A Novel Six-Rhodopsin System in a Single Archaeon

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.Microbial rhodopsins comprise a large family of seven-transmembrane helical proteins that either mediate light-driven ion transport to harvest solar energy or serve as receptors to mediate phototaxis (13) and possibly photoadaptation (32). In archaea, four rhodopsins responding to different wavelengths of light with distinct functions in Halobacterium salinarum have been identified and characterized (20, 32). These consist of two light-driven ion transporters that pump protons (bacteriorhodopsin [BR]) (21) or chloride (halorhodopsin [HR]) (19, 28) and two sensory rhodopsins that mediate both attractant and repellent phototaxis (sensory rhodopsin I [SRI]) (4, 33) or only repellent phototaxis (sensory rhodopsin II [SRII]) (35).The two ion-transporting rhodopsins perform light-driven outward proton transport to create a proton-electrochemical potential or inward chloride transport to maintain the osmotic and pH homeostasis of the cell. The photoactivated sensory rhodopsins, on the other hand, undergo light-triggered conformational changes to relay signals to their cognate transducers and consequently activate signaling cascades in a manner similar to that of the two-component system involved in eubacterial chemotaxis (1, 22) to control flagellum rotation and thus swimming direction. Our current understanding of microbial rhodopsins as both ion transporters and photosensory receptors has been based primarily on these four known rhodopsins.A recently completed genome project for Haloarcula marismortui (3) proposed the existence of six opsin-related genes, the greatest number ever found in a single archaeon. This proposal immediately raised three questions. (i) Are these six rhodopsins biologically expressed and functionally active? (ii) What is the maximum-absorbance-wavelength (λ_max) distribution pattern of these six rhodopsins? (iii) Do the two extra rhodopsins, compared to the four in H. salinarum, perform new functions or have new features beyond those of the known system? This study demonstrates several features of this system that are unique compared to the features of currently known systems, and it confirms the six-rhodopsin system and the presence of two additional isochromatic rhodopsins comprising a novel light-driven proton transporter system and a new type of sensory-like rhodopsin signaling complex with a unique transducer.     

Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

Integrin-growth factor receptor cross-talk plays a role in growth factor signaling, but the specifics are unclear. In a current model, integrins and growth factor receptors independently bind to their ligands (extracellular matrix and growth factors, respectively). We discovered that neuregulin-1 (NRG1), either as an isolated EGF-like domain or as a native multi-domain form, binds to integrins αvβ3 (with a K_D of 1.36 × 10⁻⁷ m) and α6β4. Docking simulation predicted that three Lys residues at positions 180, 184, and 186 of the EGF-like domain are involved in integrin binding. Mutating these residues to Glu individually or in combination markedly suppressed integrin binding and ErbB3 phosphorylation. Mutating all three Lys residues to Glu (the 3KE mutation) did not affect the ability of NRG1 to bind to ErbB3 but markedly reduced the ability of NRG1 to induce ErbB3 phosphorylation and AKT and Erk1/2 activation in MCF-7 and T47D human breast cancer cells. This suggests that direct integrin binding to NRG1 is critical for NRG1/ErbB signaling. Notably, stimulation of cells with WT NRG1 induced co-precipitation of ErbB3 with α6β4 and with αvβ3 to a much lower extent. This suggests that WT NRG1 induces integrin-NRG1-ErbB3 ternary complex formation. In contrast, the 3KE mutant was much less effective in inducing ternary complex formation than WT NRG1, suggesting that this process depends on the ability of NRG1 to bind to integrins. These results suggest that direct NRG1-integrin interaction mediates integrin-ErbB cross-talk and that α6β4 plays a major role in NRG-ErbB signaling in these cancer cells.     

Shrunken methodology to genome-wide SNPs selection and construction of SNPs networks

Background

Recent development of high-resolution single nucleotide polymorphism (SNP) arrays allows detailed assessment of genome-wide human genome variations. There is increasing recognition of the importance of SNPs for medicine and developmental biology. However, SNP data set typically has a large number of SNPs (e.g., 400 thousand SNPs in genome-wide Parkinson disease data set) and a few hundred of samples. Conventional classification methods may not be effective when applied to such genome-wide SNP data.

Results

In this paper, we use shrunken dissimilarity measure to analyze and select relevant SNPs for classification problems. Examples of HapMap data and Parkinson disease (PD) data are given to demonstrate the effectiveness of the proposed method, and illustrate it has a potential to become a useful analysis tool for SNP data sets. We use Parkinson disease data as an example, and perform a whole genome analysis. For the 367440 SNPs with less than 1% missing percentage from all 22 chromosomes, we can select 357 SNPs from this data set. For the unique genes that those SNPs are located in, a gene-gene similarity value is computed using GOSemSim and gene pairs that has a similarity value being greater than a threshold are selected to construct several groups of genes. For the SNPs that involved in these groups of genes, a statistical software PLINK is employed to compute the pair-wise SNP-SNP interactions, and SNPs with significance of P < 0.01 are chosen to identify SNPs networks based on their P values. Here SNPs networks are constructed based on Gene Ontology knowledge, and therefore each SNP network plays a role in the biological process. An analysis shows that such networks have relationships directly or indirectly to Parkinson disease.

Conclusions

Experimental results show that our approach is suitable to handle genetic variations, and provide useful knowledge in a genome-wide SNP study.

     

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines

Background

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/Principal Findings

Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/Significance

The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.     

Situational awareness and health protective responses to pandemic influenza A (H1N1) in Hong Kong: a cross-sectional study

Background

Whether information sources influence health protective behaviours during influenza pandemics or other emerging infectious disease epidemics is uncertain.

Methodology

Data from cross-sectional telephone interviews of 1,001 Hong Kong adults in June, 2009 were tested against theory and data-derived hypothesized associations between trust in (formal/informal) information, understanding, self-efficacy, perceived susceptibility and worry, and hand hygiene and social distancing using Structural Equation Modelling with multigroup comparisons.

Principal Findings

Trust in formal (government/media) information about influenza was associated with greater reported understanding of A/H1N1 cause (β = 0.36) and A/H1N1 prevention self-efficacy (β = 0.25), which in turn were associated with more hand hygiene (β = 0.19 and β = 0.23, respectively). Trust in informal (interpersonal) information was negatively associated with perceived personal A/H1N1 susceptibility (β = −0.21), which was negatively associated with perceived self-efficacy (β = −0.42) but positively associated with influenza worry (β = 0.44). Trust in informal information was positively associated with influenza worry (β = 0.16) which was in turn associated with greater social distancing (β = 0.36). Multigroup comparisons showed gender differences regarding paths from trust in formal information to understanding of A/H1N1 cause, trust in informal information to understanding of A/H1N1 cause, and understanding of A/H1N1 cause to perceived self-efficacy.

Conclusions/Significance

Trust in government/media information was more strongly associated with greater self-efficacy and handwashing, whereas trust in informal information was strongly associated with perceived health threat and avoidance behaviour. Risk communication should consider the effect of gender differences.     

Distinct kinetics of memory B-cell and plasma-cell responses in peripheral blood following a blood-stage Plasmodium chabaudi infection in mice

B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC). To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(-) IgM(-) CD19(+) B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1)-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+) plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25) or secondary (day 10) infection. CD138(+) plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood.     

Nitrate respiration protects hypoxic Mycobacterium tuberculosis against acid- and reactive nitrogen species stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection.