Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.     

Intramolecular Isopeptide Bonds Give Thermodynamic and Proteolytic Stability to the Major Pilin Protein of Streptococcus pyogenes

The pili expressed by Streptococcus pyogenes and certain other Gram-positive bacterial pathogens are based on a polymeric backbone in which individual pilin subunits are joined end-to-end by covalent isopeptide bonds through the action of sortase enzymes. The crystal structure of the major pilin of S. pyogenes, Spy0128, revealed that each domain of the two domain protein contained an intramolecular isopeptide bond cross-link joining a Lys side chain to an Asn side chain. In the present work, mutagenesis was used to create mutant proteins that lacked either one isopeptide bond (E117A, N168A, and E258A mutants) or both isopeptide bonds (E117A/E258A). Both the thermal stability and proteolytic stability of Spy0128 were severely compromised by loss of the isopeptide bonds. Unfolding experiments, monitored by circular dichroism, revealed a transition temperature T_m of 85 °C for the wild type protein. In contrast, mutants with only one isopeptide bond showed biphasic unfolding, with the domain lacking an isopeptide bond having a T_m that was ∼30 °C lower than the unaltered domain. High resolution crystal structures of the E117A and N168A mutants showed that the loss of an isopeptide bond did not change the overall pilin structure but caused local disturbance of the protein core that was greater for E117A than for N168A. These effects on stability appear also to be important for pilus assembly.The stability of a globular protein is in general a fine balance between large numbers of weak, noncovalent stabilizing forces (hydrophobic interactions, hydrogen bonds, and ion pairs) and the destabilizing loss of conformational entropy (1). Stability can be greatly enhanced, however, by the presence of a small number of strategically placed covalent bonds, for example those provided by disulfide bonds between Cys residues or by a bound metal ion that bonds to several amino acid side chains. Indeed, some striking examples have been reported in which the introduction of disulfide bonds that link sequentially distant parts of a polypeptide chain can result in large increases in protein stability (2).Our recent discovery of cross-linking isopeptide bonds within the protein subunits of the pili expressed by the Gram-positive organism Streptococcus pyogenes (3) has raised questions as to how these bonds are formed and what they contribute to stability. These pili, which are extremely thin (2–5 nm in diameter) but can extend up to 4 μm from the bacterial cell surface (4), are formed as covalently linked polymers through the action of cysteine transpeptidase enzymes called sortases. In this process, which is common to a number of Gram-positive bacterial pathogens, the pilus backbone is formed from a single protein subunit, the so-called major pilin, by the covalent, end-to-end polymerization of major pilin subunits, generating an assembly resembling beads on a string (5–7). Polymerization requires recognition, by the sortase, of an LPXTG-type sequence motif near the pilin C terminus, cleavage after the Thr residue, and transfer to a specific Lys residue in the next pilin subunit. The resulting amide bond, between the terminal COOH of one subunit and the Lys ϵ-amino group of the next subunit, is referred to as an isopeptide bond.Surprisingly, the three-dimensional structure of Spy0128, the major pilin from the M1 strain of S. pyogenes (group A streptococcus; GAS),² revealed the presence of additional isopeptide bonds as internal cross-links within the protein. These bonds, which were confirmed by mass spectrometry, joined lysine and asparagine side chains (3). One such bond was found within each domain of the two domain protein and, in a similar location, between the first and last strands