the 90-kDa heat shock protein complex. Altering the distance between FLP and the EBD domain in the fusion proteins, by reducing the included length of the estrogen receptor D domain, affects ligand efficacy. A FLP-EBD with no D domain shows reduced inducibility by agonists and, unexpectedly, complete insensitivity to induction by all antagonists tested. A FLP-EBD including some D domain shows a ligand-inducible phenotype intermediate to those displayed by FLP-EBDs containing all or none of the D domain. Thus, we observed a tethered interference between FLP and the EBD domains that differs depending on the distance between the two domains, the conformations induced by agonists or antagonists, and which presents a previously undetectable distinction between estrogen agonists and antagonists in yeast.