RabGEFs are a major determinant for specific Rab membrane targeting

Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Frequent Occurrence of Mixed Enterocytozoon bieneusi Infections in Humans

Enterocytozoon bieneusi (phylum Microsporidia) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb E. bieneusi genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal E. bieneusi isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped E. bieneusi DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous E. bieneusi populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of E. bieneusi is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.     

Trends in cation, nitrogen, sulfate and hydrogen ion concentrations in precipitation in the United States and Europe from 1978 to 2010: a new look at an old problem

Industrial emissions of SO₂ and NO_x, resulting in the formation and deposition of sulfuric and nitric acids, affect the health of both terrestrial and aquatic ecosystems. Since the mid-late 20th century, legislation to control acid rain precursors in both Europe and the US has led to significant declines in both SO₄–S and H⁺ in precipitation and streams. However, several authors noted that declines in streamwater SO₄–S did not result in stoichiometric reductions in stream H⁺, and suggested that observed reductions in base cation inputs in precipitation could lessen the effect of air pollution control on improving stream pH. We examined long-term precipitation chemistry (1978–2010) from nearly 30 sites in the US and Europe that are variably affected by acid deposition and that have a variety of industrial and land-use histories to (1) quantify trends in SO₄–S, H⁺, NH₄–N, Ca, and NO₃–N, (2) assess stoichiometry between H⁺ and SO₄–S before and after 1990, and (3) examine regional synchrony of trends. We expected that although the overall efforts of developed countries to reduce air pollution and acid rain by the mid-late 20th century would tend to synchronize precipitation chemistry among regions, geographically varied patterns of fossil fuel use and pollution control measures would produce important asynchronies among European countries and the United States. We also expected that control of particulate versus gaseous emission, along with trends in NH₃ emissions, would be the two most significant factors affecting the stoichiometry between SO₄–S and H⁺. Relationships among H⁺, SO₄–S, NH₄–N, and cations differed markedly between the US and Europe. Controlling for SO₄–S levels, H⁺ in precipitation was significantly lower in Europe than in the US, because (1) alkaline dust loading from the Sahara/Sahel was greater in Europe than the US, and (2) emission of NH₃, which neutralizes acidity upon conversion to NH₄ ⁺, is generally significantly higher in Europe than in the US. Trends in SO₄–S and H⁺ in precipitation were close to stoichometric in the US throughout the period of record, but not in Europe, especially eastern Europe. Ca in precipitation declined significantly before, but not after 1990 in most of the US, but Ca declined in eastern Europe even after 1990. SO₄–S in precipitation was only weakly related to fossil fuel consumption. The stoichiometry of SO₄–S and H⁺ may be explained in part by emission controls, which varied over time and among regions. Control of particulate emissions reduces alkaline particles that neutralize acid precursors as well as S-containing particulates, reducing SO₄–S and Ca more steeply than H⁺, consistent with trends in the northeastern US and Europe before 1990. In contrast, control of gaseous SO₂ emissions results in a stoichiometric relationship between SO₄–S and H⁺, consistent with trends in the US and many western European countries, especially after 1991. However, in many European countries, declining NH₃ emissions contributed to the lack of stoichiometry between SO₄–S and H⁺.Recent reductions in NO_x emissions have also contributed to declines in H⁺ in precipitation. Future changes in precipitation acidity are likely to depend on multiple factors including trends in NO_x and NH₃ emission controls, naturally occurring dust, and fossil fuel use, with significant implications for the health of both terrestrial and aquatic ecosystems.     

Detection and validation of volatile metabolic patterns over different strains of two human pathogenic bacteria during their growth in a complex medium using multi-capillary column-ion mobility spectrometry (MCC-IMS)

Headspace analyses over microbial cultures using multi-capillary column-ion mobility spectrometry (MCC-IMS) could lead to a faster, safe and cost-effective method for the identification of pathogens. Recent studies have shown that MCC-IMS allows identification of bacteria and fungi, but no information is available from when on during their growth a differentiation between bacteria is possible. Therefore, we analysed the headspace over human pathogenic reference strains of Escherichia coli and Pseudomonas aeruginosa at four time points during their growth in a complex fluid medium. In order to validate our findings and to answer the question if the results of one bacterial strain can be transferred to other strains of the same species, we also analysed the headspace over cultures from isolates of random clinical origin. We detected 19 different volatile organic compounds (VOCs) that appeared or changed their signal intensity during bacterial growth. These included six VOCs exclusively changing over E. coli cultures and seven exclusively changing over P. aeruginosa cultures. Most changes occurred in the late logarithmic or static growth phases. We did not find differences in timing or trends in signal intensity between VOC patterns of different strains of one species. Our results show that differentiation of human pathogenic bacteria by headspace analyses using MCC-IMS technology is best possible during the late phases of bacterial growth. Our findings also show that VOC patterns of a bacterial strain can be transferred to other strains of the same species.