of the domain (Fig. 1). Isopeptide bonds have until now been recognized for their importance in the intermolecular cross-linking of a variety of proteins, such as in ubiquitination (8), transglutamination (9), and sortase-mediated cell wall anchoring of surface proteins (10), as well as pilus polymerization. In this context, the presence of the intramolecular isopeptide bonds in Spy0128 seemed highly unusual. There has been speculation in the past that such internal bonds could exist, but no mechanism has been put forward, and none has previously been proven to exist.Open in a separate windowFIGURE 1.Overall structure and topology of Spy0128 with internal isopeptide bonds. A, the structure of Spy0128 is shown in ribbon representation with the N terminus (N) and C terminus (C) marked. The isopeptide bond in each of the N and C domains is shown as spheres with residue labels next to them. The residue forming the intersubunit bond, Lys¹⁶¹, is shown in stick mode. The sortase recognition motif, EVPTG, is shown as a dotted line at the C terminus of the structure. B, the topology diagram of Spy0128 is color-coded in rainbow from blue (N terminus) to red (C terminus). The position of Lys¹⁶¹ is indicated by the red arrowhead, and the positions of the isopeptide bonds are indicated by black bars. C, the N domain isopeptide bond of Spy0128 between Lys³⁶ and Asn¹⁶⁸ and the catalytic residue Glu¹¹⁷ are shown in ball-and-stick mode.The locations of the two internal Lys-Asn isopeptide bonds in Spy0128 immediately suggested that they are self-generated, each being the result of an intramolecular reaction. Mutagenesis showed that bond formation was in each case dependent on an adjacent Glu residue, Glu¹¹⁷ in the N-terminal domain and Glu²⁵⁸ in the C-terminal domain; mutation of either of these Glu residues to Ala abrogated the formation of the associated isopeptide bond (3). A close parallel exists in the self-generated Lys-Asn isopeptide bonds that form during maturation of the protein coat of the bacteriophage HK97, where a nearby Glu residue is essential for the reaction, and the capsid subunits become covalently linked to form interlocked circular rings referred to as chain mail (11, 12).Further investigations have shown that similar isopeptide bonds can be found as internal cross-links in other proteins. Examination of previously determined structures of a minor pilin GBS52 from Streptococcus agalactiae (13) and the CnaA and CnaB domains of a collagen-binding adhesin from Staphylococcus aureus (14–16) revealed constellations of Lys-Asn-Glu/Asp residues similar to those that form the internal cross-links in Spy0128, and examination of the electron density confirmed their probable presence in those cases where the x-ray data were available (3). Sequence comparisons showed that similar domains, with corresponding Lys-Asn-Glu/Asp residues, are present in many cell surface proteins of Gram-positive bacteria. Recently the Bacillus cereus major pilin BcpA was shown, by mass spectral analyses, to contain internal isopeptide bonds similar to those in Spy0128 (7), and sequence comparisons point to similar bonds in the major pilins of other species. These data suggest that isopeptide bond cross-links could be important features in many surface proteins involved in adhesive functions, where stability against physical and chemical stresses is important.Here we describe the preparation of mutants of Spy0128 that lack one or both of the internal isopeptide bonds, using mass spectrometry to confirm their absence. We show by x-ray crystallography that the overall structure of Spy0128 is not significantly affected by the loss of an isopeptide bond. We also show, however, that the proteolytic and thermal stability of Spy0128 is severely compromised when the internal isopeptide bonds are removed and thereby establish their important stabilizing role in proteins of this type.     

