Immune Cell-Type Specific Ablation of Adapter Protein ADAP Differentially Modulates EAE.

The cytosolic adhesion and degranulation-promoting adapter protein ADAP is expressed in various hematopoietic cells including T cells, NK cells, myeloid cells, and platelets but absent in mature B cells. The role of ADAP in T cell activation, proliferation and integrin activation is well-accepted. We previously demonstrated that conventional ADAP knockout mice show a significantly attenuated course of experimental autoimmune encephalomyelitis (EAE). To dissect the impact of different ADAP expressing cell populations on the reduced EAE severity, here, we generated lineage-specific conditional knockout mice. ADAP was deleted in T cells, myeloid cells, NK cells and platelets, respectively. Specific loss of ADAP was confirmed on the protein level. Detailed immunophenotyping was performed to assess the consequence of deletion of ADAP with regard to the maturation and distribution of immune cells in primary and secondary lymphoid organs. The analysis showed equivalent results as for conventional ADAP knockout mice: impaired thymocyte development in ADAPfl/fl Lck-Cre mice, normal NK cell and myeloid cell distribution in ADAPfl/fl NKp46-Cre mice and ADAPfl/fl LysM-Cre mice, respectively as well as thrombocytopenia in ADAPfl/fl PF4-Cre mice. Active EAE was induced in these animals by immunization with the myelin oligodendrocyte glycoprotein35-55 peptide. The clinical course of EAE was significantly milder in mice with loss of ADAP in T cells, myeloid cells and NK cells compared to ADAP-sufficient control littermates. Surprisingly, specific deletion of ADAP in platelets resulted in a more exacerbated disease. These data show that T cell-independent as well as T cell-dependent mechanisms are responsible for the complex phenotype observed in conventional ADAP knockout mice.

Characterization of Mice with a Platelet-Specific Deletion of the Adapter Molecule ADAP.

The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knockout mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knockout mice (ADAPfl/fl PF4-Cretg) (PF4, platelet factor 4). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even under stimulation conditions. ADAPfl/fl PF4-Cretg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and transforming growth factor ß1 (TGF-ß1). In vitro, platelets from these mice revealed reduced P-selectin expression, lower levels of TGF-ß1 release, diminished integrin αIIbß3 activation, and decreased fibrinogen binding after stimulation with podoplanin, the ligand of C-type lectin-like receptor 2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of phospholipase Cγ2 (PLCγ2) and extracellular signal-regulated kinase 1/2 (ERK1/2). Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-ß1 early after T cell transfer reduced EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo.

Molecular Marker Study of Particulate Organic Matter in Southern Ontario Air.

To study the origins of airborne particulate organic matter in southern Ontario, molecular marker concentrations were studied at Hamilton, Simcoe, and York Gateway Tunnel, representing industrial, rural, and heavy traffic sites, respectively. Airborne particulate matter smaller than 10 µm in aerodynamic diameter was collected on quartz filters, and the collected samples were analyzed for total carbons, 5-6 ring PAHs, hopanes, n-alkanes (C20 to C34), and oxygenated aromatic compounds. Results showed that PAH concentrations at all three sites were highly correlated, indicating vehicular emissions as the major source. Meanwhile, in the scatter plots of α,ß-hopane and trisnorhopane, concentrations displayed different trends for Hamilton and Simcoe. The slopes of the linear regressions for Hamilton and the tunnel were statistically the same, while the slope for Simcoe was significantly different from those. Comparison with literature values revealed that the trend observed at Simcoe was explained by the influence from coal combustion. We also found that the majority of oxygenated aromatic compounds at both sites were in the similar level, possibly implying secondary products contained in the southern Ontario air. Regardless of some discrepancies, absolute principal component analysis applied to the datasets could reproduce those findings.

A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities.

To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121-137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.

Laboratory studies of carbon kinetic isotope effects on the production mechanism of particulate phenolic compounds formed by toluene photooxidation: a tool to constrain reaction pathways.