The effect of hOGG1 Ser326Cys polymorphism on cancer risk: evidence from a meta-analysis

Background

Human oxoguanine glycosylase 1 (hOGG1) in base excision repair (BER) pathway plays a vital role in DNA repair. Numerous epidemiological studies have evaluated the association between hOGG1 Ser326Cys polymorphism and the risk of cancer. However, the results of these studies on the association remain conflicting. To derive a more precise estimation of the association, we conducted a meta-analysis.

Methodology/Principal Findings

A comprehensive search was conducted to identify the eligible studies of hOGG1 Ser326Cys polymorphism and cancer risk. We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the association. We found that the hOGG1 Ser326Cys polymorphism was significantly associated with overall cancer risk (Cys/Cys vs. Ser/Ser: OR = 1.19, 95%CI = 1.09–1.30, P<0.001; Cys/Cys vs. Cys/Ser+Ser/Ser: OR = 1.16, 95%CI = 1.08–1.26, P<0.001). Moreover, in subgroup analyses by cancer types, the stronger significant association between hOGG1 Ser326Cys polymorphism and lung cancer risk was found (Cys/Cys vs. Ser/Ser: OR = 1.29, 95%CI = 1.16–1.44, P<0.001; Cys/Cys vs. Cys/Ser+Ser/Ser: OR = 1.22, 95%CI = 1.12–1.33, P<0.001). The significant effects of hOGG1 Ser326Cys polymorphism on colorectal, breast, bladder, prostate, esophageal, and gastric cancer were not detected. In addition, in subgroup analyses by ethnicities, we found that the hOGG1 Ser326Cys polymorphism was associated with overall cancer risk in Asians (Cys/Cys vs. Ser/Ser: OR = 1.21, 95%CI = 1.10–1.33, P<0.001).

Conclusions

This meta-analysis showed that hOGG1 326Cys allele might be a low-penetrant risk factor for lung cancer.     

Reduced expression of transcription factor AP-2α is associated with gastric adenocarcinoma prognosis

Background

This study aims to investigate the expression and prognostic significance of activator protein 2α (AP-2α) in gastric adenocarcinoma.

Methodology/Principal Findings

AP-2α expression was analyzed using real-time quantitative PCR (RT-qPCR), western blotting, and immunohistochemical staining methods on tissue samples from a consecutive series of 481 gastric adenocarcinoma patients who underwent resections between 2003 and 2006. The relationship between AP-2α expression, clinicopathological factors, and patient survival was investigated. RT- qPCR results showed that the expression of AP-2α mRNA was reduced in tumor tissue samples, compared with expression in matched adjacent non-tumor tissue samples (P = 0.009); this finding was confirmed by western blotting analysis (P = 0.012). Immunohistochemical staining data indicated that AP-2α expression was significantly decreased in 196 of 481 (40.7%) gastric adenocarcinoma cases; reduced AP-2α expression was also observed in patients with poorly differentiated tumors (P = 0.001) and total gastric carcinomas (P = 0.002), as well as in patients who underwent palliative tumor resection (P = 0.004). Additionally, reduced expression of AP-2α was more commonly observed in tumors that were staged as T4a/b (P = 0.018), N3 (P = 0.006), and M1 (P = 0.008). Kaplan-Meier survival curves revealed that reduced expression of AP-2α was associated with poor prognosis in gastric adenocarcinoma patients (P<0.001). Multivariate Cox analysis identified AP-2α expression as an independent prognostic factor for overall survival (HR = 1.512, 95% CI = 1.127–2.029, P = 0.006).

Conclusions/Significance

Our data suggest that AP-2α plays an important role in tumor progression and that reduced AP-2α expression independently predicts an unfavorable prognosis in gastric adenocarcinoma patients.     

Changes in temperature have opposing effects on current amplitude in α7 and α4β2 nicotinic acetylcholine receptors

We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (nAChR) subtypes: the rapidly desensitizing homomeric α7 nAChR, the more slowly desensitizing heteromeric α4β2 nAChR and on α7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in Xenopus oocytes at room temperature (RT; 21°C) were compared to those recorded at either physiological temperature (37°C) or at lower temperature (4°C). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with α4β2 nAChRs was increased at high temperature (125±9%, n = 6, P<0.01) and reduced at low temperature (47±5%, n = 6, P<0.01), whereas the amplitude of α7 responses was reduced at high temperature (27±7%, n = 11, P<0.001) and increased at low temperatures (224±16%, n = 10, P<0.001). In contrast to the effects of temperature on α4β2 and wild type α7 nAChRs, the amplitude of α7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when α7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonist-evoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of receptor desensitization on all subtypes examined. In all cases, higher temperature resulted in increased rates of desensitization. Thus, it appears that the differing effects of temperature on the amplitudes of whole-cell responses cannot be explained by temperature-induced changes in receptor desensitization rates.     

Increased gut permeability and microbiota change associate with mesenteric fat inflammation and metabolic dysfunction in diet-induced obese mice

We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80⁺) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P<0.001) and 40% (P = 0.025) respectively and TNF-α mRNA level was 6.6-fold higher (P = 0.037). HFD reduced Lactobacillus (75%; P<0.001) but increased Oscillibacter (279%; P = 0.004) in fecal microbiota. Correlations were found between abundances of Lactobacillus (r = 0.52; P = 0.013) and Oscillibacter (r = −0.55; P = 0.007) with transepithelial resistance of the proximal colon. HFD increased macrophage infiltration (58%; P = 0.020), TNF-α (2.5-fold, P<0.001) and IL-6 mRNA levels (2.5-fold; P = 0.008) in mesenteric fat. Increased macrophage infiltration in epididymal fat was also observed with HFD feeding (71%; P = 0.006) but neither TNF-α nor IL-6 was altered. Perirenal and subcutaneous adipose tissue showed no signs of inflammation in HFD mice. The current results implicate gut dysfunction, and attendant inflammation of contiguous adipose, as salient features of the metabolic dysregulation of diet-induced obesity.     

The association between OGG1 Ser326Cys polymorphism and lung cancer susceptibility: a meta-analysis of 27 studies

Background

Numerous studies have investigated association of OGG1 Ser326Cys polymorphism with lung cancer susceptibility; however, the findings are inconsistent. Therefore, we performed a meta-analysis based on 27 publications encompass 9663 cases and 11348 controls to comprehensively evaluate such associations.

Methods

We searched publications from MEDLINE and EMBASE which were assessing the associations between OGG1 Ser326Cys polymorphism and lung cancer risk. We calculated pooled odds ratio (OR) and 95% confidence interval (CI) by using either fixed-effects or random-effects model. We used genotype based mRNA expression data from HapMap for SNP rs1052133 in normal cell lines among 270 subjects with four different ethnicities.

Results

The results showed that individuals carrying the Cys/Cys genotype did not have significantly increased risk for lung cancer (OR = 1.15, 95% CI = 0.98–1.36) when compared with the Ser/Ser genotype; similarly, no significant association was found in recessive, dominant or heterozygous co-dominant model (Ser/Cys vs. Cys/Cys). However, markedly increased risks were found in relatively large sample size (Ser/Ser vs. Cys/Cys: OR = 1.29, 95% CI = 1.13–1.48, and recessive model: OR = 1.19, 95% CI = 1.07–1.32). As to histological types, we found the Cys/Cys was associated with adenocarcinoma risk (Ser/Ser vs. Cys/Cys: OR = 1.32, 95% CI = 1.12–1.56; Ser/Cys vs. Cys/Cys: OR = 1.19, 95% CI = 1.04–1.37, and recessive model OR = 1.23, 95% CI = 1.08–1.40). No significant difference of OGG1 mRNA expression was found among genotypes between different ethnicities.

Conclusions

Despite some limitations, this meta-analysis established solid statistical evidence for an association between the OGG1 Cys/Cys genotype and lung cancer risk, particularly for studies with large sample size and adenocarcinoma, but this association warrants additional validation in larger and well designed studies.     

Intraepithelial γδ T cells remain increased in the duodenum of AIDS patients despite antiretroviral treatment