In this study, we examined compound-specific stable carbon isotope ratios for phenolic compounds in secondary organic aerosol (SOA) formed by photooxidation of isotope-label-free toluene. SOA generated by photooxidation of toluene using a continuous-flow reactor and an 8 m(3) indoor smog chamber was collected on filters, which were extracted with acetonitrile for compound-specific analysis. Eight phenolic compounds were identified in the extracts using a gas chromatograph coupled with a mass spectrometer, and their compound-specific stable carbon isotope ratios were determined using a gas chromatograph coupled with a combustion furnace followed by an isotope ratio mass spectrometer. The majority of products, including methylnitrophenols and methylnitrocatechols, were isotopically depleted by 5-6‰ compared to the initial isotope ratio of toluene, whereas the isotope ratio for 4-nitrophenol remained identical to that of toluene. On the basis of the reaction mechanisms proposed in previous reports, stable carbon isotope ratios of these products were calculated. By comparing the observed isotope ratios with the predicted isotope ratios, we explored possible production pathways for the particulate phenolic compounds.

Diarrhoea prevalence in children under five years of age in rural Burundi: an assessment of social and behavioural factors at the household level.

BACKGROUND: Diarrhoea is the second leading cause of child mortality worldwide. Low- and middle-income countries are particularly burdened with this both preventable and treatable condition. Targeted interventions include the provision of safe water, the use of sanitation facilities and hygiene education, but are implemented with varying local success. OBJECTIVE: To determine the prevalence of and factors associated with diarrhoea in children under five years of age in rural Burundi. DESIGN: A cross-sectional survey was conducted among 551 rural households in northwestern Burundi. Areas of inquiry included 1) socio-demographic information, 2) diarrhoea period prevalence and treatment, 3) behaviour and knowledge, 4) socio-economic indicators, 5) access to water and water chain as well as 6) sanitation and personal/children's hygiene. RESULTS: A total of 903 children were enrolled. The overall diarrhoea prevalence was 32.6%. Forty-six per cent (n=255) of households collected drinking water from improved water sources and only 3% (n=17) had access to improved sanitation. We found a lower prevalence of diarrhoea in children whose primary caretakers received hygiene education (17.9%), boiled water prior to its utilisation (19.4%) and were aged 40 or older (17.9%). Diarrhoea was associated with factors such as the mother's age being less than 25 and the conviction that diarrhoea could not be prevented. No gender differences were detected regarding diarrhoea prevalence or the caretaker's decision to treat. CONCLUSIONS: Diarrhoea prevalence can be reduced through hygiene education and point-of use household water treatment such as boiling. In order to maximise the impact on children's health in the given rural setting, future interventions must assure systematic and regular hygiene education at the household and community level.

Compound-specific stable carbon isotope ratios of phenols and nitrophenols derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide.

We developed an analytical method for measuring compound-specific stable carbon isotope ratios (δ(13)C) of phenols and nitrophenols in filter samples of particulate organic matter. The method was tested on 13 phenols derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), together with four nonphenolic compounds. The data obtained by our method required two specific corrections for the determination of valid δ(13)C values: (1) for nitro compounds, the routine correction with use of m/z 46 for the contribution of (12)C(17)O(16)O molecules) to m/z 45 was modified due to impact of NO2 on the m/z 46 trace, and (2) for the derivatized phenols, measured δ(13)C values were corrected for the shift in δ(13)C due to the addition of carbon atoms from the BSTFA moiety. Analysis of standard-spiked filters showed that overall there was a small compound-dependent bias in the δ(13)C values: the average bias±the standard error of the mean of -0.21±0.1‰ for the standard compounds tested, except 3-methylcatechol, methylhydroquinone, 4-methyl-2-nitrophenol, and 2,6-dimethyl-4-nitrophenol, whereas the average biases±the standard errors of the mean for those were +1.2±0.3‰, +1.2±0.2‰, -1.2±0.2‰, and -1.4±0.5‰, respectively, when the injected mass of a derivatized compound exceeded 15 ngC. In situations where such small biases and uncertainties are acceptable, the method described here could be used to obtain valuable information about δ(13)C values. We also analyzed a real filter sample to demonstrate the practical applicability of the method.