Intraepithelial lymphocytes (IELs) bearing the γδ T-cell receptor are a unique intestinal subset whose function remains elusive. Here, we examine how they behave in AIDS and during various regimens of antiretroviral treatment in order to obtain mechanistic insight into their adaptive or innate functional in vivo properties. IELs were studied by multimarker two-colour immunofluorescence in situ staining. Consecutive duodenal biopsies were obtained from advanced infection-prone HIV⁺ patients (n = 30). The systemic adaptive immune status was monitored by determining T-cell subsets and immunoglobulins in peripheral blood. The γδ IEL ratio (median 14.5%, range 1.5–56.3%) was significantly increased (p<0.02) compared with that in clinically healthy HIV⁻ control subjects (n = 11, median 2.8%; range 0.3–38%), although the number of γδ IELs per mucosal length unit (U) only tended to be increased (4.0/U in HIV⁺ versus 3.2/U in HIV⁻subjects). Notably, the total number of CD3⁺ IELs was significantly reduced in AIDS (p<0.0001, 39.6/U in HIV⁺ versus 86.4/U in HIV⁻ subjects). Almost 100% of the γδ IELs were CD8⁻ and they often expressed the Vδ1/Jδ1-encoded epitope (median 65.2%). HIV⁺ patients on highly active antiretroviral therapy only tended to have a lower ratio of γδ IELs (median 12.8%) than those receiving no treatment (median 14.3%) or 1 nucleoside analogue (NA) (median 23.5%) or 2 NAs (median 13.0%). This minimal variation among therapy groups, contrasting the treatment response of systemic and local adaptive immunity, harmonizes with the novel idea derived from animal experiments that γδ T cells are largely innate cells in first-line microbial defence.     

Age-related differences in naturally acquired T cell memory to Plasmodium falciparum merozoite surface protein 1

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP1₄₂), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP1₄₂ driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5–1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP1₄₂. However, adults had higher proportions of MSP1₄₂ driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA⁻ CD62L⁻) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP1₄₂ driven IFN-γ secreting naïve-like, transitional (CD45RA⁺ CD62L⁺) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.     

Circumsporozoite-specific T cell responses in children vaccinated with RTS,S/AS01E and protection against P falciparum clinical malaria

Background

RTS,S/AS01_E is the lead candidate pre-erythrocytic malaria vaccine. In Phase IIb field trials the safety profile was acceptable and the efficacy was 53% (95%CI 31%–72%) for protecting children against clinical malaria caused by P. falciparum. We studied CS-specific T cell responses in order to identify correlates of protection.

Methods and Findings

We used intracellular cytokine staining (for IL2, IFNγ, and TNFα), ex-vivo ELISPOTs (IFNγ and IL2) and IFNγ cultured ELISPOT assays to characterize the CS-specific cellular responses in 407 children (5–17 months of age) in a phase IIb randomized controlled trial of RTS,S/AS01_E ({"type":"clinical-trial","attrs":{"text":"NCT00380393","term_id":"NCT00380393"}}NCT00380393). RTS,S/ AS01_E vaccinees had higher frequencies of CS-specific CD4+ T cells producing IFNγ, TNFα or IL2 compared to control vaccinees. In a multivariable analysis TNFα⁺ CD4⁺ T cells were independently associated with a reduced risk for clinical malaria among RTS,S/AS01_E vaccinees (HR = 0.64, 95%CI 0.49–0.86, p = 0.002). There was a non-significant tendency towards reduced risk among control vaccinees (HR = 0.80, 95%CI 0.62–1.03, p = 0.084), albeit with lower CS-specific T cell frequencies and higher rates of clinical malaria. When data from both RTS,S/AS01_E vaccinees and control vaccinees were combined (with adjusting for vaccination group), the HR was 0.74 (95%CI 0.62–0.89, p = 0.001). After a Bonferroni correction for multiple comparisons (n-18), the finding was still significant at p = 0.018. There was no significant correlation between cultured or ex vivo ELISPOT data and protection from clinical malaria. The combination of TNFα⁺ CD4⁺ T cells and anti-CS antibody statistically accounted for the protective effect of vaccination in a Cox regression model.

Conclusions

RTS,S/AS01_E induces CS-specific Th1 T cell responses in young children living in a malaria endemic area. The combination of anti-CS antibody concentrations titers and CS-specific TNFα⁺ CD4⁺ T cells could account for the level of protection conferred by RTS,S/AS01_E. The correlation between CS-specific TNFα⁺ CD4⁺ T cells and protection needs confirmation in other datasets.     

Structure of the full-length major pilin from Streptococcus pneumoniae: implications for isopeptide bond formation in gram-positive bacterial pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili.     

Tumor necrosis factor-alpha g308α gene polymorphism and essential hypertension: a meta-analysis involving 2244 participants

Background

The tumor necrosis factor-alpha (TNFα) G308A gene polymorphism has been implicated in susceptibility to essential hypertension (EH), but study results are still controversial.

Objective and Methods

The present meta-analysis is performed to investigate the relationship between the TNFα G308A gene polymorphism and EH. Electronic databases were searched and seven separate studies on the association of the TNF α G308A gene polymorphism with EH were analyzed. The meta-analysis involved 1092 EH patients and 1152 controls. The pooled odds ratios (ORs) and their corresponding 95% confidence interval (CI) were calculated by a fixed or random effect model.

Results

A significant relationship between the TNFα G308A gene polymorphism and EH was found in an allelic genetic model (OR: 1.45, 95% CI: 1.17 to 1.80, P = 0.0008), a recessive genetic model (OR: 3.181, 95% CI: 1.204 to 8.408, P = 0.02), and a homozygote model (OR: 3.454, 95% CI: 1.286 to 9.278, P = 0.014). No significant association between them was detected in both a dominant genetic model (OR: 1.55, 95% CI: 0.99 to 2.42, P = 0.06) or a heterozygote genetic model (OR: 1.45, 95% CI: 0.90 to 2.33, P = 0.13).

Conclusion

The TNFα G308A gene polymorphism is associated with EH susceptibility.     

Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R² = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase_134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase_134-143 peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R² = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase_134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase_134-143 peptide.     

Infliximab Induces Clonal Expansion of γδ-T Cells in Crohn's Disease: A Predictor of Lymphoma Risk?

Background

Concominant with the widespread use of combined immunotherapy in the management of Crohn''s disease (CD), the incidence of hepato-splenic gamma-delta (γδ)-T cell lymphoma has increased sharply in CD patients. Malignant transformation of lymphocytes is believed to be a multistep process resulting in the selection of malignant γδ-T cell clones. We hypothesised that repeated infusion of anti-TNF-α agents may induce clonal selection and that concurrent treatment with immunomodulators further predisposes patients to γδ-T cell expansion.

Methodology/Principal Findings

We investigated dynamic changes in the γδ-T cells of patient with CD following treatment with infliximab (Remicade®; n = 20) or adalimumab (Humira®; n = 26) using flow cytometry. In patients with a high γδ-T cell level, the γδ-T cells were assessed for clonality. Of these 46 CD patients, 35 had a γδ-T cells level (mean 1.6%) comparable to healthy individuals (mean 2.2%), and 11 CD patients (24%) exhibited an increased level of γδ-T cells (5–15%). In the 18 patients also receiving thiopurines or methotrexate, the average baseline γδ-T cell level was 4.4%. In three male CD patients with a high baseline value, the γδ-T cell population increased dramatically following infliximab therapy. A fourth male patient also on infliximab monotherapy presented with 20% γδ-T cells, which increased to 25% shortly after treatment and was 36% between infusions. Clonality studies revealed an oligoclonal γδ-T cell pattern with dominant γδ-T cell clones. In support of our clinical findings, in vitro experiments showed a dose-dependent proliferative effect of anti-TNF-α agents on γδ-T cells.

Conclusion/Significance

CD patients treated with immunomodulators had constitutively high levels of γδ-T cells. Infliximab exacerbated clonal γδ-T cell expansion in vivo and induced γδ-T cell proliferation in vitro. Overall, young, male CD patients with high baseline γδ-T cell levels may be at an increased risk of developing malignant γδ-T cell lymphomas following treatment with anti-TNF-α agents.     

Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M_W = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.     

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

Streptococcus pyogenes and other Gram‐positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild‐type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125^Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Factors affecting intention to receive and self-reported receipt of 2009 pandemic (H1N1) vaccine in Hong Kong: a longitudinal study

Background

Vaccination was a core component for mitigating the 2009 influenza pandemic (pH1N1). However, a vaccination program''s efficacy largely depends on population compliance. We examined general population decision-making for pH1N1 vaccination using a modified Theory of Planned Behaviour (TBP).