HIV-1 Nef disrupts membrane-microdomain-associated anterograde transport for plasma membrane delivery of selected Src family kinases.

HIV-1 Nef, an essential factor in AIDS pathogenesis, boosts virus replication in vivo. As one of its activities in CD4(+) T-lymphocytes, Nef potently retargets the Src family kinase (SFK) Lck but not closely related Fyn from the plasma membrane to recycling endosomes and the trans-Golgi network to tailor T-cell activation and optimize virus replication. Investigating the underlying mechanism we find Lck retargeting involves removal of the kinase from membrane microdomains. Moreover, Nef interferes with rapid vesicular transport of Lck to block anterograde transport and plasma membrane delivery of newly synthesized Lck. The sensitivity of Lck to Nef does not depend on functional domains of Lck but requires membrane insertion of the kinase. Surprisingly, the short N-terminal SH4 domain membrane anchor of Lck is necessary and sufficient to confer sensitivity to Nef-mediated anterograde transport block and microdomain extraction. In contrast, the SH4 domain of Fyn is inert to Nef-mediated manipulation. Nef thus interferes with a specialized membrane microdomain-associated pathway for plasma membrane delivery of newly synthesized Lck whose specificity is determined by the affinity of cargo for these sorting platforms. These results provide new insight into the mechanism of Nef action and the pathways used for SFK plasma membrane delivery.

HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network-associated Lck signaling.

The Nef protein of HIV-1 facilitates viral replication and disease progression in vivo. Nef disturbs the organization of immunological synapses between infected CD4(+) T lymphocytes and antigen-presenting B-lymphocytes to interfere with TCR proximal signaling. Paradoxically, Nef enhances distal TCR signaling in infected CD4(+) T lymphocytes, an effect thought to be involved in its role in AIDS pathogenesis. Using quantitative confocal microscopy and cell fractionation of Nef-expressing cells and HIV-1-infected primary human T lymphocytes, we found that Nef induces intracellular compartmentalization of TCR signaling to adjust TCR responses to antigenic stimulation. Nef reroutes kinase-active pools of the TCR signaling master switch Lck away from the plasma membrane (PM) to the trans-Golgi network (TGN), thereby preventing the recruitment of active Lck to the immunological synapse after TCR engagement and limiting signal initiation at the PM. Instead, Nef triggers Lck-dependent activation of TGN-associated Ras-Erk signaling to promote the production of the T lymphocyte survival factor IL-2 and to enhance virus spread. Overexpression of the Lck PM transporter Unc119 restores Nef-induced subversions of Lck trafficking and TCR signaling. Nef therefore hijacks Lck sorting to selectively activate TGN-associated arms of compartmentalized TCR signaling. By tailoring T-lymphocyte responses to antigenic stimulation, Nef optimizes the environment for HIV-1 replication.

Nef does not inhibit F-actin remodelling and HIV-1 cell-cell transmission at the T lymphocyte virological synapse.

Nef, a HIV-1 pathogenesis factor, elevates virus replication in vivo and thus progression to AIDS by incompletely defined mechanisms. As one of its biological properties, Nef enhances the infectivity of cell-free HIV-1 particles in single round infections, however it fails to provide a significant and amplifying growth advantage for HIV-1 on such virus producing cells. A major difference between HIV-1 cell-free single round infections and virus replication kinetics on T lymphocytes consists in the predominant role of cell-associated virus transmission rather than cell-free infection during multiple round virus replication. HIV-1 cell-to-cell transmission occurs across close cell contacts also referred to as virological synapse (VS) and involves polarization of the F-actin cytoskeleton, formation of F-actin rich membrane bridges as well as virus budding to cell-cell contacts. Since Nef potently interferes with triggered actin remodelling in several cell systems to reduce e.g. cell motility and signal transduction, we set out here to address whether Nef also affects organization and possibly function of the T lymphocyte VS. We find that in addition to increasing infectivity of cell-free virions, Nef can also moderately enhance single rounds of HIV-1 cell-cell transmission between Jurkat T lymphocytes. This occurs without affecting cell conjugation efficiencies or polarization of F-actin and HIV-1 p24Gag at the VS, identifying actin remodelling at the VS as an example of Nef-insensitive host cell actin rearrangements. However, Nef-mediated enhancement of single round cell-free infection or cell-to-cell transmission does not potentiate over multiple rounds of infection. These results suggest that Nef affects cell-free and cell-associated HIV-1 infection by the same mechanism acting on the intrinsic infectivity of HIV-1 particles. They further indicate that the high efficacy of cell-to-cell transmission can compensate such infectivity defects. Nef therefore selectively interferes with actin remodelling processes involved in antiviral host cell defense while actin driven processes that promote virus propagation remain unaltered.

Lentiviral Nef proteins utilize PAK2-mediated deregulation of cofilin as a general strategy to interfere with actin remodeling.

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1(SF2) (HIV-1(SF2)) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

Inhibition of T-cell receptor-induced actin remodeling and relocalization of Lck are evolutionarily conserved activities of lentiviral Nef proteins.

Nef, an important pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), elevates virus replication in vivo. Among other activities, Nef affects T-cell receptor (TCR) signaling via several mechanisms. For HIV-1 Nef these include alteration of the organization and function of the immunological synapse (IS) such as relocalization of the Lck kinase, as well as early inhibition of TCR/CD3 complex (TCR-CD3)-mediated actin rearrangements and tyrosine phosphorylation. Although most SIV and HIV-2 Nef alleles (group 2) potently downregulate cell surface TCR-CD3, this activity was lost in the viral lineage that gave rise to HIV-1 and its SIV counterparts (group 1). To address the contribution of TCR-CD3 downregulation to Nef effects on TCR signal initiation, we compared the activities of 18 group 1 and group 2 Nef proteins, as well as SIV Nef mutants with defects in TCR-CD3 downmodulation. We found that alteration of Lck's subcellular localization is largely conserved and occurs independently of actin remodeling inhibition or TCR-CD3 downregulation. Surprisingly, Nef proteins of both groups also strongly reduced TCR-induced actin remodeling and tyrosine phosphorylation on TCR-stimulatory surfaces and TCR-CD3 downmodulation competence by group 2 Nef proteins only slightly elevated these effects. Furthermore, Nef proteins from HIV-1 and SIV reduced conjugation between infected primary human T lymphocytes and Raji B cells and potently prevented F-actin polarization at the IS independently of their ability to downmodulate TCR-CD3. These results establish alterations of early TCR signaling events at the IS, including F-actin remodeling and relocalization of Lck, as evolutionary conserved activities of highly divergent lentiviral Nef proteins.

Measurements of the 12C/13C kinetic isotope effects in the gas-phase reactions of light alkanes with chlorine atoms.

The carbon kinetic isotope effects (KIEs) of the reactions of several light non-methane hydrocarbons (NMHC) with Cl atoms were determined at room temperature and ambient pressure. All measured KIEs, defined as the ratio of the Cl reaction rate constants of the light isotopologue over that of the heavy isotopologue (Clk12/Clk13) are greater than unity or normal KIEs. For simplicity, measured KIEs are reported in per mil according to Clepsilon=(Clk12/Clk13 -1)x1000 per thousand unless noted otherwise. The following average KIEs were obtained (all in per thousand): 10.73+/-0.20 (ethane), 6.44+/-0.14 (propane), 6.18+/-0.18 (methylpropane), 3.94+/-0.01 (n-butane), 1.79+/-0.42 (methylbutane), 3.22+/-0.17 (n-pentane), 2.02+/-0.40 (n-hexane), 2.06+/-0.19 (n-heptane), 1.54+/-0.15 (n-octane), 3.04+/-0.09 (cyclopentane), 2.30+/-0.09 (cyclohexane), and 2.56+/-0.25 (methylcyclopentane). Measurements of the 12C/13C KIEs for the Cl atom reactions of the C2-C8 n-alkanes were also made at 348 K, and no significant temperature dependence was observed. To our knowledge, these 12C/13C KIE measurements for alkanes+Cl reactions are the first of their kind. Simultaneous to the KIE measurement, the rate constant for the reaction of each alkane with Cl atoms was measured using a relative rate method. Our measurements agree with published values within+/-20%. The measured rate constant for methylcyclopentane, for which no literature value is available, is (2.83+/-0.11)x10-10 cm3 molecule-1 s-1, 1sigma standard error. The Clepsilon values presented here for the C2-C8 alkanes are an order of magnitude smaller than reported methane Clepsilon values (Geophys. Res. Lett., 2000, 27, 1715), in contrast to reported OHepsilon values for methane (J. Geophys. Res. (Atmos.), 2001, 106, 23, 127) and C2-C8 alkanes (J. Phys. Chem. A, 2004, 108, 11537), which are all smaller than 10 per thousand. This has important implications for atmospheric modeling of saturated NMHC stable carbon isotope ratios. 13C-structure reactivity relationship values (13C-SRR) for alkane-Cl reactions have been determined and are similar to previously reported values for alkane-OH reactions.