Methodology

We conducted a longitudinal study, collecting data before and after the introduction of pH1N1 vaccine in Hong Kong. Structural equation modeling (SEM) tested if a modified TPB had explanatory utility for vaccine uptake among adults.

Principal Findings

Among 896 subjects who completed both the baseline and the follow-up surveys, 7% (67/896) reported being “likely/very likely/certain” to be vaccinated (intent) but two months later only 0.8% (7/896) reported having received pH1N1 vaccination. Perception of low risk from pH1N1 (60%) and concerns regarding adverse effects of the vaccine (37%) were primary justifications for avoiding pH1N1 vaccination. Greater perceived vaccine benefits (β = 0.15), less concerns regarding vaccine side-effects (β = −0.20), greater adherence to social norms of vaccination (β = 0.39), anticipated higher regret if not vaccinated (β = 0.47), perceived higher self-efficacy for vaccination (β = 0.12) and history of seasonal influenza vaccination (β = 0.12) were associated with higher intention to receive the pH1N1 vaccine, which in turn predicted self-reported vaccination uptake (β = 0.30). Social norm (β = 0.70), anticipated regret (β = 0.19) and vaccination intention (β = 0.31) were positively associated with, and accounted for 70% of variance in vaccination planning, which, in turn subsequently predicted self-reported vaccination uptake (β = 0.36) accounting for 36% of variance in reported vaccination behaviour.

Conclusions/Significance

Perceived low risk from pH1N1 and perceived high risk from pH1N1 vaccine inhibited pH1N1 vaccine uptake. Both the TPB and the additional components contributed to intended vaccination uptake but social norms and anticipated regret predominantly associated with vaccination intention and planning. Vaccination planning is a more significant proximal determinant of uptake of pH1N1 vaccine than is intention. Intention alone is an unreliable predictor of future vaccine uptake.     

Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer''s disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.     

Are ‘Endurance’ Alleles ‘Survival’ Alleles? Insights from the ACTN3 R577X Polymorphism

Exercise phenotypes have played a key role for ensuring survival over human evolution. We speculated that some genetic variants that influence exercise phenotypes could be associated with exceptional survival (i.e. reaching ≥100years of age). Owing to its effects on muscle structure/function, a potential candidate is the Arg(R)577Ter(X) polymorphism (rs1815739) in ACTN3, the structural gene encoding the skeletal muscle protein α-actinin-3. We compared the ACTN3 R577X genotype/allele frequencies between the following groups of ethnically-matched (Spanish) individuals: centenarians (cases, n = 64; 57 female; age range: 100–108 years), young healthy controls (n = 283, 67 females, 216 males; 21±2 years), and humans who are at the two end-points of exercise capacity phenotypes, i.e. muscle endurance (50 male professional road cyclists) and muscle power (63 male jumpers/sprinters). Although there were no differences in genotype/allele frequencies between centenarians (RR:28.8%; RX:47.5%; XX:23.7%), and controls (RR:31.8%; RX:49.8%; XX:18.4%) or endurance athletes (RR:28.0%; RX:46%; XX:26.0%), we observed a significantly higher frequency of the X allele (P = 0.019) and XX genotype (P = 0.011) in centenarians compared with power athletes (RR:47.6%; RX:36.5%;XX:15.9%). Notably, the frequency of the null XX (α-actinin-3 deficient) genotype in centenarians was the highest ever reported in non-athletic Caucasian populations. In conclusion, despite there were no significant differences with the younger, control population, overall the ACTN3 genotype of centenarians resembles that of world-class elite endurance athletes and differs from that of elite power athletes. Our preliminary data would suggest a certain ‘survival’ advantage brought about by α-actinin-3 deficiency and the ‘endurance’/oxidative muscle phenotype that is commonly associated with this condition.