Measurements of low-molecular-mass carboxylic acids in atmospheric aerosols by capillary electrophoresis.

Capillary electrophoresis (CE) methods for the determination of low-molecular-mass (LMM) carboxylic acids in airborne particular matter have been developed. The separations of 22 LMM carboxylic acids, including acids derived from the oxidation of biogenic hydrocarbons, are performed using a background electrolyte consisting of 3.0mM 2,6-naphthalenedicarboxylic acid and 18.0mM 2,2-bis (hydroxymethyl)-2,2',2"-nitrilotriethanol (Bis-tris) in 16% (v/v) 1-propanol within 10 min. Using a combination of a buffer mixed with an organic solvent and electroosmotic flow modifier, a minimum of peak overlaps is achieved with migration time variation of less than 1% and peak area ratio (relative to an internal standard) variation of less than 5% within 1 day. The detection limits for the aliphatic LMM acids that can be determined by this method are in the range of 30-140 micro g/L. Furthermore, a simple method for efficient extraction of LMM organic acids from particulate atmospheric matter collected on quartz fiber filters using high-volume samplers is developed. Combining the extraction procedure with a reduction of the extract to approximately 0.2 mL allows for the measurement of LLM in atmospheric particulate organic matter at concentrations well below 1 ng.m(-3). Repeat analysis of filters collected in tunnels, urban, suburban, and forested areas demonstrate that the procedure allows for measurements of aliphatic and aromatic LMM acids within a variability of 10-25%.

Determination of aromatic acids and nitrophenols in atmospheric aerosols by capillary electrophoresis.

A capillary zone electrophoresis method is developed for the determination of aromatic organic acids and nitrophenols in atmospheric aerosols. The procedure is based on sampling atmospheric particulate matter on quartz fiber filters and the extraction and analysis of the extracts by capillary electrophoresis. Separation conditions are optimized by varying the pH and acetonitrile content of the electrolyte buffer. Separations in a 20% acetonitrile-20 mM borate mixture (pH 9.9) are able to resolve all of the geometric isomers of hydroxybenzoic acid, phthalic acid, benzenetricarboxylic acid, and nitrophenol as well as 1,2,4,5-benzenetricarboxylic acid, m-toluic acid, and sulfosalicylic acid. A buffer consisting of 11% acetonitrile-20 mM borate (pH 9.9) is found to be most suitable for the analysis of atmospheric aerosol samples. Detection limits are in the order of 40 to 130 ng/mL. Intersample migration time reproducibility is generally better than 1.5%, with day-to-day variations under 3%. A general extraction scheme using diethyl ether-HCl in combination with a preconcentration step is developed. Recoveries of spiked standards range from 59% to 102%, with relative standard deviations ranging from 2% to 17% for five determinations. The method is applied towards the analysis of ambient aerosol samples as well as vehicle emission studies with promising results, thus showing it to be a potential complement to already existing methodology for the analysis of organic acids and nitrophenols in atmospheric aerosols.